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C-MYC: More than just a matter of life and death
Abstract
Deregulated expression of c-MYC occurs in a broad range of human cancers and is often associated with poor prognosis, indicating a key role for this oncogene in tumour progression. However, as established human tumours often bear multiple genetic lesions, it is difficult to determine whether c-MYC is instrumental in the initiation/progression of the tumour, or indeed whether inactivating c-MYC would lead to tumour regression. Regulatable transgenic mouse models of oncogenesis have shed light on these issues and provide hope for effective cancer therapies.
... There are three members of the Myc proto-oncogene family, c-Myc, MYCL, and MYCN, which encode the c-Myc, L-Myc, and N-Myc proteins, respectively, and play key roles in cellular proliferation, apoptosis, and differentiation (Pelengaris et al., 2002;Adhikary and Eilers, 2005;Dang, 2012). The Myc family are at the crossroads of many growth-promoting signal transduction pathways and constitutes an immediate early response downstream of many ligand-membrane receptor complexes (Kelly et al., 1983;Armelin et al., 1984). ...
... The Myc family are at the crossroads of many growth-promoting signal transduction pathways and constitutes an immediate early response downstream of many ligand-membrane receptor complexes (Kelly et al., 1983;Armelin et al., 1984). All three Myc family members perform their transcriptional regulation function by forming Myc/MAX heterodimers that bind to the transcriptional regulatory region of many target genes (Pelengaris et al., 2002;Adhikary and Eilers, 2005;Meyer and Penn, 2008). Moreover, Myc family members can amplify transcription by interacting with the promoter regions of downstream target genes (Lin et al., 2012;Nie et al., 2012;Nie et al., 2020). ...
... MAX is a member of the basic helix-loop-helix leucine zipper (BR/HLH/LZ) family that can homodimerize or heterodimerize with other BR/HLH/LZ proteins (Fig 4), in particular with the Myc protein (Pelengaris et al., 2002;Adhikary and Eilers, 2005;Meyer and Penn, 2008;Cascón and Robledo, 2012;Suzuki et al., 2017). MAX is commonly expressed as protein isoforms that migrate at 21-and 22-kDa in SDS-PAGE gels (Blackwood et al., 1992). ...
Cancer is a leading cause of death worldwide resulting in ~10 million deaths in 2020. Major oncogenic effectors are the Myc proto-oncogene family that consists of three members including c-Myc, N-Myc, and L-Myc. As a pertinent example of the role of the Myc family in tumorigenesis, amplification of MYCN in childhood neuroblastoma strongly correlates with poor patient prognosis. Complexes between Myc oncoproteins and their partners such as hypoxia-inducible factor-1α (HIF-1α) and Myc-associated protein X (MAX) results in proliferation arrest and pro-proliferative effects, respectively. Interactions with other proteins are also important for N-Myc activity. For instance, the enhancer of zest homolog 2 (EZH2) binds directly to N-Myc to stabilize it by acting as a competitor against the ubiquitin ligase, SCFFBXW7, which prevents proteasomal degradation. Heat shock protein 90 may also be involved in N-Myc stabilization since it binds to EZH2 and prevents its degradation. N-Myc downstream-regulated gene 1 (NDRG1) is down-regulated by N-Myc, and participates in the regulation of cellular proliferation via associating with other proteins, such as glycogen synthase kinase-3β (GSK3β) and low-density lipoprotein receptor-related protein 6 (LRP6). These molecular interactions provide a better understanding of the biological roles of N-Myc and NDRG1, which can be potentially used as therapeutic targets. In addition to directly targeting these proteins, disrupting their key interactions may also be a promising strategy for anti-cancer drug development. This review examines the interactions between the Myc proteins and other molecules, with a special focus on the relationship between N-Myc and NDRG1 and possible therapeutic interventions. Significance Statement Neuroblastoma (NB) is one of the most common childhood solid tumors, with a dismal 5-year survival rate. This problem makes it imperative to discover new and more effective therapeutics. The molecular interactions between major oncogenic drivers of the Myc family and other key proteins e.g, the metastasis suppressor, NDRG1, may potentially be used as targets for anti-neuroblastoma drug development. In addition to directly targeting these proteins, disrupting their key molecular interactions may also be promising for drug discovery.
... In vitro experiments demonstrated that EphA2 loss of function significantly curtailed MDA-MB-231 cell proliferation and induced G1/S cell cycle phase arrest [34]. Mechanistically, EphA2 has been shown to regulate the CDK2/Cyclin E1/2 complex via the proteasome-mediated degradation of p27ˆKIP1, thereby inhibiting cancer cell growth [34,35]. Additionally, tropomyosin-related kinase A (TrkA) recruits EphA-2, which subsequently enhances BCA cell invasion; conversely, TrkA inhibition reverses cancer cell migration [36,37]. ...
... In vitro experiments demonstrated that EphA2 loss of function significantly curtailed MDA-MB-231 cell proliferation and induced G1/S cell cycle phase arrest [34]. Mechanistically, EphA2 has been shown to regulate the CDK2/Cyclin E1/2 complex via the proteasome-mediated degradation of p27^KIP1, thereby inhibiting cancer cell growth [34,35]. Additionally, tropomyosin-related kinase A (TrkA) recruits EphA-2, which subsequently enhances BCA cell invasion; conversely, TrkA inhibition reverses cancer cell migration [36,37]. ...
Breast cancer (BCA) remains the leading cause of cancer-related mortality among women worldwide. This review delves into the therapeutic challenges of BCA, emphasizing the roles of interleukin-13 receptor α2 (IL-13Rα2) and erythropoietin-producing hepatocellular receptor A2 (EphA2) in tumor progression and resistance. Highlighting their overexpression in BCA, particularly in aggressive subtypes, such as Her-2-enriched and triple-negative breast cancer (TNBC), we discuss the potential of these receptors as targets for chimeric antigen receptor T-cell (CAR-T) therapies. We examine the structural and functional roles of IL-13Rα2 and EphA2, their pathological significance in BCA, and the promising therapeutic avenues their targeting presents. With an in-depth analysis of current immunotherapeutic strategies, including the limitations of existing treatments and the potential of dual antigen-targeting CAR T-cell therapies, this review aims to summarize potential future novel, more effective therapeutic interventions for BCA. Through a thorough examination of preclinical and clinical studies, it underlines the urgent need for targeted therapies in combating the high mortality rates associated with Her-2-enriched and TNBC subtypes and discusses the potential role of IL-13Rα2 and EphA2 as promising candidates for the development of CAR T-cell therapies.
... In OS, Myc is known to play a key role 14,15 , though the kinetic of its of deregulation is not well defined. Myc may contribute to tumorigenesis by several ways including overstimulating cell growth and metabolism and/or by causing genomic instability 16 , probably through its ability to induce DNA damage, promote gross chromosomal rearrangements, induce inappropriate cell cycle progression and impair DNA repair 17 . Moreover, Myc has been reported to regulate the pre-replicative complex of the cell cycle including MCM5 and MCM7 among other proteins 18,19 . ...
... Differential gene expression for the 16 samples was explored using the R package (v1. 16.1) and analyzed using DESeq2 (v1.28.1). ...
Osteosarcoma (OS) is an aggressive bone tumor that primarily affects children and adolescents. This malignant tumor is highly aggressive, associated with poor clinical outcomes, and metastasizes mainly to the lungs. Due to its rarity and biological heterogeneity, limited studies of its molecular basis exist, thus hindering the development of effective therapies. WW domain-containing oxidoreductase (WWOX) spans one of the most active and common fragile sites that is frequently altered in human OS. The combined deletion of Wwox and Trp53 using Osterix1-Cre transgenic mice has been shown to accelerate OS development. In this study, we generated a traceable OS mouse model, SKO-Trp53 or DKO-Wwox/Trp53, expressing a tdTomato reporter. By tracking tomato expression at different time points, we detected the early presence of tdTomato-positive cells in the bone marrow (BM) mesenchymal stem cells (MSCs) of non-OS bearing mice, young BM (yBM). We found that DKO yBM cells, but not SKO yBM cells, exhibited tumorigenic traits both in vitro and in vivo. Molecular and cellular characterization of these DKO yBM cells revealed their resemblance to OS tumor cells. Interestingly, one of the observed significant transcriptomic changes in DKO yBM was the upregulation of Myc and its target genes, as compared to SKO yBM cells. Intriguingly, Myc-chromatin immunoprecipitation sequencing (ChIP-Seq) revealed increased enrichment on Myc targets, which were upregulated in DKO yBM cells. Restoration of WWOX in DKO-yBM cells reduced Myc protein levels. As a prototype target, we demonstrated upregulation of MCM7, a known Myc target, in DKO yBM relative to SKO yBM. Inhibition of MCM7 expression using Simvastatin resulted in reduced proliferation and tumor cell growth of DKO yBM cells. Our findings revealed BM-MSCs as a platform to study OS and Myc and its targets as WWOX effectors and early molecular events during osteosarcomagenesis.
... The proto-oncogenic c-Myc is an essential regulator in multiple biological processes, such as cell cycle and cellular metabolism. With a C-terminal basic helix-loop-helix zipper (bHLH-Zip) domain, the disordered c-Myc can specifically interact with its partner protein Max, forming a heterodimeric structure that regulates gene expression 18,24,25 . Overexpression of c-Myc causes disorder in cell proliferation and signaling pathways 23 , resulting in a wide range of human cancers 25 . ...
... useful information for designing molecules that bind with them 28 . A number of compounds that target c-Myc have been reported 18,24,25,[29][30][31] , such as 10074-A4, which was discovered by high-throughput screening, and PKUMDL-YC-1205, which was found through a "multi-conformational-affinity" computational strategy 32 . These compounds were shown to directly bind the disordered bHLH-Zip domain of c-Myc. ...
Intrinsically disordered proteins (IDPs) play crucial roles in cellular processes and hold promise as drug targets. However, the dynamic nature of IDPs remains poorly understood. Here, we construct a single-molecule electrical nanocircuit based on silicon nanowire field-effect transistors (SiNW-FETs) and functionalize it with an individual disordered c-Myc bHLH-LZ domain to enable label-free, in situ, and long-term measurements at the single-molecule level. We use the device to study c-Myc interaction with Max and/or small molecule inhibitors. We observe the self-folding/unfolding process of c-Myc and reveal its interaction mechanism with Max and inhibitors through ultrasensitive real-time monitoring. We capture a relatively stable encounter intermediate ensemble of c-Myc during its transition from the unbound state to the fully folded state. The c-Myc/Max and c-Myc/inhibitor dissociation constants derived are consistent with other ensemble experiments. These proof-of-concept results provide an understanding of the IDP-binding/folding mechanism and represent a promising nanotechnology for IDP conformation/interaction studies and drug discovery.
... Song and colleagues previously reported that EPHA2 promotes growth in basal-like/TNBCs through p27/KIP1 inhibition-dependent and c-Myc-dependent mechanisms, which activate cyclin/CDK pathways to promote S phase progression (Song et al., 2017). c-Myc regulates the gene transcription of proteins that sequester p27/KIP1 to enable activation of cyclin E2-associated CDKs for basal-like/TNBC proliferation (Pelengaris et al., 2002). However, our transcriptomic analysis showed that in cells with EPHA2-AS1/2 silencing and AHCC ® treatment, cyclin E2 mRNA expression was suppressed, whereas c-Myc mRNA expression was amplified and p27/ KIP1 mRNA expression was not elevated but reduced (see Supplementary Table S7). ...
