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Unique Anti-Activator Protein-1 Activity of Retinoic Acid Receptor ??
Abstract
The anticancer effects of retinoids are mainly mediated by two classes of nuclear receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs), which are encoded by three distinct genes (alpha, beta, and gamma). Recent studies have demonstrated that RARbeta plays a critical role in mediating anticancer effects of retinoids. However, how RARbeta exerts its potent anticancer effects remains largely unknown. In this study, we investigated anti-Activator Protein-1 (AP-1) activity of RARbeta. In a transient transfection assay, all three RAR subtypes, RARalpha, RARbeta, and RARgamma, could effectively inhibit phorbol ester 12-O-tetradecanoylphorbol-13-acetate-induced AP-1 activity and the activity of oncogenes c-Jun and c-Fos on AP-1 containing reporter genes in the presence of retinoic acid (RA). However, RARbeta showed a strong RA-independent inhibition of AP-1 activity, whereas inhibition of AP-1 activity by RARalpha and RARgamma was RA dependent. By using several hybrid receptors that contain either the COOH-terminal portion or the NH2-terminal portion of RARbeta, we demonstrated that the NH2-terminal portion of RARbeta, the A/B domain, was mainly responsible for the RA-independent inhibition of AP-1 activity. This activity was not attributable to constitutive AF-1 activity of RARbeta, because it did not activate several RA response element-containing reporter genes. In addition, inhibition of histone deacetylase activity by trichostatin A did not overcome the inhibitory effect of RARbeta. In cancer cells, stable transfection of RARbeta exhibited strong inhibition of AP-1 activity, even in the absence of RA. Moreover, expression of endogenous AP-1-responsive gene collagenase I was strongly repressed in cancer cells stably transfected with RARbeta. In studying the antitransforming activity of RARbeta, we observed that the growth of breast cancer MDA-MB231 cells in soft agar was significantly repressed in a RA-independent manner when cells were stably transfected with RARbeta but not RARalpha. Together, our results demonstrate that RARbeta may exert its potent anticancer effect in part through its unique anti-AP-1 activity.
... Given that in MDA-MB231 cells RARs were incapable of trans-repress AP-1 sites upon ATRA treatment, next we investigated whether RARβ could restore this ability. Although both RARβ and RARγ levels are practically undetectable in this cell line, we chose to express RARβ since this retinoid receptor has been described as the main responsible of trans-repressing activities (Lin et al. 2000). As shown in Fig. 6a, we could induce an important increase in RARβ expression upon transfection. ...
... The RARβ receptor has been described as the main responsible of AP-1 inhibition (Karamouzis et al. 2004;Lin et al. 2000;Yang et al. 1997), thus we next analyzed the expression of all RAR components in both cell models. ...
PurposeRetinoids have proved to be effective for hematologic malignancies treatment but till nowadays, their use as single agent for the solid tumor’s management is still controversial. All-trans retinoic acid (ATRA), the main active metabolite of vitamin A, exerts non-genomic interactions with different members of the protein kinase C (PKC) family, recognized modulators of different tumor progression pathways. To determine whether a group of patients could become benefited employing a retinoid therapy, in this study we have evaluated whether PKCα expression (a poor prognosis marker in breast cancer) could sensitizes mammary cells to ATRA treatment.MethodsPKCα overexpression was achieved by stable transfection and confirmed by western blot. Transfected PKC functionality was determined by nuclear translocation-induction and confocal microscopy. In vitro proliferation was evaluated by cell counting and cell cycle distribution was analyzed by flow cytometry. In vivo studies were performed to evaluate orthotopic tumor growth and experimental lung colonization. Retinoic acid response elements (RARE) and AP1 sites-dependent activity was studied by gene reporter assays and retinoic acid receptors (RARs) were measured by RT-qPCR.
ResultsOur findings suggest that high PKCα levels improve the differentiation response to ATRA in a RAR signaling-dependent manner. Moreover, RARβ expression appears to be critical to induce ATRA sensitization, throughout AP1 trans-repression.
Conclusion
Here we propose that retinoids could lead a highly personalized anticancer treatment, bringing benefits to patients with aggressive breast tumors resulting from high PKCα expression but, an adequate expression of the RARβ receptor is required to ensure the effect on this process.
... Exogenous expression of RARb2 in cancer cells presenting low or absent RARb2 expression restored RA induced G1 phase inhibition and caused decreased tumorigenicity (191). This expression of RARb2 results both in RA-dependent and RA-independent apoptosis as well as growth arrest even in breast cancer cell lines with scanty amounts of RARa, the first effector of RARb P2 promoter (185,190,195). Moreover, F9 teratocarcinoma cells RARb2 null could not undergo growth arrest in the presence of RA, indicating that RARb2 is required for the growth inhibitory action of RA (194). ...
Persistent infection with high-risk human papillomaviruses (HR-HPVs) is involved in cervical cancer (CC), a major cause of cancer mortality worldwide. Infection occurs primarily at the transformation zone (TZ), the most estrogen-and retinoid-sensitive region of the cervix. Development of CC affects a small percentage of HR-HPV-infected women and often takes decades after infection, suggesting that HR-HPV is a necessary but not sufficient cause of CC. Thus, other cofactors are necessary for progression from cervical HR-HPV infection to cancer such as long-term use of hormonal contraceptives, multiparity, smoking, as well as micronutrient depletion and in particular retinoid deficiency, which alters epithelial differentiation, cellular growth and apoptosis of malignant cells. Therefore, early detection of HR-HPV and management of precancerous lesions together with a profound understanding of additional risk factors could be a strategy to avoid this disease. In this review we focus on the synergic effect of estrogens, retinoid deficiency and HR-HPVs in the development of CC. These risk factors may act in concert to induce neoplastic transformation in squamous epithelium of the cervix, setting the stage for secondary genetic or epigenetic events leading to cervical cancer. Ó 2009 IMSS. Published by Elsevier Inc.
... RARB (retinoic acid receptor beta) is a tumor suppressor protein involved in regulation of cell proliferation and differentiation, cell cycle progression, and apoptosis (Alvarez et al., 2007). RARB can act as a potent repressor of transcriptional activity of the AP-1 protein complex (Lin et al., 2000;Yang et al., 1997). Thus, the proteins encoded by PTEN, RARB, APC, and CDKN2A that are negative regulators of AP-1, TCF, and E2F might be indirectly involved in the DNMT1 downregulation (Bigey et al., 2000;Qin et al., 2011). ...
Clofarabine (2-chloro-2’-fluoro-2’-deoxyarabinosyladenine, ClF), a second-generation 2’-deoxyadenosine analog, possesses manifold anti-cancer activities. Our previous reports and some of others demonstrate the potential capacity of ClF to regulate the epigenetic machinery. The study presented here is the first to investigate the influence of ClF on modulators of the DNA methylation machinery, including DNMT1 and CDKN1A, in acute lymphoblastic leukemia (ALL) cells. ClF effects on promoter methylation and transcriptional activity of hypermethylated and silenced tumor suppressor genes (TSGs), including APC, CDKN2A, PTEN, and RARB, have been tested as well. Methylation level of the proximal promoter region of APC, CDKN2A, PTEN, and RARB, as well as expression of those TSGs, DNMT1 and CDKN1A, were estimated by using a methylation-sensitive restriction analysis and qPCR, respectively. The Nalm-6 cell line was used as an experimental in vitro model of ALL cells. We observed ClF-mediated inhibition of cellular viability and apoptosis induction of Nalm-6 cells with an increased percentage of cells positive for active Caspase-3. Interestingly, exposure of Nalm-6 cells to CIF at 20 nM concentration for three days has led to a significant DNMT1 downregulation, accompanied by robust CDKN1A upregulation. ClF caused hypomethylation of APC, CDKN2A, and PTEN, with a concomitant increase in their transcript levels. Taken together, our results demonstrate the ability of ClF to reactivate DNA methylation-silenced TSGs in ALL cells. This may implicate translational significance of our findings and support ClF application as a new epigenetic modulator in the anti-leukemia therapy.
... Indeed, in this paper the authors stated that RARβ strongly inhibit AP-1 activity in ATRA-independent fashion, whereas inhibition of AP-1 activity by RARα and RARγ was ATRA-dependent. Therefore, RARβ could act as tumor suppressor even in the absence of ATRA, and RARβ loss expression gives AP-1 over-expression, which could abrogate the growth-inhibitory effects of ATRA through RARα and RARγ, resulting in retinoid resistance [222]. However, retinoids showed also the capacity to inhibit the AP-1 responsive genes in ATRA-sensitive MCF-7 cell line by inhibiting MKK6/p38 and MEK/ERK signaling pathway [223]. ...
Although the use of oral administration of pharmacological all-trans retinoic acid (ATRA) concentration in acute promyelocytic leukaemia (APL) patients was approved for over 20 years and used as standard therapy still to date, the same use in solid cancers is still controversial. In the present review the literature about the top five lethal solid cancers (lung, stomach, liver, breast, and colon cancer), as defined by The Global Cancer Observatory of World Health Organization, and retinoic acids (ATRA, 9-cis retinoic acid, and 13-cis retinoic acid, RA) was compared. The action of retinoic acids in inhibiting the cell proliferation was found in several cell pathways and compartments: from membrane and cytoplasmic signaling, to metabolic enzymes, to gene expression. However, in parallel in the most aggressive phenotypes several escape routes have evolved conferring retinoic acids-resistance. The comparison between different solid cancer types pointed out that for some cancer types several information are still lacking. Moreover, even though some pathways and escape routes are the same between the cancer types, sometimes they can differently respond to retinoic acid therapy, so that generalization cannot be made. Further studies on molecular pathways are needed to perform combinatorial trials that allow overcoming retinoic acids resistance.
... The target genes include RAR-β, TGF-β, IGFBP-3, p21 WAF13 , Hoxa1, and TIG-3 which are involved in regulation of cell cycle, differentiation, and apoptosis (Falk et al. 1991;Gucev et al. 1996;Liu et al. 1996;Marshall et al. 1996;DiSepio et al. 1998;Sun et al. 2000). Thus, the RAR-β is specifically known as a key transcription factor which modulates the expression of cell regulatory proteins (Liu et al. 2014) and is also a well-accepted tumor suppressor (Lin et al. 2000b) as it is involved in the regulation of cell growth and induction of apoptosis (Li et al. 1998). A study in human medulloblastoma cell line also highlighted that among the studied types of RARs, the RAR-β is a tumor suppressor gene which itself is one of the target genes expressed during ATRA-RAR interaction (Chang et al. 2007). ...
Targeting the specific molecular proteins or genes which are responsible for the suppression of cancer growth is currently an emerging molecular method to treat cancer. ATRA treatment is now considered as a molecular targeted therapy for many cancers. As ATRA exhibits its therapeutic effect through its receptors, this study was focused to investigate the effect and action of liposomal-ATRA treatment on the expression of RAR-β protein which is also a tumor suppressor. The liposomal-ATRA was developed with cationic DOTAP and cholesterol by thin-film formation method. The benzo(a)pyrene(50 mg/kg b.wt)-induced mice were treated with free and liposomal-ATRA(0.60 mg/kg b.wt). The RAR-β protein expression in lung and liver tissue samples were analyzed by immunohistochemistry (IHC) and western blotting (WB) on the 30th and 120th days. Almost nil expression of RAR-β protein was observed in B(a)P cancer control and liposome alone-treated groups. A comparatively elevated expression was seen in the free ATRA-treated group (IHC score—2+ in lung on the 120th day with band density of 14.46 ± 1.24% in WB). Interestingly, the liposomal-ATRA treatment demonstrated a significantly (p ≤ 0.01) higher RAR-β expression in lung (35.20 ± 3.398% band intensity and score 4+ in the 120th day) than that of in ATRA alone treatment. This study results indicate that the RAR-β protein expression was suppressed by B(a)P during cancer induction even on the 30th day itself. The treatment could reactivate the suppression and the lipo-ATRA treatment could show significantly higher RAR-β expression on the 120th day, which implies that it accumulated more ATRA in target site and sustained it for enhanced action.
... RARB is a tumor suppressor protein that modulates cell proliferation and differentiation, cell cycle progression, and apoptosis [27]. RARB can act as an effective suppressor of transcriptional activity of AP-1 (activator protein 1) protein complex [28,29]. PTEN encodes protein involved in downregulation of intracellular oncogenic signaling pathways, such as phosphoinositide 3-kinase (PI3K)/AKT and mitogen-activated protein kinase (MAPK)/AP-1 [30,31]. ...
An epigenetic component, especially aberrant DNA methylation pattern, has been shown to be frequently involved in sporadic breast cancer development. A growing body of literature demonstrates that combination of agents, i.e. nucleoside analogues with dietary phytochemicals, may provide enhanced therapeutic effects in epigenetic reprogramming of cancer cells. Clofarabine (2-chloro-2′-fluoro-2′-deoxyarabinosyladenine, ClF), a second-generation 2′-deoxyadenosine analogue, has numerous anti-cancer effects, including potential capacity to regulate epigenetic processes. Our present study is the first to investigate the combinatorial effects of ClF (used at IC50 concentration) with epigallocatechin-3-gallate (EGCG, tea catechin) or genistein (soy phytoestrogen), at physiological concentrations, on breast cancer cell growth, apoptosis, and epigenetic regulation of retinoic acid receptor beta (RARB) transcriptional activity. In MCF7 and MDA-MB-231 cells, RARB promoter methylation and expression of RARB, modifiers of DNA methylation reaction (DNMT1, CDKN1A, TP53), and potential regulator of RARB transcription, PTEN, were estimated using methylation-sensitive restriction analysis (MSRA) and quantitative real-time polymerase chain reaction (qPCR), respectively. The combinatorial exposures synergistically or additively inhibited the growth and induced apoptosis of breast cancer cells, followed by RARB hypomethylation with concomitant multiple increase in RARB, PTEN, and CDKN1A transcript levels. Taken together, our results demonstrate the ability of ClF-based combinations with polyphenols to promote cancer cell death and reactivate DNA methylation-silenced tumor suppressor genes in breast cancer cells with different invasive potential.
... Overexpression of AP-1 is found to be associated with squamous metaplasia and with increased expression of PCNA in the lung [8]. Mechanistically, the RARβ DNA binding domain was important for inhibition of AP-1 activity [32]. In addition, it is found that RARβ has unique anti-AP-1 activity in that it exhibits strong inhibition of AP-1 activity via protein-protein interaction in the absence of the ligand retinoic acid [33,34]. ...
We have previously demonstrated that cigarette smoke is associated with a significant reduction of retinoic acid in rat lungs and the formation of tracheal precancerous lesions. However, the underlying mechanism of cancer risk induced by vitamin A deficiency is unclear. The purpose of this study was to determine whether the cigarette smoke-induced depletion of vitamin A is related to changes in lung cancer risk-related molecular markers.
We investigated the roles of the retinoic acid receptors (RARs) as well as other biomarkers for potential cancer risk in the lungs of rats exposed to cigarette smoke. Twenty-four male weanling rats were fed a purified diet and divided equally into four groups. Three experimental groups were exposed to increasing doses of cigarette smoke from 20, 40 or 60 commercial cigarettes/day for 5 days/week. After 6 weeks, the retinoic acid concentrations in the lung tissue as measured via high performance liquid chromatography (HPLC) significantly decreased (P < 0.01) in cigarette smoke exposed groups. Western Blot analysis revealed that cigarette smoke exposure increased lung protein expression of RAR α in a threshold manner and decreased RAR β and RAR γ expression in a dose-dependent fashion. Protein expressions of cyclin E and proliferating cell nuclear antigen (PCNA) were increased significantly in a dose-dependent manner in cigarette smoke exposed-groups. Additionally, there was a significant increase in protein expression of cJun and cyclin D1 demonstrating a threshold effect similar to that exhibited by RARα, suggesting a potential independent signaling pathway for RARα in lung carcinogenesis.
Findings from this study suggest that cigarette smoke-induced lung retinoic acid depletion may involve two independent pathways, RARα- and RARβ-mediated, responsible for the increased cancer risk associated with cigarette smoke-induced vitamin A deficiency.
... Several in vitro studies have revealed that RARB inhibits proliferation through inhibition of the transcription factor activator protein-1 (AP-1) via both liganddependent and ligand-independent mechanisms (van der Burg et al, 1995;Lee et al, 1996;Lin et al, 2000;De-Castro Arce et al, 2004). AP-1 is a heterodimeric protein containing proteins from 2 major families, Jun (c-Jun, JunB, JunD) and Fos (c-Fos, FosB, Fra-1, Fra-2) (Angel & Karin, 1991). ...
... The dimeric combination of the AP-1 family determines the genes that are regulated by AP-1 (15,16). Cellular growth, proliferation, survival, differentiation, and environmental stress signals induce formation of the AP-1 transcriptional complex and modulate AP-1 transcriptional activity (14,17). Activating AP-1 may contribute to breast and other tumor cell growth, invasive ability, and metastasis (18,19). ...
LY6K is a cancer biomarker and a therapeutic target that induces invasion and metastasis. However, the molecular mechanisms
that determine human LY6K transcription are completely unknown. To elucidate the mechanisms involved in human LY6K gene regulation and expression, multiple cis-elements were predicted using TRANSFAC software, and the LY6K regulatory region was identified using the luciferase assay in the human LY6K gene promoter. We performed ChIP, EMSA, and supershift assays to investigate the transcription factor activity on the LY6K promoter, and the effect of a SNP and CpG site methylation on AP-1 transcription factor binding affinity. AP-1 and the CREB
transcription factor bound to LY6K promoter within −550/−1, which was essential for LY6K expression, but only the AP-1 heterodimer, JunD, and Fra-1, modulates
LY6K gene transcriptional level. A decrease in LY6K was associated with the SNP242 C allele, a polymorphic G/C-SNP at the 242
nucleotide in the LY6K promoter region (rs2585175), or methylation of the CpG site, which was closely located with the AP-1 site by interfering
with binding of the AP-1 transcription factor to the LY6K promoter. Our findings reveal an important role for AP-1 activation in promoting LY6K gene expression that regulates cell mobility of breast cancer cells, whereas the SNP242 C allele or methylation of the CpG
site may reduce the risk of invasion or metastasis by interfering AP-1 activation.
