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Molecular Mechanisms of Prostate Cancer
Abstract
During the last ten years our knowledge of genetic alterations in prostate cancer has significantly increased. For example, several chromosomal loci possibly harboring predisposing or somatically mutated genes have been suggested. Still, we lack the comprehensive molecular model for the development and progression of prostate cancer. Only a few genes have been found to be aberrant in a significant proportion of prostate cancer. These include GSTP1, PTEN, TP53, and AR. Thus, they are natural targets for new treatment strategies.
... Since hypermethylation androgens and thereby regulates growth, function and of GSTP1 is common in prostate cancer and neoplasia it differentiation of prostate cells [8]. It is expressed in could be used as a diagnostic marker [15,16]. primary prostate cancer as well as in advanced Gain or loss of specific alleles but also over hormone-refractory tumors and in metastatic lesions expression of tumor growth-promoting genes, so-called [9,10]. ...
... cell cycle and upon DNA damage either induces cell cycle The MSR1 gene encodes subunits of a macrophage-arrest for DNA repair or induces apoptosis. Thus, loss of scavenger receptor that is involved in recognition of p53 function increases cell survival [16]. ...
... cell survival [15,16]. At a late stage of disease when the cancer has become increased co activator expression and activation of metastatic patients are usually treated with androgen several kinases that may directly or indirectly sensitize AR withdrawal. ...
Prostate cancer is the second most common cause of death from malignancy in men. Gene therapy comes as a powerful approach to specifically treat advanced prostate cancer. Cancer gene therapy may be defined as the transfer of recombinant DNA into human cells to achieve an antitumor effect. The prostate is ideal for gene therapy. It is an accessory organ, offers unique prostate-specific antigen and prostate-specific membrane antigen. Viral and non-viral means are being used to transfer the genetic material into tumor cells. Summarizing, viruses are obviously potential vectors yielding high transfection efficiencies with the risk of increased genetic instability of transfected host cells and induces strong immunological responses. Suicide gene therapy using genetically engineered mesenchymal stem cells or neural stem cells has the advantage of being safe, because prodrug administration not only eliminates tumor cells but consequently kills the more resistant therapeutic stem cells as well. The number of clinical trials utilizing gene therapy methods for PCA is increasing.The main limiting factors for the development of an effective gene therapy are efficiency of gene transfer, selectivity of tumor targeting and the immunogenic properties of the vectors as well as general safety considerations.
... NajËeπÊe se zamje-Êuje aneuploidija kromosoma 7 i 8. NumeriËke kromosomske aberacije koreliraju s progresijom raka prostate, agresivnijim ponaπanjem tumora, nepovoljnom prognozom, odnosno s kraÊim vremenom preaeivljavanja. 1,5,9,18 Analize onkogena i tumor-supresorskih gena u raka prostate Molekularne analize tumora usmjerene su ka traaeenju konkretnih gena u svezi tumorogeneze i drugih bioloπkih svojstava kao metastazibilnosti, hormonske neovisnosti i sliËnih. ...
... U sluËaju HPC1 izgleda da je nasljeivanje tog autosomalno dominantnog alela odgovorno za 5 do 10% sluËajeva raka prostate, odnosno za 45% raka prostate u muπkaraca mlaih od 45 godina. 1,5,9,11,18 Drugi molekularni istraaeivaËki pristupi kreÊu od analize uklju-Ëenosti onkogena i tumor supresorskih gena naenih u drugih neoplazmi. Kako su ti geni okarakterizirani u drugih neoplazmi, postoje odgovarajuÊe probe za njihovu izolaciju i analizu, pa je tako istraaeivana njihova uloga i u raku prostate. ...
... Koncentracija PTEN je Ëesto reducirana, a osobito u karcinoma visokog gradusa ili stadija. 5,[18][19][20] Istraaeivanje ovisnosti na androgene Stanice karcinoma prostate najËeπÊe posjeduju hormonske-androgene receptore, a muπki spolni hormoni imaju vaaeno djelovanje na rast stanica karcinoma i njegovu progresiju. Stoga u sistemskom lijeËenju pacijenata s uznapredovalim rakom prostate prevladavaju postupci i lijekovi usmjereni ablaciji androgena. ...
Rak prostate nastaje kao posljedica akumulacije mnogobrojnih genetskih oštećenja. Neka od njih su naslijeđena, uglavnom kao sklonost malignoj transformaciji stanica epitela uslijed poremećenih mehanizama obrane od oštećenja gena. Većina njih uglavnom se stječe tijekom života, što uvjetuje i najčešću pojavu tog karcinoma u zrelijoj dobi. Od čimbenika okoline koji utječu na razvoj i progresiju karcinoma važni su kronična upala i infekcija, prehrana i neki lijekovi. Oni ne samo da utječu na pojavu karcinomskih stanica već djeluju i na stupanj njihove zloćudnosti u smislu invazivnosti, metastaziranja i time progresije bolesti odnosno njene prognoze. Pritom važnu ulogu ima pojava hormonske neovisnosti stanica raka. Točna etiopatogeneza raka prostate na molekularnoj razini još nije u potpunosti razjašnjena, ali napredovanjem znanosti i pronalaženjem novih metoda molekularne analize otvaraju se vrata i novim mogućnostima preciznijeg i specifičnog liječenja
... Regarding the molecular pathogenesis of PrC, it has been observed that certain genetic alterations may provoke the transformation of normal prostatic cells into cancerous cells. In this context, it has been reported that certain mutations in key specific genes such as PTEN, TP53, E-cadherin and β-catenin (5) are important in this transformation process. In addition, it has been demonstrated that several growth factors, such as insulin growth factor I (IGF-I), transforming growth factor α and β (TGFα or β) and members of the fibroblast growth factor (FGF) family, may be involved in the proliferation and metastasis of PrC (6). ...
... The molecular factors involved in the development of PrC are various and as the transformation process in the cell evolves, it acquires a malignant phenotype with the ability to invade and generate metastasis in different parts of the body, but with a preference to form metastatic lesions in the bones (5,6). For this reason, many compounds have been used to try and control the progression of prostate cancer. ...
In the early stages, prostate cancer is androgen‑ dependent; therefore, medical castration has shown significant results during the initial stages of this pathology. Despite this early effect, advanced prostate cancer is resilient to such treatment. Recent evidence shows that derivatives of Cannabis sativa and its analogs may exert a protective effect against different types of oncologic pathologies. The purpose of the present study was to detect the presence of cannabinoid receptors (CB1 and CB2) on cancer cells with a prostatic origin and to evaluate the effect of the in vitro use of synthetic analogs. In order to do this, we used a commercial cell line and primary cultures derived from prostate cancer and benign prostatic hyperplasia. The presence of the CB1 and CB2 receptors was determined by immunohistochemistry where we showed a higher expression of these receptors in later stages of the disease (samples with a high Gleason score). Later, treatments were conducted using anandamide, 2-arachidonoyl glycerol and a synthetic analog of anandamide, methanandamide. Using the MTT assay, we proved that the treatments produced a cell growth inhibitory effect on all the different prostate cancer cultures. This effect was demonstrated to be dose-dependent. The use of a specific CB1 receptor blocker (SR141716) confirmed that this effect was produced primarily from the activation of the CB1 receptor. In order to understand the MTT assay results, we determined cell cycle distribution by flow cytometry, which showed no variation at the different cell cycle stages in all the cultures after treatment. Treatment with endocannabinoids resulted in an increase in the percentage of apoptotic cells as determined by Annexin V assays and caused an increase in the levels of activated caspase-3 and a reduction in the levels of Bcl-2 confirming that the reduction in cell viability noted in the MTT assay was caused by the activation of the apoptotic pathway. Finally, we observed that endocannabinoid treatment activated the Erk pathway and at the same time, produced a decrease in the activation levels of the Akt pathway. Based on these results, we suggest that endocannabinoids may be a beneficial option for the treatment of prostate cancer that has become nonresponsive to common therapies.
... The nuclear receptor exerts its action by binding to cytoplasmic hormones and translocating to the nucleus where it interacts with basal transcription machinery to trigger the transcription of the target genes. AR mutations in un-treated prostate cancer are rare (112). Most metastatic androgen-independent prostate cancers express high levels of androgen-receptor gene transcripts. ...
... Examples of point mutations such as CAA-to-CGA or GCC-to-ACC were sporadically detected in the AR gene in metastatic cells of prostate cancer. AR mutations are more common in patients that have been treated for prostate cancer with anti-androgens (112). Some studies have shown that the CAG repeat polymorphism length has an inverse relationship with transcriptional activity of the AR (5, 111). ...
Prostate cancer is a prevalent disease with a high impact on patients' morbidity and mortality. Despite efforts to profile prostate cancer, the genetic alterations and biological processes that correlate with disease progression remain partially elusive. The purpose of this study is to review the recent evidence relating to the initiation and progression of prostate cancer in relation to the familial correlation of the disease, the genetic aberrations resulting in prostate cancer and the new molecular biology data regarding prostate cancer.
A Medline database search identified all the existing publications on the molecular events associated with the pathogenesis and evolution of prostate cancer. Particular emphasis was given on the specific genetic phenomena associated with prostate cancer.
Like other cancers, prostate cancer is caused by an accumulation of genetic alterations in a cell that drives it to malignant growth. Specific genes and gene alterations have been suggested to play a role in its development and progression. Aneuploidy, loss of heterozygosity, gene mutations, hypermethylation and inactivation of specific tumour suppressor genes such as GSTpi, APC, MDR1, GPX3 and others have been detected in prostate cancers, but generally only at a low or moderate frequency. The androgen receptor (AR) signalling pathway may play a crucial role in the early development of prostate cancer, as well as in the development of androgen-independent disease that fails to respond to hormone deprivation therapies. Other alterations linked to the transition to hormone-independence include amplification of MYC and increased expression of ERBB2 and BCL2. Inflammatory changes may also contribute to the development of prostate cancer.
The identification of specific molecular markers for prostate cancer may lead to its earliest detection and better prediction of its behavior. The better understanding of the molecular events affecting prostate cancer progression may result in the introduction of new drugs to target these events thus providing a potential cure and a tool for prevention of this very common disease.
... Although the chromosomal aberrations commonly associated with different stages of prostate cancer are well known, only a few individual genes have been shown to be involved in a significant proportion of prostate cancers (reviewed in Ref. 1). Identification of genetically or epigenetically altered genes in prostate cancer has been hampered by the lack of good model systems. ...
... However, 4 interesting matches were identified: 6q14.1-q14.3 and 10q26.12-q26.3, which are commonly lost, as well as 8q11.21 and 8q24.13-q24.3, which are frequently gained in prostate carcinomas. 1 Target genes of these alterations in human prostate cancer have been searched for more than a decade, but they are still not known. For example, for the 8q24.13-q24.3 ...
Lack of good models has complicated investigations on the mechanisms of prostate cancer. By far, the most commonly used transgenic mouse model of prostate cancer is TRAMP, which, however, has not been fully characterized for genetic and epigenetic aberrations. Here, we screened TRAMP-derived C2 cell line for the alterations using different microarray approaches, and compared it to human prostate cancer. TRAMP-C2 had relatively few genomic copy number alterations according to array comparative genomic hybridization (aCGH). However, the gene copy number and expression were significantly correlated (p < 0.001). Screening genes for promoter hypermethylation using demethylation treatment with 5-aza-2'-deoxycytidine and subsequent expression profiling indicated 43 putatively epigenetically silenced genes. Further studies revealed that clusterin is methylated in the TRAMP-C2 cell line, as well as in the human prostate cancer cell line LNCaP. Its expression was found to be significantly reduced (p < 0.01) in untreated and hormone-refractory human prostate carcinomas. Together with known function of clusterin, the data suggest an epigenetic component in the regulation of clusterin in prostate cancer.
... At the chromosomal level losses of genetic material are more common than gains or amplifications in prostate cancer. The two most common regions with deletions are 8p and 13q (Porkka and Visakorpi, 2004). The most promising target gene for the 8p loss is a homeobox gene NKX3.1 (He et al., 1997). ...
... The retinoblastoma gene (RB1) at 13q14 is a strong candidate as a cancer suppressor gene in this area, but controversial results on the loss of the RB1 locus have been reported (Latil et al., 1999; Chen et al., 2001; Ueda et al., 1999; Hosoki et al., 2002). Other regions showing losses in prostate cancer are 6q, 10q, 16q and 18q, indicating the presence of possible tumor suppressor genes in these regions (Porkka and Visakorpi, 2004). PTEN gene on 10q23 has been found to be mutated in the prostate cancer cell lines LNCaP and DU-145 (Li et al., 1997). ...
... (PCAP) (Berthon, Valeri et al. 1998), 8p22-23 (Xu, Zheng et al. 2001), 16q23 (Suarez, Lin et al. 2000), 17p12-p13 (Tavtigian, Simard et al. 2001), 19q13 (Witte, Goddard et al. 2000), 20q13 (HPC20) (Berry, Schroeder et al. 2000), and Xq27-q28 (HPCX) (Xu, Meyers et al. 1998) that may harbor high-penetrance prostate cancer susceptibility genes. However, none of the loci have been verified indisputably by a second independent study confirming the tremendous heterogeneity in the predisposition of prostate cancer (Porkka and Visakorpi 2004). Three candidate susceptibility genes have also been identified. ...
... The prostate tumors carrying this mutated gene have reduced RNASEL enzyme activity. RNASEL is an endoribonuclease involved in the mediation of the antiviral and proapoptotic activities of the interferon-regulated 2-5A system (Porkka and Visakorpi 2004). The third identified prostate cancer susceptibility gene is the macrophage scavenger receptor 1 (MSR1) gene, located at 8p22-23 (Xu, Zheng et al. 2002). ...
Advanced stage head and neck cancers (HNC) with distant metastasis, as well as prostate cancers (PC), are devastating diseases currently lacking efficient treatment options. One promising developmental approach in cancer treatment is the use of oncolytic adenoviruses, especially in combination therapy with conventional cancer therapies. The safety of the approach has been tested in many clinical trials. However, antitumor efficacy needs to be improved in order to establish oncolytic viruses as a viable treatment alternative. To be able to test in vivo the effects on anti-tumor efficiency of a multimodal combination therapy of oncolytic adenoviruses with the standard therapeutic combination of radiotherapy, chemotherapy and Cetuximab monoclonal antibody (mAb), a xenograft HNC tumor model was developed. This model mimics the typical clinical situation as it is initially sensitive to cetuximab, but resistance develops eventually. Surprisingly, but in agreement with recent findings for chemotherapy and radiotherapy, a higher proportion of cells positive for HNC cancer stem cell markers were found in the tumors refractory to cetuximab. In vitro as well as in vivo results found in this study support the multimodal combination therapy of oncolytic adenoviruses with chemotherapy, radiotherapy and monoclonal antibody therapy to achieve increased anti-tumor efficiency and even complete tumor eradication with lower treatment doses required. In this study, it was found that capsid modified oncolytic viruses have increased gene transfer to cancer cells as well as an increased antitumor effect. In order to elucidate the mechanism of how oncolytic viruses promote radiosensitization of tumor cells in vivo, replicative deficient viruses expressing several promising radiosensitizing viral proteins were tested. The results of this study indicated that oncolytic adenoviruses promote radiosensitization by delaying the repair of DNA double strand breaks in tumor cells. Based on the promising data of the first study, two tumor double-targeted oncolytic adenoviruses armed with the fusion suicide gene FCU1 or with a fully human mAb specific for human Cytotoxic T Lymphocyte-Associated Antigen 4 (CTLA-4) were produced. FCU1 encodes a bifunctional fusion protein that efficiently catalyzes the direct conversion of 5-FC, a relatively nontoxic antifungal agent, into the toxic metabolites 5-fluorouracil and 5-fluorouridine monophosphate, bypassing the natural resistance of certain human tumor cells to 5-fluorouracil. Anti-CTLA4 mAb promotes direct killing of tumor cells via apoptosis and most importantly immune system activation against the tumors. These armed oncolytic viruses present increased anti-tumor efficacy both in vitro and in vivo. Furthermore, by taking advantage of the unique tumor targeted gene transfer of oncolytic adenoviruses, functional high tumor titers but low systemic concentrations of the armed proteins were generated. In addition, supernatants of tumor cells infected with Ad5/3-24aCTLA4, which contain anti-CTLA4 mAb, were able to effectively immunomodulate peripheral blood mononuclear cells (PBMC) of cancer patients with advanced tumors. -- In conclusion, the results presented in this thesis suggest that genetically engineered oncolytic adenoviruses have great potential in the treatment of advanced and metastatic HNC and PC. Pitkälle levinneisiin pään ja kaulan alueen syöpiin sekä eturauhassyöpään ei ole olemassa tehokkaita parantavia hoitomuotoja. Onkolyyttisten virusten käyttö yhdessä perinteisten hoitojen kanssa on lupaava kokeellinen hoitomuoto ja sen turvallisuus on osoitettu useissa kliinisissä kokeissa. Kasvainta tuhoavaa vaikutusta pitää kuitenkin tehostaa riittävän hoitovasteen saavuttamiseksi. Tässä työssä kehitettiin pään ja kaulan alueen syövän eläinmalli, jonka avulla tutkittiin onkolyyttisten virusten, solunsalpaajien ja monoklonaalisen vasta-aineen (Setuksimab) kasvainta tuhoavaa yhteisvaikutusta. Tämä malli jäljittelee kliinistä tilannetta hyvin, sillä alun perin setuksimabille herkät syöpäsolut kehittivät nopeasti hoitoresistenssin. Kuten viimeaikaiset tutkimukset koskien solunsalpaajia ja sädehoitoa ovat osoittaneet, erityisesti syöpäsolut jotka ilmentävät syövän kantasoluille tyypillisiä pintaproteiineja olivat resistenttejä setuksimabille. Multimodaalihoito käyttäen onkolyyttisiä viruksia, solunsalpaajia ja monoklonaalista vasta-ainetta paransi hoidon kasvainta tuhoavaa vaikutusta, pienensi tarvittavia hoitoannoksia ja johti jopa kasvainten häviämiseen. Onkolyyttisten virusten kykyä herkistää syöpäsolut sädehoidolle tutkittiin eläinmallissa siirtämällä syöpäsoluihin ei-replikoituvien virusten avulla useita sädehoidolle herkistäviä viruksen proteiinejä. Tulokset osoittivat, että onkolyyttiset adenovirukset herkistävät syöpäsolut sädehoidolle viivyttämällä DNA:n kaksoiskierteeseen syntyvien katkosten korjausta. Tässä työssä kehitettiin kaksi syöpäsoluihin kohdennettua onkolyyttistä adenovirusta joista toinen oli aseistettu FCU1 itsemurhageenillä ja toinen ihmisen sytotoksiseen T lymfosyyttiin liittyvän antigeeni 4:n (CTLA-4) monoklonaalisella vasta-aineella. FCU1 koodaa proteiiniä joka katalysoi ei-myrkyllisen 5-fluorosytosiinin (5-FC) muuntumista myrkyllisiksi metaboliiteiksi, 5-fluorourasiiliksi ja 5-fluori uridiini monofosfaatiksi. CTLA-4:n monoklonaalinen vasta-aine edistää syopäsolujen apoptoottista kuolemista ja aktivoi elimistön immuunijärjestelmän kasvainta tuhoavaa vaikutusta. Molemmilla viruksilla osoitettiin olevan parempi kasvainta tuhoava vaikutus verrattuna muokkaamattomiin viruksiin. Onkolyyttisten virusten avulla siirtogeenin ilmentyminen voidaan kohdentaa syöpäsoluihin. Kasvaimessa todettiin suuri pitoisuus proteiinia, kun taas proteiinin pitoisuus veressä pysyi alhaisena. Tämän lisäksi, Ad5/3-24aCTLA4 viruksella infektoitujen solujen kasvatusliuoksella, joka sisälsi CTLA4:n monoklonaalista vasta-ainettta, pystyttiin aktivoimaan edennyttä syöpää sairastavien potilaiden verestä eristettyjä mononukleaarisia valkosoluja (PBMC). Yhteenvetona, tulokset osoittavat että geneettisesti muokattuja adenoviruksia voidaan käyttää edenneiden pään ja kaulan alueiden syöpien sekä eturauhassyövän hoidossa.