Ephrin type-A receptor 2 (EPHA2) is a receptor tyrosine kinase that is overexpressed in a variety of cancers, including breast cancer. EPHA2 expression may be causally related to tumorigenesis; therefore, it is important to understand how EPHA2 expression is regulated. We previously reported that EPHA2 antisense RNA (EPHA2-AS), a natural antisense transcript, is an important modulator of EPHA2 mRNA levels and hence production of EPHA2 protein. EPHA2-AS encodes two splice variants, EPHA2-AS1 and EPHA2-AS2. The two variants are constitutively expressed in a concordant manner with EPHA2 mRNA in human breast adenocarcinoma cell lines and in patient samples, with the highest levels detected in the basal-like/triple-negative molecular subtype of breast cancer cells. In this study, we investigated the mechanism of EPHA2-AS1/2 in triple-negative breast cancer using MDA-MB-231 cells. We performed RNA-seq transcriptome analyses of MDA-MB-231 cells treated with AHCC®, which suppressed expression of EPHA2-AS1/2 and EPHA2 mRNA, and EPHA2-AS1/2-silenced MDA-MB-231 cells. Bioinformatics analyses identified 545 overlapping differentially expressed genes that were significantly up- or down-regulated by these treatments. Subsequent functional enrichment analyses of the overlapping genes in combination with in vitro assays indicated that EPHA2-AS1/2 may promote the proliferation and migration of MDA-MB-231 cells through the EPHA2-dependent Ras signaling pathways mediated by MAPK8/JNK1, MAPK9/JNK2-NFATC2/NFAT1 (proliferation and migration) and JUND (migration). These results thus suggest that EPHA2-AS1/2 may represent a potential molecular target for triple-negative breast cancer treatment.
... 18 The elevated or deregulated MYC levels drive many oncogenic processes, including metabolic reprogramming and non-stop cell cycle progression, but MYC also sensitizes cells to diverse inducers of extrinsic or intrinsic programmed cell death pathways. [19][20][21][22] These early findings have laid the conceptual foundation for MYC-dependent SL (MYC SL) therapeutic strategies, which seek to specifically harness MYC-generated vulnerability pathways as opposed to drugging the MYC protein directly. [23][24][25] A previous study exploring therapeutic opportunities through the MYC SL concept revealed that in several mouse models of MYC high BC, the MYChigh tumors in vivo are specifically vulnerable to combination treatment with venetoclax and metformin. ...
Background
Combining cytotoxic chemotherapy or novel anticancer drugs with T-cell modulators holds great promise in treating advanced cancers. However, the response varies depending on the tumor immune microenvironment (TIME). Therefore, there is a clear need for pharmacologically tractable models of the TIME to dissect its influence on mono- and combination treatment response at the individual level.
Methods
Here we establish a patient-derived explant culture (PDEC) model of breast cancer, which retains the immune contexture of the primary tumor, recapitulating cytokine profiles and CD8+T cell cytotoxic activity.
Results
We explored the immunomodulatory action of a synthetic lethal BCL2 inhibitor venetoclax+metformin drug combination ex vivo, discovering metformin cannot overcome the lymphocyte-depleting action of venetoclax. Instead, metformin promotes dendritic cell maturation through inhibition of mitochondrial complex I, increasing their capacity to co-stimulate CD4+T cells and thus facilitating antitumor immunity.
Conclusions
Our results establish PDECs as a feasible model to identify immunomodulatory functions of anticancer drugs in the context of patient-specific TIME.
... Contrariwise, in tumor cells, the function of c-Myc is frequently enhanced, often due to mutations in the gene itself or more commonly through the induction of c-Myc expression via upstream oncogenic pathways. Regardless of its oncogenic properties, c-Myc has a neutralizing ability to stimulate apoptosis through different pathways (Kelly & Rickinson, 2007;Pelengaris, Khan, & Evan, 2002). This contradiction likely accounts for the exceptional role of c-Myc as the primary oncogene in early detected tumors. ...
Tumor formation is supported by metabolic reprogramming, characterized by increase nutrient uptake, glycolysis and glutaminolysis. The c-Myc proto-oncogene is a transcription factor, upregulated in most cancers and several reports showed the role of c-Myc in other metabolic pathways such as glucose, amino acid, and nucleotide metabolism. In this short report, we tried to summarize the existing takeaway points from studies conducted in different cancer types with respect to c-Myc and lipid and serine metabolism. Here, we report that c-Myc can activate both lipid and serine metabolism against the backdrop of tumor formation, and different therapies like aspirin and lomitapide target the links between c-Myc and metabolism to slow down tumor progression and invasion. We also report diverse upstream regulators that influence c-Myc in different cancers, and interestingly components of the lipid metabolism (like lipid phosphate phosphatase and leptin) and serine metabolism can also act upstream of c-Myc in certain occasions. Finally, we also summarize the existing knowledge on the involvement of epigenetic pathways and non-coding RNAs in regulating lipid and serine metabolism and c-Myc in tumor cells. Identification of non-coding factors and epigenetic mechanisms present a promising avenue of study that could empower researchers with novel anticancer treatment targeting c-Myc and lipid and serine metabolism pathways!
... 2-25 = fractions obtained from polysome profile assay corresponding to each ribosomal subunit investigated. apoptosis, and cell cycle regulation (Pelengaris et al., 2002). It has been described that HNRNPC interaction with c-myc mRNA in G2/M increases its translation in an IRES-dependent manner when cap-dependent translation is partially inhibited. ...
Background Information
The dual‐specificity phosphatase 3 (DUSP3) regulates cell cycle progression, proliferation, senescence, and DNA repair pathways under genotoxic stress. This phosphatase interacts with HNRNPC protein suggesting an involvement in the regulation of HNRNPC‐ribonucleoprotein complex stability. In this work, we investigate the impact of DUSP3 depletion on functions of HNRNPC aiming to suggest new roles for this enzyme.
Results
The DUSP3 knockdown results in the tyrosine hyperphosphorylation state of HNRNPC increasing its RNA binding ability. HNRNPC is present in the cytoplasm where it interacts with IRES trans‐acting factors (ITAF) complex, which recruits the 40S ribosome on mRNA during protein synthesis, thus facilitating the translation of mRNAs containing IRES sequence in response to specific stimuli. In accordance with that, we found that DUSP3 is present in the 40S, monosomes and polysomes interacting with HNRNPC, just like other previously identified DUSP3 substrates/interacting partners such as PABP and NCL proteins. By downregulating DUSP3, Tyr‐phosphorylated HNRNPC preferentially binds to IRES‐containing mRNAs within ITAF complexes preferentially in synchronized or stressed cells, as evidenced by the higher levels of proteins such as c‐MYC and XIAP, but not their mRNAs such as measured by qPCR. Under DUSP3 absence, this increased phosphorylated‐HNRNPC/RNA interaction reduces HNRNPC‐p53 binding in presence of RNAs releasing p53 for specialized cellular responses. Similarly, to HNRNPC, PABP physically interacts with DUSP3 in an RNA‐dependent manner.
Conclusions and Significance
Overall, DUSP3 can modulate cellular responses to genotoxic stimuli at the translational level by maintaining the stability of HNRNPC‐ITAF complexes and regulating the intensity and specificity of RNA interactions with RRM‐domain proteins.
... Some studies have suggested that aberrant overexpression of MYC is common in NSCLC [40]. The c-Myc oncogene family, encoding nuclear phosphoproteins, has vital functions in cell proliferation, loss of differentiation, apoptosis, tumorigenesis, cancer cell reprogramming and chemoresistance [41][42][43]. Moreover, c-Myc is believed to exert dual effects by both stimulating and inhibiting specific components of the cell cycle machinery and is correlated with two distinct genetic pathways that control cell progression through the G1 phase [42]. ...
... We chose two tumor-associated proteins as antitumor targets, including (1) programmed death-ligand 1 (PD-L1), a transmembrane protein that is highly expressed in melanoma cells and helps tumor cells escape immune attack 39 ; and (2) c-Myc, an oncogenic transcription factor. 40 After initially confirming that the OptoD 2 Trim-TPD system can achieve targeted degradation of PD-L1 and c-Myc in light-illuminated mouse melanoma B16F10 Luci cells in vitro ( Figure S6A), we targeted protein degradation by the Opto-D 2 Trim-TPD system in mice bearing B16F10 Luci tumors. ...
... Cyclin D1, along with other positive cell cycle regulators such as Rb, is known to be highly expressed in foetal cardiomyocytes but significantly downregulated in neonatal cardiomyocytes, coinciding with postnatal cell cycle arrest (Zhao et al., 2020). c-MYC (downregulated with maturation) forms a heterodimeric complex with the protein MAX which activates a variety of known target genes essential for cell-cycle progression and growth including Cyclin D2, CDK2, and the translation initiation factors eIF4 and eIF2 (Pelengaris et al., 2002). ...
Heart maturation and remodelling during the foetal and early postnatal period are critical for proper survival and growth of the foetus, yet our knowledge of the molecular processes involved are lacking for many cardiac cell types. To gain a deeper understanding of the transcriptional dynamics of the heart during the perinatal period in the mouse, we performed single-cell RNA-seq using Drop-seq. We sampled hearts over the course of foetal to early postnatal stages (E14.5 E16.5, E18.5, P0, P4, P7) to establish a catalogue of 49,769 cardiac cells. Gene regulatory network and pathway activity analyses underscored that maturation of the mouse heart is strongly associated with regulation of cell growth and proliferation via pathways such as TGFβ. We additionally identified a common, cell-type independent transcriptomic signature of imprinted genes changing over time. The lncRNA H19 in particular was found to significantly decrease over time in multiple cardiac cell types, with the exception of endocardial cells. Our dataset further provides insights for often underrepresented cardiac cell types such as valvular interstitial cells, vascular smooth muscle cells, and pericytes of the coronary vasculature. We envision this dataset to serve as a resource for better understanding mouse heart maturation at the transcriptomic level during perinatal development, and to help bridge the gap between early developmental and adult heart stages for single-cell transcriptomics.
... In OS, Myc is known to play a key role [14,15], though the kinetic of its deregulation is not well defined. Myc may contribute to tumorigenesis by several ways including overstimulating cell growth and metabolism and/or by causing genomic instability [16], probably through its ability to induce DNA damage, promote gross chromosomal rearrangements, induce inappropriate cell cycle progression and impair DNA repair [17]. Moreover, Myc has been reported to regulate the prereplicative complex of the cell cycle including MCM5 and MCM7 among other proteins [18,19]. ...
Osteosarcoma is an aggressive bone tumor that primarily affects children and adolescents. This malignancy is highly aggressive, associated with poor clinical outcomes, and primarily metastasizes to the lungs. Due to its rarity and biological heterogeneity, limited studies on its molecular basis exist, hindering the development of effective therapies. The WW domain-containing oxidoreductase (WWOX) is frequently altered in human osteosarcoma. Combined deletion of Wwox and Trp53 using Osterix1-Cre transgenic mice has been shown to accelerate osteosarcoma development. In this study, we generated a traceable osteosarcoma mouse model harboring the deletion of Trp53 alone (single-knockout) or combined deletion of Wwox/Trp53 (double-knockout) and expressing a tdTomato reporter. By tracking Tomato expression at different time points, we detected the early presence of tdTomato-positive cells in the bone marrow mesenchymal stem cells of non-osteosarcoma-bearing mice (young BM). We found that double-knockout young BM cells, but not single-knockout young BM cells, exhibited tumorigenic traits both in vitro and in vivo. Molecular and cellular characterization of these double-knockout young BM cells revealed their resemblance to osteosarcoma tumor cells. Interestingly, one of the observed significant transcriptomic changes in double-knockout young BM cells was the upregulation of Myc and its target genes compared to single-knockout young BM cells. Intriguingly, Myc-chromatin immunoprecipitation sequencing revealed its increased enrichment on Myc targets, which were upregulated in double-knockout young BM cells. Restoration of WWOX in double-knockout young BM cells reduced Myc protein levels. As a prototype target, we demonstrated the upregulation of MCM7, a known Myc target, in double-knockout young BM relative to single-knockout young BM cells. Inhibition of MCM7 expression using simvastatin resulted in reduced proliferation and tumor cell growth of double-knockout young BM cells. Our findings reveal BM mesenchymal stem cells as a platform to study osteosarcoma and Myc and its targets as WWOX effectors and early molecular events during osteosarcomagenesis.