... Blocking AP-1 activity augmented antitumoral efficacy in HNSCC cells (98). In vitro and in vivo evidence demonstrated that the antitumor effect of retinoid was enhanced by inhibiting AP-1 activity (100,101). Retinoid has appeared a promising chemopreventive drug in several clinical trials ( Table 1). The combination of AP-1 inhibitor with retinoid or other chemopreventive compounds could be a useful tool for cancer chemoprevention. ...
Head and neck squamous cell carcinoma (HNSCC) is one of the most fatal cancers worldwide. Despite advances in the management of HNSCC, the overall survival for patients has not improved significantly due to advanced stages at diagnosis, high recurrence rate after surgical removal, and second primary tumor development, which underscore the importance of novel strategies for cancer prevention. Cancer chemoprevention, the use of natural or synthetic compounds to prevent, arrest, or reverse the process of carcinogenesis at its earliest stages, aims to reverse premalignancies and prevent second primary tumors. Genomics and proteomics information including initial mutation, cancer promotion, progression, and susceptibility has brought molecularly targeted therapies for drug development. The development of preventive approaches using specific natural or synthetic compounds, or both, requires a depth of understanding of the cross-talk between cancer signaling pathways and networks to retain or enhance chemopreventive activity while reducing known toxic effects. Many natural dietary compounds have been identified with multiple molecular targets, effective in the prevention and treatment of cancer. This review describes recent advances in the understanding of the complex signaling networks driving cancer progression and of molecularly targeted natural compounds under preclinical and clinical investigation.
... Exogenous expression of RARb2 in cancer cells presenting low or absent RARb2 expression restored RA induced G1 phase inhibition and caused decreased tumorigenicity (191). This expression of RARb2 results both in RA-dependent and RA-independent apoptosis as well as growth arrest even in breast cancer cell lines with scanty amounts of RARa, the first effector of RARb P2 promoter (185,190,195). Moreover, F9 teratocarcinoma cellsFigure 2. Inactivation of AP1 by RARb. ...
Persistent infection with high-risk human papillomaviruses (HR-HPVs) is involved in cervical cancer (CC), a major cause of cancer mortality worldwide. Infection occurs primarily at the transformation zone (TZ), the most estrogen- and retinoid-sensitive region of the cervix. Development of CC affects a small percentage of HR-HPV-infected women and often takes decades after infection, suggesting that HR-HPV is a necessary but not sufficient cause of CC. Thus, other cofactors are necessary for progression from cervical HR-HPV infection to cancer such as long-term use of hormonal contraceptives, multiparity, smoking, as well as micronutrient depletion and in particular retinoid deficiency, which alters epithelial differentiation, cellular growth and apoptosis of malignant cells. Therefore, early detection of HR-HPV and management of precancerous lesions together with a profound understanding of additional risk factors could be a strategy to avoid this disease. In this review we focus on the synergic effect of estrogens, retinoid deficiency and HR-HPVs in the development of CC. These risk factors may act in concert to induce neoplastic transformation in squamous epithelium of the cervix, setting the stage for secondary genetic or epigenetic events leading to cervical cancer.
... Analysis of RARβ protein domains shows that both the N-terminal portion (A/B and C domains) and the C-terminal portion (E/F domain) are involved in the inhibition of AP-1 transcriptional activity. Additionally, theA/B domain of RARβ is responsible for retinoic acid independent repression ofAP-1 transcriptional activity (Lin et al., 2000). Consequently analysis of RARβ2 and RARβ4 AP-1 trans-repression activity reveal that RARβ4 has not anti-AP-1 activity. ...
In many types of cancer, including cervical cancer, the nuclear retinoic receptor ß2 (RARß2) gene is epigenetically modified and unable to be significantly induced. RARß2 can act as a tumor suppressor, since loss of its expression is associated with human tumor progression. One mechanism of RARß2-mediated growth inhibition is based on its ability to constitutively repress the AP-1 transcription factor, but this mechanism has never been demonstrated in cervical cancer cells. In this thesis the biological consequences of reconstitute RARß2 receptor expression in cervical cancer cells was investigated. For this purpose, human papillomavirus HPV-18 positive HeLa cells were stably transfected with RARß2 cDNA under the control of the ß-actin promoter. The characterization of the RARß2 tranfectants revealed a strongly reduced AP-1 binding to the corresponding specific oligonucleotides, even in the absence of atRA treatment. In HeLa cells, the AP-1 reduction correlates with diminished HPV-18 oncogene transcription and slower cellular growth. Western blot analysis demonstrated that the only member of the AP-1 family consistently reduced in HeLa RARß2 clones was c-Jun, despite ongoing gene expression. In order to understand by which mechanism c-Jun is reduced, and since the phosphorylation of c-Jun is important for protein stabilization, HeLa RARß2 clones were treated with different c-Jun-N-terminal kinase (JNK) stimulators. The treatments resulted in a c-Jun increase at RNA and protein levels, and led to a reconstitution of AP-1 binding similar to non-transfected HeLa controls. However, the reconstitution of AP-1 binding levels did not have an inductor effect on the HPV-18 oncogenes, the expression of which has been postulated to be AP-1 dependent. Other classical AP-1 regulated genes such as metalloproteinases were up regulated as expected. In conclusion, the data of this thesis indicate that RARß2 induced a destabilization and accelerated degradation of c-Jun at the protein level responsible for the AP-1 reduction. The mechanism of AP-1 trans-repression in HeLa cells is different from that postulated for other cancer cell models. All changes produced in HeLa cells after RARß2 constitutive expression lead to reduced cell proliferation, which can be associated with the RARß2 tumor suppressor function.
... Its activity is determined by the composition of the dimers, which include members of the fos (c-fos, FosB, Fra-1 and Fra-2) and jun (c-jun, junB and junD) families, and which is controlled by a complex network of phosphorylations. The inhibition of AP-1-driven transactivation by RARs is the prototype of transrepression and has been revealed in the context of several genes such as the collagenase and stromelysin genes [Lafyatis et al., 1990;Lin et al., 2000;Nicholson et al., 1990]. Generally, AP-1 inhibition is driven by liganded RARs, though AP-1 has been found to be inhibited in a ligand-independent manner by the RARβ subtype [De-Castro Arce et al., 2007;De-Castro Arce et al., 2004]. ...
Nuclear retinoic acid receptors (RARs) are transcriptional regulators controlling the expression of specific subsets of genes in a ligand-dependent manner. The basic mechanism for switching on transcription of cognate target genes involves RAR binding at specific response elements and a network of interactions with coregulatory protein complexes, the assembly of which is directed by the C-terminal ligand-binding domain of RARs. In addition to this scenario, new roles for the N-terminal domain and the ubiquitin-proteasome system recently emerged. Moreover, the functions of RARs are not limited to the regulation of cognate target genes, as they can transrepress other gene pathways. Finally, RARs are also involved in nongenomic biological activities such as the activation of translation and of kinase cascades. Here we will review these mechanisms, focusing on how kinase signaling and the proteasome pathway cooperate to influence the dynamics of RAR transcriptional activity.
... However, it is also possible that other nuclear retinoid receptors are suppressed in the lung (79,84). Also, there may be defects downstream of nuclear receptors that block retinoid activity (85). Abnormalities in the balance of the orphan receptors COUP and Nur77 (86) or co-factors essential for retinoid signaling (87,88) could also explain aberrant retinoid signaling in NSCLC. ...
Promising data have suggested that retinoid chemoprevention may help to control second primary tumors (SPTs), recurrence, and mortality of stage I non-small-cell lung cancer (NSCLC) patients.
We carried out a National Cancer Institute (NCI) Intergroup phase III trial (NCI #I91-0001) with 1166 patients with pathologic stage I NSCLC (6 weeks to 3 years from definitive resection and no prior radiotherapy or chemotherapy). Patients were randomly assigned to receive a placebo or the retinoid isotretinoin (30 mg/day) for 3 years in a double-blind fashion. Patients were stratified at randomization by tumor stage, histology, and smoking status. The primary endpoint (time to SPT) and the secondary endpoints (times to recurrence and death) were analyzed by log-rank test and the Cox proportional hazards model. All statistical tests were two-sided.
After a median follow-up of 3.5 years, there were no statistically significant differences between the placebo and isotretinoin arms with respect to the time to SPTs, recurrences, or mortality. The unadjusted hazard ratio (HR) of isotretinoin versus placebo was 1.08 (95% confidence interval [CI] = 0.78 to 1.49) for SPTs, 0.99 (95% CI = 0.76 to 1.29) for recurrence, and 1.07 (95% CI = 0.84 to 1.35) for mortality. Multivariate analyses showed that the rate of SPTs was not affected by any stratification factor. Rate of recurrence was affected by tumor stage (HR for T(2) versus T(1) = 1.77 [95% CI = 1.35 to 2.31]) and a treatment-by-smoking interaction (HR for treatment-by-current-versus-never-smoking status = 3.11 [95% CI = 1.00 to 9.71]). Mortality was affected by tumor stage (HR for T(2) versus T(1) = 1.39 [95% CI = 1.10 to 1.77]), histology (HR for squamous versus nonsquamous = 1.31 [95% CI = 1.03 to 1.68]), and a treatment-by-smoking interaction (HR for treatment-by-current-versus-never-smoking = 4.39 [95% CI = 1.11 to 17.29]). Mucocutaneous toxicity (P<.001) and noncompliance (40% versus 25% at 3 years) were higher in the isotretinoin arm than in the placebo arm.
Isotretinoin treatment did not improve the overall rates of SPTs, recurrences, or mortality in stage I NSCLC. Secondary multivariate and subset analyses suggested that isotretinoin was harmful in current smokers and beneficial in never smokers.
... 15,16 Recent studies have indicated that RAR plays a critical role in mediating the growth inhibitory effect of retinoids in many different types of cancer cells, [17][18][19][20][21][22][23][24][25][26][27][28] in part through its potent apoptosisinducing effect 20 and anti-AP-1 activity. 29 Expression of RAR is highly induced by trans-RA through the RARE (RARE) present in its promoter, 30 -32 which is mainly activated by RAR/RXR heterodimers. 33 In addition to RAR and RXR, several orphan receptors, such as COUP-TF (also called ARP-1 and ear-3) 34,35 and TR3 (also called NGFI-B and nur77), 36 -38 have been implicated in the regulation of RAR expression and the retinoid responses. ...
Epidemiological and animal studies have demonstrated that vitamin A and its natural and synthetic derivatives, retinoids, are effective agents in preventing the development of tobacco-associated cancers. Unfortunately, clinical trials of retinoids on cigarette smokers have shown lack of efficacy in preventing lung cancer. In our study, we investigated the effect of nicotine on the anti-cancer activity of all trans-retinoic acid (trans-RA) in human lung cancer cells. Our results demonstrated that nicotine could abrogate the growth inhibitory effect of trans-RA by suppressing its ability to induce the expression of RA receptor beta (RAR beta), a tumor suppressor. The inhibitory effect of nicotine was accompanied with induction of orphan receptor TR3. Inhibition of TR3 expression by overexpression of TR3 anti-sense RNA in H460 lung cancer cells strongly prevented the suppressive effect of nicotine on trans-RA activity. Treatment with nicotine or the cotransfection of TR3 expression vector inhibited the induction of RAR beta promoter activity by trans-RA in transient transfection assays. The inhibition of RAR beta promoter activity was due to the interaction of TR3 with orphan receptor COUP-TF, resulting in inhibition of COUP-TF DNA binding and transactivation on the RAR beta promoter. Furthermore, we found that nicotine failed to suppress the effect of a retinoid X receptor (RXR)-selective retinoid SR11237 on inducing both growth inhibition and RAR beta promoter activity, due to the ability of SR11237 to activate the RAR beta promoter through the RXR/TR3 heterodimer. Together, our results demonstrate that nicotine suppresses the growth inhibitory effects of trans-RA by inhibiting RAR beta expression through its induction of TR3 expression and suggest that RXR-selective retinoids may be more effective than classical retinoids for preventing and treating tobacco-associated cancers.
... How RARβ exerts its potent tumor-suppressive effects remains to be elucidated. A recent study demonstrated that RARβ can potently inhibit AP-1 activity (Lin et al. 2000b) and induce apoptosis of various cancer cells. The proapoptotic effect of RARβ was implicated in the finding that the expression of RARβ in the developing mouse limb is highly restricted to the mesenchyme of the interdigital regions destined to undergo apoptosis (Dolle et al. 1989, Mendelsohn et al. 1991, Kochhar et al. 1993, Soprano et al. 1993a). ...
Apoptosis represents an effective way to eliminate cancer cells. Unfortunately, advanced prostate tumors eventually progress to androgen-independent tumors, which are resistant to current therapeutic approaches that act by triggering apoptosis. Vitamin A and its natural and synthetic analogs (retinoids) induce apoptosis in prostate cancer cells in vitro and in animal models, mainly through induction of retinoic acid receptor-beta (RARbeta). Expression levels of RARbeta, however, are significantly reduced in hormone-independent prostate cancer cells. Recently, a new class of synthetic retinoids related to 6-[3-(1-adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN) (also called CD437) that effectively induces apoptosis of both hormone-dependent and -independent prostate cancer cells in a retinoid receptor-independent manner was identified and has drawn a lot of attention in the field. The apoptotic effect of AHPN requires expression of orphan receptor TR3 (also called nur77 or NGFI-B). Paradoxically, TR3 expression is also induced by androgen and other mitogenic agents in prostate cancer cells to confer their proliferation. The recent finding that TR3 migrates from the nucleus to mitochondria to trigger apoptosis in response to AHPN suggests that the opposing biological activities of TR3 are regulated by its subcellular localization. Thus, agents that induce translocalization of TR3 from the nucleus to mitochondria will have improved efficacy against prostate cancer. TR3, therefore, represents an unexplored molecule that may be an ideal target for developing new agents for prostate cancer therapy.
... Downregulation of RAR-β expression may lead to an increase in the activity of anti-activator protein (AP-1) associated with enhanced expression of the proto-oncogenes c-fos and c-jun resulting in hyperproliferation and expansion of malignant clones. 23,24 Decrease in RAR-β expression may be a key mechanism by which SCCs and other types of tumours escape the control of normal growth and differentiation. This would impair heterodimerization with other retinoid X receptors (RXRs). ...
The effect of aqueous garlic (Allium sativum Linn.) on retinoic acid receptor beta (RARbeta) mRNA expression was investigated in male Syrian hamsters during 12-dimethyl enz[a]anthracene (DMBA)-induced hamster buccal pouch (HBP) carcinogenesis. RARbeta mRNA expression was analysed by slot blotted hybridization with radiolabelled RAR-beta probe. In DMBA-induced HBP tumours, decreased expression of RARbeta mRNA was observed. Administration of garlic (250mg/kg body weight) to animals painted with DMBA restored RARbeta mRNA expression to normal pattern suggesting that this may be one of the mechanisms by which garlic exerts its chemopreventive effects.
... TR3 cDNA (1 to 151) was amplified using 5-GAA GAT CTT CAT GCC CTG TAT CCA AGC CCA ATA TG-3 (forward primer) and 5-CGG AAT TCC GGA GCT GGG GCG GCT GGA AGC TGG G-3 (reverse primer), digested with BglII and EcoR1, and ligated into BglII-and EcoR1-digested pGFP-N2 vector or pGEX-3X vector to generate GFP-TR3-1-151 and pGEX-TR3-1-151, respectively. The construction of the reporter plasmids NurRE-tk-CAT, ERE-tk-CAT, TREpal-tk-CAT, GREtk-CAT, and 73-Col-CAT and the receptor expression plasmids pECE-ER, pECE-TR, and pECE-GR were described previously (33,34,36,37,38,62). NBRE-tk-CAT was obtained by inserting two copies of the NBRE oligonucleotide (AAAGGTCA) into the BamHI site of pBLCAT 2 . ...
TR3, also known as NGFI-B or nur77, is an immediate-early response gene and an orphan member of the steroid/thyroid/retinoid receptor superfamily. We previously reported that TR3 expression was induced by apoptotic stimuli and was required for their apoptotic effect in lung cancer cells. Here, we present evidence that TR3 was also induced by epidermal growth factor (EGF) and serum and was required for their mitogenic effect in lung cancer cells. Ectopic expression of TR3 in both H460 and Calu-6 lung cancer cell lines promoted their cell cycle progression and BrdU incorporation, while inhibition of TR3 expression by the small interfering RNA approach suppressed the mitogenic effect of EGF and serum. Analysis of TR3 mutants showed that both TR3 DNA binding and transactivation were required for its mitogenic effect. In contrast, they were dispensable for its apoptotic activity. Furthermore, confocal microscopy analysis demonstrated that TR3 functioned in the nucleus to induce cell proliferation, whereas it acted on mitochondria to induce apoptosis. In examining the signaling that regulates the mitogenic function of TR3, we observed that coexpression of constitutive-active MEKK1 inhibited TR3 transcriptional activity and TR3-induced proliferation. The inhibitory effect of MEKK1 was mediated through activation of Jun N-terminal kinase, which efficiently phosphorylated TR3, resulting in loss of its DNA binding. Together, our results demonstrate that TR3 is capable of inducing both proliferation and apoptosis in the same cells depending on the stimuli and its cellular localization.
... Another possible mechanism relates to the apparent ability of RARs to inhibit the activity of the transcription factor AP-1 (36). RARh exhibits a strong ATRA-independent inhibition of AP-1 activity, whereas inhibition of AP-1 activity by RARs a and g is ATRA dependent (37). In HepG2 cells, we found that ACR inhibits both AP-1 and c-Fos promoter activity in a dosedependent manner and we also found similar effects of ACR in a human squamous cell carcinoma cell line (unpublished data). ...