... PCa is the most frequently diagnosed cancer in men in the Western world. The course of PCa is largely driven by androgen receptor (AR) and, accordingly, androgen ablation therapy is a cornerstone of current therapies (1,2). A large proportion of patients with advanced PCa become resistant to ADT resulting in lethal castration resistant prostate cancer (CRPC). ...
Androgen deprivation therapy (ADT) remains a key approach in the treatment of prostate cancer (PCa). However, PCa inevitably relapses and becomes ADT resistant. Besides androgens, there is evidence that thyroid hormone thyroxine (T4) and its active form 3,5,3’-triiodo-L-thyronine (T3) are involved in the progression of PCa. Epidemiologic evidence indicates a higher incidence of PCa in men with elevated thyroid hormone levels. The thyroid hormone binding protein μ-Crystallin (CRYM) mediates intracellular thyroid hormone action by sequestering T3 and blocks its binding to cognate receptors (TRa/TRb) in target tissues. We show in this study that low CRYM expression levels in PCa patient samples are associated with early BCR and poor prognosis. Moreover, we found a disease stage-specific expression of CRYM in PCa. CRYM counteracted thyroid and androgen signaling and blocked intracellular choline uptake. CRYM inversely correlated with [18F]fluoromethylcholine (FMC) levels in PET/MRI imaging of PCa patients. Our data suggest CRYM as a novel antagonist of T3 and androgen-mediated signalling. The role of CRYM could therefore be an essential control mechanism for the prevention of aggressive PCa growth.
Highlights
Thyroid and androgen hormone driven pathways in prostate cancer (PCa) are antagonized by μ- Crystallin (CRYM).
[18F]fluoromethylcholine uptake and prognostic values in PCa correlate with CRYM protein levels.
Reduced CRYM expression predicts early biochemical recurrence (BCR) in PCa patients.
... Checkpoint kinase2 (CHEK2) gene monitors checkpoints of the cell cycle that regulate cell division. Mutation in CHEK2 gene results in the development of cancer types like colorectal [15], breast [16], and prostate [17]. When subjected to genomic stress, p53 and CHEK2 are activated by ataxia telangiectasia mutated (ATM) protein, and thereby involved in DNA repair, regulate cell cycle and senescence [18,19]. ...
Purpose: To investigate the potential role of checkpoint kinase2 (CHEK2) in regulating gastric cancer stem cells. Methods: The abnormal features of cellular arrangements in early and advanced stages of gastric cancer were analysed using histology. Immunohistochemistry and Western blotting were used to evaluate the expressions of cancer stem cell markers CD44 and CHEK2 at different stages of gastric cancer. Results: When compared with the control tissue, the size of the cellular nucleus was enlarged as the gastric tumour developed to more advanced stages, and the expression of CD44 increased exponentially. The expression of CHEK2, a protein that regulates cell cycle and apoptosis was aberrantly up-regulated at the early stage of cancer, but as the tumour advanced, its expression became down-regulated. Conclusion: These results show that the higher expression of CHEK2 at the early stages of gastric cancer exerts control over gastric cancer stem cell proliferation. Thus, CHECK2 is a potential target for early treatment of gastric cancer. © Pharmacotherapy Group, Faculty of Pharmacy, University of Benin, Benin City, 300001 Nigeria and 2018 The authors.
... HPC2/ELAC2 (17p12) genes. The involvement of these 3 putative susceptibility genes was very infrequent in sporadic CaP (Porkka and Visakorpi, 2004 19. It has been proposed that these candidate genes and their products may be used to screen CaP and potentially be used as therapeutic targets. ...
... Changes in some genes expression of a significant rate have been detected in PCa, i.e. GSTP1, PTEN, NkX3.1, TP53, AR, CDH1, and CTNNB1 [98]. By microarray gene expression profiling, a discovery of PCa biomarkers: AMACR, EZH2, TMPRSS2-ERG, mi-R-221, and miR-141 was possible [99]. ...
Although the worldwide urological community generally accepts the existing protocol of TRUS-guided (transrectal ultrasound-guided) random prostate biopsy, there is strong evidence that sextant, extended, and saturation protocols are not sufficiently accurate (with ranges from 28-78%). Moreover, the number of repeated biopsies remains extremely high (33%) as a consequence of using an imperfect diagnostic tool. An overview of current literature concerning common practice in prostatic biopsies has revealed discrepancies in indications, technique, number of cores collected, and pathological examination standards. This fact has prompted many authors to search for methods of improving the existing standard. The latest developments in the field are primarily related to MRI-guided (magnetic resonance-guided) targeted prostate biopsy, which is currently a promising tool in diagnosing prostate cancer. However, genetically supported molecular biopsy seems to be a highly promising avenue for developing the future biopsy standard.
... Ultimately, all prostate cancers become hormone-refractory (HRPC), i.e. androgen-independent 10 . The switch to androgen-independent tumors has been associated with an increase in the mutational frequency of the androgen receptor (AR) and in nearly 30% of androgen-independent tumors duplication of AR can be found 11 . Metastatic HRPC is the most important cause of morbidity and mortality and there are no effective treatments available. ...
... Prostate cancer (PCa) is the second most common cause of cancer related deaths in men in the United States, and is one of the leading causes of sickness and death in men in the U.S. and Western Europe [8,9]. Despite this high prevalence, the molecular mechanisms of PCa progression still remain largely unknown, due in part to heterogeneity during tumor development [10]. PCa is initially androgen-dependent, making androgen deprivation therapy the first line of defense in combating the disease [11]. ...
Background
Insertional mutagenesis screens have been used with great success to identify oncogenes and tumor suppressor genes. Typically, these screens use gammaretroviruses (gammaRV) or transposons as insertional mutagens. However, insertional mutations from replication-competent gammaRVs or transposons that occur later during oncogenesis can produce passenger mutations that do not drive cancer progression. Here, we utilized a replication-incompetent lentiviral vector (LV) to perform an insertional mutagenesis screen to identify genes in the progression to androgen-independent prostate cancer (AIPC).
Methods
Prostate cancer cells were mutagenized with a LV to enrich for clones with a selective advantage in an androgen-deficient environment provided by a dysregulated gene(s) near the vector integration site. We performed our screen using an in vitro AIPC model and also an in vivo xenotransplant model for AIPC. Our approach identified proviral integration sites utilizing a shuttle vector that allows for rapid rescue of plasmids in E. coli that contain LV long terminal repeat (LTR)-chromosome junctions. This shuttle vector approach does not require PCR amplification and has several advantages over PCR-based techniques.
Results
Proviral integrations were enriched near prostate cancer susceptibility in cells grown in androgen-deficient medium (p < 0.001), and five candidate genes that influence AIPC were identified; ATPAF1, GCOM1, MEX3D, PTRF, and TRPM4. Additionally, we showed that RNAi knockdown of ATPAF1 significantly reduces growth (p < 0.05) in androgen-deficient conditions.
Conclusions
Our approach has proven effective for use in PCa, identifying a known prostate cancer gene, PTRF, and also several genes not previously associated with prostate cancer. The replication-incompetent shuttle vector approach has broad potential applications for cancer gene discovery, and for interrogating diverse biological and disease processes.
... Prostate cancer (PCa) is the second most common cause of cancer related deaths in men in the United States, and is one of the leading causes of sickness and death in men in the U.S. and Western Europe [8,9]. Despite this high prevalence, the molecular mechanisms of PCa progression still remain largely unknown, due in part to heterogeneity during tumor development [10]. PCa is initially androgen-dependent, making androgen deprivation therapy the first line of defense in combating the disease [11]. ...
BACKGROUND:Insertional mutagenesis screens have been used with great success to identify oncogenes and tumor suppressor genes. Typically, these screens use gammaretroviruses (gammaRV) or transposons as insertional mutagens. However, insertional mutations from replication-competent gammaRVs or transposons that occur later during oncogenesis can produce passenger mutations that do not drive cancer progression. Here, we utilized a replication-incompetent lentiviral vector (LV) to perform an insertional mutagenesis screen to identify genes in the progression to androgen-independent prostate cancer (AIPC).METHODS:Prostate cancer cells were mutagenized with a LV to enrich for clones with a selective advantage in an androgen-deficient environment provided by a dysregulated gene(s) near the vector integration site. We performed our screen using an in vitro AIPC model and also an in vivo xenotransplant model for AIPC. Our approach identified proviral integration sites utilizing a shuttle vector that allows for rapid rescue of plasmids in E. coli that contain LV long terminal repeat (LTR)-chromosome junctions. This shuttle vector approach does not require PCR amplification and has several advantages over PCR-based techniques.RESULTS:Proviral integrations were enriched near prostate cancer susceptibility in cells grown in androgen-deficient medium (p < 0.001), and five candidate genes that influence AIPC were identified; ATPAF1, GCOM1, MEX3D, PTRF, and TRPM4. Additionally, we showed that RNAi knockdown of ATPAF1 significantly reduces growth (p < 0.05) in androgen-deficient conditions.CONCLUSIONS:Our approach has proven effective for use in PCa, identifying a known prostate cancer gene, PTRF, and also several genes not previously associated with prostate cancer. The replication-incompetent shuttle vector approach has broad potential applications for cancer gene discovery, and for interrogating diverse biological and disease processes.
... Prostate cancer is driven by androgen receptor signaling [12], and is likely also influenced by basic cellular processes that contribute to other cancers [35,36]. Therefore there are two classes of potential targets. ...
Genome-wide association studies (GWAS) have revolutionized the field of cancer genetics, but the causal links between increased genetic risk and onset/progression of disease processes remain to be identified. Here we report the first step in such an endeavor for prostate cancer. We provide a comprehensive annotation of the 77 known risk loci, based upon highly correlated variants in biologically relevant chromatin annotations--we identified 727 such potentially functional SNPs. We also provide a detailed account of possible protein disruption, microRNA target sequence disruption and regulatory response element disruption of all correlated SNPs at r(2) ≥ 0.88%. 88% of the 727 SNPs fall within putative enhancers, and many alter critical residues in the response elements of transcription factors known to be involved in prostate biology. We define as risk enhancers those regions with enhancer chromatin biofeatures in prostate-derived cell lines with prostate-cancer correlated SNPs. To aid the identification of these enhancers, we performed genomewide ChIP-seq for H3K27-acetylation, a mark of actively engaged enhancers, as well as the transcription factor TCF7L2. We analyzed in depth three variants in risk enhancers, two of which show significantly altered androgen sensitivity in LNCaP cells. This includes rs4907792, that is in linkage disequilibrium (r(2) = 0.91) with an eQTL for NUDT11 (on the X chromosome) in prostate tissue, and rs10486567, the index SNP in intron 3 of the JAZF1 gene on chromosome 7. Rs4907792 is within a critical residue of a strong consensus androgen response element that is interrupted in the protective allele, resulting in a 56% decrease in its androgen sensitivity, whereas rs10486567 affects both NKX3-1 and FOXA-AR motifs where the risk allele results in a 39% increase in basal activity and a 28% fold-increase in androgen stimulated enhancer activity. Identification of such enhancer variants and their potential target genes represents a preliminary step in connecting risk to disease process.
... The researches about the molecular genetics and growth regulation of the prostate cancer have made remarkable progress (Singh et al., 2012). For example, large-scale genomic rearrangements and gene mutation found in prostate cancer may be the molecular mechanism of the cancer (Porkkaand Visakorpi, 2004). Abundant of reports about the regulatory functions of transcription factors and microRNAs in cancer have Department of Urology, the 452 nd Hospital of PLA, Chengdu, China *For correspondence: xuyahong452@hotmail.com Abstract Purpose: Prostate cancer caused by the abnormal disorderly growth of prostatic acinar cells is the most prevalent cancer of men in western countries. ...
Purpose:
Prostate cancer caused by the abnormal disorderly growth of prostatic acinar cells is the most prevalent cancer of men in western countries. We aimed to screen out differentially expressed genes (DEGs) and explore small molecule drugs for prostate cancer.
Materials and methods:
The GSE3824 gene expression profile of prostate cancer was downloaded from Gene Expression Omnibus database which including 21 normal samples and 18 prostate cancer cells. The DEGs were identified by Limma package in R language and gene ontology and pathway enrichment analyses were performed. In addition, potential regulatory microRNAs and the target sites of the transcription factors were screened out based on the molecular signature database. In addition, the DEGs were mapped to the connectivity map database to identify potential small molecule drugs.
Results:
A total of 6,588 genes were filtered as DEGs between normal and prostate cancer samples. Examples such as ITGB6, ITGB3, ITGAV and ITGA2 may induce prostate cancer through actions on the focal adhesion pathway. Furthermore, the transcription factor, SP1, and its target genes ARHGAP26 and USF1 were identified. The most significant microRNA, MIR-506, was screened and found to regulate genes including ITGB1 and ITGB3. Additionally, small molecules MS-275, 8-azaguanine and pyrvinium were discovered to have the potential to repair the disordered metabolic pathways, abd furthermore to remedy prostate cancer.
Conclusions:
The results of our analysis bear on the mechanism of prostate cancer and allow screening for small molecular drugs for this cancer. The findings have the potential for future use in the clinic for treatment of prostate cancer.
... È ormai riconosciuto che la patologia neoplastica è prima di tutto una patologia genetica (9). Per il CaP studi genetici ed epidemiologici hanno messo in evidenza la presenza di una predisposizione ereditaria (10)(11)(12)(13)(14)(15), e studi di citogenetica, di analisi di perdita dell'eterozigosi (LOH) e di ibridazione genomica comparativa hanno evidenziato numerose alterazioni somatiche (16,17). ...
PURPOSE.Uro-oncological translational research requires clinical data and human biological tissues collected within a biological tissue bank (BTB). We are hereby outlining ethic-legal, methodological and technical issues of a BTB establishment process, focusing particularly on prostate cancer and Italian setting. MATERIALSANDMETHODS. Review of literature data, and national and international regulations and guidelines; direct field experience of urological BTB; counseling of the different pro- fessionals involved. RESULTS. Within a BTB establishment process, it is of utmost importance to protect the donors' privacy and rights through the programmatic adoption of the following procedures: 1) informed consent; 2) confidentiality protection thanks to anonymity of biological speci- mens and use of an "honest broker" method; 3) identification of a single responsible re- searcher; 4) dedicated and protected location; 5) approval of the Ethical Committee. There are two main organizational models of BTB: "systematic", i.e. collecting specimens from all patients and through the same methodology; "project-driven", i.e. prospectively selecting patients for a specific study and using the specific methods required by researchers. In the preliminary step it is necessary to establish detailed protocols of sampling and crio- conservation techniques, and methods of validation and quality control. For prostate tis- sue sampling, several techniques have been described such as specimens of alternate slices, macro dissection, Tru-Cut. CONCLUSIONS. Today BTBs are necessary in order to support molecular and translational re- search in uro-oncology, and to overcome the limits of the research based only on clinical- pathological data. Ethic-legal and methodological issues related to BTBs are still requiring specific legislation and standardization of techniques. (Urologia 2007; 74: 61-72)
... The PTEN tumor suppressor gene is one of the most frequently mutated/deleted genes in human prostate cancer, especially in advanced or metastatic forms [1]. Our murine Pten prostate cancer model mimics human prostate cancer development and provides an ideal experimental tool for characterization of the molecular events in disease initiation and progression [2]. ...
Both PTEN and Nkx3.1 play important role in prostate cancer control. Preliminary results from Pten prostate model indicated that reduced Nkx3.1 expression is correlated with Cre-mediated Pten deletion. The goal of this study is to investigate how these two proteins interplay in prostate cancer development. We demonstrate that restoration of Nkx3.1 into Pten null prostate epithelium can lead to increases cell apoptosis, decreased cell proliferation and prevention of tumor initiation. The anti-tumor effect of Nkx3.1 is correlated with stabilization p53, inhibition Akt activation and AR function. This novel finding was published in Cancer cell. We generated and characterize the primary cell lines from Pten prostate cancer model, which will facilitate us to study the molecular mechanism of hormone refractory prostate cancer.
... Invasion and metastasis of different types of cancers are highly complex and coordinated events. These steps include detachment of cancer cells from primary tumour, directional migration, invasion into the micro-vasculature (micro-metastasis), extravasation and finally spreading of tumours to distant tissues (Porkka and Visakorpi, 2004). The initial, as well as, the rate-limiting step in this process is the cellular recognition of various extracellular matrix (ECM) proteins, resulting in the spreading and acquisition of migratory properties in response to chemotactic stimuli. ...