... promoted proliferation of islet cells, including β cells (P < 0.01, Fig. 8, L and M), with a fourfold increase in BrdU + β cells in CD47 −/− islets (Fig. 8N). To establish the mechanism for enhanced proliferation, we considered transcription factor c-Myc because it is a major regulator of cell growth and proliferation, including in islets (29,30). We, and others, have shown that loss of CD47 permits sustained proliferation of primary murine cells, and CD47 knockdown or blockade acutely increases mRNA and protein expression of c-Myc (and other stem cell transcription factors) in vitro and in vivo (17,31,32). ...
Diabetes is a global public health burden and is characterized clinically by relative or absolute insulin deficiency. Therapeutic agents that stimulate insulin secretion and improve insulin sensitivity are in high demand as treatment options. CD47 is a cell surface glycoprotein implicated in multiple cellular functions including recognition of self, angiogenesis, and nitric oxide signaling; however, its role in the regulation of insulin secretion remains unknown. Here, we demonstrate that CD47 receptor signaling inhibits insulin release from human as well as mouse pancreatic β cells and that it can be pharmacologically exploited to boost insulin secretion in both models. CD47 depletion stimulated insulin granule exocytosis via activation of the Rho GTPase Cdc42 in β cells and improved glucose clearance and insulin sensitivity in vivo. CD47 blockade enhanced syngeneic islet transplantation efficiency and expedited the return to euglycemia in streptozotocin-induced diabetic mice. Further, anti-CD47 antibody treatment delayed the onset of diabetes in nonobese diabetic (NOD) mice and protected them from overt diabetes. Our findings identify CD47 as a regulator of insulin secretion, and its manipulation in β cells offers a therapeutic opportunity for diabetes and islet transplantation by correcting insulin deficiency.
... Computational simulation also excluded a statistical drift of the cell populations due to prevalent apoptosis and alterations of cell cycle dynamics. Clonal competition, a concept introduced by Moreno and coworkers [9], was the only explanation that resisted simulation, and transcriptional analyses of the successful clones concordantly showed that activation of the c-MYC pathway makes the game consistent with what we know about this driver of cell cycle progression, apoptosis, cellular transformation [10] and cell competition [11]. ...
Before a tumor is diagnosed and surgically removed, it has been growing for many months or even years [...]
... Interestingly, Myc was reported to promote G1-S transition by controlling the activities of complexes Cyclin D-CDK4 and Cyclin E-CDK2 complexes. 34 Furthermore, immunoblotting results demonstrated that Myc levels were remarkedly decreased in the MDIG-KO mice, regardless of whether they were quiescent or proliferating ( Fig. 3d and Supplementary Fig. S7c). In contrast, other transcriptionally deregulated genes showed little difference at the protein levels between the WT and MDIG-KO groups ( Supplementary Fig. S7d). ...
The mineral dust-induced gene (MDIG) comprises a conserved JmjC domain and has the ability to demethylate histone H3 lysine 9 trimethylation (H3K9me3). Previous studies have indicated the significance of MDIG in promoting cell proliferation by modulating cell-cycle transition. However, its involvement in liver regeneration has not been extensively investigated. In this study, we generated mice with liver-specific knockout of MDIG and applied partial hepatectomy or carbon tetrachloride mouse models to investigate the biological contribution of MDIG in liver regeneration. The MDIG levels showed initial upregulation followed by downregulation as the recovery progressed. Genetic MDIG deficiency resulted in dramatically impaired liver regeneration and delayed cell cycle progression. However, the MDIG-deleted liver was eventually restored over a long latency. RNA-seq analysis revealed Myc as a crucial effector downstream of MDIG. However, ATAC-seq identified the reduced chromatin accessibility of OTX2 locus in MDIG-ablated regenerating liver, with unaltered chromatin accessibility of Myc locus. Mechanistically, MDIG altered chromatin accessibility to allow transcription by demethylating H3K9me3 at the OTX2 promoter region. As a consequence, the transcription factor OTX2 binding at the Myc promoter region was decreased in MDIG-deficient hepatocytes, which in turn repressed Myc expression. Reciprocally, Myc enhanced MDIG expression by regulating MDIG promoter activity, forming a positive feedback loop to sustain hepatocyte proliferation. Altogether, our results prove the essential role of MDIG in facilitating liver regeneration via regulating histone methylation to alter chromatin accessibility and provide valuable insights into the epi-transcriptomic regulation during liver regeneration.
... Chromosomal translocations dysregulating MYC (8q24) were reported in 5-15% of GCB DLBCL cases (17), while MYC protein detection in 5-40% of DLBCL cases (30)(31)(32). MYC gene translocation was associated with a very poor outcome in DLBCL (33)(34)(35)(36). MYC translocation in DLBCL is usually associated with other gene abnormalities, e.g., BCL2 or BCL6 rearrangements (37,38). ...
Background
The clinical and genetic heterogeneity of diffuse large B-cell lymphoma (DLBCL) presents distinct challenges in predicting response to therapy and overall prognosis. The main objective of this study was to assess the application of the immunohistochemistry- and interphase fluorescence in situ hybridization (FISH)-based molecular markers in the diagnosis of DLBCL and its prognostic value in patients treated with rituximab-based immunochemotherapy.
Methods
This is a multicenter, retrospective study, which analyzed data from 7 Hungarian hematology centers. Eligible patients were adults, had a histologically confirmed diagnosis of DLBCL, were treated with rituximab-based immunochemotherapy in the first line, and had available clinicopathological data including International Prognostic Index (IPI). On the specimens, immunohistochemistry and FISH methods were performed. Germinal center B-cell like (GCB) and non-GCB subtypes were classified by the Hans algorithm. Outcomes included overall survival (OS), event-free survival (EFS), and EFS at 2 years (EFS24). For survival analysis, we used Kaplan-Meier curves with the log-rank test and multivariate Cox regression.
Results
A total of 247 DLBCL cases were included. Cases were positive for MYC, BCL2, BCL6, and MUM1 expression in 52.1%, 66.2%, 72.6%, and 77.8%, respectively. BCL6 translocation, BCL2 gene copy number (GCN) gain, IGH::MYC translocation, MYC GCN gain, IGH::BCL2 translocation, and BCL6 GCN gain were detected in 21.4%, 14.1%, 7.3%, 1.8%, 7.3%, and 0.9%, respectively. At a median follow-up of 52 months, 140 patients (56.7%) had disease progression or relapse. The Kaplan-Meier estimate for EFS24 was 56.2% (CI: 50.4–62.8%). In univariate analysis, only IPI and BCL6 expression were significant predictors of both OS and EFS, whereas MUM1 predicted EFS only. In multivariate analysis, the IPI score was a significant independent negative, whereas MIB-1 and BCL6 protein expressions were significant independent positive predictors of both OS and EFS.
Conclusion
In our study, we found that only IPI, BCL6 protein expression and MIB-1 protein expression are independent predictors of survival outcomes in DLBCL. We did not find any difference in survival by GCB vs. non-GCB subtypes. These findings may improve prognostication in DLBCL and can contribute to designing further research in the area.
... Roghayeh et al. discovered that the downregulation of c-MYC expression in breast cancer cell culture by inducing higher differentiation of cancer cells (Pourbagher et al., 2020). More than 70% of human cancers have abnormal MYC expression, which is associated with poor prognosis and aggressiveness (Pelengaris et al., 2002;Vita and Henriksson, 2006). Therefore, owing to the characteristics of MYC overexpression in malignancies and its widespread role in transcription regulation, it is regarded as an ideal therapeutic target. ...
Cancer is a major public health issue globally and is one of the leading causes of death. Although available treatments improve the survival rate of some cases, many advanced tumors are insensitive to these treatments. Cancer cell differentiation reverts the malignant phenotype to its original state and may even induce differentiation into cell types found in other tissues. Leveraging differentiation-inducing therapy in high-grade tumor masses offers a less aggressive strategy to curb tumor progression and heightens chemotherapy sensitivity. Differentiation-inducing therapy has been demonstrated to be effective in a variety of tumor cells. For example, differentiation therapy has become the first choice for acute promyelocytic leukemia, with the cure rate of more than 90%. Although an appealing concept, the mechanism and clinical drugs used in differentiation therapy are still in their nascent stage, warranting further investigation. In this review, we examine the current differentiation-inducing therapeutic approach and discuss the clinical applications as well as the underlying biological basis of differentiation-inducing agents.
... RNA-seq analysis showed that the KLF4 gene was upregulated in OKC compared to DF, and immunohistochemistry showed strong nuclear expression of the KLF4 protein, mainly in intermediate layers, and focally weak expression in the basal layer of OKC epithelia [37]. c-Myc is a proto-oncoprotein that acts as a transcription factor involved in cellular proliferation, apoptosis, and inhibition of differentiation [66]. Immunohistochemical expression of c-Myc was observed in the epithelial cells of most AMBL, AOT, and OKC cases, and in half or less than half of cases of RC and DC, respectively [45]. ...
Background:
Stem cells have been associated with self-renewing and plasticity and have been investigated in various odontogenic lesions in association with their pathogenesis and biological behavior. We aim to provide a systematic review of stem cell markers' expression in odontogenic tumors and cysts.
Methods:
The literature was searched through the MEDLINE/PubMed, EMBASE via OVID, Web of Science, and CINHAL via EBSCO databases for original studies evaluating stem cell markers' expression in different odontogenic tumors/cysts, or an odontogenic disease group and a control group. The studies' risk of bias (RoB) was assessed via a Joanna Briggs Institute Critical Appraisal Tool. Meta-analysis was conducted for markers evaluated in the same pair of odontogenic tumors/cysts in at least two studies.
Results:
29 studies reported the expression of stem cell markers, e.g., SOX2, OCT4, NANOG, CD44, ALDH1, BMI1, and CD105, in various odontogenic lesions, through immunohistochemistry/immunofluorescence, polymerase chain reaction, flow cytometry, microarrays, and RNA-sequencing. Low, moderate, and high RoBs were observed in seven, nine, and thirteen studies, respectively. Meta-analysis revealed a remarkable discriminative ability of SOX2 for ameloblastic carcinomas or odontogenic keratocysts over ameloblastomas.
Conclusion:
Stem cells might be linked to the pathogenesis and clinical behavior of odontogenic pathologies and represent a potential target for future individualized therapies.
... Estas se encargan de bloquear selectivamente la unión de las histonas acetiladas y de BRD2 (proteína bromodominio 2), BRD3 y BRD4, reduciendo la sobreexpresión del gen c-Myc en cánceres (Gómez et al., 2022). La regulación de este gen se ve afectado en más del 70% de los cánceres humanos (Madden et al., 2021), como en los cánceres de mama, de colon, carcinoma de cuello uterino, osteosarcomas, melanomas, glioblastomas y leucemias mieloides (Pelengaris et al., 2002). ...
La epigenética estudia los cambios en la expresión de los genes sin modificaciones en las secuencias del ADN. Por lo tanto, relaciona las influencias ambientales y genéticas que determinan un fenotipo determinado, el cual está relacionado con la aparición de enfermedades. El cáncer es una enfermedad asociada a un descontrol en la división celular y determinada por factores genéticos y ambientales. La terapia epigenética puede modificar las alteraciones que sufre el ADN y reducir o interrumpir el desarrollo del cáncer. En esta revisión se discutirán los conceptos de epigenética y cáncer, así como su relación y las terapias epigenéticas que actualmente están en estudio como tratamiento terapéutico, siendo una de las mejores alternativas terapéuticas en un futuro cercano.
... According to a previous review [47], c-Myc dysregulation can either promote proliferation or induce apoptosis depending on the in vivo cellular context. There are usually two main conditions under which c-Myc would induce apoptosis. ...