Acyclic retinoid (ACR), a novel synthetic retinoid, has recently been demonstrated by us to inhibit the in vitro growth of human hepatoma cells, and this effect was associated with decreased expression of cell cycle-related molecules. These results, taken together with previous in vitro and clinical studies with ACR, suggest that this agent may be useful in the chemoprevention and therapy of hepatoma and possibly other human malignancies. In the present study, we further examined the molecular effects of ACR on the HepG2 human hepatoma cell line, focusing on the expression of nuclear retinoid receptors and the cell cycle inhibitor protein p21CIP1. Reverse transcription-PCR assays and Western blot analyses indicated that these cells express retinoic acid receptors (RARs) α, β, and γ, retinoid X receptors (RXRs) α and β, and peroxisome proliferator-activated receptors (PPAR) γ mRNA. Treatment with ACR caused a rapid induction within 3 h of RARβ mRNA and the related protein, but there was no significant change in the levels of the mRNA or proteins for RARs α and γ, RXRs α and β, and PPARγ. There was also a rapid increase in p21CIP1 mRNA and protein in HepG2 cells treated with ACR, and this induction occurred via a p53-independent mechanism. In transient transfection reporter assays, we cotransfected the retinoic acid response element-chloramphenicol acetyltransferase (CAT) reporter gene into HepG2 cells together with a RARβ expression vector. RARβ expression markedly stimulated CAT activity (up to about 4-fold) after the addition of ACR. However, CAT activity in the presence of ACR was only about 2-fold higher than that in the absence of ACR, when cells were cotransfected with RARs α and γ or RXRα. These findings suggest that the growth inhibitory effects of ACR are mediated at least in part through RARβ and that both RARβ and p21CIP1 play critical roles in the molecular mechanisms of growth inhibition induced by ACR.
... Furthermore, in tumor-derived cell lines resistant to retinoic acid-induced growth arrest, increasing or restoring RAR expression by ectopic expression confers response to retinoic acid (Si et al., 1996;Wu et al., 1998;Faria et al., 1999;Wan et al., 1999;Weber et al., 1999). The growth inhibitory action of RAR may in part reflect its ability to inhibit the transcriptional activity of AP-1, the fos and Jun heterodimer whose activity is typically up-regulated with cell proliferation or tumor progression (Lin et al., 2000). These considerations motivate interest in the potential antiproliferative effects of RAR agonists. ...
Retinoic acid receptor (RAR)beta is perceived to function as a tumor suppressor gene in various contexts where its absence is associated with tumorigenicity and its presence causes cell cycle arrest. Tazarotene is a prodrug selective for RARbeta/gamma, thereby motivating interest in determining whether tazarotene might activate putative tumor suppressor activity. Using HL-60 human myeloblastic leukemia cells, a cell line that undergoes G0 cell cycle arrest and myeloid differentiation in response to retinoic acid (RA), tazarotene failed to cause extracellular signal-regulated kinase (ERK) activation, a requirement for retinoic acid (RA)-induced G0 arrest and differentiation; retinoblastoma (RB) hypophosphorylation, another characteristic of RA-induced G0 arrest and cell differentiation; G0 arrest; or differentiation into mature myeloid cells. However, when used in combination with a retinoid X receptor (RXR)-selective ligand, tazarotene caused ERK activation, RB tumor suppressor protein hypophosphorylation, G0 arrest, and myeloid differentiation. The kinetics of G0 arrest and differentiation was similar to that of RA. Dose-response studies showed that diminishing tazarotene progressively diminished both induced cell differentiation and G0 arrest, where the doses for cellular effects were consistent with the transcriptional transactivation data. For either tazarotene or an RARalpha-selective ligand, diminishing the coadministered RXR-selective ligand diminished both induced differentiation and G0 arrest. Tazarotene could propel either early or late portions of the period leading to differentiation and G0 arrest and was interchangeable with an RARalpha-selective ligand. Tazarotene used with RXR-selective ligand may thus be a useful antineoplastic agent in differentiation induction therapy as exemplified by the prototypical RA treatment of acute promyelocytic leukemia.
Systemic sclerosis (SSc) is an autoimmune connective tissue disease that leads to irreversible fibrosis of the skin and the internal organs. The etiology of SSc is complex, its pathophysiology is poorly understood, and clinical therapeutic options are restricted. Thus, research into medications and targets for treating fibrosis is essential and urgent. Fos-related antigen 2 (Fra2) is a transcription factor that is a member of the activator protein-1 family. Fra2 transgenic mice were shown to have spontaneous fibrosis. All-trans retinoic acid (ATRA) is a vitamin A intermediate metabolite and ligand for the retinoic acid receptor (RAR), which possesses anti-inflammatory and anti-proliferative properties. Recent research has demonstrated that ATRA also has an anti-fibrotic effect. However, the exact mechanism is not fully understood. Interestingly, we identified potential binding sites for the transcription factor RARα to the promoter region of the FRA2 gene through JASPAR and PROMO databases. In this study, the pro-fibrotic effect of Fra2 in SSc is confirmed. SSc dermal fibroblasts and bleomycin-induced fibrotic tissues of SSc animals exhibit increased levels of Fra2. Inhibition of Fra2 expression in SSc dermal fibroblasts with Fra2 siRNA markedly decreased collagen I expression. ATRA reduced the expressions of Fra2, collagen I, and α-smooth muscle actin(α-SMA) in SSc dermal fibroblasts and bleomycin-induced fibrotic tissues of SSc mice. In addition, chromatin immunoprecipitation and dual-luciferase assays demonstrated that retinoic acid receptor RARα binds to the FRA2 promoter and modulates its transcriptional activity. ATRA decreases collagen I expression both in vivo and in vitro via the reduction of Fra2 expression. This work establishes the rationale for expanding the use of ATRA in the treatment of SSc and indicates that Fra2 can be used as an anti-fibrotic target.
Cisplatin's toxicity in renal tubular epithelial cells limits the therapeutic efficacy of this antineoplastic drug. In cultured human proximal tubular HK-2 cells (PTC) a prostaglandin uptake transporter (PGT)-dependent increase in intracellular prostaglandin E2 (iPGE2) mediates cisplatin's toxicity (i.e. increased cell death and loss of cell proliferation) so that it is prevented by PGT inhibitors. Here we found in cisplatin-treated PTC that 4,4′-diisothiocyanostilbene-2,2′-disulfonic acid (DIDS), a PGT inhibitor, prevented cisplatin's toxicity but not the increase in iPGE2. Because expression of retinoic acid receptor-β (RAR-β) is dependent on iPGE2 and because RAR-β is a regulator of cell survival and proliferation, we hypothesized that RAR-β might mediate the protective effect of DIDS against cisplatin's toxicity in PTC. Our results confirmed this hypothesis because: i) protection of PTC by DIDS was abolished by RAR-β antagonist LE-135; ii) DIDS increased the expression of RAR-β in PTC and prevented its decrease in cisplatin-treated PTC but not in cisplatin-treated human cervical adenocarcinoma HeLa cells in which DIDS failed to prevent cisplatin's toxicity; iii) while RAR-β expression decreased in cisplatin-treated PTC, RAR-β over-expression prevented cisplatin's toxicity. RAR-β agonist CH55 or RAR pan-agonist all-trans retinoic acid did not prevent cisplatin's toxicity, which suggests that RAR-β does not protect PTC through activation of gene transcription. In conclusion, RAR-β might be a new player in cisplatin-induced proximal tubular injury and the preservation of its expression in proximal tubules through treatment with DIDS might represent a novel strategy in the prevention of cisplatin's nephrotoxicity without compromising cisplatin's chemotherapeutic effect on cancer cells.
Considerable evidence demonstrates that retinoids (retinol, retinoic acid, and retinylester, which are also the important metabolites from provitamin A carotenoids), may beeffective in the prevention and treatment of a variety of human chronic diseases, includingcancer. Substantial work has been done investigating the mechanisms by which tobaccosmoke and excessive alcohol intake interfere with retinoid metabolism and signaling.Exposure to cigarette smoke subjects tissues to increased reactive oxygen species, which caninduce cytochrome P450 enzymes and result in the degradation of retinoic acid, the bioactiveform of vitamin A. Further, oxidative stress can result in cleavage of β-carotene at positionsother than the central double bond, decreasing the production of retinoic acid from this vitamin A precursor. This leads to aberrant retinoid signaling through nuclear retinoid receptors,while at the same time cigarette smoke also causes dysregulated signaling through themitogen-activated protein kinase signaling pathways. Alcohol acts as a competitive inhibitorof vitamin A oxidation to retinoic acid involving alcohol dehydrogenases and acetaldehydedehydrogenases, induces cytochrome P450 enzymes (particularly CYP2E1) that degraderetinol and retinoic acid, and alters retinoid homeostasis by increasing vitamin A mobiliza-tion from liver to extrahepatic tissues. Moreover, this alcohol-impaired retinoid homeostasisinterferes with retinoic acid signaling by decreasing target gene expression and interferingwith retinoic acid cross-talk with the mitogen-activated protein kinase pathways. The overalleffect of both cigarette smoke and chronic, excessive alcohol intake is dysregulated apopto-sis and uncontrolled cellular proliferation, which can act to promote the process of carcino-genesis. Nutritional interventions that serve to restore normal retinoid signaling andfunctioning may offer protection at the cellular level and represent a means to modify cancerrisk in high-risk human populations.
In the past several decades, intensive research in this field has uncovered a surprising number of regulatory factors and their associated enzymatic properties to reveal the network of complexes that function in activation and repression of the transcriptional programs mediated by nuclear receptors (NR). These factors and their associated complexes have been extensively characterized both biochemically and functionally [34, 87, 94]. Several principles have emerged: (1) It is widely recognized that ligand-dependent cofactor complexes mediating repression and activation exhibit ligand-dependent exchange. (2) These complexes mediate modifications of chromatin structure consequent to their binding at regulatory elements, particularly at promoter and enhancer Enhancer sites. (3) The concept about the rapid exchange of coregulatory complexes at regulatory sites has been suggested [88]. Key questions in the NR field have included: (a) What are the cofactors and exchange complexes used to mediate the ligand and signaling network-dependent switches in gene regulation programs; (b) Do long non-coding RNAs (lncRNAs) serve as regulatory "factors" for ligand-dependent gene programs, and do enhancers actually regulate transcription units encoding enhancer Enhancer non-coding RNAs (eRNAs) Enhancer RNA that might have functional significance; (c) What is the relationship between DNA damage repair machinery and transcriptional machinery? (d) Do Retinoic Acid Receptors (RAR) also regulate Pol III-dependent, non-coding repeat transcriptional units in stem cells? and (e) How have new technologies such as deep sequencing altered our ability to investigate transcriptional regulatory mechanisms utilized by NRs?
Clofarabine (2-chloro-2'-fluoro-2'-deoxyarabinosyladenine, ClF) is a second-generation 2'-deoxyadenosine analogue that is structurally related to cladribine (2-chloro-2'-deoxyadenosine, 2CdA) and fludarabine (9-beta-D-arabinosyl-2-fluoroadenine, F-ara-A). It demonstrates potent antitumour activity at much lower doses than parent compounds with high therapeutic efficacy in paediatric blood cancers. Our previous studies in breast cancer cells indicate that 2CdA and F-ara-A are involved in epigenetic regulation of gene transcription. We therefore investigated whether ClF influences methylation and expression of selected tumour suppressor genes, such as adenomatous polyposis coli (APC), phosphatase and tensin homologue (PTEN), and retinoic acid receptor beta 2 (RARbeta2), as well as expression of p53, p21 and DNA methyltransferase 1 (DNMT1) in MCF-7 and MDA-MB-231 breast cancer cell lines with different invasive potential. Promoter methylation and gene expression were estimated using methylation-sensitive restriction analysis (MSRA) and real-time PCR, respectively. ClF demonstrated potent growth inhibitory activity in MCF-7 and MDA-MB-231 cells after 96h treatment with IC50 determined as equal to 640nM and 50nM, respectively. In both breast cancer cell lines, ClF led to hypomethylation and up-regulation of APC, PTEN and RARbeta2 as well as increase in p21 expression. Only in non-invasive MCF-7 cells, these changes were associated with down-regulation of DNMT1. Our results provide first evidence of ClF implications in epigenetic regulation of transcriptional activity of selected tumour suppressor genes in breast cancer. It seems to be a new important element of ClF anticancer activity and may indicate its potential efficacy in epigenetic therapy of solid tumours, especially at early stages of carcinogenesis.
Recent advances have suggested that nongenomic action of retinoids and their receptors represents an important mechanism by which they exert therapeutic effects. Here we review rapid biological responses triggered by retinoids and related molecules by focusing on novel nongenomic mechanisms involving cytoplasmic action of nuclear receptors Nur77 and retinoid X receptor (RXR) in the regulation of apoptosis and inflammation. A class of apoptosis-inducing compounds related to atypical retinoid AHPN/CD437 has emerged as promising therapeutic agents. They rapidly induce mitochondrial targeting of Nur77 and RXR and conversion of Bcl-2 from a cellular protector to a killer. Thus, understanding the nongenomic action of Nur77 and RXR may provide a rational basis to guide the design and development of new therapeutic agents for cancer, and skin, cardiovascular, metabolic and central nervous disorders.
To uncover the mechanisms relating to the anticancer effect of retinoic acids in gastric cancer cells, the mediation of activator protein-1 (AP-1) activity repression by retinoic acid receptors (RARs) was investigated. All-trans retinoic acid (ATRA) inhibited AP-1 activity in BGC-823 cells (RARα+, RARβ+), but not in MKN-45 cells (RARαlo, RARβ−). Transient transfection of RARβ expression vector into MKN-45 cells significantly resulted in direct repression of AP-1 activity in a receptor concentration-dependent manner, and this could be strengthened by ATRA. Stable transfection of RARβ into MKN-45 cells directly inhibited cell growth and colony formation, and ATRA also enhanced these effects. Transient transfection of RARα into MKN-45 cells however, displayed receptor concentration-dependent AP-1 activity inhibition only in the presence of ATRA. Stable transfection of RARα into MKN-45 cells resulted in ATRA-dependent inhibition of cell growth and colony formation. For AP-1 binding activity induced by TPA, the repressive effect of ATRA was only observed in BGC-823 and RARα and RARβ stably transfected MKN-45 cells, but not in intact MKN-45 cells. This indicates the necessity for sufficient cellular RARα and/or RARβ in order for AP-1 activity repression to occur. Deletion of DNA binding domain (DBD) of RARβ, but not ligand binding domain (LBD), eliminated the anti-AP-1 function of RARβ. It is therefore concluded that both RARα and RARβ are mediators in the anticancer function of ATRA via AP-1 activity inhibition, and that RARβ, not RARα, can inhibit AP-1 activity to a certain extent directly by itself. Thus DBD, not LBD, is critical for anti-AP-1 activity.
The mandibular condyle formation during temporomandibular joint (TMJ) development exhibits endochondral bone formation, and the elongation process is dependent on the normal cartilage proliferation and differentiation. Retinoids are important for maturation of growth-plate chondrocytes, but the identity of their downstream effectors remains unclear. In this study, we carried out a series of studies at the cellular, biochemical, and molecular levels to determine whether, and if so how, retinoid signaling is related to the expression and function of Indian hedgehog (Ihh) in chondrocyte proliferation. First we analyzed the RA receptor (RAR) and Ihh expression pattern in E18 mandibular condyle. RARα and RARβ mRNA were characterized in the perichondrium around the condyle, whereas RARγ mRNA was expressed in the immature and prehypertrophic chondrocytes and the expression was overlapped with Ihh gene expression. Next we established a high-density culture model of chick cephalic chondrocytes in the prehypertrophic stage. We found that all-trans retinoic acid (RA) induced Ihh mRNA gene expression in this system. The RA pan-antagonist Ro 41-5253 inhibited both endogenous and RA-induced Ihh mRNA in a dose-dependent manner. The Ihh mRNA expression induced by RA required de novo protein synthesis, and was mediated by RARγ. Immunoblots showed that the prehypertrophic chondrocytes contained sizable levels of phosphorylated p38 mitogen-activated protein (MAP) kinase that were time- and dose-dependently increased by the RA treatment. Experimental p38 inhibition led to a severe drop in baseline and RA-stimulated Ihh expression. Exogenous recombinant Ihh stimulated the proliferation of proliferating chondrocytes, whereas RA inhibited the proliferation of these chondrocytes through p38 MAPK. Retinoids appear to play a primary role in controlling both the expression and function of Ihh in prehypertrophic chondrocytes and do so via p38 MAP kinase.
Silencing of gene expression by aberrant methylation at the CpG islands is common in human tumors, including colorectal cancer. This epigenetic alteration affects promoter of genes having crucial cellular functions such as tumor suppressor, DNA repair, apoptosis, cell adhesion, etc. We investigated the methylation status in the promoter regions of the RARβ2, RASSF1A, DAPKinase, and CDH1 genes in 73 colorectal carcinoma and 43 paired normal tissues of Tunisian patients using methylation-specific PCR assays. The association between methylation status and the clinicopathological features was evaluated. To determine whether aberrant methylation affects gene expression, we performed immunohistochemistry analysis for E-cadherin and COX-2, a target gene of RARβ2. The methylation frequencies vary from 80.8% for RARβ2 to 35.6% for RASSF1A while in non-tumor-paired samples; the frequencies of methylation are significantly lower for all the fourth genes tested. The methylation status did not correlate with any of the clinical features considered; however, aberrant methylation of RARβ2 was associated with a shortened overall patients' survival (p log rank = 0.026); nevertheless, it needs to be confirmed on larger sample size. Moreover, a significant inverse association was observed between methylation status of RARβ2 and COX-2 protein expression in tumor specimen (p = 0.014). On the other hand, we found that loss of E-cadherin expression was significantly associated with aberrant methylation of the CDH1 promoter (p = 0.005). Our findings showed that RARβ2 was frequently methylated in colorectal cancer and correlated with a worse prognosis and high expression of COX-2 suggesting a link between these two proteins in colorectal carcinogenesis. We also showed that epigenetic alteration of CDH1 is a major mechanism of the loss of E-cadherin protein expression in primary colorectal tumors.