Activation of Akt and increased expression of integrin β(3) are the two most important changes that have been linked to the attainment of metastatic potential by prostate cancer cells. However, a direct link between Akt activity and inside-out activation of integrin β(3) in mediating prostate cancer cell metastatic properties is not established.
Using functional and biochemical approaches, we examined the role of Akt1 in the affinity modulation of integrin β(3) in prostate cancer cells.
Although expression of murine TRAMP and human PC3 cells with constitutively active Akt1 (CA-Akt1) enhanced their affinity for integrin α(v)β(3) specific ligands and motility on various extracellular matrix proteins, the reverse was observed with the expression of dominant-negative Akt1 (DN-Akt1). Although enhanced motility and transendothelial migration of CA-Akt1-expressing cells were blunted by co-expression with DN-integrin β(3) or upon pre-treatment with integrin β(3)-blocking antibodies (LM 609), impaired motility and transendothelial migration of DN-Akt1-expressing cells were rescued by pre-treatment of prostate cancer cells with integrin β(3)-activating antibodies, AP7.4.
Our data is the first to demonstrate a link between Akt1 activity and affinity modulation of integrin β(3) in the regulation of prostate cancer cell motility, transendothelial migration and chemotaxis to metastatic stimuli.
... These tumors are heterogenous and still Lukas Kenner and Zoran Culig contributed equally to this work. ß 2009 Wiley-Liss, Inc. contain active AR signaling456. Thus, identification of novel androgen-regulated genes implicated in prostate cancer development and progression is of great interest. ...
BACKGROUND
Androgen receptor (AR) signaling is implicated in prostate cancer progression. Therefore, identification of AR downstream genes is potentially important for selection of novel markers and therapy targets in prostate cancer.METHODS
Expression of a thyroid hormone T3-binding protein µ-crystallin (CRYM) mRNA and protein in cell lines was evaluated by real-time PCR and Western blot, respectively. CRYM expression in vivo was analyzed in patients' samples by immunohistochemistry. The effects of androgen and T3 on proliferation of MDA PCa 2b cells were assessed by 3H-thymidine uptake assay.RESULTSCRYM expression was detected in AR-positive LNCaP and MDA PCa 2b cells. In MDA PCA 2b cells, CRYM was regulated by androgens. Androgen-induced CRYM expression was diminished by antiandrogens or AR siRNA. Inhibition of transcription by α-amanitin caused a reduction in CRYM mRNA. The lack of CRYM expression was noted in LAPC-4 cells and in AR-negative prostate cancer cell lines PC-3 and DU-145. CRYM protein was increased in cancer tissue and decreased in samples from patients after hormonal therapy. In samples from patients with therapy-refractory cancer CRYM was not detectable. We also found that androgens and T3 have additive effects on stimulation of MDA PCa 2b cells proliferation.CONCLUSIONCRYM is a novel androgen-regulated gene whose expression is elevated in prostate cancer but down-regulated in castration therapy-resistant tumors. Prostate 69:1109–1118, 2009. © 2009 Wiley-Liss, Inc.
... Gene copy number gain is a common mechanism by which genes achieve overexpression in tumors. For prostate cancer, amplifications of specific genes, such as c-MYC, HER2/neu, and androgen receptor, have been shown to be of prognostic relevance (1). However, most of the chromosomal regions found amplified in prostate cancer by comparative genomic hybridization (CGH) do not harbor known oncogenes. ...
Gain at chromosome 3q25-q26 has been reported to commonly occur in prostate cancer. To map the 3q25-q26 amplification unit and to identify the candidate genes of amplification, we did fluorescence in situ hybridization and quantitative real-time PCR for gene copy number and mRNA expression measurements in prostate cancer cell lines and prostate cancer samples from radical prostatectomy specimens. The minimal overlapping region of DNA copy number gains in the cell lines could be narrowed down to 700 kb at 3q26.2. Of all positional and functional candidates in this region, the gene TLOC1/SEC62 revealed the highest frequency (50%) of copy number gains in the prostate cancer samples and was found to be up-regulated at the mRNA level in all samples analyzed. TLOC1/Sec62 protein was also shown to be overexpressed by Western blot analysis. Intriguingly, the TLOC1/SEC62 gene copy number was increased in prostate tumors from patients who had a lower risk of and a longer time to progression following radical prostatectomy. These findings make TLOC1/SEC62 the best candidate within the 3q amplification unit in prostate cancer. TLOC1/Sec62 protein is a component of the endoplasmic reticulum protein translocation machinery, whose function during prostate carcinogenesis remains to be determined.
... Gene copy number gain is a common mechanism by which genes achieve overexpression in tumors. For prostate cancer, amplifications of specific genes, such as c-MYC, HER2/neu, and androgen receptor, have been shown to be of prognostic relevance (1). However, most of the chromosomal regions found amplified in prostate cancer by comparative genomic hybridization (CGH) do not harbor known oncogenes. ...
... Interestingly, in humans, TP53 mutations rarely occur in primary prostatic carcinomas, yet they are found much more frequently (upwards of 50%) in advanced metastatic and hormone refractory metastatic prostate cancer. 69 In terms of genetically engineered mouse models, Zhang et al. described a rat C(3)1 promoter-driven MYC transgenic mouse. 70 In these mice, forced overexpression of mouse Myc was observed in the ventral prostates as well as the testes, the latter because of an insufficiency in promoter specificity. ...
Prostate cancer, the majority of which is adenocarcinoma, is the most common epithelial cancer affecting a majority of elderly men in Western nations. Its manifestation, however, varies from clinically asymptomatic insidious neoplasms that progress slowly and do not threaten life to one that is highly aggressive with a propensity for metastatic spread and lethality if not treated in time. A number of somatic genetic and epigenetic alterations occur in prostate cancer cells. Some of these changes, such as loss of the tumor suppressors PTEN and p53, are linked to disease progression. Others, such as ETS gene fusions, appear to be linked more with early phases of the disease, such as invasion. Alterations in chromosome 8q24 in the region of MYC have also been linked to disease aggressiveness for many years. However, a number of recent studies in human tissues have indicated that MYC appears to be activated at the earliest phases of prostate cancer (e.g., in tumor-initiating cells) in prostatic intraepithelial neoplasia, a key precursor lesion to invasive prostatic adenocarcinoma. The initiation and early progression of prostate cancer can be recapitulated in genetically engineered mouse models, permitting a richer understanding of the cause and effects of loss of tumor suppressors and activation of MYC. The combination of studies using human tissues and mouse models paints an emerging molecular picture of prostate cancer development and early progression. This picture reveals that MYC contributes to disease initiation and progression by stimulating an embryonic stem cell-like signature characterized by an enrichment of genes involved in ribosome biogenesis and by repressing differentiation. These insights pave the way to potential novel therapeutic concepts based on MYC biology.
... Despite the extensive clinical and experimental studies over the recent decades, the pathogenesis of PC remains unanswered (reviewed in [1,2]). The interaction of genetics and the environment and its influence on the molecular mechanisms responsible in the development and progression of malignant prostate cells are largely unknown [24][25][26]. There is a great need to explore the role of differential gene expression that leads to altered cellular metabolism as an essential factor in prostate malignancy [27]. ...
Zinc (Zn) is essential for a very large number and variety of cellular functions but is also potentially toxic. Zn homeostasis is therefore dynamically maintained by a variety of transporters and other proteins distributed in distinct cellular and subcellular compartments. Zn transport is mediated by two major protein families: the Zip family, which mediates Zn influx, and the ZnTs which are primarily linked to Zn sequestration into intracellular compartments and are, thereby, involved in lowering cytoplasmic Zn free ion concentrations. In the prostate epithelial cell, the accumulation of high cellular zinc is a specialized function that is necessary for these cells to carry out the major physiological functions of production and secretion of prostatic fluids. The loss of Zn accumulation is the most consistent and persistent characteristic of prostate malignancy. Currently, there are no direct methods to determine the relative Zn levels in various cell types of prostate gland (i.e. stroma, glandular epithelia, acini, and muscular) and no reliable ways to compare the Zn in normal versus malignant areas of the gland. Here we report a new method to show a differential Zn staining method that correlates with various stages of prostate cancer development in situ and expression of a human Zn transporter1-hZIP1 -in situ by in situ reverse transcriptase-polymerase chain reaction hybridization (ISRTPCR) that correlate with the relative Zn levels determined by the differential Zn staining method. By utilizing these methods, we show for the first time that: (1) the relative Zn levels are very low to absent in the malignant glands, (2) normal glands show high Zn levels in both glandular epithelia as well as in stromal tissues, (3) the Zn levels begin to decrease in pre-malignant glands and precedes the development of malignancy, and (4) the expression of human Zn transporter1 (hZIP1) appears to correlate with the Zn levels in the prostate glands and may be the major Zn regulator in this organ.
... Chromosomal CGH and loss of heterozygosity (LOH) studies have verified that in early-stage tumours genomic DNA losses prevail over gains, with 6q, 8p, 10q, 13q, 16q and 17p being implicated most often in these alterations. In hormone-refractory prostate cancer, LOH frequency raises three-to fourfold and chromosomal gains also become prevalent: amplifications at 7p, 7q, 8q and Xq are common late events associated with an aggressive phenotype (Visakorpi et al. 1995, Nupponen et al. 1998a,b, Porkka & Visakorpi 2004, Saramaki et al. 2006). In particular, gain of the 8q region is the most frequent (80–90% of cases) and huge chromosomal alteration in advanced prostate tumours, involving nearly the whole 8q arm. ...
MicroRNAs (miRNAs) are a class of small, non-coding, single-stranded RNAs that negatively regulate gene expression by mainly
binding to 3′ untranslated region (UTR) of target mRNAs at the post-transcriptional level. Recent studies have demonstrated
that aberrant expressions of miRNAs are closely associated with the development, invasion, metastasis and prognosis of various
cancers including prostate cancer (PCa). The proposed molecular mechanisms that underlie the aberrant expression of miRNAs
result from gene changes, epigenetic modification and alteration of Dicer abundance. Although up to 50 miRNAs have been reported
to be significantly expressed in human PCa, only a small number of them were experimentally shown to make contribution to
the pathogenesis of PCa. The aim of this review is to describe the mechanisms of several known miRNAs, summarize recent studies
on the relevance of altered expression of oncogenic miRNAs (e.g. miR-221/-222, miR-21, and miR-125b) and tumor suppressor
miRNAs (e.g. miR-101, miR-126*, miR-146a, miR-330, miR-34 cluster, and miR-200 family) for PCa. Additionally, their potential
clinical applications and prospects in PCa, such as biomarkers and clinical therapies, are also briefly discussed.
... The hereditary prostate cancer locus-1 (HPC1) was the first locust identified1213, offering proof of a hereditary link to prostate cancer. Several other genes linked to family inheritance were discovered afterwards, such as EPAC2, RNASEL, MSR1, CHEK2, CAPZB, vitamin D receptor, PON1, and BRCA21314151617. In addition, genetic predisposition can also stem from ethnic origins. ...
Increasing evidence suggests that oxidative stress plays a causative role in prostate cancer initiation and progression. Furthermore, enhanced levels of systemic oxidative stress detected in lymphocytes are also associated with prostate cancer risk. Due to mtDNA's high sensitivity to oxidative stress, we hypothesize that mtDNA may serve as a surrogate to oxidative stress in cancer cells and lymphocytes. First, we improved the sensitivity of a novel method for mtDNA damage analysis and proposed a standardized protocol. Secondly, we developed an approach for quantifying systemic oxidative stress using mtDNA responses in circulating lymphocytes. Finally, we demonstrated differential mtDNA responses induced by oxidative stress in two isogenic prostate cancer cell lines with different metastatic potential. The more metastatic C4-2 was more susceptible to oxidative stress and prone to mtDNA damage and copy number change. These results offer new insights into prostate cancer progression. De plus en plus d'études suggèrent que le stress oxydatif joue un rôle déterminant dans l'initiation et la progression du cancer de la prostate. Aussi, des niveaux élevés de stress oxydatif systémique mesurés dans les lymphocytes ont été associés à un risque accru du cancer de la prostate. Puisque le génome mitochondrial (ADNmt) est très sensible au stress oxydatif, nous postulons que ce dernier peut servir de marqueur pour le stress oxydatif dans les cellules cancéreuses ainsi que dans les lymphocytes. Dans un premier temps, nous avons accru la sensibilité d'une nouvelle méthode d'analyse du dommage de l'ADNmt. Ceci nous a permis de proposer un protocole de mesure standardisé. Deuxièmement, nous avons développé une méthode afin de quantifier le stress oxydatif systémique en utilisant les réponses de l'ADNmt des lymphocytes. Finalement, nous avons démontré que deux lignées cellulaires prostatiques isogéniques, LNCaP et C4-2, ayant un potentiel métastatique distinct, répondaient différemment au stress oxydatif induit expérimentalement. La lignée C4-2, à potentiel plus métastatique, était plus sensible au stress oxydatif, se traduisant par une plus grande susceptibilité de l'ADNmt à être endommagé. Ces résultats apportent donc une nouvelle avenue dans la compréhension de la progression du cancer de la prostate.
Immunosensors have emerged as vital tools in cancer diagnostics, providing simplified and rapid detection of biomarkers that are necessary for timely diagnosis. The objective of using an electrochemical immunosensor is to detect cancers at early stages, so that obtained biological information can be analyzed using artificial intelligence (AI) for deciding an appropriate treatment, avoiding false diagnosis, and preventing patient fatalities. The focus of this article is on four major reproductive cancers—breast, ovarian, cervical, and prostate cancers. Specifically, it explores the identification and optimization of biomarkers crucial for precise detection in these cancers. Examining a decade of research, the review delves into nanotechnology-assisted electrochemical immunosensors (affinity biosensors), outlining advancements and emphasizing their potential in reproductive cancer diagnostics. Furthermore, the review contemplates avenues for enhancing sensor characteristics to pave the way for their application in field diagnosis, with a forward-looking perspective on AI-assisted diagnostics for the next generation of personalized healthcare. In navigating the landscape of reproductive cancer diagnostics, the integration of advanced technologies promises to transform our approach, offering improved accuracy and outcomes for patients.
The circadian rhythm is regulated by an intrinsic time-tracking system, composed both of a central and a peripheral clock, which influences the cycles of activities and sleep of an individual over 24hours. At the molecular level, the circadian rhythm begins when two basic helix-loop-helix/Per-ARNT-SIM (bHLH-PAS) proteins, BMAL-1 and CLOCK, interact with each other to produce BMAL-1/CLOCK heterodimers in the cytoplasm. The BMAL-1/CLOCK target genes encode for the repressor components of the clock, cryptochrome (Cry1 and Cry2) and the Period proteins (Per1, Per2 and Per3). It has been recently demonstrated that the disruption of circadian rhythm is associated with an increased risk of developing obesity and obesity-related diseases. In addition, it has been demonstrated that the disruption of the circadian rhythm plays a key role in tumorigenesis. Further, an association between the circadian rhythm disruptions and an increased incidence and progression of several types of cancer (e.g., breast, prostate, colorectal and thyroid cancer) has been found. As the perturbation of circadian rhythm has adverse metabolic consequences (e.g., obesity) and at the same time tumor promoter functions, this manuscript has the aim to report how the aberrant circadian rhythms affect the development and prognosis of different types of obesity-related cancers (breast, prostate, colon rectal and thyroid cancer) focusing on both human studies and on molecular aspects.
Androgen deprivation therapy (ADT) remains a key approach in the treatment of prostate cancer (PCa). However, PCa inevitably relapses and becomes ADT resistant. Besides androgens, there is evidence that thyroid hormone thyroxine (T4) and its active form 3,5,3′‐triiodo‐l‐thyronine (T3) are involved in the progression of PCa. Epidemiologic evidences show a higher incidence of PCa in men with elevated thyroid hormone levels. The thyroid hormone binding protein μ‐Crystallin (CRYM) mediates intracellular thyroid hormone action by sequestering T3 and blocks its binding to cognate receptors (TRα/TRβ) in target tissues. We show in our study that low CRYM expression levels in PCa patients are associated with early biochemical recurrence and poor prognosis. Moreover, we found a disease stage‐specific expression of CRYM in PCa. CRYM counteracted thyroid and androgen signaling and blocked intracellular choline uptake. CRYM inversely correlated with [18F]fluoromethylcholine (FMC) levels in positron emission tomography/magnetic resonance imaging of PCa patients. Our data suggest CRYM as a novel antagonist of T3‐ and androgen‐mediated signaling in PCa. The role of CRYM could therefore be an essential control mechanism for the prevention of aggressive PCa growth.