Neural stem cells (NSCs) play crucial roles in neurological disorders and tissue injury repair through exerting paracrine effects. However, the effects of NSC-derived factors on glioma progression remain unclear. This study aimed to evaluate the effects of human NSC-conditioned medium (NSC-CM) on the behaviour of glioma cells using an in vitro co-culture system. Cell counting kit-8 and 5-ethynyl-2'-deoxyuridine assays revealed that NSC-CM inhibited glioma cell proliferation and growth in a foetal bovine serum (FBS)-independent manner. In addition, our wound healing assay demonstrated that NSC-CM repressed glioma cell migration, while results from transwell and 3D spheroid invasion assays indicated that NSC-CM also reduced the invasion capacity of glioma cells. Flow cytometry showed that NSC-CM prevented cell cycle progression from the G1 to S phase and promoted apoptosis. Western blotting was used to show that the expression of Wnt/β-catenin pathway-related proteins, including β-catenin, c-Myc, cyclin D1, CD44, and Met, was remarkably decreased in NSC-CM-treated glioma cells. Furthermore, the addition of a Wnt/β-catenin pathway activator, CHIR99021, significantly induced the expression of β-catenin and Met and increased the proliferative and invasive capabilities of control medium-treated glioma cells but not those of NSC-CM-treated glioma cells. The use of enzyme-linked immunosorbent assays (ELISA) revealed the secretion of some anti-tumour factors in human and rat NSCs, including interferon-α and dickkopf-1. Our data suggest that NSC-CM partially inhibits glioma cell progression by downregulating Wnt/β-catenin signalling. This study may serve as a basis for developing future anti-glioma therapies based on NSC derivatives.
... The oncogenic transcription factor MYC coordinately regulates the cell cycle, proliferation and metabolism in many cancer types, including in cervical cancer as exemplified by HeLa cells 47,48 . Notably, the MYC IRES also forms a thermodynamically stable structure comprising two hairpins and three internal loops (Fig. 5a) [19][20][21][22] and, fortuitously, one internal loop, 5′UUCG/3′ACCC, is targetable with a molecule from our database, named MYC-binder (Fig. 5b). ...
Target occupancy is often insufficient to elicit biological activity, particularly for RNA, compounded by the longstanding challenges surrounding the molecular recognition of RNA structures by small molecules. Here we studied molecular recognition patterns between a natural-product-inspired small-molecule collection and three-dimensionally folded RNA structures. Mapping these interaction landscapes across the human transcriptome defined structure–activity relationships. Although RNA-binding compounds that bind to functional sites were expected to elicit a biological response, most identified interactions were predicted to be biologically inert as they bind elsewhere. We reasoned that, for such cases, an alternative strategy to modulate RNA biology is to cleave the target through a ribonuclease-targeting chimera, where an RNA-binding molecule is appended to a heterocycle that binds to and locally activates RNase L¹. Overlay of the substrate specificity for RNase L with the binding landscape of small molecules revealed many favourable candidate binders that might be bioactive when converted into degraders. We provide a proof of concept, designing selective degraders for the precursor to the disease-associated microRNA-155 (pre-miR-155), JUN mRNA and MYC mRNA. Thus, small-molecule RNA-targeted degradation can be leveraged to convert strong, yet inactive, binding interactions into potent and specific modulators of RNA function.
... ; https://doi.org/10.1101/2023.05. 10.539944 doi: bioRxiv preprint deregulated MYC levels drive many oncogenic processes, including metabolic reprogramming and non-stop cell cycle progression, but MYC also sensitizes cells to diverse inducers of extrinsic or intrinsic programmed cell death pathways [19][20][21][22] . These early findings have laid the conceptual foundation for MYC-dependent synthetic lethal (MYC SL) therapeutic strategies, which seek to specifically harness MYC generated vulnerability pathways as opposed to drugging the MYC protein directly [23][24][25] . ...
Combining cytotoxic chemotherapy or novel anticancer drugs with T-cell modulators holds great promise in treating advanced cancers. However, the response varies depending on the tumor immune microenvironment (TIME). Therefore, there is a clear need for pharmacologically tractable models of the TIME to dissect its influence on mono- and combination treatment response at the individual level. Here we establish a Patient-Derived Explant Culture (PDEC) model of breast cancer, which retains the immune contexture of the primary tumor, recapitulating cytokine profiles and CD8+ T cell cytotoxic activity. We explored the immunomodulatory action of a synthetic lethal BCL2 inhibitor venetoclax + metformin drug combination ex vivo, discovering metformin cannot overcome the lymphocyte-depleting action of venetoclax. Instead, metformin promotes dendritic cell maturation through inhibition of mitochondrial complex I, increasing their capacity to co-stimulate CD4+ T cells and thus facilitating anti-tumor immunity. Our results establish PDECs as a feasible model to identify immunomodulatory functions of anticancer drugs in the context of patient-specific TIME.
... GPBAR1dependent PKC activation further stimulates the NF-κB pathway and the autocrine activation of ERK1/2, causing the release of pro-inflammatory cytokines such as interleukin-1β (IL-1β), IL-6, and TNF-α. SRC-dependent activation of ERK1/2 in the M1-like phenotype further promotes the c-MYC upregulation and results in cell death [57]. ...
Bile Acid-Activated Receptors (BARs) such as a G-protein-coupled receptor (GPBAR1) and the farnesol X receptor (FXR) are activated by bile acids (BAs) and have been implicated in the regulation of microbiota-host immunity in the intestine. The mechanistic roles of these receptors in immune signaling suggest they may also influence the development of metabolic disorders. In this perspective, we provide a summary of recent literature describing the main regulatory pathways and mechanisms of BARs and how they affect both innate and adaptive immune system, cell proliferation, and signaling in the context of inflammatory diseases. We also discuss new approaches for therapy and summarizes clinical projects on BAs for the treatment of diseases. In parallel, some drugs that are classically used for other therapeutic purposes and BAR activity have recently been proposed as regulators of immune cells phenotype. Another strategy consists of using specific strains of gut bacteria to regulate BA production in the intestine. This article is protected by copyright. All rights reserved.
... The MYC family member c-Myc [205] is a transcription factor crucial to many cell functions, including cell growth and metabolism, proliferation, and apoptosis. The activity of c-Myc is tightly related to other pathways, including the Ras/Phosphoinositide 3-kinase (PI3K)/AKT/GSK3, Ras/Raf/ERK, and Wnt pathways [206][207][208][209][210]. Dysregulation of Myc was described in the tumorigenesis or progression of different cancers [211][212][213][214][215][216]. ...
Illnesses following the degeneration of the nervous system can occur due to aging or genetic mutations and represent a clinical concern. In neurodegenerative diseases, loss of neuronal structure and functions mainly causes cognitive impairment, representing an increasing social burden. In neurodegenerative diseases, the progressive loss of vulnerable populations of neurons in specific regions of the central nervous system was traced to different pathological events, such as misfolded proteins’ accumulation, abnormalities in proteasomes or phagosomes, as well as anomalies in lysosomes or mitochondria. Many research efforts identified important events involved in neurodegeneration, but the complex pathogenesis of neurodegenerative diseases is far from being fully elucidated. More recently, insights into the signal transduction pathways acting in the nervous system contributed to unveiling some molecular mechanisms triggering neurodegeneration. Abnormalities in the intra- or inter-cellular signaling were described to be involved in the pathogenesis of neurodegenerative disease. Understanding the signal transduction pathways that impact the nervous system homeostasis can offer a wide panel of potential targets for modulating therapeutic approaches. The present review will discuss the main signal transduction pathways involved in neurodegenerative disorders.
... Moreover, MYC regulates different targets depending on cell type. Still, the broad spectrum of MYC-controlled processes means that direct targeting of MYC can cause undesirable side effects and toxicity to normal cells that depend on MYC function 14 . Moreover, MYC is an intrinsically disordered protein that lacks defined targetable structures for small molecule inhibitors 10 . ...
Group 3 medulloblastoma (G3 MB) carries the worst prognosis of all MB subgroups. MYC oncoprotein is elevated in G3 MB tumors; however, the mechanisms that support MYC abundance remain unclear. Using metabolic and mechanistic profiling, we pinpoint a role for mitochondrial metabolism in regulating MYC. Complex-I inhibition decreases MYC abundance in G3 MB, attenuates the expression of MYC-downstream targets, induces differentiation, and prolongs male animal survival. Mechanistically, complex-I inhibition increases inactivating acetylation of antioxidant enzyme SOD2 at K68 and K122, triggering the accumulation of mitochondrial reactive oxygen species that promotes MYC oxidation and degradation in a mitochondrial pyruvate carrier (MPC)-dependent manner. MPC inhibition blocks the acetylation of SOD2 and oxidation of MYC, restoring MYC abundance and self-renewal capacity in G3 MB cells following complex-I inhibition. Identification of this MPC-SOD2 signaling axis reveals a role for metabolism in regulating MYC protein abundance that has clinical implications for treating G3 MB.
... c-Myc is an important transcription factor that can be tightly regulated in normal cells but is overexpressed in cancer cells and plays multiple roles [19]. Specifically, c-Myc, a proto-oncogene, is involved in cell growth, proliferation, the cell cycle, metabolism, and apoptosis in cancer cells [20]. It has now been found that c-Myc is amplified in various types of cancer, such as colonic carcinoma [21], breast carcinoma [22,23], and lung cancer [24]. ...
We hypothesized that Euonymus sachalinensis (ES) induces apoptosis by inhibiting the expression of c-Myc in colon cancer cells, and this study proved that the methanol extract of ES has anticancer effects in colon cancer cells. ES belongs to the Celastraceae family and is well known for its medicinal properties. Extracts of species belonging to this family have been used to treat diverse diseases, including rheumatoid arthritis, chronic nephritis, allergic conjunctivitis, rhinitis, and asthma. However, ES has been targeted because there are currently few studies on the efficacy of ES for various diseases, including cancer. ES lowers cell viability in colon cancer cells and reduces the expression of c-Myc protein. We confirm that the protein level of apoptotic factors such as PARP and Caspase 3 decrease when ES is treated with Western blot, and confirm that DNA fragments occur through TUNEL assay. In addition, it is confirmed that the protein level of oncogenes CNOT2 and MID1IP1 decrease when ES is treated. We have also found that ES enhances the chemo-sensitivity of 5-FU in 5-FU-resistant cells. Therefore, we confirm that ES has anticancer effects by inducing apoptotic cell death and regulating the oncogenes CNOT2 and MID1IP1, suggesting its potential for use in the treatment of colon cancer.
... Curiously, IPA predicted PR, NRF1, and MYC as potential upstream regulators involved in BCO pathogenesis. Although the opposite regulation was detected by qPCR, the role of MYC in the apoptotic pathways is confounding and not fully understood [128][129][130] . MYC is a proto-oncogene, which encodes for a nuclear phosphoprotein that plays a key role in cellular transformation and apoptosis 129,131,132 . ...
Bacterial Chondronecrosis with Osteomyelitis (BCO) is a specific cause of lameness in commercial fast-growing broiler (meat-type) chickens and represents significant economic, health, and wellbeing burdens. However, the molecular mechanisms underlying the pathogenesis remain poorly understood. This study represents the first comprehensive characterization of the proximal tibia proteome from healthy and BCO chickens. Among a total of 547 proteins identified, 222 were differentially expressed (DE) with 158 up- and 64 down-regulated proteins in tibia of BCO vs. normal chickens. Biological function analysis using Ingenuity Pathways showed that the DE proteins were associated with a variety of diseases including cell death, organismal injury, skeletal and muscular disorder, immunological and inflammatory diseases. Canonical pathway and protein–protein interaction network analysis indicated that these DE proteins were involved in stress response, unfolded protein response, ribosomal protein dysfunction, and actin cytoskeleton signaling. Further, we identified proteins involved in bone resorption (osteoclast-stimulating factor 1, OSFT1) and bone structural integrity (collagen alpha-2 (I) chain, COL2A1), as potential key proteins involved in bone attrition. These results provide new insights by identifying key protein candidates involved in BCO and will have significant impact in understanding BCO pathogenesis.
... [1,2] Transcriptional c-Myc protein acts as a master regulator of various genes which can regulate the cell proliferation, differentiation, metabolism, and apoptosis. [3][4][5] As c-Myc protein is over-expressed in ubiquitous human cancers, such as gastric cancer, lung cancer, colon cancer, breast cancer, hepatoma, neoplasms, and myeloid leukemia, it is a very attractive anticancer target. [6,7] Therefore, monitoring of c-Myc protein as the biomarker plays a critical role in prevention or diagnosis of cancers. ...