TAC-101, 4-[3,5-bis(trimethylsilyl)benzamido] benzoic acid, is a synthetic ligand for retinoic acid receptor (RAR)-alpha. Here, we demonstrate the contribution of TAC-101-induced AP-1 interference to stabilization of tumor growth. TAC-101 induced transcriptional activation of RAR, resulting in marked elevation of RARbeta, a representative retinoid response marker, and it also significantly repressed the transcriptional activity of AP-1 in JHH-7 cells. In contrast to JHH-7, JHH-6 is another RARalpha-expressing human hepatocellular carcinoma (HCC) cell line with constitutive activation of AP-1, but it is retinoid insensitive and did not respond to the TAC-101-induced RAR signal. TAC-101 did not inhibit AP-1 activity of the JHH-6 cell line, showing that AP-1 interference by TAC-101 must be in parallel with RAR activation. Interleukin-8 (IL-8), one of the AP-1-regulated factors which correlate with a poor prognosis in HCC patients, was found to be overexpressed in JHH-7 cells. TAC-101 reduced IL-8 production without cytotoxicity and inhibited the progression of HCC in the orthotopic mouse model with decreased tumor IL-8 level. These results suggest that downregulation of the extracellular biomarker for AP-1 interference via the induction of retinoid signals will enhance the pharmacological effect of TAC-101 on HCC and it could be useful as a surrogate biomarker of therapeutic efficacy.
The oncogene erbB2 is overexpressed in 20% to 30% human breast cancers and is most commonly overexpressed in estrogen receptor (ER)-negative breast cancers. Transgenic mice expressing erbB2 develop ER-negative mammary tumors, mimicking human breast carcinogenesis. Previously, we have shown that activator protein 1 (AP-1) regulates proliferation of ER-negative breast cancer cells. We hypothesized that blockade of AP-1 in mouse mammary epithelial cells will suppress ER-negative tumorigenesis induced by erbB2. Trigenic erbB2 mice were generated by crossing a bigenic pUHD-Tam67/MMTV-rtTA mouse to a MMTV-erbB2 mouse. The resulting trigenic mice develop tumors and express a doxycycline-inducible c-Jun dominant negative mutant (Tam67) in the mammary glands. In vivo AP-1 blockade by Tam67 expression started delayed mammary tumor formation in MMTV-erbB2 mice by more than 11 weeks. By 52 weeks of age, 100% (18 of 18) of the untreated animals had developed mammary tumors, whereas 56% (9 of 16) of the doxycycline-treated trigenic mice developed tumors. In addition, the tumors that arose in the AP-1-blocked erbB2 mice failed to express Tam67. Twenty-five percent of the doxycycline-treated MMTV-erbB2 mice survived more than 72 weeks of age without developing mammary tumors. Examination of normal-appearing mammary glands from these mice showed that AP-1 blockade by Tam67 also significantly prevents the development of premalignant lesions in these glands. The expression of erbB2 either in normal mammary tissue or in mammary tumors was not altered. Our results show that blocking the AP-1 signaling in mammary cells suppresses erbB2-induced transformation, and show that the AP-1 transcription factor is a critical transducer of erbB2. These results provide a scientific rationale to develop targeted drugs that inhibit AP-1 to prevent the development of ER-negative breast cancer.
Chemoprevention of cancer represents a challenge for oncology during this new millennium. Substantial advances have been accomplished in the last decade, especially for primary and secondary prevention of breast cancer. In addition to tamoxifen, raloxifene and other selective estrogen receptor modulators, retinoids are among the most promising agents, given their ability to inhibit mammary carcinogenesis in preclinical models.
Fenretinide, the synthetic amide of retinoic acid, inhibits cell growth mostly through the induction of apoptosis with mechanisms which may partly involve the retinoid receptors. Because it has a favourable toxicological profile, fenretinide has been extensively investigated in clinical trials. A large randomised phase III trial for secondary breast cancer prevention has been recently carried out in Italy. Results showed a reduction of second breast malignancies in premenopausal women. In addition, a significant decrease of circulating insulin-like growth factor (IGF)-1, a known risk factor for premenopausal breast cancer, was observed after 1 year of fenretinide administration in premenopausal women with breast cancer.
Ongoing studies on the validation of the circulating IGF-1 as a surrogate endpoint biomarker of fenretinide activity and on the effectiveness of the combination with low dose tamoxifen may provide further insight into the future clinical application of fenretinide.
Retinoids are compounds with pleiotropic functions and a relatively selective targeting of certain skin structures. They are vitamins, because retinol (vitamin A) is not synthesized in the body and must be derived from diet, but also hormones with intracrine activity, because retinol is transformed into molecules that bind to nuclear receptors, exhibit their activity, and are subsequently inactivated. Retinoids exert their effects on target cells by binding and activating nuclear retinoid receptors. Retinoid receptors bind their ligands in form of dimers. Heterodimers can be formed between two different retinoid receptor molecules but also between retinoid X receptors and the vitamin D receptor as well as the triiodothyronin receptor. This fact indicates complex interactions between retinoids and further hormonal signal transduction molecules. Interaction of retinoid receptors with transcriptional factors activated by other signal transduction mechanisms, e.g. AP-1, may provide dissociation of the retinoid effects. Retinoids can exhibit agonistic activity but also be neutral antagonists and inverse agonists. Topical and oral retinol, tretinoin, isotretinoin, and bexarotene, topical alitretinoin, retinaldehyde, motretinide, adapalene, tazarotene, and systemin acitretin compose the list of launched retinoids. Psoriasis and related disorders, congenital disorders of keratinization, acne, photoaging and hypovitaminosis A are classical approved indications of retinoid treatment, whereas cutaneous T-cell lymphoma, AIDS-associated Kaposi's sarcoma, acute promyelocytic leukemia and actinic lentigines were currently confirmed. In addition, retinoids have been successfully used in several other dermatoses, e.g. epithelial precanceroses and tumors, seborrhea, rosacea and acneiform dermatoses, lichen planus, eosinophilic folliculitis, condylomata accuminata, lichen sclerosus and atrophicus. Highly receptor selective molecules, retinoic acid receptor-beta-inducers, AP-1 complex antagonists, and inverse agonists will be probably lead the retinoid development in the near future. New, more effective and less toxic retinoids, alone or in combination with other drugs and new delivery systems may provide therapeutic solutions for benign and malignant proliferative skin diseases, such as psoriasis and non-melanoma tumors, cancer chemoprevention and differentiation therapy.
Retinoids, derivatives of vitamin A, have strong anti-inflammatory and antiproliferative properties. We previously demonstrated that the pan-agonists all-transretinoic acid (RA) and isotretinoin (13-cis RA) alleviate renal damage in rat acute glomerulonephritis (GN) induced by anti-Thy-1.1 mAb OX-7.
The present study examined the effects of low dose and high dose treatment with isotretinoin in the chronic glomerulonephritis model, Thy-GN. Thy-GN was induced by a single intravenous injection of monoclonal antibody (mAb) 1-22-3 in uninephrectomized Wistar rats (N = 7 to 10 per group). Control and nephritic groups were treated with vehicle (veh), low dose isotretinoin (2 mg/kg body wt), or high dose isotretinoin (10 mg/kg body wt). The experiment was terminated 60 days after induction of Thy-GN.
In animals with Thy-GN, isotretinoin abrogated the increase in blood pressure and significantly reduced albuminuria. Glomerulosclerosis index, glomerular and interstitial cell counts, as well as the area of the interstitial space were significantly lower in nephritic rats treated with low and high dose isotretinoin compared to vehicle-treated nephritic controls. Treatment with isotretinoin also significantly reduced the number of glomerular and interstitial macrophages. The increase of transforming growth factor (TGF)-beta1, TGF receptor II and prepro-endothelin-1 gene expression in vehicle-treated nephritic rats was significantly attenuated by isotretinoin.
Treatment with isotretinoin significantly reduces glomerular and interstitial damage in rats with chronic glomerulonephritis as indicated by different functional and histological markers. Retinoids may provide a novel therapeutic option for the treatment of glomerulonephritis.
Since retinoic acid receptor (RAR)-beta mRNA is frequently lost during esophageal carcinogenesis and esophageal cancer cells that do not express RAR-beta are resistant to retinoic acid (RA), we stably transfected RAR-beta expression vector into an esophageal cancer cell line TE-8 and an antisense RAR-beta into TE-3 cells. Transfection of RAR-beta decreased cell growth and colony formation and induced apoptosis in TE-8 cells. Antisense RAR-beta-transfected TE-3 cells had a shorter doubling time and became resistant to RA. Induction of RAR-beta decreased COX-2 expression in RAR-beta transfected TE-8 cells, whereas antisense RAR-beta transfected TE-3 cells increased COX-2 expression. The inhibitory effect of RAR-beta on COX-2 expression was further enhanced in the presence of RA, which was blocked by an RAR antagonist. The synthetic retinoid N-(4-hydroxyphenyl)retinamide, which does not bind effectively to RAR-beta, had no effect on COX-2 suppression. Furthermore, RA blocked bile acid-induced COX-2 expression and prostaglandin E(2) production only in the RAR-beta positive cells. Our data demonstrated that anticancer effect of RAR-beta may be related to its ability to suppress COX-2 expression and support that the loss of RAR-beta expression may contribute to esophageal carcinogenesis.
Retinoids have a reputation for being both detrimental and beneficial: they are teratogens, but they also have tumour-suppressive capacity. Cell biology and genetics have significantly improved our understanding of the mechanisms that underlie the anti-proliferative action of retinoids. Recent elucidation of the pathways that are activated by retinoids will help us to exploit the beneficial aspects of this powerful class of compounds for cancer therapy and prevention.
Loss of expression of retinoic acid receptor beta2 (RARbeta2), a potent tumor suppressor gene, is commonly observed during breast carcinogenesis. RARbeta2 silencing can be traced to epigenetic chromatin changes affecting the RARbeta P2 promoter. Here we show that retinoic acid therapy fails to induce RARbeta2 in primary breast tumors, which carry a methylated RARbeta P2 promoter. DNA methylation leads to repressive chromatin deacetylation at RARbeta P2. By inducing an appropriate level of histone reacetylation at RARbeta P2 we could reactivate endogenous RARbeta2 transcription from unmethylated as well as methylated RARbeta P2 in breast cancer cell lines and xenograft tumors, and obtain significant growth inhibition both in vitro and in vivo. This study may have translational implications for breast cancer and other cancers carrying an epigenetically silenced RARbeta P2 promoter.
To uncover the mechanisms relating to the anticancer effect of retinoic acids in gastric cancer cells, the mediation of activator protein-1 (AP-1) activity repression by retinoic acid receptors (RARs) was investigated. All-trans retinoic acid (ATRA) inhibited AP-1 activity in BGC-823 cells (RARalpha(+), RARbeta(+)), but not in MKN-45 cells (RARalpha(lo), RARbeta(-)). Transient transfection of RARbeta expression vector into MKN-45 cells significantly resulted in direct repression of AP-1 activity in a receptor concentration-dependent manner, and this could be strengthened by ATRA. Stable transfection of RARbeta into MKN-45 cells directly inhibited cell growth and colony formation, and ATRA also enhanced these effects. Transient transfection of RARalpha into MKN-45 cells however, displayed receptor concentration-dependent AP-1 activity inhibition only in the presence of ATRA. Stable transfection of RARalpha into MKN-45 cells resulted in ATRA-dependent inhibition of cell growth and colony formation. For AP-1 binding activity induced by TPA, the repressive effect of ATRA was only observed in BGC-823 and RARalpha and RARbeta stably transfected MKN-45 cells, but not in intact MKN-45 cells. This indicates the necessity for sufficient cellular RARalpha and/or RARbeta in order for AP-1 activity repression to occur. Deletion of DNA binding domain (DBD) of RARbeta, but not ligand binding domain (LBD), eliminated the anti-AP-1 function of RARbeta. It is therefore concluded that both RARalpha and RARbeta are mediators in the anticancer function of ATRA via AP-1 activity inhibition, and that RARbeta, not RARalpha, can inhibit AP-1 activity to a certain extent directly by itself. Thus DBD, not LBD, is critical for anti-AP-1 activity.
AP-1 transcription factors play a critical role in signal transduction pathways in many cells. We have investigated the role of AP-1 in controlling proliferative signals in breast cells, and have previously shown that AP-1 complexes are activated by peptide and steroid growth factors in both normal and malignant breast cells. In this study, we investigated the role of AP-1 in transducing proliferative signals induced by peptide and steroid growth factors. We used MCF-7 clones that express a specific inhibitor of AP-1, a dominant-negative cJun mutant (TAM67), under the control of an inducible promoter to investigate the role of AP-1 in regulating breast cancer growth. In the presence of doxycycline (Dox), the AP-1 inhibitor was not expressed, and the MCF-7 clones proliferated normally in response to serum stimulation. However, when Dox was withdrawn, TAM67 was expressed, AP-1 activity was inhibited, and serum-induced proliferation was blocked. We next investigated whether the mitogenic response to specific growth factors also requires AP-1. MCF-7 Tet-Off-TAM67 cells were grown in the presence of increasing concentrations of IGF-1, EGF, heregulin-beta, bFGF, or estrogen under un-induced and induced conditions. These studies showed that the AP-1 inhibitor completely blocked proliferation in response to the peptide growth factors (IGF-1, EGF, heregulin-beta, and bFGF), and partially blocked the response to estrogen. To investigate the effect of AP-1 blockade on in vivo tumor growth, we injected the MCF-7 Tet-Off TAM67 cells into nude mice receiving doxycycline to suppress the expression of the AP-1 inhibitor. After the mice developed tumors, they were randomized to either continue to receive Dox or not. In mice not receiving Dox, the expression of TAM67 was induced, and tumor growth was inhibited, while the tumors in mice receiving Dox continued to grow. Analysis of the tumors from these mice showed that the expression of TAM67 caused reduced proliferation of the breast cancer cells without inducing apoptosis. These results demonstrate that AP-1 blockade supresses mitogenic signals from multiple different peptide growth factors as well as estrogen, and inhibits the growth of MCF-7 breast cancer cells both in vitro and in vivo. These results suggest that novel agents specifically targeting AP-1 or its activating kinases could be promising agents for the treatment of breast cancer.
Chronic and excessive alcohol intake is associated with an increased incidence of a variety of cancers (e.g., liver, oral cavity, esophagus, colorectal and breast). Long-term alcohol intake results in impaired nutritional status of retinoic acid (RA), the most active derivative of vitamin A, which may provide a promoting environment for tumor formation. Recent studies demonstrate that chronic alcohol-induced hepatocellular proliferation, which may convert hepatocytes from a state of resistance to a carcinogen to a state of high susceptibility, is due to alcohol-impaired RA metabolism and signaling and crosstalk with the Jun N-terminal kinases-dependent signaling pathway. Further, the restoration of hepatic RA homeostasis by treatment with either RA supplementation or inhibitors of RA catabolism can suppress alcohol-induced hepatocyte hyperproliferation and restore alcohol-deregulated apoptosis, thereby reducing the risk of alcohol-promoted hepatocellular carcinogenesis. These studies indicate the importance of RA actions in the prevention and/or treatment of alcohol-related carcinogenic process in the liver and other organs.
Vitamin A, as an essential micronutrient, is involved in higher animals in embryonic development and postnatal growth, reproduction and maintenance of normal skin, immunity and vision. Recently, studies in vivo and in cell lines have shown that vitamin A and its active metabolite, retinoic acid, regulate the expression of fatty acid desaturases including stearoyl-CoA desaturase and delta-5 desaturase. Whereas the former desaturase catalyzes the formation of monounsaturated from saturated fatty acids, the latter enzyme is involved in the desaturation pathway of dietary essential fatty acids for production of polyunsaturated fatty acids. The reaction products of these desaturases serve as critical regulators in a wide range of physiological processes which include fetal growth and development, reproduction, cell differentiation, immune and inflammatory responses.
Mouse rdh1 encodes retinol dehydrogenase type 1 (RDH1), a short-chain dehydrogenase, which recognizes as substrates all-trans-retinol, 9-cis-retinol, 5alpha-androstan-3,17-diol and 5alpha-androstan-3-ol-17-one. RDH1 is the most efficient known mouse short-chain dehydrogenase that catalyzes dehydrogenation of all-trans-retinol, and contributes to a reconstituted path of all-trans-retinoic acid biosynthesis, when coexpressed in reporter cells with any one of three retinal dehydrogenases. Rdh1 shows widespread, if not ubiquitous, mRNA expression in the mouse beginning no later than embryo day 7. Here we report genomic organization, chromosomal localization and analysis of a minimum promoter of mouse rdh1. Rdh1 consists of four exons and three introns and spans approximately 14412 bp. Rdh1 is a single copy gene that maps to chromosome 10D3 with rdh5-9, but no known disorder maps precisely to rdh1. Rdh1 has three transcription start sites in kidney and one start site in liver. The rdh1 5'-region between -424 and +43 induces transcription maximally in COS7, mouse kidney RAG, and mouse liver NMu3Li cells. This section has no TATA box, but has a CCAAT box beginning 65 bp upstream of the major transcription start site, which is required for transcription of transfected reporter constructs. An AP1 binding site at -119 also activates transfected reporter constructs, and mediates 2-O-tetradecanoylphorbol-13-acetate (TPA) induced transcription. All-trans-retinoic acid antagonizes the TPA affect; however, no RARE or RXRE was found in the proximal promoter region, consistent with indirect regulation by all-trans-retinoic acid.