Das Prostatakarzinom (PCa) stellt die zweithäufigste krebsbedingte Todesursache bei Männern in Deutschland dar. Seine heterogenen Verlaufsformen erschweren es, eine optimale Therapieentscheidung zu treffen, denn die derzeit bekannten klinischen und molekularen Prognosemarker sind trotz intensiver Forschungsbemühungen nicht ausreichend in der Lage den Krankheitsverlauf vorherzusagen. Große Hoffnungen auf brauchbare prognostische Marker werden seit ihrer Entdeckung in miRNAs gesetzt, kleine genregulatorische, nicht-kodierende RNAs. MiRNAs regulieren im Rahmen einer posttranskriptionellen Inhibierung die Expression einer Vielzahl relevanter Zielgene. Für einige miRNAs ist bereits belegt, dass ihre differentielle Expression in verschiedenen Tumorentitäten mit der Genese und in einzelnen Fällen auch mit der Prognose assoziiert ist. Diese Arbeit sollte untersuchen, welches globale miRNA-Expressionsprofil in einem Kollektiv von Hochrisiko-Prostatakarzinomen (HR-PCa) vorliegt und welche miRNAs im HR-PCa aberrant exprimiert sind. Zudem sollte sie klären, ob Assoziationen der so identifizierten miRNAs mit Prognosegruppen des PCa vorliegen. Somit sollten erste Hinweise auf prognostisch relevante miRNAs und deren mögliche Bedeutung für die Tumorgenese aber auch für die Progression des PCa erbracht werden. Hierzu wurde die Expression von 640 miRNAs mittels Microarray-Analysen in Proben eines HR-PCa-Kollektivs (n=14) bestimmt und anschließend die Expression von acht tumorassoziierten miRNAs mittels qRT-PCR in einem erweiterten HR-PCa-Kollektiv (n=23) evaluiert. Um eine Grundlage für weitere molekulare Analysen vorzubereiten, wurde eine Zielgensuche in drei verschiedenen Datenbanken für elf potentielle Onkomirs durchgeführt. Im Vergleich zum nicht-tumorös veränderten Referenzgewebe wurden mittels Microarray-Analyse im HR-PCa 52 miRNAs als signifikant unterschiedlich exprimiert detektiert und es zeigte sich eine ausgeprägte Herunterregulation der globalen miRNA-Expression im HR-PCa. Mit diesen 52 miRNAs konnte in einer Clusteranalyse das Referenzgewebe von HR-PCa unterschieden werden. Bei 21 tumorspezifischen miRNAs zeigte sich eine Überlappung mit Daten bereits publizierter Studien. Hierunter fanden sich die als Onkomirs beschriebenen miRNAs miR-let-7a, miR-126 und miR-16 mit jeweils möglichen Zielgenen wie z.B. MAP4K3, EGFR und ESSRA. 15 miRNAs waren – im Gegensatz zur Expression in Kollektiven mit konventionellem Risikoprofil – im HR-PCa gegenüber nicht-malignem Referenzgewebe signifikant unterschiedlich exprimiert, darunter miR-515-5p mit den vorhergesagten Zielgenen C13orf34 und CDCA7. Die vorliegenden qRT-PCR-Analysen zeigten eine deutliche und häufige Herunterregulation von miR-221, -125b und -29a im HR-PCa. Als mögliche Zielgene wurden z.B. FOS und IRF2 für miR-221, EIF2C2 für miR-125b sowie MYBL2 und TRAF4 für miR-29a vorhergesagt. Mit den genannten drei miRNAs konnte das HR-PCa vom nicht-malignen Referenzgewebe unterschieden werden. Anhand eines Expressionsprofiles von 24 miRNAs war eine partielle Trennung der Kollektive nach Gleason-Score möglich. Die miRNAs miR-147 und miR-515-3p waren in den Microarray-Analysen in Prognosegruppen nach dem Gleason-Score signifikant unterschiedlich exprimiert. Eine mittels qRT-PCR determinierte niedrige Expression von miR-221 konnte mit hohem Gleason-Score assoziiert werden. Die signifikant unterschiedliche Expression von miR-422a in Prognosegruppen des PCa konnte in den Validierungsexperimenten nicht bestätigt werden. Die miRNAs miR-147, miR-515-3p bzw. miR-221 sind mit Blick auf ihr Potential als Prognosefaktoren Kandidaten für weitere Untersuchungen. Als potentielle Zielgene wurden z.B. RGS3, CDKN1B bzw. FOS/IRF2 vorhergesagt. Die Bedeutung einzelner miRNAs als mögliche prognostische Marker sollte in größeren Kollektiven und anhand von funktionellen Untersuchungen weiter geklärt werden. Die vorliegende Arbeit stellt eine Grundlage dar, um in weiterführenden Untersuchungen die hier im HR-PCa aberrant exprimierten miRNAs als brauchbare prognostische Marker für das PCa zu bestätigen und deren molekulare Funktionen im Rahmen der Genese des HR-PCa zu definieren.
Purpose
Uro-oncological translational research requires clinical data and human biological tissues collected within a biological tissue bank (BTB). We are hereby outlining ethic-legal, methodological and technical issues of a BTB establishment process, focusing particularly on prostate cancer and Italian setting.
Materials and Methods
Review of literature data, and national and international regulations and guidelines; direct field experience of urological BTB; counseling of the different professionals involved.
Results
Within a BTB establishment process, it is of utmost importance to protect the donors’ privacy and rights through the programmatic adoption of the following procedures: 1) informed consent; 2) confidentiality protection thanks to anonymity of biological specimens and use of an “honest broker” method; 3) identification of a single responsible researcher; 4) dedicated and protected location; 5) approval of the Ethical Committee. There are two main organizational models of BTB: “systematic”, i.e. collecting specimens from all patients and through the same methodology; “project-driven”, i.e. prospectively selecting patients for a specific study and using the specific methods required by researchers. In the preliminary step it is necessary to establish detailed protocols of sampling and crioconservation techniques, and methods of validation and quality control. For prostate tissue sampling, several techniques have been described such as specimens of alternate slices, macro dissection, Tru-Cut.
Conclusions
Today BTBs are necessary in order to support molecular and translational research in uro-oncology and to overcome the limits of the research based only on clinical-pathological data. Ethic-legal and methodological issues related to BTBs are still requiring specific legislation and standardization of techniques.
The phosphatidylinositol-3-kinase (PI3K)/Akt/mTOR pathway is one of the most frequently activated signaling pathways in prostate cancer cells, and loss of the tumor suppressor PTEN and amplification of PIK3CA are the two most commonly detected mechanisms for the activation of these pathways. Aberrant activation of PI3K/Akt/mTOR has been implicated not only in the survival and metastasis of prostate cancer cells but also in the development of drug resistance. As such, selective inactivation of this pathway may provide opportunities to attack prostate cancer from all fronts. However, while preclinical studies examining specific inhibitors of PI3K or mTOR have yielded promising results, the evidence from clinical trials is less convincing. Emerging evidence from the analyses of some solid tumors suggests that a class of dual PI3K/mTOR inhibitors, which bind to and inactivate both PI3K and mTOR, may achieve better anti-cancer outcomes. In this review, we will summarize the mechanisms of action of these inhibitors, their effectiveness when used alone or in combination with other chemotherapeutic compounds, and their potential to serve as the next generation therapies for prostate cancer patients, particularly those who are resistant to the frontline chemotherapeutic drugs.
Biopsy of the prostate is a common procedure with minor complications that are usually self-limited. However, if one considers that millions of men undergo biopsy worldwide, one realizes that although complication rate is low, the number of patients suffering from biopsy complications should not be underestimated and can be a clinically relevant problem for healthcare professionals. In this review, the authors present diagnosis and management of postbiopsy of prostate complications. Bleeding is the most common complication observed after prostate biopsy, but the use of aspirin or nonsteroidal anti-inflammatory drugs is not an absolute contraindication to prostate biopsy. Emerging resistance to ciprofloxacin is the most probable cause of the increasing risk of infectious complications after prostate biopsy. Even though extremely rare, fatal complications are possible and were described in case reports.
Androgens exert their key function in development and maintenance of the male phenotype via the androgen receptor (AR). Ligand-activated ARs also play a role in prostate cancer. Despite initial success of treatment by testosterone depletion or blocking of androgen binding to the AR using antiandrogens, eventually all tumors escape to a therapy resistant stage. Development of novel therapies by other antagonistic ligands or compounds that target events subsequent to ligand binding is very important. Here, we validate a fluorescence resonance energy transfer (FRET) based imaging assay for ligand-induced AR activity, based on the conformational change in the AR caused by interaction between the FQNLF motif in the N-terminal domain and the cofactor binding groove in the ligand-binding domain (N/C-interaction). We test the assay using known agonistic and antagonistic ligands on wild type AR and specific AR mutants. Our data show a strong correlation between the ligand-induced AR N/C-interaction and transcriptional activity in wild type AR, but also in AR mutants with broadened ligand responsiveness. Moreover, we explore additional readouts of this assay that contribute to the understanding of the working mechanism of the ligands. Together, we present a sensitive assay that can be used to quantitatively assess the activity of agonistic and antagonistic AR ligands. © 2013 International Society for Advancement of Cytometry.
In the award (W81XWH-08-1-0626), the stated goals are to study the zinc transporters that may be disparately expressed in the prostate tissues of African American (AA) males as compared to European American (EA) males. This may lead to the identification of the potential molecular targets for preventive or therapeutic measures, nutritional, environmental or life style factors for their potential relationship to the incidence or progression of prostate cancer in various racial groups. The following tasks are to be carried out in a synergistic fashion between the laboratories of Dr. Bagasra (Claflin University) and Dr. Balla (University of Illinois, Chicago). The prostate tissues, pre-made tissue microarrays (TMAs) and primary cell lines will be obtained from the NCI -initiated "Cooperative Prostate Cancer Tissue Resource (CPCTR)" by Dr. Balla, one of the P.I.s of the present proposal and of the CPCTR. The main tissue microarray to be used is composed of agematched and the Gleason score-matched prostate tissues with representative tissues from 150 African Americans males and 150 European American males. Other TMAs will be used to address questions of whether hZIP gene and protein expression is associated with poor outcome and/or Gleason grade. Significant progress has been made in each of the three TASKS proposed in the essence of one peer-reviewed publication as well as numerous oral and poster presentations. We currently have three additional publications in preparation for peer reviewed journals.
To determine if AR gene silencing in prostate cancer cells via RNA interference mechanism leads to disruption of androgen-independent progression. We generated a recombinant AAV for long-term expression of a hairpin-structured AR siRNA in vivo. Then we determined the essential role of the androgen receptor in androgen-independent growth of prostate cancer. We demonstrated that knocking down AR expression abolished tumor growth and blocked androgen-independent transition in LNCaP and LAPC-4 cell-derived xenografts. In addition, we observed that AR silencing resulted in a significant decrease of androgen-independent tumor growth in C4-2 cell-derived xenografts in castrated mice. We also demonstrated that knocking down AR expression by systemic delivery of the AAV particle eradicated CWR22RV1 androgen-independent xenograft tumors but not AR-null PC-3 xenograft tumors. We analyzed the mechanisms for AR siRNA-induced cell death and identified the anti-apoptotic proteins SGK1 and Bcl-xL, as the downstream effecters of AR-mediated survival pathway. Most of these results were published (Appendix II-VI).
Prostate cancer (PCa) is a highly prevalent disease in older men of the western world and overall greatly affects the quality of life of elderly people.
Understanding the mechanism of prostate cancer onset and metastasis is the key to treating this disease successfully and increasing survivability.
In this study, we constructed crosstalk networks among prostate cancer (primary and metastatic) related pathways by integrating protein-protein interactions (PPI) and KEGG (Kyoto encyclopedia of genes and genomes) pathways information. Total 11 pathways crosstalk with each other in primary prostate cancer and 7 pathways crosstalk with each other in metastatic prostate cancer.
Among these pathways, Notch signaling pathway and chemokine signaling pathway were found regulate multiple processes during prostate cancer progression.
Results from these studies will provide the groundwork for a combination therapy approach targeting multiple pathways which will likely be more effective than targeting one pathway alone.
An important requirement for many gene therapy applications is to direct therapeutic genes specifically to target cells. Here we describe an improved vector targeting method that does not depend on the use of a known cell-targeting ligand. It entails screening a library of constitutively produced retroviruses with random amino acid substitutions in the cell-targeting region of the envelope proteins for their ability to mediate gene delivery to a target cell. By screening such a library on the ras-transformed 143B human cell line, we have isolated an envelope protein that preferentially targets 143B cells and 293T cells expressing the SV40 T antigen via a novel, unidentified receptor. Furthermore, retroviruses expressing the library-derived envelope protein can be concentrated by centrifugation. This is the first demonstration of a novel concept in vector targeting: the selection of productive retroviral entry via an alternate receptor with modified cellular tropism in the absence of a known cell-targeting moiety. The method is, in principle, applicable even to cells that have not been well characterized, and therefore potentially suitable for targeting many diverse cell types.
Prostate cancer is the most frequently diagnosed cancer in men. However, current diagnostic techniques (including serum PSA level measurement) lack sufficient specificity and sensitivity to determine the aggressiveness of the disease and to identify appropriate treatment. Additional reliable biomarkers are needed that can facilitate early diagnosis of prostate cancer, determine the patient's prognosis and predict responses to a given therapeutic intervention. To achieve clinical utility, biomarkers require the potential for commercialization, and such an investment is generally only made if a financial return is promised. Patenting is one way to protect the intellectual property surrounding such biomarkers and several patented biomarkers are being developed, although few have been validated in large-scale clinical trials.
Despite progress in diagnosis and treatment, prostate cancer is still one of the most frequent lethal diseases in men in Western countries. Today, an increasing number of prostate cancers are detected through elevated serum prostate-specific antigen (PSA) levels. PSA detection is very sensitive. Determining free versus total PSA serum level has enabled the achievement of better specificity (Balk et al. 2003), however, this tool remains imperfect. Many patients undergo unnecessary diagnostic procedures, experiencing physiological and psychological stress. Similarly, current techniques such as imaging and biopsies are not optimal thus, hopes are high for the discovery of new molecular markers for prostate cancer. Studying prostate cancer progression may reveal new insights into the molecular mechanisms of cancer development to help improve prevention, provide better tools for diagnosis, as well as for prognosis and treatment.
Objective: To diagnose prostatic cancer (CaP) with cDNA macroarray. Methods: Total RNA was isolated from patients with prostate cancer and from normal people, and poly(A) RNA was further purified. Then
differentially expressed genes were analysed in CaP and normal prostate by cDNA macroarray system. Results: There were differential expressions of nine prostate-associated specific genes in CaP as compared with normal prostate, among
which, 7 were significantly up-regulated and 2 were down-regulated. Conclusion: As a diagnostic approach at molecular level, the cDNA macroarray is supposed to elevate the detection rate of CaP.
The aim of the present study is to evaluate the effects of quercetin, a dietary flavonoid, on human prostate adenocarcinoma PC-3 cells. Lactate dehydrogenase (LDH) release, microculture tetrazolium test (MTT assay) and real-time PCR array were employed to evaluate the effects of quercetin on cell cytotoxicity, cell proliferation and expression of various genes in PC-3 cell line. Quercetin inhibited cell proliferation and modulated the expression of genes involved in DNA repair, matrix degradation and tumor invasion, angiogenesis, apoptosis, cell cycle, metabolism and glycolysis. No cytotoxicity of quercetin on PC-3 cells was observed. Taken together, as shown by the issues of the current study, the manifold inhibitory effects of quercetin on PC-3 cells may introduce quercetin as an efficacious anticancer agent in order to be used in the future nutritional transcriptomic investigations and multi-target therapy to overcome the therapeutic impediments against prostate cancer.
Once prostate cancer becomes castration resistant, cancer cells may rapidly gain the ability to invade and to metastasize to lymph nodes and distant organs. The progression through hormone-dependent to hormone-independent/castration-resistant and metastatic PCa is poorly understood. In this review paper, we provide an overview on the cellular and molecular mechanisms underlying the process of tumor cell invasion and metastasis in prostate cancer. We specifically present the most recent findings on the role of multiple cellular signaling pathways including androgen receptor (AR), mitogen-activated protein kinases (MAPK), Akt, transforming growth factor b (TGFb interleukin-6 (IL-6) and vascular endothelial growth factor (VEGF) in the development of hormone-independent/castration-resistant prostate cancer. In addition, we also discuss the recent findings on signatures of gene expression during prostate cancer progression. Our overviews on the novel findings will help to gain better understanding of the complexity of molecular mechanisms that may play an essential role in the development of castration-resistant and metastatic prostate cancer. It will also shed light on the identification of specific targets and the design of effective therapeutic drug candidates.
Adenovirus-mediated gene therapy is a potential complement to standard cancer treatments. Advantages are that vectors can be used to target tumors and that replicating viruses lead to increased therapeutic dosage.
In this thesis, an oncolytic serotype 5 adenovirus (Ad5), Ad[i/PPT-E1A, E3], was developed where viral replication is controlled by the insulator-shielded (i) prostate-specific PPT promoter. The adenoviral E3 region was inserted for its immune regulatory and lysis functions. Ad[i/PPT-E1A, E3] had improved cytotoxic abilities both in vitro and in a prostate cancer xenograft mouse model compared to a virus lacking the E3 region. To further improve adenoviral vectors, the histone deacetylase inhibitor (HDACi) FK228 was studied. FK228 has been proposed to enhance the effect of adenoviral therapy by upregulation of CAR, the primary receptor for Ad5 infection. In the present study, we observed that FK228 promotes transgene expression even better when administered after viral transduction, indicating a post-transductional enhancement of transgene expression. Another interesting finding was that FK228 reduced transgene expression from the PPT promoter in the prostate cancer cell line LNCaP. This is explained by the fact that different HDACi have the ability to provoke a neuroendocrine phenotype of LNCaP.
A potential drawback with adenoviral gene therapy is the rapid clearance of the virus from the circulation. Viral particles have been coated with polyethylene glycol (PEG) to evade immune recognition, a strategy that works well in mouse models. However, less is known about the effects of adenoviral PEGylation in human blood. We have studied cell interactions and immune responses to PEGylated and uncoated Ad5 vectors in human whole blood using a blood loop model with constant blood flow. Limited effects of PEGylation were observed in human blood, which were associated with the neutralizing ability of the donor blood. An important finding that donors with high neutralizing ability in whole blood do not necessarily have neutralizing antibodies against the virus strongly implies that neutralization should be measured in whole blood.
One mechanism of prostate tumors for escape from androgen ablation therapies is mutation of the androgen receptor (AR). We
investigated the unique properties of the AR L701H mutant, which is strongly stimulated by cortisol, by a systematic structure-function
analysis. Most amino acid substitutions at position 701 did not affect AR activation by 5α-dihydrotestosterone. Further analysis
of the AR Leu701 variants showed that AR L701M and AR L701Q, like AR L701H, had changed ligand responsiveness. AR L701M was strongly activated
by progesterone but not by cortisol, whereas the opposite was observed for AR L701Q and AR L701H. Next, we analyzed a panel
of structurally related steroids to study which of the OH groups at positions 11β, 17α, and 21, which discriminate cortisol
from progesterone, underlie the differential responses to both hormones. The results showed that the 17α-OH group was essential
for activation of AR L701H and AR L701Q, whereas its absence was important for activation of AR L701M. Modeling indicated
a conserved H-bonding network involving the steroidal 17α-OH group, His701 or Gln701, and the backbone of Ser778. This network is absent in Leu701 and in other mutants. A hydrophobic leucine or methionine at position 701 is unfavorable for the 17α-OH group. Our results
indicate that the specific amino acid residue at position 701, its interaction with the backbone of Ser778, and the steroidal 17α-hydroxyl group of the ligand are all important for the distinct transcriptional responses to progesterone
and cortisol of AR mutants, including the prostate cancer mutant L701H.