Monitoring of trace c‐Myc protein as the biomarker of ubiquitous cancers is critical in achieving predictive medical diagnostics. However, qualitative and quantitative detection of c‐Myc protein with superior single selectivity and sensitivity is still challenging. Herein, a bioinspired photonic sensing microchip for single recognition of c‐Myc protein is outlined with two synergistic aspects involving chemical and physical design criteria. Chemical design uses specific molecularly imprinted polymer (MIP) with exquisite complementarity in its chemical functions and spatial geometries to targeted c‐Myc protein, leading to excellent sensitivity and selectivity for single identification. Physical design involves optical geometrical double‐reflection polarization rotation and multilayer interference of the fabricated periodic photonic architecture inspired by Papilio palinurus butterfly wings to enhance the spectral diversity of reflectance. Therefore, a one‐of‐a‐kind sensing platform integrates the advantages of MIP and bioinspired photonic structure is demonstrated to actualize distinctive signal conversion and amplification for qualitative and quantitative detection of trace c‐Myc protein, accompanied with superior sensitivity (detection limit is 0.014 µg mL⁻¹), selectivity, stability, anti‐interference ability as well as rapid response/recovery time. This sensor microchip uniquely ventures into the territory of functionally combining bioinspired photonic structure with MIP absorbers, proven promising for prevention or diagnosis of cancers in medical field.
... Phosphorylation of EphA2 at S897 mediates the activation of the AKT, STAT3, SOX-2, and c-MYC signaling pathways, which is crucial to nasopharyngeal carcinoma stem cell formation [33] . EphA2 has been reported to play an important role in the proliferation, migration, and invasion [34][35][36][37] of BLBC. However, it remains unclear whether EphA2 is associated with BLBC cell stemness and chemo-therapy resistance. ...
Ephrin type-A receptor 2 (EphA2) is a member of the tyrosine receptor kinases, a family of membrane proteins recognized as potential anticancer targets. EphA2 highly expressed in a variety of human cancers, playing roles in proliferation, migration, and invasion. However, whether and how EphA2 regulates basal-like breast cancer (BLBC) cell stemness and chemoresistance has not been revealed. Here, KLF5 was proven to be a direct transcription factor for EphA2 in BLBC cells, and its expression was positively correlated in clinical samples from breast cancer patients. The inflammatory factor TNF-α could promote BLBC cell stemness partially by activating the KLF5-EphA2 axis. Moreover, phosphorylation of EphA2 at S897 (EphA2 pS897) induced by TNF-α and PTX/DDP contributes to chemoresistance of BLBC. Furthermore, the EphA2 inhibitor ALW-II-41-27 could effectively reduce EphA2 pS897 and tumor cell stemness in vitro and significantly enhance the sensitivity of xenografts to the chemotherapeutic drugs PTX and DDP in vivo. Clinically, tumor samples from breast patients with less response to neoadjuvant chemotherapy showed a high level of EphA2 pS897 expression. In conclusion, KLF5-EphA2 promotes stemness and drug resistance in BLBC and could be a potential target for the treatment of BLBC.
... The related proteins are c-myc (MYC), n-myc, and l-myc. They all play critical roles in cell differentiation, proliferation, and survival [60][61][62][63][64]. It appears that suppression of miR-196α significantly increases HOXB7 expression and tamoxifen resistance. ...
Myeloid cells are abundant and plastic immune cell subsets in the liver, to which pro-tumorigenic, inflammatory and immunosuppressive roles have been assigned in the course of tumorigenesis. Yet several aspects underlying their dynamic alterations in hepatocellular carcinoma (HCC) progression remain elusive, including the impact of distinct genetic mutations in shaping a cancer-permissive tumor microenvironment (TME). Here, in newly generated, clinically-relevant somatic female HCC mouse models, we identify cancer genetics’ specific and stage-dependent alterations of the liver TME associated with distinct histopathological and malignant HCC features. Mitogen-activated protein kinase (MAPK)-activated, NrasG12D-driven tumors exhibit a mixed phenotype of prominent inflammation and immunosuppression in a T cell-excluded TME. Mechanistically, we report a NrasG12D cancer cell-driven, MEK-ERK1/2-SP1-dependent GM-CSF secretion enabling the accumulation of immunosuppressive and proinflammatory monocyte-derived Ly6Clow cells. GM-CSF blockade curbs the accumulation of these cells, reduces inflammation, induces cancer cell death and prolongs animal survival. Furthermore, GM-CSF neutralization synergizes with a vascular endothelial growth factor (VEGF) inhibitor to restrain HCC outgrowth. These findings underscore the profound alterations of the myeloid TME consequential to MAPK pathway activation intensity and the potential of GM-CSF inhibition as a myeloid-centric therapy tailored to subsets of HCC patients.
Doxorubicin (DOX) is a chemotherapy agent commonly used to treat triple-negative breast cancer (TNBC), but it has insufficient efficacy against the disease and considerable toxicity due to its off-target delivery. To improve the specificity of DOX for TNBC, we encapsulated it in poly(lactic-co-glycolic acid) (PLGA) nanoparticles (NPs) coated with antibodies against Frizzled7 (FZD7), a receptor that is overexpressed on TNBC cells and which is a key activator of the Wnt signaling pathway. In vitro studies show that DOX encapsulation does not hinder its ability to localize to the nucleus in human TNBC cell cultures and that DOX delivered via NPs induces apoptosis and DNA damage via H2A.X phosphorylation to the same degree as freely delivered DOX. FZD7-targeted NPs delivering DOX caused significantly greater inhibition of metabolic activity and led to a smaller cell population following treatment when compared to freely delivered DOX or DOX-loaded NPs coated only with poly(ethylene glycol) (PEG). The FZD7 antibodies additionally provided significant levels of Wnt pathway inhibition, as demonstrated by an increase in β-catenin phosphorylation, indicative of β-catenin destruction and downregulation. These results show that FZD7-targeted platforms have great promise for improving the therapeutic window of otherwise toxic chemotherapies like DOX in TNBC and other cancers that display the overexpression of FZD7 receptors.
The universally active transcription factor c-Myc is essential for the regulation of global gene expression and has an impact on many biological functions, including cell division, proliferation, and death. Approximately 70% of human malignancies are caused by dysregulation of c-Myc, contributing to tumor initiation and maintenance. As a result, the therapeutic targeting of c-Myc has attracted considerable interest in the development of cancer drugs. Extensive in vivo studies have demonstrated that inhibition of c-Myc leads to substantial anti-proliferative effects and sustained tumor regression, while remaining reversible in healthy tissues. Despite its pivotal role in cancer progression, the lack of druggable binding pockets and complex protein–protein interaction (PPI) interfaces has traditionally deemed c-Myc as an “undruggable” target. Nevertheless, alternative strategies, such as disrupting the Myc/Max complex, inhibiting Myc transcription and/or translation, destabilizing Myc protein, and exploring synthetic lethality associated with Myc overexpression, have been explored to achieve desirable anti-tumor effects. This review provides a comprehensive overview of recent advancements in targeting oncogenic c-Myc, specifically focusing on its potential as a therapeutic target for cancer treatment. We discuss the underlying mechanisms of c-Myc dysregulation, its impact on cellular pathways, and the challenges associated with developing effective pharmacological inhibitors. Furthermore, we summarize emerging strategies and technologies that have shown promise in tackling the complex network of c-Myc interactions, aiming to develop conceptually innovative and efficient anticancer therapies that can be applied to a wide range of tumors.
Triple negative breast cancer (TNBC) subtype is characterized with higher EMT/stemness properties and immune suppressive tumor microenvironment (TME). Women with advanced TNBC exhibit aggressive disease and have limited treatment options. Although immune suppressive TME is implicated in driving aggressive properties of basal/TNBC subtype and therapy resistance, effectively targeting it remains a challenge. Minnelide, a prodrug of triptolide currently being tested in clinical trials, has shown anti-tumorigenic activity in multiple malignancies via targeting super enhancers, Myc and anti-apoptotic pathways such as HSP70. Distinct super-enhancer landscape drives cancer stem cells (CSC) in TNBC subtype while inducing immune suppressive TME. We show that Minnelide selectively targets CSCs in human and murine TNBC cell lines compared to cell lines of luminal subtype by targeting Myc and HSP70. Minnelide in combination with cyclophosphamide significantly reduces the tumor growth and eliminates metastasis by reprogramming the tumor microenvironment and enhancing cytotoxic T cell infiltration in 4T1 tumor-bearing mice. Resection of residual tumors following the combination treatment leads to complete eradication of disseminated tumor cells as all mice are free of local and distant recurrences. All control mice showed recurrences within 3 weeks of post-resection while single Minnelide treatment delayed recurrence and one mouse was free of tumor. We provide evidence that Minnelide targets tumor intrinsic pathways and reprograms the immune suppressive microenvironment. Our studies also suggest that Minnelide in combination with cyclophosphamide may lead to durable responses in patients with basal/TNBC subtype warranting its clinical investigation.
There has been an extensive amount of work examining and developing cancer vaccines. However, the term itself can at times be confusing, for unlike classic vaccines which target pathogens using the immune system, many cancer vaccines target the cancer cells themselves, the effect of the pathogen, if you will, rather than the cause. We examine some of the current approaches.
Opioid use disorder (OUD) is a public health crisis currently being exacerbated by increased rates of use and overdose of synthetic opioids, primarily fentanyl. Therefore, the identification of novel biomarkers and treatment strategies to reduce problematic fentanyl use and relapse to fentanyl taking is critical. In recent years, there has been a growing body of work demonstrating that the gut microbiome can serve as a potent modulator of the behavioral and transcriptional responses to both stimulants and opioids. Here, we advance this work to define how manipulations of the microbiome drive fentanyl intake and fentanyl-seeking in a translationally relevant drug self-administration model. Depletion of the microbiome of male rats with broad spectrum antibiotics leads to increased drug administration on increased fixed ratio, progressive ratio, and drug seeking after abstinence. Utilizing 16S sequencing of microbiome contents from these animals, specific populations of bacteria from the gut microbiome correlate closely with levels of drug taking. Additionally, global proteomic analysis of the nucleus accumbens following microbiome manipulation and fentanyl administration to define how microbiome status alters the functional proteomic landscape in this key limbic substructure. These data demonstrate that an altered microbiome leads to marked changes in the synaptic proteome in response to repeated fentanyl treatment. Finally, behavioral effects of microbiome depletion are reversible by upplementation of the microbiome derived short-chain fatty acid metabolites. Taken together, these findings establish clear relevance for gut-brain signaling in models of OUD and lay foundations for further translational work in this space.
The protein c-Myc is a transcription factor that remains largely intrinsically disordered and is known to be involved in various biological processes and is overexpressed in various cancers, making it an attractive drug target. However, intrinsically disordered proteins such as c-Myc do not show funnel-like basins in their free-energy landscapes; this makes their druggability a challenge. For the first time, we propose a heterodimer model of c-Myc/Max in full length in this work. We used Gaussian-accelerated molecular dynamics (GaMD) simulations to explore the behavior of c-Myc and its various regions, including the transactivation domain (TAD) and the basic helix–loop–helix-leucine-zipper (bHLH-Zipper) motif in three different conformational states: (a) monomeric c-Myc, (b) c-Myc when bound to its partner protein, Max, and (c) when Max was removed after binding. We analyzed the GaMD trajectories using root-mean-square deviation (RMSD), radius of gyration, root-mean-square fluctuation, and free-energy landscape (FEL) calculations to elaborate the behaviors of these regions. The results showed that the monomeric c-Myc structure showed a higher RMSD fluctuation as compared with the c-Myc/Max heterodimer in the bHLH-Zipper motif. This indicated that the bHLH-Zipper motif of c-Myc is more stable when it is bound to Max. The TAD region in both monomeric and Max-bound states showed similar plasticity in terms of RMSD. We also conducted residue decomposition calculations and showed that the c-Myc and Max interaction could be driven mainly by electrostatic interactions and the residues Arg299, Ile403, and Leu420 seemed to play important roles in the interaction. Our work provides insights into the behavior of c-Myc and its regions that could support the development of drugs that target c-Myc and other intrinsically disordered proteins.