All-trans retinoic acid (ATRA) can down regulate the anti-apoptotic protein Bcl-2 and the cell cycle proteins cyclin D1 and cdk2 in estrogen receptor-positive breast cancer cells. We show here that retinoids can also reduce expression of the inhibitor of apoptosis protein, survivin. Here we have compared the regulation of these proteins in MCF-7 and ZR-75 breast cancer cells by natural and synthetic retinoids selective for the RA receptors (RARs) alpha, beta, and gamma then correlated these with growth inhibition, induction of apoptosis and chemosensitization to Taxol. In both cell lines ATRA and 9-cis RA induced the most profound decreases in cyclin D1 and cdk2 expression and also mediated the largest growth inhibition. The RARalpha agonist, Ro 40-6055 also strongly downregulated these proteins although did not produce an equivalent decrease in S-phase cells. Only ATRA induced RARbeta expression. ATRA, 9-cis RA and 4-HPR initiated the highest level of apoptosis as determined by mitochondrial Bax translocation, while only ATRA and 9-cis RA strongly reduced Bcl-2 and survivin protein expression. Enumeration of dead cells over 96 h correlated well with downregulation of both survivin and Bcl-2. Simultaneous retinoid-mediated reduction of both these proteins also predicted optimal Taxol sensitization. 4-HPR was much weaker than the natural retinoids with respect to Taxol sensitization, consistent with the proposed requirement for reduced Bcl-2 in this synergy. Neither the extent of cell cycle protein regulation nor AP-1 inhibition fully predicted the antiproliferative effect of the synthetic retinoids suggesting that growth inhibition requires regulation of a spectrum of RAR-regulated gene products in addition even to pivotal cell cycle proteins.
To study the role of hypermethylation in the loss of retinoic acid receptor beta(2) (RARbeta(2)) in esophageal squamous cell carcinoma (ESCC).
The role of hypermethylation in RARbeta(2) gene silencing in 6 ESCC cell lines was determined by methylation-specific PCR (MSP), and its methylation status was compared with RARbeta(2) mRNA expression by RT-PCR. The MSP results were confirmed by bisulfite sequencing of RARbeta(2) promoter regions.
Methylation was detected in 4 of the 6 cell lines, and the expression of RARbeta(2) was markedly downregulated in 3 of the 4 methylated cell lines. The expression of RARbeta(2) was restored in one RARbeta(2) -downregulated cell line with the partial demethylation of promoter region of RARbeta(2) after 5-aza-2'-deoxycytidine (5-aza-dc) treatment.
The methylation of the 5' region may play an important role in the downregulation of RARbeta(2) in some ESCC cell lines, suggesting that multiple mechanisms contribute to the loss of RARbeta(2) expression in ESCC cell lines. This study may have clinical applications for treatment and prevention of ESCC.
Multiple studies performed in in vitro and in vivo settings have confirmed the cancer therapeutic and cancer preventive capacity of retinoids and rexinoids. These compounds mediate their actions through the retinoid and rexinoid receptors, respectively, which exist in multiple isoforms and form a plethora of distinct heterodimers. Despite their apparent anticancer potential, with one exception the molecular basis of this activity has remained largely elusive. The exception concerns acute promyelocytic leukemia (APL), the prototype of retinoic acid-dependent differentiation therapy, for which both the molecular nature of the disease and the mechanism of action of retinoids are well understood. However, retinoids and rexinoids are active beyond the borderlines of the well-defined chromosomal translocation that gives rise to curable APL. In this context, particularly interesting is that retinoic acid induces a member of the tumor necrosis factor family, tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or Apo2L. This ligand is exceptional in that it is capable of inducing apoptosis in cancer cells but not in normal cells. It is possible that this connection to the TRAIL signaling pathway contributes to the anti-tumor activity of retinoids and rexinoids. This review focuses on what is presently known about the regulation of cell life and death by the retinoid/rexinoid and TRAIL signaling pathways.
The anticancer effects of retinoids are mainly mediated by their nuclear receptors. Recent studies have demonstrated that retinoic acid receptor beta (RARbeta) plays a pivotal role from the early stages of laryngeal carcinogenesis; however, the exact mechanism of this detrimental effect has not yet been elucidated. One of the best-documented actions of retinoid receptors is the transrepression of activator protein-1 (AP-1) transcription factor activity, although this complex interplay has not been clarified. The present report is the first systematic morphological evaluation of the cross-talk of RARbeta and AP-1 transcription factor in a large series of human laryngeal tissues containing normal epithelium, premalignant lesions (hyperplasia and/or dysplasia) and squamous cell carcinoma. Immunohistochemical methodology was performed on formalin-fixed, paraffin-embedded sections by using a panel of monoclonal and polyclonal antibodies against RARbeta and the AP-1 components c-Jun, p-c-Jun (phosphorylated, active c-Jun) and c-Fos proteins. Their expression was screened and compared in 154 patients with various laryngeal histological entities. Nuclear expression of RARbeta, c-Jun, p-c-Jun and c-Fos was detected in 81 (89.2%), 48 (52.8%), 66 (72.6%) and 73 (80.3%), respectively, out of 91 specimens with normal-appearing laryngeal epithelium; in 86 (87.8%), 94 (95.9%), 94 (95.9%) and 94 (95.9%), respectively, out of 98 specimens with hyperplastic laryngeal epithelium; in 58 (56.8%), 92 (90.2%), 96 (94.1%) and 96 (94.1%), respectively, out of 102 specimens with dysplastic laryngeal epithelium; in 10 (22.3%), 41 (91.2%), 44 (97.8%) and 41 (91.2%), respectively, out of 45 specimens with well-differentiated squamous cell carcinoma; in 13 (30.3%), 37 (86%), 39 (90.7%) and 41 (95.3%), respectively, out of 43 specimens with moderately-differentiated squamous cell carcinoma; and in 8 (66.7%), 10 (83.3%), 12 (100%) and 12 (100%), respectively, out of 12 specimens with poorly-differentiated squamous cell laryngeal carcinoma. Statistical analysis and correlation of the intensity of nuclear immunostaining of the studied proteins among the various histological entities revealed statistically significant results. The progressive upregulation of the AP-1 transcription factor constituents and downregulation of the RARbeta protein detected from the onset of laryngeal tumorigenesis suggests an important role for the immediate-early AP-1/RARbeta on/off "switch" in the process of laryngeal carcinogenesis.
Myelofibrosis with myeloid metaplasia (MMM) is a clonal stem-cell disorder that leads to ineffective hematopoiesis, bone marrow fibrosis, and extramedullary hematopoiesis. The molecular mechanisms underlying the development of this myeloproliferative syndrome are currently unknown. In order to identify tumor suppressor genes that may be involved in the disease process, we performed an analysis for loss of heterozygosity (LOH) in CD34+ cells from 29 patients with MMM. We observed a frequency of allelic loss on chromosomal arm 3p in 24% of cases. Detailed mapping of 3p revealed a distinct region of deletion at 3p24. Among the genes known to map within this region is the retinoic acid receptor-beta (RARbeta2) gene. To determine whether RARbeta2 gene activity is diminished in this disease, we analysed its expression in CD34+ cells from 17 patients with MMM using quantitative PCR. Our results indicate that expression of RARbeta2 is significantly decreased in 100% of patient samples compared to that in CD34+ cells from 10 normal individuals. Since allelic loss at 3p24 occurs in <25% of patients, we investigated the contribution of epigenetic modifications to RARbeta2 inactivity. Using methylation-specific PCR, we found hypermethylation of RARbeta2 in 16 of 18 patients (89%), while the methylated form of the gene was absent in CD34+ cells from nine normal individuals. Our results suggest that RARbeta2 acts as a tumor suppressor gene in MMM and that epigenetic changes are the most significant determinants of RARbeta2 gene activity in these patients.
The AP-1 transcription factor is a central component of signal transduction pathways in many cells, although the exact role of AP-1 in controlling cell growth and malignant transformation is unknown. We have previously shown that AP-1 complexes are activated by peptide and steroid growth factors in both normal and malignant breast cells, and that blocking AP-1 by overexpressing a dominant-negative form of cJun (cJun-DN, TAM67) inhibits breast cancer cell growth both in vivo and in vitro. We hypothesized that TAM67 inhibits cell growth by altering the expression of cell cycle regulatory proteins, thus causing a cell cycle block. In the present study, we used clones of MCF7 breast cancer cells that express TAM67 under the control of an inducible promoter. First, we determined the effect of AP-1 blockade on cell growth, then we performed 3H-thymidine incorporation and flow cytometry assays to investigate whether TAM67 inhibits the cell cycle. We observed that in the presence of serum TAM67 inhibited cell growth and caused a block in the G1 phase of the cell cycle. Next, we performed Western-blotting and CDK kinase assays to determine the effects of TAM67 on retinoblastoma (Rb) phosphorylation, the expression of cell cycle regulatory proteins, and CDK activity. We discovered that TAM67 inhibited Rb phosphorylation and reduced E2F activity. We also found that TAM67 decreased the expression of D and E cyclins, reduced CDK2 and CDK4 activity, and increased the CDK inhibitor p27. The studies of gene expression at the RNA level showed that TAM67 decreased cyclin Ds mRNA expression. Our study suggests that in the presence of serum, TAM67 inhibits breast cancer growth predominantly by inducing inhibitors of cyclin-dependent kinases (such as p27) and by reducing the expression of the cyclins involved in transitioning from G1 into S phase of the cell cycle. These studies lay the foundation for future attempt to develop new agents for the treatment and prevention of breast cancer.
The Vitamin A metabolite, retinoic acid, has been shown to have chemopreventive and therapeutic activity for certain cancers such as head and neck, cervical, neuroblastoma and promyelocytic leukemia. Retinoic acid achieves these activities by inducing differentiation and/or growth arrest. A large number of studies have investigated the mechanism(s) by which retinoic acid alters the behavior of premalignant and tumor cells. Although much important data has been obtained, the exact signaling pathways required for retinoic acid to exert its biological effects remains elusive. In this review, we outline the role and function of retinoid nuclear receptors, followed by a discussion of how major signaling pathways are affected in different tumor types by retinoids. We conclude by examining the effect of retinoic acid on G1 cell cycle regulatory proteins in various tumors.
Retinoids, a family of vitamin A metabolites or analogs, play an important role in regulating cell proliferation, differentiation, and apoptosis.
The biological importance of retinoids in the kidney and the potential of retinoids in the treatment of renal diseases are reviewed.
Vitamin A deficiency and mutations of retinoid nuclear receptors cause abnormalities in fetal kidneys, which might predispose to adult diseases such as hypertension. Further, the therapeutic value of retinoids in animal models of kidney diseases, such as lupus nephritis, puromycin aminonucleoside nephrosis, anti-glomerular basement membrane nephritis, mesangioproliferative nephritis, and acute renal allograft rejection has been unveiled recently. Retinoids target mesangial cells, podocytes, tubular epithelial cells, interstitial fibroblasts, as well as lymphocytes and macrophages. The anti-inflammation, anti-coagulation effects, and the proliferation- and immunity-modulating actions of retinoids, have been widely appreciated. Our recent in vitro data revealed a direct antifibrotic effect and a cytoprotective effect of retinoids in various renal cell types. In animal studies, the adverse effects of retinoids are generally minimal; however, the clinical use of retinoids in other diseases points to some major side effects. In addition, in vitro, retinoids can induce lipid accumulation in smooth muscle cells and macrophages and increase expression of some proinflammatory molecules, indicating that their clinical toxicity profile in the setting of renal diseases needs to be better understood.
Retinoids not only are important in renal development, but also show promise as a new generation of renal medication and deserve to be tested in clinical trials to clarify their full potential.
Retinoids and analogs of vitamin D3 may achieve greater in vivo applications if the toxic side effects encountered at pharmacologically active doses could be alleviated. These seco-steroid hormones often act in concert, and therefore, we attempted to dissect these interactions by isolating combinations of receptor-selective retinoids and a potent vitamin D3 analog[ 1α,25(OH)2-16ene-23-yne-26,27,F6-19nor-D3, code name LH] that were potent inhibitors of prostate cancer cell growth at low, physiologically safer doses.
Using a panel of prostate cancer cell lines representing progressively more transformed phenotypes, we found that the LNCaP cell line (least transformed) was either additively or synergistically inhibited in its clonal growth by LH and various naturally occurring and receptor-selective retinoids, the most potent combination being with a retinoic acid receptor (RAR)βγ-selective retinoid (SR11262). The effect was not found with either PC-3 (intermediate transformation) or DU-145 (most transformed). We also undertook RT-PCR to examine the subtypes of RARs present, and we found that PC-3 and DU-145 did not express RARβ. Stable expression of RARβ into the RARβ-negative PC-3 cells resulted in increased sensitivity to SR11262 and LH proportional to the amount of RARβ expressed.
This study indicates that RARβ may play an important role in synergistically controlling cell proliferation, and expression is lost with increased prostate cancer cell transformation. Simultaneous administration of a potent vitamin D3 analog and receptor-selective retinoids may have therapeutic potential for the treatment of androgen-dependent and -independent prostate cancer.
In the developing mouse, retinoic acid receptors (RARs) beta and gamma 1 are expressed in characteristic spatiotemporal patterns which are correlated with different developmental fates of the respective tissues. Understanding the cues that regulate the expression of the various RARs may therefore provide insights into the process of tissue diversification. Transcription of RAR beta is rapidly upregulated through a retinoic acid-responsive element (here referred to as the beta RARE) in its promoter. Like RAR alpha and RAR beta, RAR gamma 1 has been implicated in the activation of the beta RARE. Therefore, it is puzzling that RAR beta and RAR gamma 1 appear to be expressed in reciprocal patterns. In the present report, we show that RAR gamma 1, one of the two predominant RAR gamma isoforms, can inhibit the activity of RAR gamma 2, RAR beta, and endogenous RAR on the beta RARE. In contrast, the three RAR gamma isoforms tested and RAR beta activated a palindromic thyroid hormone response element with similar levels of efficiency. The differential activity of RAR gamma 1 compared with that of RAR beta appears to reside in both the N-terminal and the C-terminal halves of RAR gamma 1. RAR gamma 1-mediated inhibition of other RARs may involve competition for the response element as well as direct interaction with other receptors and might be part of a regulatory system contributing to the characteristic tissue distribution of the various RARs.
We present evidence that retinoic acid can down-regulate transcriptional activation by the nuclear protooncogene c-jun. All three members of the retinoic acid receptor (RAR) subfamily (RAR alpha, RAR beta, and RAR gamma) can repress transcriptional induction of the human collagenase gene or a heterologous promoter that contains the collagenase promoter AP-1-binding site. In contrast, the retinoid X receptor fails to repress Jun/AP-1 activity, demonstrating a significant difference between the two regulatory systems through which retinoids exert their transcriptional control. Analysis of RAR alpha mutants in transfection studies reveals that the DNA-binding domain is important for the inhibition of Jun/AP-1 activity, even though the RAR does not bind the collagenase AP-1 site. Rather, gel-retardation assays reveal that bacterially expressed full-length RAR alpha inhibits binding of Jun protein to target DNA. These data suggest that the RAR alpha may form a nonproductive complex with c-Jun and provides a simple mechanisms by which retinoic acid may limit cell growth and possibly malignant progression.
Retinoids such as retinoic acid (RA) are potent anti-arthritic and anti-neoplastic agents. We investigated the mechanism by which RA inhibits induction of collagenase gene transcription by inflammatory mediators, tumor promoters, and proto-oncogenes. We found that the RA receptors (RARs) are potent inhibitors of AP-1 activity generated either by cJun homodimers or cJun/cFos heterodimers. In addition, both cJun and cFos can inhibit RAR activity. In vitro experiments suggested that this inhibition is due to an interaction between RAR and AP-1 proteins that results in mutual loss of DNA-binding activity. The RARs need not bind to the AP-1 site, neither does AP-1 bind to RA response elements. An understanding of this antagonism between the RAR and AP-1 might help to elucidate the anti-neoplastic and anti-arthritic effects of RA as well as its effects on cell differentiation and proliferation.
Using retinoic acid receptor (RAR) reporter cells specific for either RAR alpha, beta or gamma, we have identified synthetic retinoids which specifically induce transactivation by RAR beta, while antagonizing RA-induced transactivation by RAR alpha and RAR gamma. Like RA, these synthetic retinoids allow all three RAR types to repress AP1 (c-Jun/c-Fos) activity, demonstrating that the transactivation and transrepression functions of RARs can be dissociated by properly designed ligands. Using AP1 reporter cells, we also show that glucocorticoids or vitamin D3, together with either RA or these 'dissociating' synthetic retinoids, can synergistically repress phorbol ester-induced AP1 activity. RA, but not these 'dissociating' retinoids, induced transcription of an interleukin-6 promoter-based reporter gene transiently transfected into HeLa cells together with RARs. Using Ki-ras-transformed 3T3 cells as a model system, we show that both RA and the 'dissociating' retinoids inhibit anchorage-independent cell proliferation, suggesting that retinoid-induced growth inhibition may be related to AP1 transrepression.
Retinoids are effective in the treatment and prevention of certain human cancers. Most of their actions are thought to result from changes in gene expression mediated by nuclear retinoic acid receptors and retinoid X receptors. We conducted a study to determine whether the expression of these receptors was altered in premalignant oral lesions and, if so, whether their expression could be restored by treatment with isotretinoin.
We performed in situ hybridization of retinoic acid receptors and retinoid X receptors using antisense riboprobes in specimens of oral mucosa from 7 normal subjects and specimens of premalignant oral lesions from 52 patients before treatment with isotretinoin and from 39 of the 52 patients after three months of treatment.
All the normal specimens expressed retinoic acid receptor-beta messenger RNA (mRNA). In contrast, retinoic acid receptor-beta mRNA was detected in only 21 of the 52 premalignant oral lesions (P = 0.003). Thirty-five of the 39 specimens available for evaluation after treatment expressed retinoic acid receptor-beta mRNA (P < 0.001). All normal and premalignant specimens expressed similar levels of mRNA for retinoic acid receptor-alpha and retinoic acid receptor-gamma and the three types of retinoic X receptors, alpha, beta, and gamma. The levels of retinoic acid receptor-beta mRNA increased in the specimens from 18 of the 22 patients who had responses to isotretinoin and in 8 of the 17 specimens from the patients without responses (P = 0.04).
The expression of retinoic acid receptor-beta mRNA is selectively lost in premalignant oral lesions and can be restored by treatment with isotretinoin. Restoration of the expression of retinoic acid receptor-beta mRNA is associated with a clinical response. Retinoic acid receptor-beta may have a role in mediating the response to retinoids and may be a useful intermediate biologic marker in trials of these agents for the prevention of oral carcinogenesis.