Although treatment of different types of sexual dysfunction has improved in the past decade with the introduction of phosphodiesterase type 5 inhibitors and selective serotonin reuptake inhibitors, response rates to these targeted therapies are variable. There are a number of studies in the published literature that provide proof-of-concept that genetic variation contributes to the variable response. Pharmacogenomics will most likely be one part of our therapeutic armamentarium in the future and will provide a stronger scientific basis for optimizing drug therapy on the basis of each patient's genetic constitution. This article will review English language medical literature on the state-of-the-art genetic polymorphisms of drug targets, transporters and signaling molecules as well as pharmacogenetic studies of sexual dysfunction and suggested possible applications. Collectively, the data demonstrate that pharmacogenomics in the field of sexual medicine is still in its infancy. More research will provide further intriguing new discoveries in years to come.
The Y chromosome has been suggested to play a role in prostate cancer (PCa) because the loss of this chromosome is the most common aberration in PCa. Study of short tandem repeats (STRs) could provide a means to rapidly scan genomes at known or unknown predisposing loci for some diseases. DNA samples from 281 patients with PCa at the Portuguese Institute of Oncology, Porto, Portugal, and a population control of 175 healthy controls were analyzed for region Yp11.2 using AmpFlSTR Y-Filer kit (Applied Biosystems). The results demonstrated that microvariant alleles of DYS458 are overrepresented (p = 0.026). We found that allele 12 of DYS393 and allele 19 of DYS458 could have a protective effect (p = 0.0051; odds ratio [OR] = 0.48; 95% confidence interval [95% CI] 0.27-0.38; and p = 0.0272; OR = 0.47; 95% CI 0.22-0.98). On the other hand, patients carrying allele 13 of DYS393 presented an increased risk to PCa (p = 0.015; OR = 1.97; 95% CI 1.26-3.07). These results are in concordance with the involvement of Y chromosome in PCa development. STR allele studies may add further information from the definition of a genetic profile of PCa resistance or susceptibility. As TSPY is located at region Yp11.2, this gene could play an essential role in PCa development.
Prostatakrebs ist die zweithäufigste krebsbedingte Todesursache bei Männern der westlichen Welt. Obwohl immer mehr Prostatakarzinome in frühen Stadien diagnostiziert werden, ist die Mortalitätsrate nicht entsprechend gesunken, da eine kurative Therapie fortgeschrittener, metastasierter Prostatakarzinome bisher nicht möglich ist. Die Aufklärung der biologischen Prozesse, welche an der Prostatakarzinogenese beteiligt sind, stellt die Grundlage zur Entwicklung neuer molekularer Marker und Therapeutika dar. Die Verwendung von Mikroarray-Technologien ermöglicht die Identifikation einer zunehmenden Anzahl an Kandidatengenen, deren Bedeutung für die Tumorprogression und Prognose z.B. mittels Gewebe-Mikroarrays an einem großen Tumorkollektiv validiert werden. In der vorliegenden Studie wurden Gewebe-Mikroarrays hergestellt, welche zur Untersuchung von Änderungen der Proteinexpression in unterschiedlichen Stadien der Progression des Prostatakarzinoms eingesetzt wurden. Zur Identifikation neuer Kandidatengene wurden numerische chromosomale Veränderungen von 161 Prostatakarzinomen aus sieben Genomprofil-Studien systematisch mit den Ergebnissen von vier Expressionsprofil-Studien an 61 Prostatakarzinomen verglichen. Von der resultierenden Liste an Kandidatengenen wurden diejenigen der Kandidaten, für welche geeignete Antikörper verfügbar waren, mittels Immunhistochemie an einem Gewebe-Mikroarray mit 651 Gewebeproben von 175 Prostatakrebspatienten untersucht: Fettsäuresynthase (FASN), MYC, Beta-Adrenergische Rezeptor Kinase 1 (BARK1), die katalytischen Untereinheiten der Protein Phosphatasen PP1a (PPP1CA) und PP2A (PPP2CB) sowie der Tumorsuppressor NM23-H1. In univariaten Analysen korrelierte die Immunfärbung von PP1a mit dem pathologischen Parameter Gleason Score. Die Immunfärbung von MYC korrelierte in univariaten und multivariaten Analysen invers mit pT-Stadium und Gleason Score. Zudem war eine Untergruppe von Patienten mit hohen Gleason Scores durch den Verlust der Beta-Adrenergischen Rezeptor Kinase 1 (BARK1) charakterisiert. In dieser IHC-Studie wurden somit neue molekulare Marker von potentieller diagnostischer und therapeutischer Relevanz identifiziert. Des Weiteren wurde die Bedeutung des in der IHC-Studie identifizierten Markers BARK1 bei der Progression des Prostatakarzinoms an Zelllinien analysiert (BPH-1, 22RV1, LNCaP, PC-3, DU145, HEK, Jurkat). BARK1 desensibilisiert spezifisch Agonist-gebundene Beta-Adrenergische Rezeptoren, sodass der bei fortgeschrittenen Tumoren beobachtete Verlust dieser Kinase zu einer verstärkten Signaltransduktion führen könnte. Eine Modulation der Signaltransduktion durch spezifische Agonisten (Isoproterenol, Terbutalin) und Antagonisten (ICI 118,551, ICI 89,406) beeinflusste zwar die Zellproliferation nicht. Die deutlich erhöhte Expression der Beta-Adrenergischen Rezeptor Gene (B1AR, B2AR) in Prostatazelllinien metastatischen Ursprungs (LNCaP, PC-3, DU145) sowie die durch Agonisten und Antagonisten hoch-regulierte Genexpression von B1AR und B2AR in den Zelllinien aus Fernmetastasen, PC-3 bzw. DU145, deuten jedoch auf eine Beteiligung der Beta-Adrenergischen Signaltransduktion an der Metastasierung hin. Ob Beta-Blocker die Metastasierung verhindern können, ist in weiteren Studien zu klären. Zusammenfassend konnten in dieser Studie an Prostata-Gewebe-Mikroarrays neue molekulare Marker identifiziert werden. Einer dieser Marker, BARK1, könnte über die Regulation der Beta-Adrenergischen Rezeptoren an der Progression des Prostatakarzinoms involviert sein. Die Ergebnisse dieser Studie unterstützen den Vorschlag, in der Therapie von Prostatakarzinomen Beta-Blocker einzusetzen, um die Ausbildung von Metastasen zu hemmen. Prostate cancer is the second most frequent cause of cancer-related deaths in western countries. Although the incidence of early prostate carcinoma stages increased in recent years, this did not result in a respectively decreased mortality rate because a curative therapy of advanced, metastatic stages does not exist. Elucidation of the biologic processes involved in prostate carcinogenesis provides a basis for the development of novel molecular markers and therapies. By means of microarray technologies an increasing number of candidate genes has been identified. The relevance of these genes in tumour progression and prognosis is validated in large tumour collections e.g. by using tissue microarray technology. In this study tissue microarrays were generated to analyze changes in protein expression during the progression of prostate cancer. To identify new candidate genes of diagnostic and therapeutic relevance, we performed an exhaustive gene search in seven previously described genomic profiling studies with 161 prostate tumours, and four expression profiling studies with 61 tumours. From the resulting list of candidate genes, six were selected for protein expression analysis based on the availability of antibodies applicable for paraffinized tissue: fatty acid synthase (FASN), MYC, beta-adrenergic receptor kinase 1 (BARK1), the catalytic subunits of protein phosphatases PP1a (PPP1CA) and PP2A (PPP2CB) and tumoursuppressor NM23-H1. These candidates were analysed by immunohistochemistry on a tissue microarray containing 651 cores of primary prostate cancer samples and benign prostatic hyperplasias from 175 patients. In univariate analysis, expression of PP1a was found to strongly correlate with the pathological parameter Gleason score. MYC immunostaining negatively correlated with both pT-stage and Gleason score in univariate and multivariate analysis. Furthermore, a subgroup of patients with high Gleason scores was characterized by a complete loss of BARK1 protein. In conclusion, this analysis revealed novel molecular markers of potential diagnostic and therapeutic relevance. Finally the potential role of BARK1 in prostate cancer progression was further analyzed in cell lines (BPH-1, 22RV1, LNCaP, PC-3, DU145, HEK, Jurkat). BARK1 specifically desensitizes agonist-bound beta-adrenergic receptors. Therefore, the loss of BARK1 which was observed in advanced tumours is likely to promote an enhanced signal transduction. Modulating the signal transduction intensity by specific agonists (isoproterenol, terbutaline) and antagonists (ICI 118,551, ICI 89,406) of the beta-adrenergic receptors did not influence cell proliferation. However, expression of beta-adrenergic receptor genes was found to be highly upregulated in all prostate cancer cell lines originating from metastases (LNCaP, PC-3, DU145). Furthermore, specific agonists and antagonists influenced beta-adrenergic receptors by further increasing expression of beta-adrenergic receptor genes B1AR and B2AR in PC-3 and DU145, the two cell lines originating from distant metastases. These findings indicate a role of beta-adrenergic receptors in metastasis. If metastasis may be inhibited by beta-blockers has to be elucidated in further studies. In conclusion, the present analysis on tissue microarrays revealed novel molecular markers for prostate cancer progression. Of these, BARK1 might be involved in metastasis formation by regulating beta-adrenergic receptors. These results support the notion to apply beta-blockers in prostate cancer therapy in order to prevent metastasis.
E-cadherin is a ( 'a- *-dependent cell adhesion molecule which plays an important role in normal growth and development via mediation of homotypic, homophilic cell-cell interaction. Recent studies suggest that E-cadherin may be important in neoplastic progression as well, partic ularly as a suppressor of invasion. We have previously demonstrated that the invasive phenotype of rat prostate cancer cells is associated with the decreased expression of E-cadherin (M. J. G. Bussemakers, R. J. A. Van Moorselaar, L. A. Giroldi, T. Ichikawa, J. T. Isaacs, F. M. J. Debruyne, and J. A. Schalken, Cancer Res., 52:2916-2922,1992). This is of particular interest, since the locus to which the human E-cadherin gene is mapped is frequently involved in allelic loss in prostate cancer (B. S. Carter, C. M. Ewing, W. S. Ward, B. F. Treiger, T. W. Aalders, J. A. Schalken, J. 1. Epstein, and W. B. Isaacs, Proc. Nati. Acad. Sci. USA, 87:8751-8755,1990; U. S. Bergerheim, K. Kunimi, V. P. Collins, and P. Ekman, Genes, Chromosomes Cancer, 3: 215-220, 1991). Im paired E-cadherin function is likely to be associated with aberrant ex pression of the protein. We therefore analyzed E-cadherin expression in situ by immunohistochemistry in nonmalignant and malignant speci mens of human prostatic tissue. Of 92 tumor samples of either primary or metastatic deposits of prostate cancer, 46 had reduced or absent E-cadherin staining when compared to nonmalignant prostate, which uniformly stained strongly positive. There was a statistically significant correlation between the decreased expression of E-cadherin and loss of tumor differentiation. Additionally, certain tumors within a histologi- cally similar group could be distinguished by the presence of mixed populations of E-cadherin-negative and -positive cells. The percentage of tumors with aberrant E-cadherin staining increased when clinically localized tumors were compared to either tumors with extensive local progression or metastatic deposits of prostate cancer, suggesting a cor relation between loss of E-cadherin and tumor progression. Taken to gether, these findings suggest that further exploration of E-cadherin as a candidate invasion suppressor molecule in human prostate cancer is warranted.
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Genetic changes leading to the development of prostate cancer and factors that underlie the clinical progression of the disease are poorly characterized. Here, we used comparative genomic hybridization (CGH) to screen for DNA sequence copy number changes along all chromosomes in 31 primary and 9 recurrent uncultured prostate carcinomas. The aim of the study was to identify those chromosome regions that contain genes important for the development of prostate cancer and to identify genetic markers of tumor progression. CGH analysis indicated that 74% of primary prostate carcinoma showed DNA sequence copy number changes. Losses were 5 times more common than gains and most often involved 8p (32%), 13q (32%), 6q (22%), 16q (19%), 18q (19%), and 9p (16%). Allelic loss studies with 5 polymorphic microsatellite markers for 4 different chromosomes were done from 13 samples and showed a 76% concordance with CGH results. In local recurrences that developed during endocrine therapy, there were significantly more gains (P < 0.001) and losses (P < 0.05) of DNA sequences than in primary tumors, with gains of 8q (found in 89% of recurrences versus 6% of primary tumors), X (56% versus 0%), and 7 (56% versus 10%), as well as loss of 8p (78% versus 32%), being particularly often involved. In conclusion, our CGH results indicate that losses of several chromosomal regions are common genetic changes in primary tumors, suggesting that deletional inactivation of putative tumor suppressor genes in these chromosomal sites is likely to underlie development of prostate cancer. Furthermore, the pattern of genetic changes seen in recurrent tumors with the frequent gains of 7, 8q, and X suggests that the progression of prostate cancer and development of hormone-independent growth may have a distinct genetic basis. These chromosome aberrations may have diagnostic utility as markers of prostate cancer progression.
Expression of the Ca(2+)-dependent, homotypic cell:cell adhesion molecule, E-cadherin (E-cad), suppresses tumor cell invasion and metastasis in experimental tumor models. Decreased E-cad expression is common in poorly differentiated, advanced-stage carcinomas. These data implicate E-cad as an "invasion suppressor" gene. The mechanism by which E-cad is silenced in advanced stage carcinomas is unclear. In this report, we show that: (a) the 5' CpG island of E-cad is densely methylated in E-cad-negative breast and prostate carcinoma cell lines and primary breast carcinoma tissue but is unmethylated in normal breast tissue; (b) treatment with the demethylating agent, 5-aza-2'-deoxycytidine, partially restores E-cad RNA and protein levels in E-cad-negative breast and prostate carcinoma cell lines; and (c) and E-cad promoter/CAT construct is expressed in both E-cad-positive and -negative breast and prostate carcinoma cell lines, indicating that these cells have the active transcriptional machinery necessary for E-cad expression. Our data demonstrate that frequent loss of E-cad expression in human breast and prostate carcinomas results from hypermethylation of the E-cad promoter region.
Overexpression of amplified genes is often associated with the acquisition of resistance to cancer therapeutic agents in vitro. We have identified a similar molecular mechanism in vivo for endocrine treatment failure in human prostate cancer which involves amplification of the androgen receptor (AR) gene. Comparative genomic hybridization shows that amplification of the Xq11-q13 region (the location), is common in tumours recurring during androgen deprivation therapy. We found high-level AR amplification in seven of 23 (30%) recurrent tumours, but in none of the specimens taken from the same patients prior to therapy. Our results suggest that AR amplification emerges during androgen deprivation therapy by facilitating tumour cell growth in low androgen concentrations.
Hypermethylation of regulatory sequences at the locus of the pi-class glutathione S-transferase gene GSTP1 was detected in 20 of 20 human prostatic carcinoma tissue specimens studied but not in normal tissues or prostatic tissues exhibiting benign hyperplasia. In addition, a striking decrease in GSTP1 expression was found to accompany human prostatic carcinogenesis. Immunohistochemical staining with anti-GSTP1 antibodies failed to detect the enzyme in 88 of 91 prostatic carcinomas analyzed. In vitro, GSTP1 expression was limited to human prostatic cancer cell lines containing GSTP1 alleles with hypomethylated promoter sequences; a human prostatic cancer cell line containing only hypermethylated GSTP1 promoter sequences did not express GSTP1 mRNA or polypeptides. Methylation of cytidine nucleotides in GSTP1 regulatory sequences constitutes the most common genomic alteration yet described for human prostate cancer.
A newly developed method of comparative genomic hybridization (CGH) employing quantitative statistical comparisons was applied to DNA from two different types of advanced prostate cancer tissue. Multiple CGH analyses were obtained for each chromosome in each tumor, and the results of point-by-point comparison of the mean tumor:normal color ratio to a control normal:normal color ratio in each of 1247 evenly distributed data channels constituting the entire human genome were interpreted as loss, gain, or no change in copy number in the tumor genome. Group I tissue was obtained from prostate cancer metastases from 20 patients, 19 of whom had received no prior prostate cancer treatment. This DNA also was analyzed by Southern and microsatellite allelotyping at 53 different loci on 20 different chromosome arms. CGH results agreed with allelotyping results at 92% of the informative loci studied. These samples, which contained highly enriched tumor DNA, showed the highest rates of alteration yet reported in several chromosomal regions known to be altered frequently in prostate cancer: 8q gain (85%), 8p loss (80%), 13q loss (75%), 16q loss (55%), 17p loss (50%), and 10q loss (50%). Group II tissue was obtained predominately from primary or recurrent tumor from 11 patients who had been treated with long-term androgen-deprivation therapy and developed androgen-independent metastatic disease. Quantitative CGH analysis on DNA from these tissues showed chromosomal alterations that were very similar to those found in group I, suggesting that untreated metastatic tumors contain the bulk of chromosomal alterations necessary for recurrence to occur during androgen deprivation. In the entire data set, a number of previously undetected regions of frequent loss or gain were identified, including losses of chromosomes 2q (42%), 5q (39%), 6q (39%), and 15q (39%) and gains of chromosomes 11p (52%), 1q (52%), 3q (52%), and 2p (45%). Chi-squared analysis showed a significantly higher frequency of gain of the 4q25-q28 region in tumors from African-American patients, indicating a possible oncogene whose activation may play a role in the higher rate of progression seen in this ethnic group. Additional study of these frequently altered regions may provide insight into the mechanism of prostate cancer progression and lead to important tools for tumor-specific prognosis and therapy.