Diffuse large B-cell lymphoma (DLBCL) stands as a formidable challenge in the landscape of non-Hodgkin’s lymphomas. This review illuminates the remarkable strides made in comprehending DLBCL’s molecular intricacies and devising targeted treatments. DLBCL, the most prevalent non-Hodgkin’s lymphoma, has seen transformative progress in its characterization. Genetic investigations, led by high-throughput sequencing, have unveiled recurrent mutations in genes such as MYC, BCL2, and BCL6, casting light on the underlying genetic chaos propelling DLBCL’s aggressiveness. A pivotal facet of this understanding centers on cell signaling pathways. Dysregulation of B-cell receptor (BCR) signaling, NF-κB, PI3K/Akt/mTOR, JAK/STAT, Wnt/β-Catenin, and Toll-like receptor pathways plays a critical role in DLBCL pathogenesis, offering potential therapeutic targets. DLBCL’s complex tumor microenvironment (TME) cannot be overlooked. The dynamic interplay among tumor cells, immune cells, stromal components, and the extracellular matrix profoundly influences DLBCL’s course and response to therapies. Epigenetic modifications, including DNA methylation and histone changes, add another layer of intricacy. Aberrant epigenetic regulation plays a significant role in lymphomagenesis, offering prospects for epigenetic-based therapies. Promisingly, these molecular insights have spurred the development of personalized treatments. Targeted therapies and immunotherapies, guided by genomic profiling and molecular classification, are emerging as game-changers in DLBCL management. In conclusion, this review underscores the remarkable strides in understanding DLBCL’s molecular underpinnings, spanning genetics, cell signaling, the tumor microenvironment, and epigenetics. These advances pave the way for more effective, personalized treatments, renewing hope for DLBCL patients.
Cancer is a complex and multifaceted disease with a high global incidence and mortality rate. Although cancer therapy has evolved significantly over the years, numerous challenges persist on the path to effectively combating this multifaceted disease. Natural compounds derived from plants, fungi, or marine organisms have garnered considerable attention as potential therapeutic agents in the field of cancer research. Ellagic acid (EA), a natural polyphenolic compound found in various fruits and nuts, has emerged as a potential cancer prevention and treatment agent. This review summarizes the experimental evidence supporting the role of EA in targeting key hallmarks of cancer, including proliferation, angiogenesis, apoptosis evasion, immune evasion, inflammation, genomic instability, and more. We discuss the molecular mechanisms by which EA modulates signaling pathways and molecular targets involved in these cancer hallmarks, based on in vitro and in vivo studies. The multifaceted actions of EA make it a promising candidate for cancer prevention and therapy. Understanding its impact on cancer biology can pave the way for developing novel strategies to combat this complex disease.
Introduction:
Guanine-rich DNA sequences can fold into four-stranded noncanonical secondary structures called G-quadruplexes (G4s) which are widely distributed in functional regions of the human genome, such as telomeres and gene promoter regions. Compelling evidence suggests their involvement in key genome functions such as gene expression and genome stability. Notably, the abundance of G4-forming sequences near transcription start sites suggests their potential involvement in regulating oncogenes.
Areas covered:
This review provides an overview of current knowledge on G4s in human oncogene promoters. The most representative G4-binding ligands have also been documented. The objective of this work is to present a comprehensive overview of the most promising targets for the development of novel and highly specific anticancer drugs capable of selectively impacting the expression of individual or a limited number of genes.
Expert opinion:
Modulation of G4 formation by specific ligands has been proposed as a powerful new tool to treat cancer through the control of oncogene expression. Actually, most of G4-binding small molecules seem to simultaneously target a range of gene promoter G4s, potentially influencing several critical driver genes in cancer, thus producing significant therapeutic benefits.
The MYCC and MYCN loci are each associated with two upstream open reading frames (uORFs) potentially encoding small proteins (9-21 kDa). We previously demonstrated that uORFs mrtl and MYCHEX1 of MYCC are translated, and their protein products may function to regulate the expression of the "parent" oncogene. We hypothesized that a similar relationship might exist between MYCN and its two uORFs: MYCNOT and MNOP, and investigated the uORF-encoded proteins associated with MYCN to confirm their expression and intracellular location in neuroblastoma and medulloblastoma cells and tissues. MNOP, MYCNOT, mrtl, and MYCHEX1 were readily detected via reverse transcription polymerase chain reaction and Western blot analysis in tumor cell lines. In tumor tissue, MNOP protein expression was confirmed; however, MCYNOT generated from alternative splicing MYCNΔ1b mRNA was not detected. Immunofluorescence staining of MYCNOT displayed multiple bright foci in the nucleus and diffuse staining in the cytoplasm, suggesting that this small protein may function in both the nucleus and cytoplasm. Upon JQ1 treatment, MYCN, MYCNOT, and mrtl decreased substantially or disappeared completely in three different tumor cell lines. Significant levels of apoptosis were observed in each pediatric embryonal tumor cell line but not T47D breast carcinoma cells, suggesting that response to JQ1 transcriptional inhibition is greatest in tumor cells, which depend on MYC to maintain an undifferentiated phenotype. In conclusion, both MYCN uORF-encoded proteins MNOP and MYCNOT, together with the two MYCC uORF-encoded proteins mrtl and MYCHEX1 were detected simultaneously in tumor cell lines and tumor tissues. These four distinct proteins are translated from the "5'-untranslated region" of MYCN or MYCC mRNA and display consistent distribution patterns within the cell. Additional studies to further elucidate the physiological and pathological roles of these uORF-encoded proteins are warranted, as insights gained could inform new strategies for modulating MYC-family oncogenes by targeting their uORFs.
Overview
The initiation and progression of human neoplastic diseases is a multistep process involving the accumulation of genetic changes in somatic cells. These genetic changes typically consist of the activation of cooperating oncogenes and the inactivation of tumor suppressor genes, which both appear necessary for a complete neoplastic phenotype. Oncogenes are altered versions of normal cellular genes called proto‐oncogenes, usually involved in the regulation of cell growth and activated by mutation, chromosomal rearrangement, or gene amplification. In this chapter, at first we will describe the methods that have been applied by the researchers for the discovery and the identification of oncogenes. Then we will present the genetic mechanisms of proto‐oncogenes activation (point mutations, gene amplifications, chromosomal rearrangements) with several examples, and we will describe the role played by oncogenes in the initiation and progression of various cancers.
The identification of oncogene abnormalities has provided tools for the molecular diagnosis and monitoring of cancer. Most important, oncogenes represent potential targets for new types of cancer therapies, which are continuously discovered and tested in clinical trials. The goal of these new drugs is to kill cancer cells selectively while sparing normal cells. These new therapies display an evident benefit for the treatment of several neoplastic diseases that were, before targeted therapies development, very hard to be treated and cured. However, they are not able to kill 100% of neoplastic cells, essentially due to the occurrence of mechanisms of secondary resistance. In the last part of the chapter, we will review all the genes for which a targeted therapy has been developed.
Both oxidative stress and the immune response are associated with heart failure (HF). In this study, our aim was to identify the hub genes associated with oxidative stress andimmune infiltration of HF by bioinformatics analysis and experimental verification. The expression profile of GSE36074 was obtained from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) were screened by GEO2R. The genes related to oxidative stress were extracted from GeneCards websites. Then, the functional enrichment analysis of oxidative stress-related DEGs (OSRDEGs) was performed using DAVID. In addition, we constructed a protein-protein interaction (PPI) network using the STRING database and screened for hub genes with Cytoscape software. We also used CIBERSORTx to analyze immune infiltration in mice heart tissues between the TAC and Sham groups and explored the correlation between immune cells and hub genes. Finally, the hub genes were carried out using reverse transcription quantitative PCR (RT-qPCR), immunohistochemistry (IHC) and western blot. A total of 136 OSRDEGs were found in GSE36074 . Enrichment analysis revealed that these OSRDEGs were enriched in the mitochondrion, HIF-1, FoxO, MAPK and TNF signaling pathway. The five hub genes (Mapk14, Hif1a, Myc, Hsp90ab1, and Hsp90aa1) were screened by the cytoHubba plugin. The correlation analysis between immune cells and hub genes showed that Mapk14 was positively correlated with Th2 Cells, while Hif1a and Hsp90ab1exhibited a negative correlation with Th2 Cells; Myc exhibited a negative correlation with Monocytes; whereas, Hsp90aa1 was negatively correlated with NK Resting. Finally, five hub genes were validated by RT-qPCR, IHC and western blot. Mapk14, Hif1a, Myc, Hsp90ab1, and Hsp90aa1 are hub genes of HF and may play a critical role in the oxidative stress of HF. This study may provide new targets for the treatment of HF, and the potential immunotherapies are worthy of further study.
Competition among adult brain cells has not been extensively researched. To investigate whether healthy glia can outcompete diseased human glia in the adult forebrain, we engrafted wild-type (WT) human glial progenitor cells (hGPCs) produced from human embryonic stem cells into the striata of adult mice that had been neonatally chimerized with mutant Huntingtin (mHTT)-expressing hGPCs. The WT hGPCs outcompeted and ultimately eliminated their human Huntington’s disease (HD) counterparts, repopulating the host striata with healthy glia. Single-cell RNA sequencing revealed that WT hGPCs acquired a YAP1/MYC/E2F-defined dominant competitor phenotype upon interaction with the host HD glia. WT hGPCs also outcompeted older resident isogenic WT cells that had been transplanted neonatally, suggesting that competitive success depended primarily on the relative ages of competing populations, rather than on the presence of mHTT. These data indicate that aged and diseased human glia may be broadly replaced in adult brain by younger healthy hGPCs, suggesting a therapeutic strategy for the replacement of aged and diseased human glia.
c-Myc is a transcription factor that is constitutively and aberrantly expressed in over 70 percent of human cancers. Its direct inhibition has been shown to trigger rapid tumor regression in mice with only mild and fully reversible side effects, suggesting this to be a viable therapeutic strategy for Chronic Myeloid Leukemia (1). At its core, c-Myc is a transcription factor present in the cell nucleus that acts to regulate cell growth, differentiation, metabolism and death-it is tightly regulated in the cell by a range of upstream and downstream mechanisms at the genetic, mRNA and protein level, which can become disrupted in cancer cells. Elevation in the levels of c-Myc is a widespread phenomenon in a large variety of human tumors, and there is a strong feeling that the deregulated expression of c-Myc contributes substantively to the progression of many malignancies. (2) c-Myc is also thought to play an important role in the progression of CML. Levels of c-Myc are elevated in Bcr-Abl transformed cells, and dominant inhibitor derivatives of c-Myc can effectively block Bcr-Abl transformation. (3) The observation that c-Myc levels are responsive to Bcr dosage in Bcr-Abl transformed cells suggests a direct mechanism by which c-Myc protein levels may become elevated in Bcr-Abl transformed cells suggests a direct mechanism by which c-Myc protein levels may become elevated in Bcr-Abl transformed cells. (4) The contribution of this activity to the transforming potential of Bcr-Abl, particularly within the context of CML progression, can help to elucidate the molecular mechanisms at play with relevance to Bcr-Abl therapeutic targeting and combination therapy.
Autophagy, a critical catabolic process for cell survival against different types of stress, has a role in the differentiation of various cells, such as cardiomyocytes. Adenosine 5'-monophosphate (AMP)-activated protein kinase (AMPK) is an energy-sensing protein kinase involved in the regulation of autophagy. In addition to its direct role in regulating autophagy, AMPK can also influence other cellular processes by regulating mitochondrial function, posttranslational acetylation, cardiomyocyte metabolism, mitochondrial autophagy, endoplasmic reticulum stress, and apoptosis. As AMPK is involved in the control of various cellular processes, it can influence the health and survival of cardiomyocytes. This study investigated the effects of an AMPK inducer (Metformin) and an autophagy inhibitor (Hydroxychloroquine) on the differentiation of human pluripotent stem cell-derived cardiomyocytes (hPSC-CMs). The results showed that autophagy was upregulated during cardiac differentiation. Furthermore, AMPK activation increased the expression of CM-specific markers in hPSC-CMs. Additionally, autophagy inhibition impaired cardiomyocyte differentiation by targeting autophagosome-lysosome fusion. These results indicate the significance of autophagy in cardiomyocyte differentiation. In conclusion, AMPK might be a promising target for the regulation of cardiomyocyte generation by in vitro differentiation of pluripotent stem cells.