The effects of retinoic acid (RA) are mainly mediated by its nuclear receptors, the RA receptors (RARs) and retinoid X receptors (RXRs) that regulate target gene expression by binding to specific RA-response elements (RAREs). RAR beta is the best characterized RA-responsive gene. Due to the presence of a RARE (beta RARE) in its promoter, the expression of the RAR beta 2 is markedly increased in response to RA in most epithelial tissues, including lung. Recently, it was observed that the RAR beta gene is not expressed in a number of human lung cancer cell lines, suggesting a possible correlation between abnormal expression of the RAR beta gene and lung cancer development. In this study, we investigate the RA response in human lung cancer cell lines. Here we report that the expression of the RAR beta gene cannot be regulated by RA in the majority of human lung cancer cell lines examined, while the general response to RA is intact. The nonresponsiveness of the RAR beta gene results from different defects in the response mechanism. Interestingly, we find in some cell lines a differential responsiveness of the beta RARE such that the element is inactive in its natural promoter context but active when linked to the heterologous tk promoter. Importantly, we also observe that the presence of retinoid receptors is not sufficient for the induction of the RAR beta gene. This suggests that specific factors determine the RA responsiveness in the context of its natural promoter. Our observation that the RA nonresponsiveness of the RAR beta promoter is a common feature of human lung cancer cell lines suggests that balanced RAR beta expression is an essential feature for the maintenance of a normal state of lung tissue.
Retinoids regulate many biological processes, including differentiation, morphogenesis and cell proliferation. They are also important therapeutic agents, but their clinical usefulness is limited because of side effects. Retinoid activities are mediated by specific nuclear receptors, the RARs and RXRs, which can induce transcriptional activation through specific DNA sites or by inhibiting the transcription factor AP-1 (refs 12-15), which usually mediates cell proliferation signals. Because the two types of receptor actions are mechanistically distinct, we investigated whether conformationally restricted retinoids, selective for each type of receptor action, could be identified. Here we describe a new class of retinoids that selectively inhibits AP-1 activity but does not activate transcription. These retinoids do not induce differentiation in F9 cells but inhibit effectively the proliferation of several tumour cell lines, and could thus serve as candidates for new retinoid therapeutic agents with reduced side effects.
Retinoids mediate their actions via RARs (retinoic acid receptors) and RXRs (retinoid X receptors). Each class of these nuclear retinoid receptors is further subdivided into three species, namely alpha, beta, and gamma. Recent studies demonstrate that estrogen receptor (ER)-positive human breast carcinoma (HBC) cell lines and tumor samples exhibit significantly higher levels of RAR alpha than their ER-negative counterparts. ER-positive HBC cell lines are sensitive to, and ER-negative cell lines are resistant to, growth inhibitory effects of retinoic acid (RA). We previously demonstrated that the expression of functional ERs in an established ER-negative cell line resulted in higher levels of RAR alpha and sensitivity to growth inhibition by RA. To further investigate the major role of RAR alpha in retinoid-mediated inhibition of growth, we transfected RAR alpha cDNA in two RA-resistant ER-negative HBC cell lines. Analyses of different clonal populations of RAR alpha transfectants from each cell line revealed growth inhibition by retinoids. Utilizing RAR- and RXR-class selective retinoids, we further demonstrated that only the RAR alpha-selective retinoids mediated the growth inhibition in these cells, while the RXR-selective retinoids were biologically inert. We thus provide evidence that the molecular mechanisms of retinoid inhibition of HBC proliferation predominantly involve RAR alpha.
Both retinoic acid (RA) treatment and dominant-negative c-Jun mutant expression effectively inhibit phorbol ester-induced AP-1 activity and induced neoplastic transformation in mouse epidermal JB6 cells. However, both reagents also target non-AP-1 molecules in addition. Because liganded retinoic acid receptors interact with and transactivate RA response elements (RAREs) on DNA, as well as interact with Jun protein to block AP-1 activity, the question arises as to which of these two activities of retinoids is responsible for antitumor-promoting activity. To address this question we generated JB6 promotion-sensitive (P+) cell lines that are stably transfected with a construct containing the collagenase promoter bearing one AP-1-binding site that drives a luciferase reporter gene. The stable collagenase-luciferase-transfected cell lines showed 1.5-3.5-fold enhanced AP-1 activity when treated with 12-0-tetradecanoyl-phorbol-13-acetate (TPA). Up to 90% of TPA-induced AP-1 activity was blocked by retinoids SR11238, SR11302, or trans-RA, but not by retinoid SR11235. Of these retinoids, only RA and SR11235 were able to transactivate RARE-dependent gene expression. Transrepression of TPA-induced AP-1 and transactivation of RARE by RA, SR11238, and SR11302 were concentration dependent at 10(-10) to 10(-6) M retinoid. When tested for activity in inhibiting tumor promoter-induced transformation in JB6 P+ cells, the retinoids specific for AP-1 transrepression were inhibitory, whereas SR11235, which only activated RARE, showed little effect. We thus conclude that the AP-1-blocking activity of retinoids is likely to be responsible for the antitumor-promoting activity. This result, together with the observation that dominant-negative Jun blocks transformation, argues for a requirement of induced AP-1 in the tumor promoter-induced transformation process.
Retinoids are known to inhibit the growth of hormone-dependent but not that of hormone-independent breast cancer cells. We
investigated the involvement of retinoic acid (RA) receptors (RARs) in the differential growth-inhibitory effects of retinoids
and the underlying mechanism. Our data demonstrate that induction of RAR beta by RA correlates with the growth-inhibitory
effect of retinoids. The hormone-independent cells acquired RA sensitivity when the RAR beta expression vector was introduced
and expressed in the cells. In addition, RA sensitivity of hormone-dependent cells was inhibited by a RAR beta-selective antagonist
and the expression of RAR beta antisense RNA. Introduction of RAR alpha also restored RA sensitivity in hormone-independent
cells, but this restoration was accomplished by the induction of endogenous RAR beta expression. Furthermore, we show that
induction of apoptosis contributes to the growth-inhibitory effect of RAR beta. Thus, RAR beta can mediate retinoid action
in breast cancer cells by promoting apoptosis. Loss of RAR beta, therefore, may contribute to the tumorigenicity of human
mammary epithelial cells.
Retinoic acid has been shown to be an anticancer agent, and a growing literature suggests that it is the nuclear retinoic acid receptor beta2 (RARbeta2) that is primarily responsible for mediating this effect, at least in some systems. To determine whether partial inactivation of RARbeta2 would predispose to lung cancer in mice, we generated three transgenic lines expressing antisense sequences. When killed at 13-3/4-18 months of age, 21/36 animals had a total of 43 pulmonary tumors superficially visible upon necropsy, whereas among 23 nontransgenic mice, only 1 had a single visible lung tumor. A twofold higher incidence of lung tumors was seen in homozygous vs. hemizygous antisense mice. The endogenous RARbeta2 message level was reduced in transgenic lung tissue and further reduced in the tumors. RARbeta4, a truncated isoform derived from the same transcript as RARbeta2, does not carry the sequence identified by the antisense construct and its message was not as strongly affected. Immunofluorescence studies showed that RARbeta was virtually undetectable in the tumors, but present in normal tissue. We conclude that RARbeta2, but probably not RARbeta4, plays an important role in suppression of murine lung tumorigenesis.
The diverse function of retinoic acid (RA) is mediated by its nuclear receptors, the retinoic acid receptors (RARs) and retinoid X receptors (RXRs). However, the RA response is often lost in cancer cells that express the receptors. Previously, it was demonstrated that the RA response is regulated by the COUP-TF orphan receptors. Here, we present evidence that nur77, another orphan receptor whose expression is highly induced by phorbol esters and growth factors, is involved in modulation of the RA response. Expression of nur77 enhances ligand-independent transactivation of RA response elements (RAREs) and desensitizes their RA responsiveness. Conversely, expression of COUP-TF sensitizes RA responsiveness of RAREs by repressing their basal transactivation activity. Unlike the effect of COUP-TFs, the function of nur77 does not require direct binding of nur77 to the RAREs, but is through interaction between nur77 and COUP-TFs. The interaction occurs in solution and results in inhibition of COUP-TF RARE binding and transcriptional activity. Unlike other nuclear receptors, a large portion of the carboxy-terminal end of nur77 is not required for its interaction with COUP-TF. In human lung cancer cell lines, COUP-TF is highly expressed in RA-sensitive cell lines while nur77 expression is associated with RA resistance. Stable expression of COUP-TF in nur77-positive, RA-resistant lung cancer cells enhances the inducibility of RARbeta gene expression and growth inhibition by RA. These observations demonstrate that a dynamic equilibrium between orphan receptors nur77 and COUP-TF, through their heterodimerization that regulates COUP-TF RARE binding, is critical for RA responsiveness of human lung cancer cells.
To investigate the role of AP-1 transcription factors in mediating retinoid-induced growth suppression of breast cells, we studied the sensitivity of MCF7 breast cancer cells with different levels of AP-1 activity to all-trans retinoic acid (atRA). AP-1 activity was increased in MCF7 cells by stably transfecting c-jun cDNA into these cells. Parental and vector-transfected MCF7 cells, which were sensitive to the growth-inhibitory effects of atRA, exhibited atRA-dependent retinoic acid receptor (RAR) transactivation and transrepression of 12-O-tetradecanoylphorbol-13-acetate-induced AP-1 activity. The c-jun-transfected MCF7 cells had increased basal AP-1 transactivation activity and increased expression of AP-1-regulated genes but were resistant to the antiproliferative effects of atRA. However, MCF7 cells transfected with a deletion mutant of c-jun, TAM-67, which lacks most of the amino-terminal transactivation domain of cJun and is unable to activate AP-1-dependent gene expression, were sensitive to the growth-inhibitory effects of atRA. These results suggest that the transactivation domain of cJun is required for induction of retinoid resistance in these breast cancer cells. atRA did not activate RAR-dependent gene transcription or transrepress 12-O-tetradecanoylphorbol-13-acetate-induced AP-1 activity in these cJun-overexpressing cells. Investigation of the RAR and retinoic acid X receptor expression level demonstrated that RAR alpha and RAR gamma RNA expression was reduced in the c-jun-transfected MCF7 cells, whereas RAR beta expression was up-regulated. However, retinoic acid responsive element DNA binding activity was intact in c-jun-transfected cells. Therefore, the mechanism by which cJun overexpression induces resistance to the growth-inhibitory effect of atRA may be through interference with atRA-dependent RAR transactivation or AP-1 transrepression, possibly through titration of essential coactivators. These results suggest that the antiproliferative effects of retinoids can be overcome by cJun overexpression.
Vitamin A and its derivatives (retinoids) have profound effects on the proliferation and differentiation of many cell types
and are involved in a diverse array of developmental and physiological regulatory processes, including those responsible for
the development of the mature nervous system. Retinoid signals are mediated by retinoic acid (RA) receptors (RARs) and retinoid
X receptors (RXRs), which show distinct spatio-temporal patterns of expression during development and in adult tissues. We
have used SK-N-BE2(c) neuroblastoma cells to study the effects of reciprocal regulation of expression of various RARs. We
show that in these cells RARγ1 acts as a repressor of RARβ2 transcription in the absence of an agonist. In the presence of RA, the expression of RARγ1 is reduced and that of RARβ2 is induced. Overexpression of RARγ1 neutralizes the effects of RA on RARβ induction. Expression of an RARγ1-specific antisense construct leads to the constitutive expression of RARβ2. Although both overexpression of RARγ1 and its reduction of expression can result in inhibition of cell proliferation, they induce different morphological changes.
Reduction of RARγ1 (and induction of RARβ) leads to increased apoptosis, whereas RARγ1 overexpression leads to differentiation in the absence of apoptosis. Thus, RARγ1 appears to control a differentiation-apoptosis switch in SK-N-BE2(c) neuroblastoma cells.
I. Introduction NUCLEAR receptors mediate the signals of a broad variety of fat-soluble hormones, including the steroid and vitamin D3 hormones, thyroid hormones, and vitamin A-derived hormones and analogs (retinoids). The receptors represent one of the largest transcription factor families known (reviewed in Refs. 1–3). Only some of the members are ligand-binding receptors, while others are “orphan receptors” for which specific ligands have not yet been identified and may not exist. These proteins may therefore function as constitutive or negative transcription factors, or coregulators of ligand-binding receptors. The receptors regulate gene transcription by interacting with specific DNA sequences (called response elements) that are usually located in the promoter regions of the responsive genes. All receptors contain a highly conserved cysteine-rich region, the DNA- binding domain (DBD) that forms two “zinc-finger” structures that allow protein-DNA as well as proteinprotein interaction (4). A second but...
This chapter discusses the cellular biology and biochemistry of the retinoids. In isolated 9-day-old rat embryos, retinoic acid prevents the formation of the pharyngeal arches, which are derived from cephalic mesenchyme; these structures later form the maxilla and the mandible. Another mesenchymal derivative, whose formation is markedly suppressed by retinoic acid in rat embryos, is the yolk sac circulation. Retinoids can exert a powerful influence on cell differentiation in many different types of epithelia. The effects of retinoids on differentiation of epithelia in organ culture result from a combination of complex cellular responses and interactions of different cell types in the explant. One of the most illuminating examples of the ability of retinoids to promote differentiation is the effect of retinoids on mouse embryonal carcinoma cells. These undifferentiated stem cells of teratocarcinomas are multipotential, that is, they can differentiate into a multiplicity of somatic cell types. Another example of the ability of retinoic acid to promote terminal differentiation of neoplastic cells to nonneoplastic cell types is the effect of retinoic acid on human promyelocytic leukemia cells.
Retinoids are important regulators of the growth and differentiation of tracheobronchial epithelial cells. To determine the mechanism of action of retinoids in these cells, we began to examine the expression of nuclear retinoic acid receptors (RARs) in normal human and rabbit tracheobronchial epithelial (HBE and RbTE, respectively) cells and in several lung carcinoma cell lines. A specific nuclear RAR-binding activity with a molecular weight of 50,000 was identified in these cells. A correlation was found between the binding of several retinoids to this RAR and their ability to inhibit transglutaminase Type I activity. Normal HBE and RbTE cells contained two RAR alpha mRNA transcripts, 2.6 and 3.5 kb in size, and one 3.1 kb RAR gamma transcript. RAR beta transcripts were undetectable in HBE cells. RAR expression was unchanged during squamous differentiation. Treatment of HBE and RbTE cells with 100 nM retinoic acid increased RAR beta mRNA expression but did not change the levels of RAR alpha and RAR gamma. In contrast, retinoic acid suppressed in these cells the level of involucrin, transglutaminase Type I, and SQ37 mRNA. In comparison with normal HBE cells, certain lung carcinoma cell lines appear to have an altered expression of RAR beta and RAR gamma. Human bronchial fibroblasts (HBF) expressed RAR alpha and RAR gamma transcripts of the same size as HBE cells. HBF cells contain low levels of a 2.9- and 3.3-kb RAR beta mRNA. Treatment of HBF cells with retinoic acid increased the level of RAR beta mRNA in a time-dependent manner; the maximal induction was about 15-fold. On the basis of these findings we hypothesize that RARs are involved in the suppression of squamous differentiation in tracheobronchial epithelial cells and that lung fibroblasts are target cells for retinoids.
One of the three human retinoic acid receptors, RAR-beta, maps to a region on the short arm of chromosome 3 frequently deleted in lung cancer. Because retinoic acid is required for normal epithelial cell growth and regulation, and loss of a retinoic acid receptor might be expected to contribute to oncogenesis, we examined RAR-beta RNA and DNA in normal lung, 33 lung cancer cell lines and nine primary lung tumors. Normally, RAR-beta is expressed as two transcripts, of sizes 3.1 kb and 2.8 kb, which are strongly induced by retinoic acid. At least 50% of the cell lines and 30% of the tumor samples show altered RAR-beta expression and/or inducibility, including examples of absence or specific loss of one of the RAR-beta transcripts. Abnormalities in the expression patterns of RAR-alpha and RAR-gamma also are found, but at a lower frequency than RAR-beta abnormalities. Southern analysis reveals alteration of the RAR-beta gene in three of the cell lines. Our data suggest that abnormalities in structure and expression of the RAR-beta gene may be involved in the pathogenesis of lung cancer.
Hepatitis B virus (HBV) is clearly involved in the aetiology of human hepatocellular carcinoma (HCC) and the finding of HBV DNA integration into human liver DNA in almost all HCCs studied suggested that these integrated viral sequences may be involved in liver oncogenesis. Several HBV integrations in different HCCs and HCC-derived cell lines have been analysed after molecular cloning without revealing any obvious role for HBV. From a comparison of a HBV integration site present in a particular HCC with the corresponding unoccupied site in the non-tumorous tissue of the same liver, we now report that HBV integration places the viral sequence next to a liver cell sequence which bears a striking resemblance to both an oncogene (v-erb-A) and the supposed DNA-binding domain of the human glucocorticoid receptor and human oestrogen receptor genes. We suggest that this gene, usually silent or transcribed at a very low level in normal hepatocytes, becomes inappropriately expressed as a consequence of HBV integration, thus contributing to the cell transformation.
We have studied the role of the AP1 transcription factor in the progression of human breast carcinomas. This progression is characterized by a loss of dependence for proliferation on mitogenic hormones, and is also linked to loss of responsiveness to the growth inhibitor retinoic acid (RA). In the hormone-dependent breast tumor cell line MCF7 mitogenic stimulation was found to be linked to an enhancement of AP1 transcriptional activity, while growth inhibition by RA was parallelled by decreased AP1 activity. AP1 binding activity to its consensus DNA sequence was rapidly reduced in RA treated cells, in the absence of any noticeable change in expression of AP1 constituents. AP1 overexpression abrogated RA repression in MCF7 cells. In hormone-independent cell lines (BT20, Hs578T, MDA-MB231, MDA-MB468) autonomous proliferation was associated with an increased background AP1 activity. Interestingly, these cells are refractory to growth inhibition by RA, which can only be partly explained by underexpression of RA receptors. In these cells RA did not repress AP1 transactivation unless RA receptors were overexpressed by means of cotransfection with an expression vector. This suggests that the high background levels of AP1 activity in the autonomously growing cells are associated with prevention of RA inhibition of AP1 activity to occur. Therefore, increased AP1 activity may not only play a role in progression of breast tumors towards hormone-insensitivity but may also contribute to the RA resistance of such cells.