Production of the potent vasoconstrictor endothelin-1 (ET-1) by human prostate cancer cells accompanies prostate cancer progression in vivo. The predominant endothelin receptor expressed by normal prostate epithelium, ETB, is not expressed by any of the established human prostate cancer cell lines, and ETB binding is decreased on prostate cancer tissues. ETB, which may mediate ET-1 clearance and may inhibit ET-1 secretion, is encoded by a gene that contains a 5' CpG island encompassing the transcriptional regulatory region. We examined this regulatory region of the ETB receptor gene (EDNRB) to determine whether hypermethylation of cytidine nucleotides accompanies decreased ETB expression in human prostate cancer. We found somatic methylation of CpG island sequences in EDNRB in 5 of 5 human prostate cancer cell lines, 15 of 21 primary prostate cancer tissues, and 8 of 14 prostate cancer metastases (70% of samples overall). Normal tissues contained only unmethylated EDNRB. Treatment of human prostatic carcinoma cell line cultures with 5-azacytidine induced ETB mRNA expression, suggesting that CpG island methylation changes might accompany the apparent transcriptional silencing of EDNRB in vivo.
The role of c-myc in prostatic carcinogenesis is poorly understood. The pathogenetic relationship between high-grade prostatic intraepithelial neoplasia (PIN), prostatic carcinoma, and metastases is not well-defined. We used fluorescence in situ hybridization (FISH) with a region-specific probe for c-myc (band 8q24) and chromosome enumeration probes for chromosomes 7, 8, 10, 12, and Y to evaluate genetic changes in matched PIN (48 foci), localized prostatic carcinoma (71 foci), and lymph node metastases (23 foci) in 25 totally embedded whole-mount stage D1 (T2-3 N1-3 M0) radical prostatectomy and pelvic lymphadenectomy specimens. The c-myc protein expression in these lesions was evaluated by immunohistochemistry. Foci with extra copies of c-myc could be divided into three groups: (a) those with simple gain of a whole chromosome 8 (no increase in c-myc copy number relative to the chromosome 8 centromere), which was identified in 42, 25, and 46% of foci of PIN, carcinoma, and metastases, respectively; (b) those with an intermediate increase in c-myc copy number relative to the chromosome 8 centromere, which was found in 8, 11, and 25% of foci of PIN, carcinoma, and metastases, respectively; and (c) those with substantial amplification of c-myc (large increases in c-myc copy number relative to the chromosome 8 centromere), which was detected in 0, 8, and 21% of foci of PIN, carcinoma, and metastases, respectively. Substantial amplification of c-myc was strongly correlated with increasing cancer nuclear grade and immunohistochemical evidence of c-myc protein overexpression. Numeric chromosomal anomalies were found in 67, 68, and 96% of foci of PIN, carcinoma, and metastases, respectively. The most frequent anomaly in PIN and carcinoma was a gain of chromosome 8, and the presence of this anomaly strongly correlated with Gleason score. Carcinoma foci usually contained more FISH anomalies than paired PIN foci, but three prostates contained one or more PIN foci with more anomalies than carcinoma. Thirteen primary tumor foci exhibited intratumor genetic heterogeneity by FISH. One or more foci of the primary tumor usually shared FISH anomalies with the matched metastases. Our FISH results indicate that: (a) gain of chromosome 8 and amplification of c-myc are potential markers of prostate carcinoma progression; (b) PIN is likely a precursor of carcinoma; (c) intraglandular and intratumoral genetic heterogeneity is relatively common; and (d) usually a single focus of cancer gives rise to metastases.
The length of a polymorphic CAG repeat sequence, occurring in the androgen receptor gene, is inversely correlated with transcriptional activity by the androgen receptor. Because heightened androgenic stimulation may increase risk of prostate cancer development and progression, we examined whether shorter CAG repeats in the androgen receptor gene are related to higher risk of prostate cancer. We conducted a nested case-control study of 587 newly diagnosed cases of prostate cancer detected between 1982 and 1995, and 588 controls without prostate cancer, within the Physician's Health Study. An association existed between fewer androgen receptor gene CAG repeats and higher risk of total prostate cancer [relative risk (RR) = 1.52; 95% confidence interval (CI) = 0.92-2.49; P trend = 0.04; for men with CAG repeat lengths < or = 18 relative to > or = 26 repeats]. In particular, a shorter CAG repeat sequence was associated with cancers characterized by extraprostatic extension or distant metastases (stage C or D) or high histologic grade (RR = 2.14; CI = 1.14-4.01; P trend = 0.001). This association was observed individually both for high stage (RR = 2.23) and high grade prostate cancer (RR = 1.89). Men with shorter repeats were at particularly high risk for distant metastatic and fatal prostate cancer. Variability in the CAG repeat length was not associated with low grade or low stage disease. These results demonstrate that a shorter CAG repeat sequence in the androgen receptor gene predicts higher grade and advanced stage of prostate cancer at diagnosis, and metastasis and mortality from the disease. The clinical implications of these results should be evaluated further.
Loss of heterozygosity at chromosome 8p21-22 is common in human prostate cancer, suggesting the presence of one or more tumor suppressor genes at this locus. A homeobox gene that is expressed specifically in adult human prostate, NKX3.1, the expression of which is regulated by androgen, maps to chromosome 8p21. Fine structure in situ mapping showed that NKX3.1 is proximal to MSR32 (macrophage scavenger receptor type II) and LPL (human lipoprotein lipase) and very close to NEFL (human neurofilament light chain) on 8p21. Single-strand conformational polymorphism analysis of 48 radical prostatectomy cancer specimens and 3 metastases for the entire coding region of NKX3.1 showed no tumor-specific sequence alterations in 50 specimens and total absence of the gene in 1 specimen known to have a biallelic deletion of 8p21. NKX3.1 was found to have a polymorphism at nucleotide 154 in codon 52 that resulted in a CGC-->TGC sequence change and an Arg-->Cys amino acid alteration (R52C). This polymorphism was present in 20% of DNA samples. If NKX3.1 is a target of the 8p21 LOH, it is not via disruption of the coding region of the gene.
The identification of cell surface antigens is critical to the development of new diagnostic and therapeutic modalities for the management of prostate cancer. Prostate stem cell antigen (PSCA) is a prostate-specific gene with 30% homology to stem cell antigen 2, a member of the Thy-1/Ly-6 family of glycosylphosphatidylinositol (GPI)-anchored cell surface antigens. PSCA encodes a 123-aa protein with an amino-terminal signal sequence, a carboxyl-terminal GPI-anchoring sequence, and multiple N-glycosylation sites. PSCA mRNA expression is prostate-specific in normal male tissues and is highly up-regulated in both androgen-dependent and -independent prostate cancer xenografts. In situ mRNA analysis localizes PSCA expression in normal prostate to the basal cell epithelium, the putative stem cell compartment of the prostate. There is moderate to strong PSCA expression in 111 of 126 (88%) prostate cancer specimens examined by in situ analysis, including high-grade prostatic intraepithelial neoplasia and androgen-dependent and androgen-independent tumors. Flow cytometric analysis demonstrates that PSCA is expressed predominantly on the cell surface and is anchored by a GPI linkage. Fluorescent in situ hybridization analysis localizes the PSCA gene to chromosome 8q24.2, a region of allelic gain in more than 80% of prostate cancers. A mouse homologue with 70% amino acid identity and similar genomic organization to human PSCA has also been identified. These results support PSCA as a target for prostate cancer diagnosis and therapy.
Beta-catenin plays essential roles in both intercellular adhesion and signal transduction. As a signaling molecule, beta-catenin supplies an activating domain to the T-cell factor/lymphoid enhancer-binding factor family of DNA-binding proteins and activates gene transcription. Posttranslational stabilization of beta-catenin, leading to elevated protein levels and constitutive gene activation, has been proposed as an important step in oncogenesis. Stabilization of beta-catenin can occur through mutation to highly conserved amino acids encoded in exon 3 of the beta-catenin gene (CTNNB1). To determine whether this pathway of malignant transformation is important in prostate cancer, we analyzed 104 prostate cancer tissue specimens, 4 prostate cancer cell lines, and 3 prostate tumor xenografts for activating mutations in exon 3 of CTNNB1. Mutations were detected in 5 of the 104 prostate cancer tissue samples. Four of the five mutations involved serine or threonine residues implicated in the degradation of beta-catenin. A fifth tumor had a mutation at codon 32, changing a highly conserved aspartic acid to a tyrosine. Mutational analysis of multiple regions from several tumor samples showed that the beta-catenin mutations were present focally and therefore may occur during tumor progression.
Loss of chromosome 10q is a frequently observed genetic defect in prostate cancer. Recently, the PTEN/MMAC1 tumor suppressor gene was identified and mapped to chromosome 10q23.3. We studied PTEN structure and expression in 4 in vitro cell lines and 11 in vivo xenografts derived from six primary and nine metastatic human prostate cancers. DNA samples were allelotyped for eight polymorphic markers within and surrounding the PTEN gene. Additionally, the nine PTEN exons were tested for deletions. In five samples (PC3, PC133, PCEW, PC295, and PC324), homozygous deletions of the PTEN gene or parts of the gene were detected. PC295 contained a small homozygous deletion encompassing PTEN exon 5. In two DNAs (PC82 and PC346), nonsense mutations were found, and in two (LNCaP and PC374), frame-shift mutations were found. Missense mutations were not detected. PTEN mRNA expression was clearly observed in all cell lines and xenografts without large homozygous deletions, showing that PTEN down-regulation is not an important mechanism of PTEN inactivation. The high frequency (60%) of PTEN mutations and deletions indicates a significant role of this tumor suppressor gene in the pathogenesis of prostate cancer.
Over 200,000 new prostate cancer cases are diagnosed in the United States each year, accounting for more than 35% of all cancer cases affecting men, and resulting in 40,000 deaths annually. Attempts to characterize genes predisposing to prostate cancer have been hampered by a high phenocopy rate, the late age of onset of the disease and, in the absence of distinguishing clinical features, the inability to stratify patients into subgroups relative to suspected genetic locus heterogeneity. We previously performed a genome-wide search for hereditary prostate cancer (HPC) genes, finding evidence of a prostate cancer susceptibility locus on chromosome 1 (termed HPC1; ref. 2). Here we present evidence for the location of a second prostate cancer susceptibility gene, which by heterogeneity estimates accounts for approximately 16% of HPC cases. This HPC locus resides on the X chromosome (Xq27-28), a finding consistent with results of previous population-based studies suggesting an X-linked mode of HPC inheritance. Linkage to Xq27-28 was observed in a combined study population of 360 prostate cancer families collected at four independent sites in North America, Finland and Sweden. A maximum two-point lod score of 4.60 was observed at DXS1113, theta=0.26, in the combined data set. Parametric multipoint and non-parametric analyses provided results consistent with the two-point analysis. Significant evidence for genetic locus heterogeneity was observed, with similar estimates of the proportion of linked families in each separate family collection. Genetic mapping of the locus represents an important initial step in the identification of an X-linked gene implicated in the aetiology of HPC.
In aging men, the prostate gland becomes hyperproliferative and displays a propensity toward carcinoma. Although this hyperproliferative process has been proposed to represent an inappropriate reactivation of an embryonic differentiation program, the regulatory genes responsible for normal prostate development and function are largely undefined. Here we show that the murine Nkx3.1 homeobox gene is the earliest known marker of prostate epithelium during embryogenesis and is subsequently expressed at all stages of prostate differentiation in vivo as well as in tissue recombinants. A null mutation for Nkx3.1 obtained by targeted gene disruption results in defects in prostate ductal morphogenesis and secretory protein production. Notably, Nkx3.1 mutant mice display prostatic epithelial hyperplasia and dysplasia that increases in severity with age. This epithelial hyperplasia and dysplasia also occurs in heterozygous mice, indicating haploinsufficiency for this phenotype. Because human NKX3.1 is known to map to a prostate cancer hot spot, we propose that NKX3.1 is a prostate-specific tumor suppressor gene and that loss of a single allele may predispose to prostate carcinogenesis. The Nkx3.1 mutant mice provide a unique animal model for examining the relationship between normal prostate differentiation and early stages of prostate carcinogenesis.
Prostate cancer is the most common malignancy in males and is the second most common cause of cancer mortality in American
men. Polymorphisms have been identified in two genes, the 17-hydroxylase cytochrome P450 gene (CYP17) and the steroid 5-reductase type II gene (SRD5A2) that are involved with androgen biosynthesis and metabolism. The CYP17 A2 allele contains a T→C transition in the 5′ promoter region that creates an additional Sp1-type (CCACC box) promoter site.
The SRD5A2 valine to leucine (V89L) polymorphism has been correlated with lower dihydroxytestosterone levels. We tested genotypes in
108 prostate cases and 167 controls along with samples (n = 340) from several different ethnic groups. The CYP17 A2 allele (combined A1/A2 and A2/A2 genotypes) occurred at a higher frequency in Caucasian patients with prostate cancer
(70%) than in Caucasian clinical control urology patients (57%), suggesting that the A2 allele may convey increased risk for
prostate cancer [odds ratio (OR) = 1.7, 95% confidence interval (CI) = 1.0–3.0]. Blacks and Caucasians had a similar frequency
of the A2 genotype (16 and 17%, respectively) while Taiwanese had the highest frequency (27%). The SRD5A2 leucine genotype was most frequent in Taiwanese (28%), intermediate in Caucasians (8.5%) and lowest in Blacks (2.5%). Genotypes
having a SRD5A2 leucine allele were somewhat more common in prostate cancer cases (56%) than in controls (49%) (OR = 1.4, 95% CI = 0.8–2.2)
but this difference was not significant. These results support the hypothesis that some allelic variants of genes involved
in androgen biosynthesis and metabolism may be associated with prostate cancer risk.
Despite the high incidence of prostate cancer, only limited data are available on genes or chromosomes specifically involved in its initiation and progression. We have applied comparative genomic hybridization to routinely processed, paraffin-embedded, tissues at different times in prostatic tumor progression to screen the tumor genome for gains and losses. Our panel included specimens derived from 56 different patients: 23 patients with primary, prostate-confined carcinomas; 18 patients with regional lymph node metastases; and 15 patients with distant metastases. Chromosome arms that most frequently showed losses, included 13q (55%), 8p (48%), 6q (43%), 5q (32%), 16q (25%), 18q (20%), 2q (18%), 4q (18%), 10q (18%), and Y (16%). Gains were often seen of chromosome arms 8q (36%), 17q (23%), Xq (23%), 7q (21%), 3q (18%), 9q (18%), 1q (16%), Xp (16%). Furthermore, specific high-level amplifications, eg, of 1q21, 1q25, and Xq12 to q13, were found in metastatic cancers. A significant accumulation of genetic changes in distant metastases was observed, eg, loss of 10q (p = 0.03) and gain of 7q (p = 0.03) sequences. In addition, investigation of a potential biomarker identified in previous studies by our group, ie, extra copies of #7 and/or #8, revealed a high prevalence of 7pq and/or 8q gain in the distant metastases (p = 0.02). Importantly, gains were observed more frequently in tumors derived from progressors after radical prostatectomy, than in nonprogressors (mean time of follow-up, 74 months). Specifically, gain of chromosome 7pq and/or 8q sequences appeared an accurate discriminator between the progressors and nonprogressors. Multivariate analysis showed a significant correlation between progressive disease and the number of chromosomes with gains. This correlation also held true when stage (p = 0.007) or grade (p = 0.002) were taken into account. Likewise, this applied for gain of chromosome 7pq and/or 8q sequences (p = 0.03 and p = 0.005 for stage or grade, respectively). Additionally, an increase in the number of chromosomes with gains per case was related to a decrease in biochemical progression-free survival (Ptrend <0.001). More specifically, the gain of 7pq and/or 8q sequences markedly reduced the biochemical progression-free survival (p < 0.001). In conclusion, this study has, firstly, documented the spectrum of chromosomal alterations in subsequent stages of prostate cancer, a number of which had not been described previously. It allowed us to identify chromosomal regions related to advanced tumor stage, ie, loss of 10q24 and gain of 7q11.2 and/or 7q31 sequences. Secondly, gain of 7pq and/or 8q was identified as a potential genetic discriminator between progressors and nonprogressors after radical surgery.
The contribution of hereditary factors to the causation of sporadic cancer is unclear. Studies of twins make it possible to estimate the overall contribution of inherited genes to the development of malignant diseases.
We combined data on 44,788 pairs of twins listed in the Swedish, Danish, and Finnish twin registries in order to assess the risks of cancer at 28 anatomical sites for the twins of persons with cancer. Statistical modeling was used to estimate the relative importance of heritable and environmental factors in causing cancer at 11 of those sites.
At least one cancer occurred in 10,803 persons among 9512 pairs of twins. An increased risk was found among the twins of affected persons for stomach, colorectal, lung, breast, and prostate cancer. Statistically significant effects of heritable factors were observed for prostate cancer (42 percent; 95 percent confidence interval, 29 to 50 percent), colorectal cancer (35 percent; 95 percent confidence interval, 10 to 48 percent), and breast cancer (27 percent; 95 percent confidence interval, 4 to 41 percent).
Inherited genetic factors make a minor contribution to susceptibility to most types of neoplasms. This finding indicates that the environment has the principal role in causing sporadic cancer. The relatively large effect of heritability in cancer at a few sites suggests major gaps in our knowledge of the genetics of cancer.
The transition from androgen-dependent to androgen-independent prostate cancer is not fully understood but appears to involve multiple genetic changes. We have identified a gene, GC79, that is more highly expressed in androgen-dependent LNCaP-FGC human prostate cancer cells than in androgen-independent LNCaP-LNO human prostate cancer cells. Physiologic levels (0.1 nM:) of androgens repress expression of GC79 messenger RNA (mRNA) in LNCaP-FGC cells. To determine the role of GC79, we cloned its complementary DNA (cDNA) and functionally characterized its product.
The differentially expressed GC79 gene was cloned from human prostate cDNA libraries, sequenced, and transfected into mammalian cells to study its function. Expression of GC79 was analyzed in various adult and fetal human tissues and in prostate glands of castrated rats. The association of GC79 expression and apoptosis was investigated in COS-1 and LNCaP cells transfected with GC79 cDNA. All statistical tests are two-sided.
Sequence analysis indicates that GC79 encodes a large, complex, multitype zinc-finger protein, containing nine C(2)H(2)-type zinc-finger domains, a cysteine-rich region, and a GATA C(4)-type zinc-finger domain. Castration-induced androgen withdrawal increased the expression of GC79 mRNA in the regressing rat ventral prostate, suggesting that the expression of GC79 mRNA is associated with the process of apoptotic cell death in the rat ventral prostate. Transfection and induction of GC79 cDNA in both COS-1 and LNCaP prostate cancer cells led to an apoptotic index that was eightfold higher (P:<.001, two-sided Student's t test) than that observed in uninduced transfected cells.