Developing c-MYC transcription inhibitors that target the G-quadruplex has generated significant interest; however, few compounds have demonstrated specificity for c-MYC G-quadruplex and cancer cells. In this study, we designed and synthesized a series of benzoazole derivatives as potential G-quadruplex ligand-based c-MYC transcription inhibitors. Surprisingly, benzoselenazole derivatives, which are rarely reported as G-quadruplex ligands, demonstrated greater c-MYC G-quadruplex selectivity and cancer cell specificity compared to their benzothiazole and benzoxazole analogues. The most promising compound, benzoselenazole m-Se3, selectively inhibited c-MYC transcription by specifically stabilizing the c-MYC G-quadruplex. This led to selective inhibition of hepatoma cell growth and proliferation by affecting the MYC target gene network, as well as effective tumor growth inhibition in hepatoma xenografts. Collectively, our study demonstrates that m-Se3 holds significant promise as a potent and selective inhibitor of c-MYC transcription for cancer treatment. Furthermore, our findings inspire the development of novel selenium-containing heterocyclic compounds as c-MYC G-quadruplex-specific ligands and transcription inhibitors.
Increasing evidence supports the connection between the progression of several cancers and BRK1. However, the clinical significance of aberrant BRK1 gene expression in cancer is unknown. This study is conducted to investigate the possibility and effect of BRK1 as a potential immunotherapy target, to deliver a better option for liver cancer immunotherapy. We explored the predictive role of BRK1 expression in a variety of cancers from different bioinformatics, including differential expression in different cancers, tumor microenvironment (TME), microsatellite instability (MSI), tumor mutational burden (TMB), immune checkpoint molecules, immune-related and cell cycle-related signalling pathways, and drug response sensitivity. Finally, we verified the expression of BRK1 in hepatocellular carcinoma using immunohistochemistry. BRK1 is overexpressed in multiple cancers and displays a negative association with prognosis and progression of disease in a wide range of main cancer types. Additionally, the expression of BRK1 is related to MSI and TMB of tumors. There was also a remarkable correlation between the expression of BRK1 and immune score, immune infiltration, immune checkpoint molecules and a stromal score of tumors. In hepatocellular carcinoma, BRK1 is associated with several signaling pathways and immune cell infiltration may affect several key immune-related regulatory genes, making it an excellent biomarker and may be a sensitive target for immune drugs.Our research suggests that BRK1 may be a potential prognostic marker and target for immunotherapy and may be associated with poor prognosis in diverse malignancies, including hepatocellular carcinoma.
The basic helix‐loop‐helix‐leucine zipper (bHLHZip) protein Max associates with members of the Myc family, as well as with the related proteins Mad (Mad1) and Mxi1. Whereas both Myc:Max and Mad:Max heterodimers bind related E‐box sequences, Myc:Max activates transcription and promotes proliferation while Mad:Max represses transcription and suppresses Myc dependent transformation. Here we report the identification and characterization of two novel Mad1‐ and Mxi1‐related proteins, Mad3 and Mad4. Mad3 and Mad4 interact with both Max and mSin3 and repress transcription from a promoter containing CACGTG binding sites. Using a rat embryo fibroblast transformation assay, we show that both Mad3 and Mad4 inhibit c‐Myc dependent cell transformation. An examination of the expression patterns of all mad genes during murine embryogenesis reveals that mad1, mad3 and mad4 are expressed primarily in growth‐arrested differentiating cells. mxi1 is also expressed in differentiating cells, but is co‐expressed with either c‐myc, N‐myc, or both in proliferating cells of the developing central nervous system and the epidermis. In the developing central nervous system and epidermis, downregulation of myc genes occurs concomitant with upregulation of mad family genes. These expression patterns, together with the demonstrated ability of Mad family proteins to interfere with the proliferation promoting activities of Myc, suggest that the regulated expression of Myc and Mad family proteins function in a concerted fashion to regulate cell growth in differentiating tissues.
Although activation of c-myc is a critical step in the development of lymphomas and other tumors, its normal function(s) in cell growth remain obscure because few myc-regulated genes are known. myc expression normally increases in response to mitogens and peaks in G_1 when additional protein synthesis is required for cell-cycle progression. Protein synthesis is controlled by the availability of translation initiation factors, including the mRNA cap binding protein (eIF-4E) and the alpha subunit of the eIF-2 complex that binds the initiator Met-tRNA. Consequently we examined eIF-4E and eIF-2alpha for evidence of regulation by c-myc. Expression of eIF-4E and eIF-2alpha correlated with c-myc expression in fibroblasts after growth stimulation. In addition, expression of eIF-4E and eIF-2alpha was increased in myc-transformed rat embryo fibroblasts but was not increased in ras-transformed cells. Transcription rates of eIF-4E and eIF-2alpha mRNAs were regulated by c-myc in cells expressing an estrogen receptor-Myc fusion protein. Finally, electrophoretic mobility-shift assays identified a sequence element in the eIF-2alpha promoter, TCCGCATGCGCG, which was specifically retarded by extracts of myc-expressing cells. c-myc is thought to deregulate the growth of cancer cells by activating transcription, suggesting that specific genes regulated by c-myc should also function as oncogenes. In previous studies these translation initiation factors could induce neoplastic growth because overexpression of eIF-4E-transformed cells and inhibition of a suppressor of eIF-2alpha (eIF-2alpha kinase) also caused malignant transformation. Our studies suggest that one important biological function of c-myc may be to increase cell growth by increasing expression of eIF-4E and eIF-2alpha.
Neuroblastomas undergo spontaneous regression at an unusually high rate. The mechanisms are not clear, but apoptosis may be involved. A large proportion of neuroblastomas is characterized by amplification of MYCN. Using human neuroblastoma cells harbouring tetracycline controlled expression of MYCN we have analysed the role of the MycN protein and IFNγ in cell death decision. Neither conditional expression of MYCN nor treatment with IFNγ alone was sufficient to trigger cell death. However, when acting in concert MycN and IFNγ efficiently triggered cell death, which was accompanied by DNA fragmentation and required caspase activity, two hallmarks of apoptosis. MycN and IFNγ may cooperate along at least two different pathways. First, IFNγ increased the CD95 cell surface expression while MycN enhanced the cellular susceptibility for the CD95 mediated death signal. Second, IFNγ treatment induced expression of BAK mRNA while MycN and IFNγ in combination increased the amount of Bax protein, another activator of apoptosis, without a concomitant increase in BAX mRNA. MycN also increased cell death in response to TRAIL and TNFα, suggesting that enforced MYCN expression in general increases the susceptibility of neuroblastoma cells towards a variety of death stimuli.
Ectopic expression of Myc induces Cdk2 kinase activity in quiescent cells and antagonizes association of p27kip1 with Cdk2. The target gene(s) by which Myc mediates this effect is largely unknown. We now show that p27 is rapidly and transiently sequestered by cyclin D2–Cdk4 complexes upon activation of Myc and that cyclin D2 is a direct target gene of Myc. The cyclin D2 promoter is repressed by Mad–Max complexes and de-repressed by Myc via a single highly conserved E-box element. Addition of trichostatin A to quiescent cells mimics activation of Myc and induces cyclin D2 expression, suggesting that cyclin D2 is repressed in a histone deacetylase-dependent manner in quiescent cells. Inhibition of cyclin D2 function in established cell lines, either by ectopic expression of p16 or by antibody injection, inhibits Myc-dependent dissociation of p27 from Cdk2 and Myc-induced cell cycle entry. Primary mouse fibroblasts that are cyclin D2-deficient undergo accelerated senescence in culture and are not immortalized by Myc; induction of apoptosis by Myc is unimpaired in such cells. Our data identify a downstream effector pathway that links Myc directly to cell cycle progression.
The c-myc gene has been implicated in three distinct genetic programs regulating cell proliferation: control of cyclin E–cdk2 kinase activity, E2F-dependent transcription and cell growth. We have now used p27-/- fibroblasts to dissect these downstream signalling pathways. In these cells, activation of Myc stimulates transcription of E2F target genes, S-phase entry and cell growth without affecting cyclin E–cdk2 kinase activity. Both cyclin D2 and E2F2, potential direct target genes of Myc, are induced in p27-/- MycER cells. Ectopic expression of E2F2, but not of cyclin D2, induces S-phase entry, but, in contrast to Myc, does not stimulate cell growth. Our results show that stimulation of cyclin E–cdk2 kinase, of E2F-dependent transcription and of cell growth by Myc can be genetically separated from each other.
Cancer is thought to arise from multiple genetic events that establish irreversible malignancy. A different mechanism might be present in certain leukaemias initiated by a chromosomal translocation. We have taken a new approach to determine if ablation of the genetic abnormality is sufficient for reversion by generating a conditional transgenic model of BCR-ABL1 (also known as BCR-ABL)-induced leukaemia. This oncogene is the result of a reciprocal translocation and is associated with different forms of leukaemia. The most common form, p210 BCR-ABL1, is found in more than 90% of patients with chronic myelogenous leukaemia (CML) and in up to 15% of adult patients with de novoacute lymphoblastic leukaemia (ALL). Efforts to establish a useful transgenic model have been hampered by embryonic lethality when the oncogene is expressed during embryogenesis, by reduced penetrance or by extremely long latency periods. One model uses the 'knock-in' approach to induce leukaemia by p190 BCR-ABL1(ref. 10). Given the limitations of models with p210, we used a different experimental approach. Lethal leukaemia developed within an acceptable time frame in all animals, and complete remission was achieved by suppression of BCR-ABL1expression, even after multiple rounds of induction and reversion. Our results demonstrate that BCR-ABL1is required for both induction and maintenance of leukaemia.
MYC affects normal and neoplastic cell proliferation by altering gene expression, but the precise pathways remain unclear. We used oligonucleotide microarray analysis of 6,416 genes and expressed sequence tags to determine changes in gene expression caused by activation of c-MYC in primary human fibroblasts. In these experiments, 27 genes were consistently induced, and 9 genes were repressed. The identity of the genes revealed that MYC may affect many aspects of cell physiology altered in transformed cells: cell growth, cell cycle, adhesion, and cytoskeletal organization. Identified targets possibly linked to MYC's effects on cell growth include the nucleolar proteins nucleolin and fibrillarin, as well as the eukaryotic initiation factor 5A. Among the cell cycle genes identified as targets, the G1 cyclin D2 and the cyclin-dependent kinase binding protein CksHs2 were induced whereas the cyclin-dependent kinase inhibitor p21Cip1 was repressed. A role for MYC in regulating cell adhesion and structure is suggested by repression of genes encoding the extracellular matrix proteins fibronectin and collagen, and the cytoskeletal protein tropomyosin. A possible mechanism for MYC-mediated apoptosis was revealed by identification of the tumor necrosis factor receptor associated protein TRAP1 as a MYC target. Finally, two immunophilins, peptidyl-prolyl cis-trans isomerase F and FKBP52, the latter of which plays a role in cell division in Arabidopsis, were up-regulated by MYC. We also explored pattern-matching methods as an alternative approach for identifying MYC target genes. The genes that displayed an expression profile most similar to endogenous Myc in microarray-based expression profiling of myeloid differentiation models were highly enriched for MYC target genes.