Retinoids modulate cellular proliferation and mediate gene function through a series of nuclear receptors. The retinoic acid nuclear receptor beta (RAR beta) plays an important role in the differentiation of a number of cell types. We now demonstrate that RAR beta expression is confined to normal mammary tissue and is not expressed in either immortalized normal or malignant cell lines. Treatment of RAR beta-transfected MDA-MB-231 cells with 1 microM all-trans-retinoic acid (RA) significantly inhibited monolayer growth of the cells which express recombinant RAR beta. RAR beta-expressing MDA-MB-231 cells formed significantly smaller and fewer colonies in soft agar than the mock-transfected cells. Addition of 1 microM RA stimulated colony size and number in the RAR beta-transfected MDA-MB-231 cells. In contrast to the RAR beta-expressing cells, colony formation by the RAR alpha-expressing cells was similar to the mock-transfected controls and the addition of 1 microM RA to the RAR alpha-transfected cells inhibited colony formation. While demonstrating decreased colony formation in agar, RAR beta-expressing MDA-MB-231 cells failed to exhibit decreased growth in SCID mice. Our results show that RAR beta functions as a negative regulator of growth in breast epithelial cells. In addition, the growth of these cells is differentially regulated by RAR alpha and RAR beta which is most likely the result of the modulation of different genes.
Reversible acetylation at the epsilon-amino group of lysines located at the conserved domain of core histones is supposed to play an important role in the regulation of chromatin structure and its transcriptional activity. One promising strategy for analyzing the precise function of histone acetylation is to block the activities of acetylating or deacetylating enzymes by specific inhibitors. Recently, two microbial metabolites, trichostatin A and trapoxin, were found to be potent inhibitors of histone deacetylases. Trichostatin A reversibly inhibits the mammalian histone deacetylase, whereas trapoxin causes inhibition through irreversible binding to the enzyme. The histone deacetylase from a trichostatin A-resistant cell line is resistant to trichostatin A, indicating that the enzyme is the primary target. Both of the agents induce a variety of biological responses of cells such as induction of differentiation and cell cycle arrest. Trichostatin A and trapoxin are useful in analyzing the role of histone acetylation in chromatin structure and function as well as in determining the genes whose activities are regulated by histone acetylation.
Tissue-remodeling processes are largely controlled by matrix metalloproteinases that degrade the extracellular components of connective tissues. In this study, gene regulation of two human matrix metalloproteinases, stromelysin and collagenase, was investigated by a reverse-transcription-coupled (RT)-PCR assay. Here, signals from both the heterogenous nuclear RNA (hnRNA) and mRNA are amplified, allowing the regulation of gene expression to be divided between transcriptional and/or post-transcriptional control. In confluent human lung fibroblast cultures, tumor-necrosis factor-alpha and 12-O-tetradecanoyl-phorbol 13-acetate induce stromelysin and collagenase genes transcriptionally. Interleukin-1 beta (IL-1 beta) induces stromelysin gene transcription but has little, if any, effect on the collagenase gene transcription in cells cultured in the presence of 10% serum. By a competitive RT-PCR assay, the IL-1 beta-reated cultures contain an average of 60 molecules of stromelysin mRNA/cell and the untreated cultures about 1.9 molecules/cell. In serum-starved cells, both IL-1 beta and serum induce transcription of the collagenase gene. Also, in serum-starved cells type II collagen can induce collagenase mRNA but not stromelysin mRNA. Inhibition of protein synthesis with cycloheximide induces stromelysin gene transcription but has no effect on the collagenase gene. These data indicate different mechanisms of regulation of the human stromelysin and collagenase genes in cultured cells.
Retinoids are important cellular, dietary factors that regulate differentiation and cellular growth. They serve as ligands for specific nuclear receptors, the retinoic acid receptors (RARs). Ligand-activated receptors regulate gene transcription through target retinoic acid-responsive elements (RAREs) found in promoter regions. We have investigated the expression of retinoic acid receptor genes (alpha, beta, gamma) and retinoid X receptor beta in normal, senescing, and tumorigenic human mammary epithelial cells. We find that most tumor cells show a loss of RAR-beta expression, but that RAR-alpha and -gamma as well as retinoid X receptor beta are variably expressed in both normal and tumor cells. RAR-beta gene expression is induced both by retinoic acid and by fenretinide in normal cells, but tumor cells fail to respond to either. In contrast, RAR-beta expression increases with serial passage in senescing cells. Paradoxically, both normal and tumor cells can trans-activate an exogenous beta-RARE, as demonstrated by reporter gene assays. Oligonucleotide mobility shift assays with the beta-RARE show a single discrete complex in normal cells, whereas tumor cells exhibit a heterogeneous set of larger complexes, which indicates that tumor cells utilize a different array of factors within the beta-RARE. Reporter gene assays with extended promoter regions indicate the presence of negative regulatory elements and/or factor binding sites that reside between -1500 and the RARE located at -59, and that the promoter is down-regulated in MCF-7 tumor cells. Our findings reveal a dichotomy: RAR-beta transcription is down-regulated in tumor cells compared with normal human mammary epithelial cells, and up-regulated in senescence.
From the data summarized here it is apparent that a mechanism exists by which several nuclear receptors, GR, TRs, and RARs, and probably RXRs, and other receptors can regulate gene expression by an 'indirect' mechanism that does not involve receptor DNA interaction but receptor AP-1 interaction. Vice versa, the AP-1 components c-Jun and/or c-Fos can also antagonize these receptors apparently involving the same mechanism. In vivo the effectiveness of the antagonism is cell line dependent, such that often only a 'one sided' antagonism is observed (82, 67), and individual receptors behave differentially in different cell lines. For example, TRs showed anti AP-1 activity in CV-1 cells whereas RARs did not (72). Two possible mechanisms involving an additional factor or factors are shown in Fig. 1. In model A, a heterodimer formed between RAR and RXR binds to an RARE in the absence (or low concentration) of AP-1 components. In the presence of c-Jun (or c-Fos) a complex is formed between RAR and c-Jun involving a third partner (X). Interaction of RAR and c-Jun with (X) stabilizes the RAR-c-Jun complex. The relatively stable trimeric complex does not bind to DNA but would require the presence of all three partners (RAR, Jun, and X). As is clear from the model, an excess of AP-1 components could thus inactivate RAR (or RXR) activity, but maintain AP-1 activity, while an excess of RAR or RXR would inhibit AP-1 activity but maintain RAR/RXR activity. The factor X could be cell type specific and its activity could also be regulated by specific signals including phosphorylation. While most of the reported data fit this model, two major findings cannot easily be accommodated. One is the observation of a positive effect of receptors on AP-1 activity as seen with ER (67, 82), thyroid hormone (84), and other steroid hormone receptors, and to some extent also with RARs in CV-1 cells (72). One could argue that an ER-Jun-X complex could still bind and activate a TRE, but one would then expect to observe a larger complex in gel shift assays, which, to the author's knowledge, has not yet been reported. The second observation that does not easily fit into this model are in vivo footprinting results showing that glucocorticoids do not alter the in vivo footprint of AP-1 (40). In this case the model would have to be modified such that the RAR-Jun-X complex can still bind DNA in vivo but is inactive in transcriptional activation. That DNA binding in vitro is inhibited could, in fact, be an artifact seen only when one of the components is present in excess, as when more than one RAR molecule interacts with each c-Jun monomer. Model B is somewhat reminiscent of the squelching/transcriptional interference model, which proposes the existence of intermediary factors that mediate the activity of transactivation functions to the basal transcription machinery and can be sequestered by other activation functions, leading to a repression of activation (85, 86). Cloning of these factors will demonstrate whether different or common factors are involved in both mechanisms. It is very clear that the indirect mechanism may affect many genes since it is not dependent on specific binding sites. At this point, studies have focused only on a very few response elements and genes. Inhibition of AP-1 activity has been mostly studied with synthetic TRE constructs or the collagenase promoter. However, the stromelysin as well as TGFβ genes appear to be inhibited by RA via the same mechanism (66, 87). Additional experiments are needed to fully elucidate the mechanism of this ligand-dependent indirect mechanism of transcriptional regulation. In addition, it will be of interest to explore whether receptor-specific ligands that predominantly function via this indirect regulatory pathway but do not enhance transcription by DNA-bound receptors can be identified. Recent results from this laboratory (unpublished) indicate that for RARs such ligands can be defined. The effectiveness of the antagonism is dependent on the receptor concentration and the concentration of c-Jun and c-Fos. In this regard it is of interest to note that c-Jun and c-Fos levels are regulated by many factors (25), and that all three RAR subtypes are regulated by RA (68- 71). The RARβ-2 isoform is particularly strongly up-regulated in many epithelial tissues by RA and could thus play a very important role in these tissues in controlling AP-1 activity. Finally, the nuclear receptors may interact with additional transcription factors via a similar mechanism.
Retinoic acid receptor beta (RAR beta), which codes for a nuclear receptor for retinoic acid, is localized in a chromosomal region frequently deleted in lung cancer cells. The gene is expressed in normal lung tissue and in the majority of the cell lines derived from lung tumors but not in most of the lines derived from lung tumors with epidermoid characteristics. To study the possible role of RAR beta in growth control of epidermoid lung tumor-derived cells, transfectants expresing RAR beta were generated from nonexpressing epidermoid tumor-derived cell lines. Four clones were derived from line CALU-1, three of which showed a 20-60% increase in doubling time in the presence of retinoic acid. Parental and control-transfected cells were unaffected or slightly stimulated. All four clones expressing RAR beta were less tumorigenic in nude mice than were the untransfected or control-transfected cells, with about a 50% incidence of take vs. 95%. When tumors did develop from RAR beta-positive cells, they showed a reduced rate of growth, an increased latency, and, in six of seven tumors tested, a much reduced level of RAR beta expression. Transfectants derived from a second tumor line, H157, also showed a markedly reduced incidence of take in nude mice. Together with the known effects of retinoic acid on differentiation and carcinogenesis, our results support the hypothesis that RAR beta functions as a tumor suppressor gene in epidermoid lung tumorigenesis.
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Guidelines for submitting commentsPolicy: Comments that contribute to the discussion of the article will be posted within approximately three business days. We do not accept anonymous comments. Please include your email address; the address will not be displayed in the posted comment. Cell Press Editors will screen the comments to ensure that they are relevant and appropriate but comments will not be edited. The ultimate decision on publication of an online comment is at the Editors' discretion. Formatting: Please include a title for the comment and your affiliation. Note that symbols (e.g. Greek letters) may not transmit properly in this form due to potential software compatibility issues. Please spell out the words in place of the symbols (e.g. replace “α” with “alpha”). Comments should be no more than 8,000 characters (including spaces ) in length. References may be included when necessary but should be kept to a minimum. Be careful if copying and pasting from a Word document. Smart quotes can cause problems in the form. If you experience difficulties, please convert to a plain text file and then copy and paste into the form.
Nuclear receptors regulate gene expression by direct activation of target genes and inhibition of AP-1. Here we report that, unexpectedly, activation by nuclear receptors requires the actions of CREB-binding protein (CBP) and that inhibition of AP-1 activity is the apparent result of competition for limiting amounts of CBP/p300 in cells. Utilizing distinct domains, CBP directly interacts with the ligand-binding domain of multiple nuclear receptors and with the p160 nuclear receptor coactivators, which upon cloning have proven to be variants of the SRC-1 protein. Because CBP represents a common factor, required in addition to distinct coactivators for function of nuclear receptors, CREB, and AP-1, we suggest that CBP/p300 serves as an integrator of multiple signal transduction pathways within the nucleus.
Retinoids, the natural and synthetic vitamin A derivatives, are known to inhibit the proliferation of lung cancer and breast cancer cells and the growth of carcinogen-induced bronchogenic squamous cell carcinoma and mammary tumors, and have been used as chemoprevention agents against both types of cancer. However, clinical trials of retinoids in patients with advanced lung cancer and breast cancer have not been successful. In studying how retinoid sensitivity is lost in cancer cells, we have found that lack of the retinoic acid receptor beta (RAR beta) gene expression and its abnormal regulation by retinoic acid (RA) are common features in human lung cancer and breast cancer cells. The absence and abnormal RA regulation of RAR beta correlates with the loss of anti-proliferation effect of RA in hormone-independent breast cancer cells, and is due to different abnormalities found in cancer cells. Furthermore, expression of RAR beta gene in hormone-independent breast cancer cells restores their RA sensitivity. These data demonstrate that RAR beta can mediate the growth inhibitory effect of RA and suggest that the lack of RAR beta may contribute to retinoid resistance in certain cancer cells.
Matrix metalloproteinases (MMPs) are expressed in normal remodeling tissues in a generally tissue-restricted pattern. Transcripts for stromelysin-1 and collagenase are expressed primarily in stromal fibroblasts, whereas transcripts for matrilysin are expressed primarily in glandular epithelial cells. These expression patterns are maintained at carcinoma tumor sites until the late stages of tumor progression at which point many epithelially-derived tumors begin to express stromal fibroblast MMPs. Coincidentally, late stage carcinomas take on other characteristics of stromal fibroblasts, indicating that these tumor cells have "transdifferentiated', that is, they have begun to exhibit characteristics of cells from a separate developmental lineage. Despite their distinct expression patterns, many of the promoters for MMP genes show the same general arrangement of the nuclear proto-oncoprotein-binding sites, AP-1 and PEA3. However, the specific interaction between these cis-elements and different combinations of Fos, Jun, and Ets proteins which recognize these sites may be important in controlling both the positive and negative regulation involved in the tissue-restricted pattern of MMP expression in normal and neoplastic tissues.
Human bronchial epithelial (HBE) cells undergo squamous differentiation in response to a variety of conditions in tissue culture, and retinoid treatment has been shown to reverse this process. Retinoids mediate their effects through the retinoic acid and retinoid X nuclear receptors (RAR and RXR, respectively), which form RAR-RXR heterodimers, RXR homodimers, and heterodimers of RXR and certain orphan receptors. These receptor dimers bind to distinct response elements, activating separate pathways. In this study, we investigated the roles of RAR and RXR signaling pathways in the inhibition of HBE squamous differentiation. After induction of squamous differentiation by confluent growth, HBE cells were treated with retinoids that selectively activate RARs (E-4-[2-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthaienyl)-1- propenyl] benzoic acid), RXRs (4-[1-(5,5,8,8-tetramethyl-5,6,7,8-tetrahydro-2-naphthyl)ethenyl]benzoic acid), or both RARs and RXRs (9-c/s retinoic acid). These retinoids inhibited the mRNA expression of the squamous differentiation markers transglutaminase type I, involucrin, keratin 5, and keratin 13, suggesting that inhibition of HBE squamous differentiation could be mediated by activation of either RAR or RXR signaling pathways. We examined the role of AP-1 as a potential effector of these retinoid pathways. AP-1 transcriptional activity was reduced markedly by these retinoids, and a concomitant, but proportionally smaller, reduction in AP-1 DNA binding was observed. Furthermore, treatment of squamous HBE cells with the retinoid SR11238, which inhibited AP-1 without activating retinoid receptor transcriptional properties, reduced the expression of transglutaminase type I and involucrin. These findings support the hypothesis that, in HBE cells, RAR and RXR signaling pathways inhibit AP-1 transcriptional activity, and this contributes to retinoid-induced reversal of HBE squamous differentiation.
Retinoic acid (RA) is essential for regulation of epithelial cell differentiation. The intracellular effects of RA are mediated by RA-binding nuclear receptors, including the RA receptors (RARs) alpha, beta, and gamma. The ligand-activated receptors induce the transcription of target genes by binding to RA-responsive elements in the promoter regions. One target gene is the RAR beta gene, which encodes a potential tumor suppressor. Loss of RA inducibility of RAR beta gene expression is assumed to play a role in the development of several types of human carcinomas, including carcinomas of the uterine cervix. We have analyzed RAR beta gene expression in normal cervical cells and in cervical carcinoma cell lines. The results show that the RAR beta mRNA levels are high and RA inducible in the primary keratinocytes, whereas they are low and not inducible or only slightly inducible by RA in all of the cervical carcinoma cell lines analyzed. The basal and the RA-induced RAR beta mRNA levels tend to increase with senescence of the normal cells. Fusion of primary ectocervical keratinocytes with HeLa cervical carcinoma cells revealed that the characteristics of RAR beta gene expression of the normal cells are dominant over that of the tumor cells. Using synthetic retinoids with receptor-preferential agonist activities and a RAR alpha-specific antagonist, we show that RAR alpha is the major endogenous RAR subtype for induction of RA-dependent RAR beta gene expression. Taken together, our results indicate that abnormal downregulation of RAR beta gene expression may be an important step in the multifactorial process of cervical carcinogenesis.
Retinoids, analogues of vitamin A, are required for the normal growth and differentiation of human bronchial epithelium. They are also able to reverse premalignant lesions and prevent second primary tumors in some patients with non-small-cell lung cancer (NSCLC). These effects are thought to result from modulation of cell growth, differentiation, or apoptosis (programmed cell death). When certain retinoid receptors in the cell nucleus (i.e., retinoic acid receptors [RARs] and retinoid X receptors [RXRs]), which mediate most retinoid actions, are suppressed, abnormal activity may result that could enhance cancer development. Purpose: This study was designed to determine whether there are abnormalities in the expression of retinoid receptors in surgical specimens from patients with NSCLC.
Transcripts of nuclear retinoid receptors were detected in formalin-fixed, paraffin-embedded specimens by use of digoxigenin-labeled riboprobes specific for RAR alpha, RAR beta, RAR gamma, RXR alpha, RXR beta, and RXR gamma for in situ hybridization to histologic specimens from 79 patients with NSCLC and as control from 17 patients with non-lung cancer. The quality and specificity of the digoxigenin-labeled probes were determined by northern blotting, and the specificity of the binding of antisense riboprobes was verified by use of sense probes as controls.