We have cloned an androgen-repressible gene, GC79, that is potentially involved in apoptosis. This finding may have implications for the development of androgen-independent prostate cancer and, ultimately, for the treatment of prostate cancer.
Chromosome 13 is one of the most frequently altered chromosomes in cancer, including carcinoma of the prostate. Two known tumor suppressor genes, RB1 and BRCA2, map to chromosome 13; however, recent reports suggest that unknown genes on 13q are more likely to be involved in the development of prostate cancer. In order more fully to define the genetic changes on chromosome 13 in prostate neoplasms, we analyzed 27 polymorphic microsatellite markers spanning the q arm for loss of heterozygosity in 40 primary tumors and in metastases from 11 other patients who died of prostate cancer. Of the 40 primary tumors, 23 (58%) showed LOH for at least one marker. Three distinct regions at q14, q21‐22, and q33, defined by markers D13S267 → D13S153, D13S166 → D13S1225, and D13S259 → D13S274, showed the most frequent LOH, suggesting their involvement in the development of prostate cancer. For the 12 patients whose tumors showed LOH at these markers, the average age at diagnosis was 58 years, which was younger than that (63 years, P < 0.05) for the 28 patients whose tumors lacked LOH. Ten of the 11 (91%) metastases showed LOH with one or more markers. Two of the three most frequently deleted regions (i.e., q14 and q21‐22) in the primary tumors and markers linked to the RB1, BRCA2, and EDNRB genes showed high frequencies (56–71%) of LOH in metastases. These results demonstrate that allelic loss on chromosome 13 at q14, q21‐22, and q33 occurs in a subset of primary prostate tumors and is a frequent event in metastatic lesions of prostate cancer. Genes Chromosomes Cancer 25:108–114, 1999. © 1999 Wiley‐Liss, Inc.
Prostate cancer is considered to be one of the most hormone‐dependent human malignancies. As a key mediator of hormonal response, the androgen receptor (AR) is believed to have an important role in the progression of prostate cancer. Mutations in the coding region of the AR gene have been found in both untreated and hormone‐refractory prostate cancer, but the frequency of such mutations at different stages of the disease is poorly documented and even contradictory results have been published. In the present study, the frequency of AR gene mutations was determined in 30 locally recurrent and two metastatic hormone‐refractory prostate tumours using the polymerase chain reaction (PCR), non‐radioactive single strand conformation polymorphism (SSCP), and sequencing. The length of the polymorphic CAG repeat, which is inversely correlated with the ability of the AR to activate transcription, was also analysed as well as the GGC repeat. Twelve samples were known to contain an AR gene amplification. Altogether, one point mutation (Gly⁶⁷⁴→Ala) and one microsatellite mutation (CAG20→CAG18) were found, both in cancers containing the AR gene amplification. The mean lengths of the polymorphic CAG and GGC repeats were similar to those observed in the normal population. These results favour the view that mutations in the AR gene are rare in hormone‐refractory prostate cancer and do not play an important role, at least, in local relapse. Instead, the amplification and consequent overexpression of the wild‐type AR gene seem to be the most common alteration involving the AR in hormone‐refractory prostate cancer. Copyright © 1999 John Wiley & Sons, Ltd.
Epidemiological investigations may allow conclusions on the etiology of prostate cancer. Such data from autopsy studies and morbidity as well as mortality statistics are based on local or national cancer registries and informations obtained from death certificates. Clear cut evidence of certain environmental factors to be involved in prostate cancer development derives from the situation in Japan: (1) high incidence of latent carcinoma despite a 10-fold lower morbidity of manifest carcinoma compared to the western hemisphere; (2) increased grade of malignancy of latent carcinoma and approximating morbidity and mortality rates to the population of the country of immigration. A comparable situation is seen in the Jewish population. In Europe an absolute increase of morbidity is encountered: Cancer Registries of Birmingham, Hamburg, Sweden, Halle (DDR). According to the UICC following incidence rates can be calculated n per 100,000 population per year): USA - 33.9; Sweden - 26.5; England - 17.4; GFR - 16.5; Japanese immigrants - 12.6; Japan - 3.8, China - 0.9. German mortality data (Cancer Atlas of the GFR), which are based on death certificates, show a steady increase between 1955 and 1975 without conclusive differences for the various 'Bundeslander'. On a world-wide basis neither studies on endocrinology nor on the socioeconomic status or sexual life, infectious as well as genetic factors have provided a valuable clue for the etiology of prostatic carcinoma.
Chromosome 13 is one of the most frequently altered chromosomes in cancer, including carcinoma of the prostate. Two known tumor suppressor genes, RB1 and BRCA2, map to chromosome 13; however, recent reports suggest that unknown genes on 13q are more likely to be involved in the development of prostate cancer. In order more fully to define the genetic changes on chromosome 13 in prostate neoplasms, we analyzed 27 polymorphic microsatellite markers spanning the q arm for loss of heterozygosity in 40 primary tumors and in metastases from 11 other patients who died of prostate cancer. Of the 40 primary tumors, 23 (58%) showed LOH for at least one marker. Three distinct regions at q14, q21-22, and q33, defined by markers D13S267 → D13S153, D13S166 → D13S1225, and D13S259 → D13S274, showed the most frequent LOH, suggesting their involvement in the development of prostate cancer. For the 12 patients whose tumors showed LOH at these markers, the average age at diagnosis was 58 years, which was younger than that (63 years, P < 0.05) for the 28 patients whose tumors lacked LOH. Ten of the 11 (91%) metastases showed LOH with one or more markers. Two of the three most frequently deleted regions (i.e., q14 and q21-22) in the primary tumors and markers linked to the RB1, BRCA2, and EDNRB genes showed high frequencies (56–71%) of LOH in metastases. These results demonstrate that allelic loss on chromosome 13 at q14, q21-22, and q33 occurs in a subset of primary prostate tumors and is a frequent event in metastatic lesions of prostate cancer. Genes Chromosomes Cancer 25:108–114, 1999. © 1999 Wiley-Liss, Inc.
Prostate cancer is considered to be one of the most hormone-dependent human malignancies. As a key mediator of hormonal response, the androgen receptor (AR) is believed to have an important role in the progression of prostate cancer. Mutations in the coding region of the AR gene have been found in both untreated and hormone-refractory prostate cancer, but the frequency of such mutations at different stages of the disease is poorly documented and even contradictory results have been published. In the present study, the frequency of AR gene mutations was determined in 30 locally recurrent and two metastatic hormone-refractory prostate tumours using the polymerase chain reaction (PCR), non-radioactive single strand conformation polymorphism (SSCP), and sequencing. The length of the polymorphic CAG repeat, which is inversely correlated with the ability of the AR to activate transcription, was also analysed as well as the GGC repeat. Twelve samples were known to contain an AR gene amplification. Altogether, one point mutation (Gly674→Ala) and one microsatellite mutation (CAG20→CAG18) were found, both in cancers containing the AR gene amplification. The mean lengths of the polymorphic CAG and GGC repeats were similar to those observed in the normal population. These results favour the view that mutations in the AR gene are rare in hormone-refractory prostate cancer and do not play an important role, at least, in local relapse. Instead, the amplification and consequent overexpression of the wild-type AR gene seem to be the most common alteration involving the AR in hormone-refractory prostate cancer. Copyright © 1999 John Wiley & Sons, Ltd.
Mutations in the p53 gene resulting in the accumulation of altered p53 proteins with prolonged half-life have been found in a large variety of human malignancies.
We studied the significance of p53 protein accumulation in prostatic carcinoma.
The material consisted of 137 paraffin-embedded, primary prostatic carcinomas. Accumulation of p53 protein was studied by immunohistochemical staining using a polyclonal p53-specific CM-1 antibody. Proliferation activity was determined by DNA flow cytometry and by immunohistochemical detection of proliferative cell nuclear antigen (PCNA) using a monoclonal PC10 antibody.
Eight (6%) of the tumors showed intense p53 staining in more than 20% of the tumor cells, 15 (11%) had only lower level immunoreactivity, and 114 (83%) showed no staining. High-level p53 accumulation was associated with high histologic grade (P less than .001), DNA aneuploidy (P less than .05), and high cell proliferation rate as defined by flow cytometric S-phase analysis (P less than .01) or PCNA expression (P less than .01). High-level p53 accumulation predicted short, progression-free interval (P less than .01) and poor survival (P less than .001), with about a 12-fold relative risk of death as compared with p53-negative cases. Low-level p53 accumulation had no prognostic significance.
Accumulation of p53 confers proliferative advantage for prostatic carcinoma cells and defines a small subgroup of highly malignant carcinomas.
Nuclear accumulation of p53 protein has been shown to be strongly associated with missense p53 mutations. Studies of nuclear accumulation of p53 protein in prostate carcinoma cells have to date been confined to material from primary tumors.
We studied the accumulation of p53 protein in specimens obtained from primary and metastatic sites of prostate carcinoma. By examining the accumulation of this protein as a function of stage, histologic grade, and androgen responsiveness of the tumor, we hoped to determine the role of p53 mutation in the progression of prostate carcinoma.
The accumulation of the p53 protein in the cell nuclei was determined by immunohistochemical methods using polyclonal antibody to human p53 CM-1. The material studied consisted of formalin-fixed, paraffin-embedded tissue obtained from primary tumors and metastases of 92 patients with prostate carcinoma. Twelve samples from 11 patients were analyzed for the presence of mutations within exons 5-8 of the p53 gene (also known as TP53) by polymerase chain reaction-single-stranded conformation polymorphism (PCR-SSCP) analysis. Sequence analysis was subsequently performed on DNA obtained by polymerase chain reaction amplification of PCR-SSCP reactions produced from six different specimens. The chi-square test, Fisher's exact test, and the Freeman Halton test were used for statistical analyses of the results.
All tumors with p53 accumulation were metastatic (stage D), poorly differentiated, and androgen independent. Nuclear accumulation of p53 protein was strongly associated with stage (D2 versus D1 versus A-C, P < .0001), grade (Gleason score 8-10 versus 5-7, P < .003), and androgen sensitivity (androgen independent versus dependent, P < .0001). Logistic regression analysis demonstrated that androgen sensitivity predicted p53 outcome better than did stage (P < .0001) or grade alone (P < .006). There was a perfect concordance between the results obtained by PCR-SSCP analysis and the p53 protein accumulation determined by immunohistochemistry in the 12 samples studied. Mutation of the p53 gene was confirmed by sequencing DNA obtained from six specimens positive in the PCR-SSCP assay.
p53 gene mutation is a late event in the progression of prostate cancer and is associated with advanced (metastatic) stage, loss of differentiation, and the transition from androgen-dependent to androgen-independent growth.
Testing of prostate cancer biopsy specimens from metastatic sites for p53 protein accumulation and gene mutation may provide useful prognostic information and could influence the recommended course of treatment.
Metastatic prostate cancer is a leading cause of cancer-related death in men. The rate of response to androgen ablation is high, but most patients relapse as a result of the outgrowth of androgen-independent tumor cells. The androgen receptor, which binds testosterone and stimulates the transcription of androgen-responsive genes, regulates the growth of prostate cells. We analyzed the androgen-receptor genes from samples of metastatic androgen-independent prostate cancers to determine whether mutations in the gene have a role in androgen independence.
Complementary DNA was synthesized from metastatic prostate cancers in 10 patients with androgen-independent prostate cancer, and the expression of the androgen-receptor gene was estimated by amplification with the polymerase chain reaction. Exons B through H of the gene were cloned, and mutations were identified by DNA sequencing. The functional effects of the mutations were assessed in cells transfected with mutant genes.
All androgen-independent tumors expressed high levels of androgen-receptor gene transcripts, relative to the levels expressed by an androgen-independent prostate-cancer cell line (LNCaP). Point mutations in the androgen-receptor gene were identified in metastatic cells from 5 of the 10 patients examined. One mutation was in the same codon as the mutation found previously in the androgen-independent prostate-cancer cell line. The mutations were not detected in the primary tumors from of the two patients. Functional studies of two of the mutant androgen receptors demonstrated that they could be activated by progesterone and estrogen.
Most metastatic androgen-independent prostate cancers express high levels of androgen-receptor gene transcripts. Mutations in androgen-receptor genes are not uncommon and may provide a selective growth advantage after androgen ablation.
Some transcription factors contain stretches of polyglutamine encoded by repeats of the trinucleotide CAG. Expansion of the CAG repeat in the androgen receptor (AR) has been correlated with the incidence and severity of X-linked spinal and bulbar muscular atrophy (Kennedy's disease). In order to understand the relationship of this mutation to AR function, we constructed ARs that varied in the position and size of the polyglutamine tract, and assayed for the abilities of these mutant receptors to bind androgen and to activate transcription of several different AR-responsive reporter genes. Elimination of the tract in both human and rat AR resulted in elevated transcriptional activation activity, strongly suggesting that the presence of the polyglutamine tract is inhibitory to transactivation. Progressive expansion of the CAG repeat in human AR caused a linear decrease of transactivation function. Importantly, expansion of the tract did not completely eliminate AR activity. We postulate that this residual AR activity may be sufficient for development of male primary and secondary sex characteristics, but may fall below a threshold level of activity necessary for normal maintenance of motor neuron function. This functional abnormality may be representative of other genetic diseases that are associated with CAG expansion mutations in open reading frames, such as spinocerebellar ataxia type I and Huntington's disease.
Inactivation of p53, a tumor suppressor gene, contributes to the genesis and/or progression of a substantial fraction of all human cancers, including > or = 50% of breast, lung, and colon carcinomas. Mutated p53 alleles typically contain missense single-base substitutions within exons 5-8 and encode abnormally stable p53 proteins that accumulate to high levels in tumor cell nuclei. To evaluate the frequency, type, and clinical significance of p53 mutation in human prostate cancer, archival tumor material from 150 prostate cancer patients was examined by immunohistochemistry (IHC) with anti-p53 antibodies. Abnormal nuclear p53 accumulation (IHC) was observed in 19 tumors (12.7%) and was strongly related to disease stage (23% of 69 stage III or IV tumors were IHC+ versus 4% of 74 stage 0-II tumors; P < 0.001, Fisher's exact test). The methods of polymerase chain reaction, single-strand conformational polymorphism, and direct sequencing were used to identify mutations, predominantly missense single-base substitutions in exons 5, 7, or 8 in 9 of 14 IHC+ cases but in none of 20 IHC- cases; 5 of these mutations were G:C-->A:T transitions at CpG dinucleotides. These data indicate that mutated p53 alleles are quite uncommon in early prostate cancers but are found in 20-25% of advanced cancers, suggesting a role for p53 mutation in the progression of at least a subset of prostate cancers.
Despite its high prevalence, very little is known regarding genetic predisposition to prostate cancer. A genome-wide scan
performed in 66 high-risk prostate cancer families has provided evidence of linkage to the long arm of chromosome 1 (1q24-25).
Analysis of an additional set of 25 North American and Swedish families with markers in this region resulted in significant
evidence of linkage in the combined set of 91 families. The data provide strong evidence of a major prostate cancer susceptibility
locus on chromosome 1.
Human prostate cancers frequently show loss of heterozygosity (LOH) at loci on the long arm of chromosome 16 (16q). In this study, we analyzed prostate cancer specimens from 48 patients (Stage B, 20 cases; Stage C, 10 cases; cancer death, 18 cases) for allelic loss on 16q, using either restriction fragment length polymorphism (RFLP)- or polymerase chain reaction (PCR)-based methods. Allelic losses were observed in 20 (42%) of 48 cases, all of which were informative with at least one locus. Detailed deletion mapping identified three distinct commonly deleted regions on this chromosome arm: q22.1-q22.3, q23.2-q24.1, and q24.3-qter. On the basis of a published sex-averaged framework map, the estimated sizes of the commonly deleted regions were 4.7 (16q22.1-q22.3), 17.2 (16q23.2-q24.1) and 8.4 cM (16q24.3-qter). Allelic losses on 16q were observed more frequently in the cancer-death cases (11 of 18; 61%) than in early-stage tumor cases (9 of 30; 30%; P < 0.05). In 7 of 11 patients from whom DNA was available from metastatic cancers as well as from normal tissues and primary tumors, the primary cancer foci had no detectable abnormality of 16q, but the metastatic tumors showed LOH. These results suggest that inactivation of tumor suppressor genes on 16q plays an important role in the progression of prostate cancer. We also analyzed exons 5-8 of the E-cadherin gene, located at 16q22.1, in tumor DNA by means of PCR-single strand conformation polymorphism and direct sequencing, but we detected no somatic mutations in this candidate gene.
Prostate cancer is an increasingly common disease for which there are few well-established risk factors. Family history data suggest a genetic component; however, the majority of prostate cancer cases cannot be explained by a single-gene model. Prostate cell division is influenced by two steroid hormones, testosterone and vitamin D, the action of each being mediated by its respective receptor. The genes for the two receptors are candidates in a multigenic model for prostate cancer susceptibility.
We examined genetic polymorphisms in two steroid receptors, the androgen receptor (AR) and the vitamin D receptor (VDR), in a case-control pilot study of prostate cancer.
Fifty-seven non-Hispanic white case patients with prostate cancer and 169 non-Hispanic white control subjects were genotyped for a previously described microsatellite (CAG repeats) in the AR gene and for a newly discovered poly-A microsatellite in the 3'-untranslated region (3'UTR) of the VDR gene. To compare genotypes with respect to prostate cancer risk, we estimated odds ratios (ORs) by using logistic regression. ORs were also estimated separately for advanced and localized cases of disease. All P values resulted from two-sided tests.
Both the AR and the VDR polymorphisms were associated, individually and after mutual adjustment, with prostate cancer. Adjusted ORs (95% confidence intervals [CIs]) for prostate cancer were 2.10 (95% CI = 1.11-3.99) for individuals carrying an AR CAG allele with fewer than 20 repeats versus an allele with 20 or more repeats and 4.61 (95% CI = 1.34-15.82) for individuals carrying at least one long (A18 to A22) VDR poly-A allele versus two short (A14 to A17) poly-A alleles. For both the AR and VDR genes, the at-risk genotypes were more strongly associated with advanced disease than with localized disease.
In this pilot study, genetic variation in both the VDR and the AR genes was associated with prostate cancer, and both genes appear to preferentially confer risk for advanced disease. These two genetic risk factors, if confirmed, are among the strongest risk factors yet identified for prostate cancer.
These results are consistent with a multigenic model of prostate cancer susceptibility. On the basis of the joint effect of several genetic loci, one might ultimately be able to construct a risk profile to predict advanced disease, so that men whose disease is unlikely to progress to an advanced stage can possibly be spared aggressive treatment.
We have isolated a prostate-specific gene (NKX3.1) in humans that is homologous to the Drosophila NK homeobox gene family. Northern blot analyses indicate that this gene is expressed at high levels in adult prostate and at a much lower level in testis, but is expressed little or not at all in several other tissues. In an androgen-dependent prostate carcinoma line, LNCaP, NKX3.1 mRNA is expressed at a basal level that was increased markedly upon androgen stimulation; the NKX3.1 mRNA was undetectable in several other human tumor cell lines including two androgen-independent prostate carcinoma lines. The NKX3.1 gene maps to chromosome band 8p21, a region frequently reported to undergo a loss of heterozygosity associated with tissue dedifferentiation and loss of androgen responsiveness during the progression of prostate cancer. Based on these data we propose that NKX3.1 is a candidate gene for playing a role in the opposing processes of androgen-driven differentiation of prostatic tissue and loss of that differentiation during the progression of prostate cancer.
There is genetic predisposition associated with >=10% of all cancer of the prostate (CaP). By means of a genomewide search on a selection of 47 French and German families, parametric and nonparametric linkage (NPL) analysis allowed identification of a locus, on chromosome 1q42.2-43, carrying a putative predisposing gene for CaP (PCaP). The primary localization was confirmed with several markers, by use of three different genetic models. We obtained a maximum two-point LOD score of 2.7 with marker D1S2785. Multipoint parametric and NPL analysis yielded maximum HLOD and NPL scores of 2.2 and 3.1, respectively, with an associated P value of . 001. Homogeneity analysis with multipoint LOD scores gave an estimate of the proportion of families with linkage to this locus of 50%, with a likelihood ratio of 157/1 in favor of heterogeneity. Furthermore, the 9/47 families with early-onset CaP at age <60 years gave multipoint LOD and NPL scores of 3.31 and 3.32, respectively, with P = .001.
Phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) is a key molecule involved in cell growth signaling. We demonstrated that overexpression of PTEN, a putative tumor suppressor,
reduced insulin-induced PtdIns(3,4,5)P3 production in human 293 cells without effecting insulin-induced phosphoinositide 3-kinase activation. Further, transfection
of the catalytically inactive mutant of PTEN (C124S) caused PtdIns(3,4,5)P3 accumulation in the absence of insulin stimulation. Purified recombinant PTEN catalyzed dephosphorylation of PtdIns(3,4,5)P3, specifically at position 3 on the inositol ring. PTEN also exhibited 3-phosphatase activity toward inositol 1,3,4,5-tetrakisphosphate.
Our results raise the possibility that PTEN acts in vivo as a phosphoinositide 3-phosphatase by regulating PtdIns(3,4,5)P3 levels. As expected, the C124S mutant of PTEN was incapable of catalyzing dephosphorylation of PtdIns(3,4,5)P3 consistent with the mechanism observed in protein-tyrosine phosphatase-catalyzed reactions.
To study the genetic basis of tumor progression, we have screened 37 hormone-refractory prostate carcinomas for genetic changes by comparative genomic hybridization (CGH). All recurrent tumors showed genetic aberrations, with a mean total number of changes per tumor of 11.4 (range, 3 to 23). The most common genetic aberrations were losses of 8p (72.5%), 13q (50%), 1p (50%), 22 (45%), 19 (45%), 10q (42.5%), and 16q (42.5%) and gains of 8q (72.5%), 7q (40%), Xq (32.5%), and 18q (32.5%). The CGH results were further validated with fluorescence in situ hybridization (FISH) using probes for pericentromeric regions of chromosomes 7, 8, and 18 as well as probes for caveolin (7q31), c-myc (8q24), and bcl-2 (18q21.3). In addition, the samples had previously been analyzed for androgen receptor gene copy number. CGH and FISH results were concordant in 78% of cases. Seventeen of twenty-two tumors showed an increased copy number of c-myc by FISH. However, only 5 of 17 (29%) of the cases showed high-level (more than threefold) amplification. Both CGH and FISH findings suggested that in most of the cases 8q gain involves the whole q-arm of the chromosome. Four of seventeen (24%) cases showed increased copy number of bcl-2 by FISH; however, no high-level amplifications were found. To evaluate the clonal relationship of the primary and recurrent tumors, six primary-recurrent tumor pairs from the same patients were studied by CGH. In three of six cases (50%), the recurrent tumor had more than one-half of the aberrations found in the corresponding primary tumor, indicating a close clonal relationship. In the rest of the cases, such a linear clonal relationship was less evident. Altogether, these results suggest that recurrent prostate carcinomas are genetically unstable. The resulting heterogeneity may well underlie the poor responsiveness of hormone-refractory tumors to treatment.
Deletion of the q23-24 region of human chromosome 10 is one of the most frequent genetic alterations in prostate cancer, suggesting that inactivation of a tumor suppressor gene in this region is involved in the development or progression of this carcinoma. A candidate gene, PTEN/MMAC1, has been identified from this chromosomal region; mutations of this gene have been found in various advanced tumors and cell lines including those of prostate cancer. To further define the role of PTEN/MMAC1 in the development of prostate cancer and its spectrum of genetic alterations, we analysed 40 pT2 or pT3 prostate tumors for allelic loss, mutations, and homozygous deletions using PCR-based methods. Six tumors showed loss of heterozygosity for one of the ten markers analysed, while one tumor showed loss of two markers. None of the markers within PTEN/MMAC1 was lost. Direct sequencing of PCR amplified exons and intron/exon junctions of all 40 tumors revealed three sequence variants, one of which was a point mutation in exon 9, while the other two were polymorphisms. Using multiplex PCR, no homozygous deletions were detected in any of the neoplasms. Our results showing a low frequency of alterations of PTEN/MMAC1 in pT2 and pT3 prostate cancers suggest that this gene plays an insignificant role in the development of most low stage carcinomas of the prostate.
Combining data from a genomic screen in 70 families with a high risk for prostate cancer (PC) with data from candidate-region mapping in these families and an additional 71 families, we have localized a potential hereditary PC-susceptibility locus to chromosome 1p36. Because an excess of cases of primary brain cancer (BC) have been observed in some studies of families with a high risk for PC, and because loss of heterozygosity at 1p36 is frequently observed in BC, we further evaluated 12 families with both a history of PC and a blood relative with primary BC. The overall LOD score in these 12 families was 3.22 at a recombination fraction (theta) of .06, with marker D1S507. On the basis of an a priori hypothesis, this group was stratified by age at diagnosis of PC. In the younger age group (mean age at diagnosis <66 years), a maximum two-point LOD score of 3.65 at straight theta = .0 was observed, with D1S407. This linkage was rejected in both early- and late-onset families without a history of BC (LOD scores -7.12 and -6.03, respectively, at straight theta = .0). After exclusion of 3 of the 12 families that had better evidence of linkage to previously described PC-susceptibility loci, linkage to the 1p36 region was suggested by a two-point LOD score of 4.74 at straight theta = .0, with marker D1S407. We conclude that a significant proportion of these families with both a high risk for PC and a family member with BC show linkage to the 1p36 region.
Despite the substantial clinical importance of prostate cancer, the molecular mechanisms underlying the development and progression of the disease are poorly understood. The aim of molecular genetics is to reveal the genetic alterations and genes that are involved in disease processes. Linkage analysis have already implicated four chromosomal loci that may harbour prostate cancer susceptibility genes. In addition, chromosomal alterations in prostate tumors have been studied using several techniques, such as comparative genomic hybridization. These analyses have indicated that losses of chromosomes 6q, 8p, 10q, 13q, and 16q, as well as gains of 7, 8q, and Xq are particularly common in prostate cancer. There is also a strong evidence, that genes, such as androgen receptor gene (AR), e-cadherin, and PTEN, are involved in the development and progression of prostate cancer. However, the target genes for most of the above mentioned chromosomal alterations as well as the genes predisposing to prostate cancer have not been cloned yet. The identification of those genes should be the utmost goal of basic research of prostate cancer, today.
Chromosome 13 is one of the most frequently altered chromosomes in cancer, including carcinoma of the prostate. Two known tumor suppressor genes, RB1 and BRCA2, map to chromosome 13; however, recent reports suggest that unknown genes on 13q are more likely to be involved in the development of prostate cancer. In order more fully to define the genetic changes on chromosome 13 in prostate neoplasms, we analyzed 27 polymorphic microsatellite markers spanning the q arm for loss of heterozygosity in 40 primary tumors and in metastases from 11 other patients who died of prostate cancer. Of the 40 primary tumors, 23 (58%) showed LOH for at least one marker. Three distinct regions at q14, q21-22, and q33, defined by markers D13S267-->D13S153, D13S166-->D13S1225, and D13S259-->D13S274, showed the most frequent LOH, suggesting their involvement in the development of prostate cancer. For the 12 patients whose tumors showed LOH at these markers, the average age at diagnosis was 58 years, which was younger than that (63 years, P < 0.05) for the 28 patients whose tumors lacked LOH. Ten of the 11 (91%) metastases showed LOH with one or more markers. Two of the three most frequently deleted regions (i.e., q14 and q21-22) in the primary tumors and markers linked to the RB1, BRCA2, and EDNRB genes showed high frequencies (56-71%) of LOH in metastases. These results demonstrate that allelic loss on chromosome 13 at q14, q21-22, and q33 occurs in a subset of primary prostate tumors and is a frequent event in metastatic lesions of prostate cancer.
Amplification at the long arm of chromosome 8 occurs in a large fraction of breast and prostate cancers. To clone the target genes for this amplification, we used suppression subtraction hybridization to identify overexpressed genes in the breast cancer cell line SK-Br-3, which harbors amplification at 8q (8q21 and 8q23-q24). A differentially expressed gene identified by SSH, the p40 subunit of eukaryotic translation initiation factor 3 (eIF3), was localized to 8q23 and found to be highly amplified and overexpressed in the breast and prostate cancer cell lines studied. High-level amplification of eIF3-p40 was found in 30% of hormone-refractory prostate tumors and in 18% of untreated primary breast tumors. In the vast majority of the cases, p40 and c-myc were amplified with equal copy numbers. Tumors with higher copy numbers of p40 than c-myc were also found. Expression of p40 mRNA was analyzed with in situ hybridization. The amplification of eIF3-p40 gene was associated with overexpression of its mRNA, as expected for a functional target gene of the amplification. These results imply that genomic aberrations of translation initiation factors, such as eIF3-p40, may contribute to the pathogenesis of breast and prostate cancer.
Prostate cancer is a very common disease in more-developed countries, but its cause is largely unknown. It is an androgen-dependent cancer, and androgens have been proposed as having a substantial role in predisposition to the disease. Thus, variations in androgen metabolism genes may affect risk of this disease.
We screened 216 African-American and 172 Hispanic men with prostate cancer, and 261 African-American and 200 Hispanic healthy men (controls), from a large prospective cohort study (the Hawaii-Los Angeles Multiethnic Cohort Study) for a mis-sense substitution in the human prostatic (or type II) steroid 5alpha-reductase (SRD5A2) gene, the product of which controls metabolic activation of testosterone to dihydrotestosterone. This mis-sense substitution results in an alanine residue at codon 49 being replaced with threonine (A49T). We also reconstructed this mutation in the SRD5A2 cDNA, and overexpressed the enzyme in mammalian tissue culture cells.
The A49T aminoacid substitution in the SRD5A2 gene increased the risk of clinically significant disease 7.2-fold in African-American men (95% CI=2.17-27.91; p=0.001) and 3.6-fold in Hispanic men (1.09-12.27; p=0.04). The mutant enzyme had a higher in-vitro Vmax than the normal enzyme (9.9 vs 1.9 nmol min(-1) mg(-1)).
The A49T variant of the SRD5A2 gene may be a significant contributor to the incidence of prostate cancer in African-American and Hispanic men in Los Angeles. We estimate that the population attributable risk due to this aminoacid substitution for clinically significant disease is about 8% in both populations. Increased conversion of testosterone to dihydrotestosterone catalysed by this variant steroid 5alpha-reductase enzyme may be the cause of the increased risk.
Analysis of a genome screen of 504 brothers with prostate cancer (CaP) who were from 230 multiplex sibships identified five regions with nominally positive linkage signals, on chromosomes 2q, 12p, 15q, 16p, and 16q. The strongest signal in these data is found on chromosome 16q, between markers D16S515 and D16S3040, a region suspected to contain a tumor-suppressor gene. On the basis of findings from previous genome screens of families with CaP, three preplanned subanalyses were carried out, in the hope of increasing the subgroup homogeneity. Subgroups were formed by dividing the sibships into a group with a positive family history (FH+) that met criteria for "hereditary" CaP (n=111) versus those which did not meet the criteria (n=119) and by dividing the families into those with a mean onset age below the median (n=115) versus those with a mean onset age above the median (n=115). A separate subanalysis was carried out for families with a history of breast cancer (CaB+ [n=53]). Analyses of these subgroups revealed a number of potentially important differences in regions that were nonsignificant when all the families were analyzed together. In particular, the subgroup without a positive family history (FH-) had a signal in a region that is proximal to the putative site of the HPC1 locus on chromosome 1, whereas the late-age-at-onset group had a signal on 4q. The CaB+ subgroup revealed a strong linkage signal at 1p35.1.
Recent studies suggest that hereditary prostate cancer is a complex disease involving multiple susceptibility genes and variable phenotypic expression. While conducting a genomewide search on 162 North American families with > or =3 members affected with prostate cancer (PRCA), we found evidence for linkage to chromosome 20q13 with two-point parametric LOD scores >1 at multiple sites, with the highest two-point LOD score of 2.69 for marker D20S196. The maximum multipoint NPL score for the entire data set was 3.02 (P=.002) at D20S887. On the basis of findings from previous reports, families were stratified by the presence (n=116) or absence (n=46) of male-to-male transmission, average age of diagnosis (<66 years, n=73; > or =66 years, n=89), and number of affected individuals (<5, n=101; > or =5, n=61) for further analysis. The strongest evidence of linkage was evident with the pedigrees having <5 family members affected with prostate cancer (multipoint NPL 3.22, P=.00079), a later average age of diagnosis (multipoint NPL 3.40, P=.0006), and no male-to-male transmission (multipoint NPL 3.94, P=.00007). The group of patients having all three of these characteristics (n=19) had a multipoint NPL score of 3.69 (P=.0001). These results demonstrate evidence for a PRCA susceptibility locus in a subset of families that is distinct from the groups more likely to be linked to previously identified loci.
The aggressiveness of prostate cancer (PCa) varies widely: some tumors progress to invasive, potentially life-threatening disease, whereas others stay latent for the remainder of an individual's lifetime. The mechanisms resulting in this variability are not yet understood, but they are likely to involve both genetic and environmental influences. To investigate genetic factors, we conducted a genomewide linkage analysis of 513 brothers with PCa, using the Gleason score, which reflects tumor histology, as a quantitative measure of PCa aggressiveness. To our knowledge, this is the first time that a measure of PCa aggressiveness has been directly investigated as a quantitative trait in a genomewide scan. We employed a generalized multipoint Haseman-Elston linkage-analysis approach that regresses the mean-corrected cross product between the brothers' Gleason scores on the estimated proportion of alleles shared by brothers identical by descent at each marker location. Our results suggest that candidate regions on chromosomes 5q, 7q, and 19q give evidence for linkage to PCa-aggressiveness genes. In particular, the strongest signals detected in these regions were at the following markers (with corresponding P values): for chromosome 5q31-33, between markers D5S1480 and D5S820 (P=.0002); for chromosome 7q32, between markers D7S3061 and D7S1804 (P=.0007); and, for chromosome 19q12, at D19S433 (P=.0004). This indicates that one or more of these candidate regions may contain genes that influence the progression of PCa from latent to invasive disease. Identification of such genes would be extremely valuable for elucidation of the mechanism underlying PCa progression and for determination of treatment in men in whom this disease has been diagnosed.
Previous cytogenetic and molecular genetic analyses suggest that the q21 band of chromosome 13 harbors a tumor suppressor gene(s) involved in prostatic carcinogenesis. The precise genetic location, however, has not been defined. In this study, we examined prostate cancer specimens and cell lines/xenograft for genetic deletions at 13q21, using the methods of tissue microdissection and duplex PCR. Deletions at 13q21 were detected in 13 of 147 (9%) prostate cancer samples. Deletion of the same region was also detected in the LNCaP cell line and the PC-82 xenograft of prostate cancer. The overlapping region of deletion in LNCaP and PC-82 spans 3.1 cM or 2.9 cR, which is equivalent to 1-3 Mb. The endothelin receptor B gene, a possible tumor suppressor gene at 13q21, was not located in the region of deletion. Among the 13 prostate neoplasms with deletion at 13q21, 5 were metastases, and 7 were poorly differentiated primary tumors. The only primary tumor that was not poorly differentiated but had deletion occurred in one of the youngest patients (49 years) at diagnosis. These results provide evidence that 13q21 may harbor an unidentified gene(s) whose inactivation occurs in some aggressive carcinomas of the prostate. In addition, this study provides a framework for the cloning and identification of the 13q21 gene(s).
beta-Catenin is a multifunctional molecule with important roles in intercellular adhesion and signal transduction. We reported previously that beta-catenin is mutated in human prostate cancer. In this study, we investigated the role of beta-catenin mutations on androgen receptor (AR) signaling. beta-Catenin significantly enhanced androgen-stimulated transcriptional activation by the AR. beta-Catenin also increased AR transcriptional activation by androstenedione and estradiol and diminished the antagonism of bicalutamide. Coimmunoprecipitation of beta-catenin with AR from LNCaP prostate cancer cells showed that the two molecules are present in the same complex. The amount of beta-catenin in complex with AR was increased by androgen. These findings implicate beta-catenin in the regulation of AR function and support a role for beta-catenin mutations in the pathogenesis of prostate cancer.