All receptors were expressed in at least 89% of control normal bronchial tissue specimens from 17 patients without a primary lung cancer and in distant normal bronchus specimens from patients with NSCLC. RAR alpha, RXR alpha, and RXR gamma were expressed in more than 95% of the NSCLC specimens. In contrast, RAR beta, RAR gamma, and RXR beta expression was detected in only 42%, 72%, and 76% of NSCLC, respectively.
These data suggest that the expression of RAR alpha, RXR alpha, and RXR gamma is not altered in NSCLC; however, expression of RAR beta and possibly also of RAR gamma and RXR beta is suppressed in a large percentage of patients with lung cancer.
The loss of expression of one or more of these nuclear retinoid receptors may be associated with lung carcinogenesis.
Retinyl methyl ether (RME) is known to prevent the development of mammary cancer. However, the mechanism by which RME exerts its anticancer effect is presently unclear. The diverse biological functions of retinoids, the vitamin A derivatives, are mainly mediated by their nuclear receptors, retinoic acid receptors (RARs) and retinoid X receptors (RXRs). RARs and RXRs are ligand-dependent transcriptional factors that either activate gene transcription through their binding to retinoic acid response elements or repress transactivation of genes containing the activator protein 1 (AP-1) binding site. Previous studies demonstrated that RME can modulate transcriptional activity of retinoid receptors on retinoic acid response elements, suggesting that regulation of retinoid receptor activity may mediate the anticancer effect of RME. In this study, we present evidence that RME can down-regulate AP-1 activity induced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, insulin, growth factors, and the nuclear proto-oncogenes c-Jun and c-Fos. Transient transfection assays demonstrate that inhibition of AP-1 activity occurs on the human collagenase promoter containing an AP-1 binding site or the thymidine kinase promoter linked with an AP-1 binding site. In HeLa cells, the inhibition is observed when RAR-alpha and/or RXR-alpha but not RAR-beta or RAR-gamma expression vectors are cotransfected, whereas the endogenous retinoid receptors in breast cancer cells T-47D and ZR-75-1 were sufficient to confer the inhibition by RME. Furthermore, using gel retardation assay, we show that 12-O-tetradecanoylphorbol-13-acetate- and epidermal growth factor-induced AP-1 binding activity in breast cancer cells is inhibited by RME. These results suggest that one of the mechanisms by which RME prevents cancer development may be due to the repression of AP-1-responsive genes.
Retinoic acid receptor beta (RAR beta) expression is lost or decreased during malignant transformation in human pancreatic adenocarcinoma. The aim of this study was to evaluate the role of RAR beta expression in the propagation of a malignant phenotype in human pancreatic carcinoma cells.
Overexpression of RAR beta in the human pancreatic carcinoma cell line DAN-G was achieved by selecting stable transfected cell clones. Genomic integration and expression were verified by Southern and Northern blotting and electrophoretic mobility shift assays. Growth was determined by cell number and xenografts transplanted into nude mice. Differentiation was examined by immunohistochemistry.
Overexpression of RAR beta in DAN-G cells inhibited cellular proliferation in vitro and in vivo. Furthermore, RAR beta overexpression resulted in induction of cellular differentiation in xenografted tumors as evidenced by increased tumor cell expression of duct cell differentiation markers carcinoembryonic antigen (CEA), CA19-9, and cytokeratin 7.
Decreased expression of RAR beta plays a key role in the maintenance of a malignant phenotype in human pancreatic adenocarcinoma and therefore represents a novel target for experimental strategies in the treatment of pancreatic cancer patients.
Retinoids and their receptors [retinoic acid receptors (RARs) and retinoid X receptors] play an important role in maintaining the balance between proliferation and apoptosis. Recently, Deng et al. [Science (Washington DC), 274: 2057-2059, 1996] reported a loss of heterozygosity on chromosome 3p24 in breast cancer specimens and the morphologically normal appearing adjacent tissue. The 3p24 locus includes, among other genes, the region coding for RAR-beta. This study was designed to determine whether there are abnormalities in the expression of retinoid receptors in surgical specimens of patients with breast cancer. In 14 patients, transcripts of nuclear retinoid receptors were detected by in situ hybridization in formalin-fixed, paraffin-embedded specimens by means of digoxigenin-labeled riboprobes specific for RAR-alpha, -beta and -gamma. We found RAR-alpha expressed in all specimens, whereas RAR-gamma was expressed in 100% of normal breast tissue but in only 11 of 14 tumorous lesions. RAR-beta was found in all cases of normal breast tissue localized distant from the tumor, but in 13 of 14 cases it was completely absent in the tumor and the morphologically normal appearing tissue adjacent to the tumor. One possibility to explain the suppression of RAR-beta is a mutation in the promoter region. Sequencing the DNA extracted from paraffin-embedded tumor tissue of the corresponding breast cancer specimens, we were not able to detect any mutation in the retinoic acid-responsive element. Our results clearly indicate a crucial role of RAR-beta in the carcinogenesis of breast cancer.
All-trans-retinoic acid (trans-RA) and other retinoids exert anticancer effects through two types of retinoid receptors, the RA receptors (RARs) and retinoid X receptors (RXRs). Previous studies demonstrated that the growth-inhibitory effects of trans-RA and related retinoids are impaired in certain estrogen-independent breast cancer cell lines due to their lower levels of RAR alpha and RARbeta. In this study, we evaluated several synthetic retinoids for their ability to induce growth inhibition and apoptosis in both trans-RA-sensitive and trans-RA-resistant breast cancer cell lines. Our results demonstrate that RXR-selective retinoids, particularly in combination with RAR-selective retinoids, could significantly induce RARbeta and inhibit the growth and induce the apoptosis of trans-RA-resistant, RAR alpha-deficient MDA-MB-231 cells but had low activity against trans-RA-sensitive ZR-75-1 cells that express high levels of RAR alpha. Using gel retardation and transient transfection assays, we found that the effects of RXR-selective retinoids on MDA-MB-231 cells were most likely mediated by RXR-nur77 heterodimers that bound to the RA response element in the RARbeta promoter and activated the RARbeta promoter in response to RXR-selective retinoids. In contrast, growth inhibition by RAR-selective retinoids in trans-RA-sensitive, RAR alpha-expressing cells most probably occurred through RXR-RAR alpha heterodimers that also bound to and activated the RARbeta promoter. In MDA-MB-231 clones stably expressing RAR alpha, both RARbeta induction and growth inhibition by RXR-selective retinoids were suppressed, while the effects of RAR-selective retinoids were enhanced. Together, our results demonstrate that activation of RXR can inhibit the growth of trans-RA-resistant MDA-MB-231 breast cancer cells and suggest that low cellular RAR alpha may regulate the signaling switch from RAR-mediated to RXR-mediated growth inhibition in breast cancer cells.
Some of the nuclear retinoic acid receptors (RARs) alpha, beta, and gamma and retinoid X receptors (RXRs) alpha, beta, and gamma are thought to mediate the effects of retinoids on cell growth, differentiation, and apoptosis and thereby prevent breast carcinogenesis. We analyzed the expression of mRNAs for the three RARs and RXR-alpha in histological sections of specimens from 70 breast cancer patients, which included adjacent normal tissue, ductal carcinoma in situ, and invasive cancer, using in situ hybridization. RARs alpha, beta, and gamma and RXR-alpha were expressed in 98.1, 98.0, 93.0, and 100% of the adjacent normal tissues. Significant decreases in the number of cases expressing RAR-beta were observed among ductal carcinoma in situ (83.1%) and invasive carcinomas (51.6%), especially among the poorly differentiated cases (77.4 and 35.7 %, respectively). No relationship was found between the expression of estrogen receptor and RAR-beta. These results implicate decreases in RAR-beta expression in breast cancer development and suggest that they are independent of estrogen receptor status.
Retinoids regulate the growth and differentiation of human tracheobronchial epithelial cells. In this study, we investigated the effects of all-trans-retinoic acid (trans-RA) and receptor class-selective retinoids on the growth and apoptosis of human lung cancer cell lines. Trans-RA significantly inhibited the growth of Calu-6 and H460 cells, accompanied by induction of RA receptor (RAR) beta expression. In contrast, it had little effect on the growth of H292, SK-MES-1 and H661 lung cancer cell lines, in which RAR beta expression was not induced. Stable expression of RAR beta in RAR beta-negative, trans-RA-resistant SK-MES-1 and H661 lung cancer cells led to recovery of trans-RA-induced growth inhibition, which occurred, however, only at low serum concentration. Using fluorescent microscopy and the terminal deoxyribonucleotidyl transferase (TdT) assay, we demonstrated that induction of apoptosis by trans-RA contributed to its growth-inhibitory effect in trans-RA-sensitive lung cancer cell lines. Analysis of RAR-selective and retinoid X receptor (RXR)-selective retionoids showed that activation of both RARs and RXRs could induce growth inhibition in trans-RA-sensitive lung cancer cells. Also, an additive synergistic effect on growth inhibition and RAR beta induction was observed when cells were treated with combinations of RAR-selective and RXR-selective retinoids. Together, our results show that expression of RAR beta plays a role in mediating retinoid response in lung cancer cells and that activation of RARs or RXRs contributes to induction of RAR beta, growth inhibition and apoptosis by retinoids.
Evidence indicates that the retinoic acid receptor beta (RARbeta) gene might be a tumor suppressor gene. Previously, we have shown that the expression of the RARbeta gene was either inhibited or downregulated in tumorigenic hepatoma cell lines such as McA-RH8994. McA-RH8994 cells expressed RARalpha and gamma and three types of retinoid X receptor (RXRalpha, beta and gamma), but not RARbeta mRNA. To further analyze the molecular mechanisms which might account for RARbeta gene inactivation, the rat RARbeta gene promoter was cloned from McA-RH8994 cells and no mutation was detected. By transient transfection, McA-RH8994 cells contained the necessary factors to activate the RARbeta gene. To study the possible roles of RARbeta in hepatoma cells, the expression of the RARbeta gene was restored in McA-RH8994 cells by stable transfection. A RARbeta positive cell line named McA-RH8994beta was characterized. The results demonstrated that expression of the RARbeta gene resulted in increased sensitivity of the hepatoma cells to the antiproliferative effect of retinoic acid (RA). Furthermore, expression of RARbeta resulted in a spontaneous differentiation of the hepatoma cells. These data indicate that RARbeta plays important roles in differentiation and antiproliferation.
The differentiation and growth suppressive effects of retinoic acid are mediated through retinoic acid nuclear receptors (RARs and RXRs), which are ligand-activated transcription factors. Recent data suggest that both altered and regulated expression of RARs are linked to retinoic acid response in a cell context-dependent manner. This study examined the antiproliferative effects of 13-cis-retinoic acid (cRA) on 12 renal cancer cell lines and correlated these findings with the basal and induced expression of RAR-alpha, -beta and -gamma. Eleven of 12 renal cancers that were either resistant to or only minimally inhibited by cRA did not basally express RAR-beta as determined by Northern blot analysis. In these cells, cRA treatment did not induce RAR-beta expression. In contrast, 1 of 12 cell lines (SK-RC-06) was >90% inhibited by cRA and basally expressed RARbeta. Furthermore, RAR-beta mRNA in SK-RC-06 cells was up-regulated by cRA treatment. Amplification of cDNA using PCR and RAR-beta isoform-specific primer pairs revealed that only SK-RC-06 cells expressed the RAR-beta1 isoform. Expression of RAR-alpha transcripts was abundant in all 12 cell lines examined, whereas low levels of RAR-gamma transcripts were detectable in 6 of 10 renal cancers. Expression of RAR-alpha and RAR-gamma was not affected by cRA. These data showing that the majority of renal cancer cell lines are resistant to cRA suggest that: (a) resistance to the antiproliferative action of cRA correlates with repressed RAR-beta mRNA expression; and (b) the antiproliferative effects of cRA in renal cancer cells are mediated through RAR-beta1.
Epidemiologic studies have demonstrated that individuals who eat more fruits and vegetables and/or have high levels of serum beta-carotene have a lower risk of cancer, especially lung cancer. However, recent human intervention studies using beta-carotene supplements have shown an increase in the risk of lung cancer among smokers and asbestos workers. In this study, we used an animal model system to evaluate the hazard associated with a combination of high-dose beta-carotene supplementation and tobacco smoking.
Ferrets were given a beta-carotene supplement, exposed to cigarette smoke, or both for 6 months. Cell proliferation and squamous metaplasia in lung tissue were assessed by examination of proliferating-cell nuclear antigen expression and histopathologic examination, respectively. beta-Carotene and retinoid concentrations in lung tissue and plasma samples were analyzed by high-performance liquid chromatography. Expression of genes for retinoic acid receptors (RARs) and activator protein-1 (encoded by the c-Jun and c-Fos genes) in lung tissue specimens was examined by western blotting.
A strong proliferative response in lung tissue and squamous metaplasia was observed in all beta-carotene-supplemented animals, and this response was enhanced by exposure to tobacco smoke. When compared with control groups, all three treatment groups had statistically significantly lower concentrations of retinoic acid in lung tissue, and they exhibited 18%-73% reductions in RARbeta gene expression; however, RARalpha and RARgamma gene expression was not reduced. Ferrets given a beta-carotene supplement and exposed to tobacco smoke had threefold to fourfold elevated expression of the c-Jun and c-Fos genes.
Diminished retinoid signaling, resulting from the suppression of RARbeta gene expression and overexpression of activator protein-1, could be a mechanism to enhance lung tumorigenesis after high-dose beta-carotene supplementation and exposure to tobacco smoke.
The preventive effects of retinoids on oral carcinogenesis may be related to their ability to modulate the growth and differentiation of human oral squamous epithelial cells. Nuclear retinoid receptors (RAR alpha, beta, and gamma, and RXR alpha, beta, and gamma) may mediate these effects by regulating gene transcription. The removal of serum from the growth medium of two head and neck squamous cell carcinoma lines 1483 and SqCC/Y1 resulted in a decrease in RAR beta mRNA level and concurrent increases in the expression of the keratin K1 and transglutaminase type I (TGase I), which are markers of differentiation of keratinizing squamous epithelial cells. All-trans-retinoic acid (tRA) or 13-cis-RA increased RAR beta and decreased K1 and TGase I mRNA levels in serum-free medium. Transcriptional activation of reporter genes by means of retinoid response elements (RARE and RXRE) indicated that the RXR-RAR pathway predominates over the RXR homodimer pathway in the 1483 cells. Among several synthetic retinoids with preference for binding to specific nuclear retinoid receptors, those that induced RAR beta also suppressed K1. The inverse association between RAR beta expression and K1 and TGase I levels implicates this receptor in suppression of keratinization in oral epithelial cells.
The role of retinoic acid receptor (RAR) expression in sensitivity to N-(4-hydroxyphenyl)retinamide (4HPR or fenretinide) as well as on the tumorigenicity of human ovarian carcinoma cells was examined. Two human ovarian cancer cell lines, A2780 and IGROV-1, with a 10-fold difference in sensitivity to 4HPR were chosen to study RAR involvement in the response to 4HPR. To determine which RAR was effective, RARalpha, beta and gamma were individually overexpressed in A2780 cells, which are the most sensitive to 4HPR. Sensitivity to 4HPR was increased in RARbeta-overexpressing clones, whereas it was slightly decreased in RARalpha transfectants (which had diminished RARbeta expression) and was unchanged in clones transfected with RARgamma. IGROV-1 cells, which are RARbeta negative, were transfected with RARbeta. Surprisingly, none of the obtained IGROV-1 RARbeta transfectants expressed RARbeta protein, in spite of RARbeta mRNA transcription. All clones were similar to the parental IGROV-1 cells in their sensitivity to 4HPR. Treatment with a pharmacologically achievable concentration of 4HPR (1 microM) led to a rapid 2-fold increase in RARbeta mRNA levels in A2780 cells, but it did not induce RARbeta expression in IGROV-1 cells. Analysis of the tumorigenicity of A2780-transfected clones revealed that overexpression of RARalpha was associated with a significant reduction in tumor takes (50% and 67%, respectively, vs. 96% for the parent line) and with a reduced growth rate. Oncogenicity was clearly decreased in only 1 of the 2 RARbeta-overexpressing clones (33% takes) and was unchanged in the 2 clones with increased RARgamma expression. Our results demonstrate that basal expression and 4HPR inducibility of RARbeta play a role in mediating 4HPR response in ovarian cancer cells. The findings of reduced oncogenicity of clones overexpressing RARalpha and of one clone overexpressing RARbeta indicate that RARalpha and RARbeta might have a tumor-suppressive effect in ovarian tumors.
Retinoids are therapeutically effective in the treatment of psoriasis, photoaging, acne, and certain cancers. Some of the therapeutic actions of retinoids can be ascribed to retinoic acid receptor (RAR)-mediated antagonism of AP1-dependent gene expression. The increased activity of transcription factor AP1, a complex of oncoproteins Jun and Fos, is associated with cell growth and proliferation. Retinoids, on the other hand, inhibit cell proliferation and affect differentiation, activities that possibly stem from an antagonism of AP1-mediated gene expression by RARs. To gain insight into the molecular mechanism of RAR-AP1 interaction, we have identified the regions of the RAR required for AP1 antagonism. We demonstrate that the AP1 antagonism domain of RAR is a complex of the core of the DNA binding domain and the hydrophobic zipper region. Further, both monomeric RAR and RAR-RXR heterodimers inhibit the expression of an AP1 reporter. CREB binding protein (CBP) has been described as a cofactor for AP1, various nuclear hormone receptor proteins including RARs, and certain other transcription factors and is required for their transactivation properties. Therefore, CBP has been proposed as a common limiting cofactor that can account for inhibition of AP1-dependent gene expression by RARs. Interestingly, however, our results along with previously reported observations suggest that in addition to CBP, there may be other limiting cofactor(s) responsible for mutual transrepression of RAR and AP1.