Expression of the myc and fos genes has been monitored in mouse primary keratinocytes after induction of terminal differentiation by calcium or tetradecanoylphorbol acetate (TPA). myc RNA levels in growing cells are very high and remain elevated even at late times after calcium-induced differentiation. Thus, keratinocytes provide the first example of normal primary cells with persistent c-myc expression irrespective of their proliferative or differentiated state. fos expression is also relatively unaffected by addition of calcium. In contrast to calcium, TPA-induced differentiation is accompanied by dramatic changes in proto-oncogene expression: marked c-fos induction and considerable although transient decrease in c-myc expression. These effects might be important for the keratinocyte response to TPA: TPA treatment of a keratinocyte cell line (RBK) resistant to this substance has no effect on c-myc expression and leads only to minimal c-fos induction. In these cells full fos induction can still be triggered by addition of fresh medium. Thus, the fos gene in normal keratinocytes is inducible through at least two independent mechanisms, only one of which has been lost during derivation of the TPA-resistant cell line.
We have shown that c-myc mRNA levels decrease more than 20-fold when F9 teratocarcinoma stem cells are induced to arrest growth and terminally differentiate to parietal endoderm after exposure to retinoic acid and cyclic AMP (Campisi et al., Cell 36:241-247, 1984). Here, we demonstrate that although growth arrest and full expression of the differentiated phenotype required about 3 days, c-myc mRNA declined abruptly between 8 and 16 h after the addition of retinoic acid and cyclic AMP. The decline was independent of cyclic AMP. We found little or no change in the level of c-myc transcription during differentiation, although two other genes showed marked transcriptional regulation. Thus, decreased c-myc mRNA is a consequence of very early posttranscriptional regulation directed by retinoic acid. Differentiation was not fundamental to this regulation. We have shown that sodium butyrate blocks expression of the differentiated phenotype if added within 8 h of retinoic acid and cyclic AMP (Levine et al., Dev. Biol. 105:443-450, 1984). However, butyrate did not inhibit the decrease in c-myc mRNA. Furthermore, F9 cells partially arrested growth without differentiating when grown in isoleucine-deficient medium. Under these conditions, c-myc mRNA levels also declined. Our results suggest that induction of differentiation-specific genes may be under retinoic acid-mediated control dissimilar from that responsible for the decay of c-myc mRNA. In addition, they raise the possibility that growth arrest may be initiated by reduced c-myc expression.
Two distinct forms of the myc oncogene were assayed for their ability to induce, in cultured rat fibroblast cells, the alterations of cellular growth controls observed upon transfer of the gene of polyoma virus encoding only the large T protein (plt). Both of these rearranged myc genes and the plt gene had been previously shown to cooperate with ras oncogenes for transformation of rat embryo fibroblasts (REF) and were thought to induce the same early step ("immortalization") of the tumoral transformation pathway. We now report that these two different oncogenes elicite the same response in the following biological assays: (i) reduction of the requirements in serum factors for growth in culture of cells of the established FR3T3 line; (ii) expression of transformed properties in low serum medium after transfer into FR3T3 cells expressing only the middle T protein of polyoma virus (MTT lines); (iii) conferring on REF cells the ability to grow as clonal colonies after seeding at low cell density; (iv) conferring on REF cells the ability to grow continuously in cell culture. These congruent phenotypes suggest that the activities of the large T and myc proteins result in the induction of the same molecular events. These results also provide simple biological assays and selective systems for oncogenes of the myc class.
Activation of conditional alleles of Myc can induce proliferation in quiescent cells. We now report that induction of Myc in density-arrested fibroblasts triggers rapid hyperphosphorylation of the retinoblastoma protein and activation of both cyclin D1- and cyclin E-associated kinase activities in the absence of significant changes in the amounts of cyclin-cdk complexes. Kinase activation by Myc is blocked by inhibitors of transcription and requires intact DNA binding and heterodimerization domains of Myc. Activation of cyclin E-cdk2 kinase in serum-starved cells occurs in two steps. The first is induced by Myc and involves the release of a 120 kDa cyclin E-cdk2 complex from a 250 kDa inactive complex that is present in starved cells. This is necessary, but not sufficient, to generate full kinase activity, as cdc25 phosphatase activity is limiting in the absence of external growth factors. In vivo cdc25 activity can be supplied by the addition of growth factors. In vitro recombinant cdc25a strongly activates the 120 kDa, but only poorly activates the 250 kDa cyclin E-cdk2 complex. Our data show that two distinct signals, one of which is supplied by Myc, are necessary for consecutive steps during growth factor-induced formation of active cyclin E-cdk2 complexes in G(o)-arrested rodent fibroblasts.
The positive effects of Myc on cellular growth and gene expression are antagonized by activities of another member of the Myc superfamily, Mad. Characterization of the mouse homolog of human mad on the structural level revealed that domains shown previously to be required in the human protein for anti-Myc repression, sequence-specific DNA-binding activity, and dimerization with its partner Max are highly conserved. Conservation is also evident on the biological level in that both human and mouse mad can antagonize the ability of c-myc to cooperate with ras in the malignant transformation of cultured cells. An analysis of c-myc and mad gene expression in the developing mouse showed contrasting patterns with respect to tissue distribution and developmental stage. Regional differences in expression were more striking on the cellular level, particularly in the mouse and human gastrointestinal system, wherein c-Myc protein was readily detected in immature proliferating cells at the base of the colonic crypts, while Mad protein distribution was restricted to the postmitotic differentiated cells in the apex of the crypts. An increasing gradient of Mad was also evident in the more differentiated subcorneal layers of the stratified squamous epithelium of the skin. Together, these observations support the view that both downregulation of Myc and accumulation of Mad may be necessary for progression of precursor cells to a growth-arrested, terminally differentiated state.
A number of proteins have been rendered functionally oestrogen-dependent by fusion with the hormone-binding domain of the
oestrogen receptor. There are, however, several significant disadvantages with such fusion proteins. First, their use In cells
in vitro requires phenol red-free medium and laborious stripping of steroid hormones from serum In order to avoid constitutive activation.
Secondly, control of oestrogen receptor fusion proteins In vivo is precluded by high endogenous levels of circulating oestrogens. Thirdly, the hormone-binding domain of the oestrogen receptor
functions as a hormone-dependent transcriptional activation domain making interpretation of fusions with transcription factors
problematical. In order to overcome these drawbacks we have used a transcrip-tlonally inactive mutant of the murine oestrogen
receptor which is unable to bind oestrogen yet retains normal affinity for the synthetic ligand, 4-hydroxyta-moxifen. When
the hormone-binding domain of this mutant oestrogen receptor is fused to the C-terminus of the c-Myc protein, Myc-induced
proliferation and apoptosis In fibroblasts becomes dependent on 4-hydroxytamoxifen, but remains refractory to 17β-oestradlol.
The c-Myc protein (Myc) is involved in cellular transformation and mitogenesis, but is also a potent inducer of programmed cell death, or apoptosis. Whether these apparently opposite functions are mediated through common or distinct molecular mechanisms remains unclear. Myc and its partner protein, Max, dimerize and bind DNA in vitro and in vivo through basic/helix-loop-helix/leucine zipper motifs (bHLH-LZ). By using complementary leucine zipper mutants (termed MycEG and MaxEG), which dimerize efficiently with each other but not with their wild-type partners, we demonstrate that both cell cycle progression and apoptosis in nontransformed rodent fibroblasts are induced by Myc-Max dimers. MycEG or MaxEG alone are inactive, but co-expression restores ability to prevent withdrawal from the cell cycle and to induce cell death upon removal of growth factors. Thus, Myc can control two alternative cell fates through dimerization with a single partner, Max.
E2F-1, the first gene product identified among a family of E2F transcription factors, is thought to play a critical role in G1/S progression of the cell cycle. Transcriptional activities of E2F are modulated during the cell cycle, mainly by the formation of complexes between E2F and several key regulators of cell cycle such as the retinoblastoma protein and related proteins. To further understand the roles of E2F in the cell cycle progression, we have overexpressed exogenous E2F-1 by using a tetracycline-controlled expression system. We have found that the induced expression of E2F-1 in Rat-2 fibroblasts promotes S-phase entry and subsequently leads to apoptosis. The apoptosis occurs in an E2F-1 dose-dependent manner. Cells resistant to the induction of apoptosis have lost the ability to express exogenous E2F-1. Cells growing in low serum are more sensitive to the E2F-1-mediated cell death. Overexpression of E2F-1 mutants that impair DNA binding or transactivation does not alter cell cycle progression or induce apoptosis. These results define a novel pathway to apoptosis and demonstrate that premature S-phase entry is associated with apoptotic cell death.
The requirements for transformation of rat embryo fibroblasts (REFs) by transfected ras and myc oncogenes were explored. Under conditions of dense monolayer culture, neither oncogene was able to transform REFs on its own. However, the introduction of a ras oncogene together with a selectable neomycin resistance marker into REFs allowed killing of the normal nontransfected cells and the outgrowth of colonies of ras transformants, 10% of which survived crisis and became tumorigenic. These cells expressed greater than 10-fold-higher levels of ras p21 than tumorigenic cells cotransfected with ras and myc oncogenes. The myc oncogene similarly was unable to induce tumorigenic conversion of REFs unless especially refractile colonies of oncogene-bearing cells, produced by use of a cotransfected selectable marker, were picked and subcultured. Tumorigenic conversion of REFs by single transfected oncogenes appears to require special culture conditions and high levels of gene expression.
We have shown that c-myc mRNA levels decrease more than 20-fold when F9 teratocarcinoma stem cells are induced to arrest growth and terminally differentiate to parietal endoderm after exposure to retinoic acid and cyclic AMP (Campisi et al., Cell 36:241-247, 1984). Here, we demonstrate that although growth arrest and full expression of the differentiated phenotype required about 3 days, c-myc mRNA declined abruptly between 8 and 16 h after the addition of retinoic acid and cyclic AMP. The decline was independent of cyclic AMP. We found little or no change in the level of c-myc transcription during differentiation, although two other genes showed marked transcriptional regulation. Thus, decreased c-myc mRNA is a consequence of very early posttranscriptional regulation directed by retinoic acid. Differentiation was not fundamental to this regulation. We have shown that sodium butyrate blocks expression of the differentiated phenotype if added within 8 h of retinoic acid and cyclic AMP (Levine et al., Dev. Biol. 105:443-450, 1984). However, butyrate did not inhibit the decrease in c-myc mRNA. Furthermore, F9 cells partially arrested growth without differentiating when grown in isoleucine-deficient medium. Under these conditions, c-myc mRNA levels also declined. Our results suggest that induction of differentiation-specific genes may be under retinoic acid-mediated control dissimilar from that responsible for the decay of c-myc mRNA. In addition, they raise the possibility that growth arrest may be initiated by reduced c-myc expression.
The tyrosine kinase P210 is the gene product of the rearranged BCR-ABL locus on the Philadelphia chromosome (Ph1), which is found in leukemic cells of patients with chronic myelogenous leukemia. It has a weakly oncogenic effect in immature murine hematopoietic cells and does not transform NIH 3T3 cells. We have found that P210 has a strikingly different effect in Rat-1 cells, another line of established rodent fibroblasts. Stable expression of P210 in Rat-1 cells caused a distinct morphological change and conferred both tumorigenicity and capacity for anchorage-independent growth. The introduction of v-myc into Rat-1 cells expressing P210 led to complete morphological transformation and enhanced tumorigenicity. No such interaction took place in NIH 3T3 cells. Thus, Rat-1 cells can be used to detect cooperation between BCR-ABL and other oncogenes and may prove useful for the identification of secondary oncogenic events in chronic myelogenous leukemia.
This chapter reviews recent knowledge on the cell cycle regulation of c-Myc expression and c-Myc activity, as well as the structural features of the c-Myc protein that give new insights into the understanding of c-Myc function during the cell cycle and embryonic development. C-myc was the first nuclear proto-oncogene discovered and has been the subject of intense investigation during the past 16 years. The initial property attributed to c-Myc was its ability to transform normal cultured cells, but a great deal of evidence since then has indicated that this proto-oncogene is involved in a variety of different cellular processes—such as proliferation, differentiation, and apoptosis. The current focus of investigation into c-Myc is, therefore, primarily concerned with deciphering the way one gene can both participate in and discriminate between these seemingly divergent activities. Embryonic development requires cellular multiplication coupled to diversification of the genetic program, and therefore additional levels of control are required to provide a tight coordination between proliferation and differentiation. The biochemical analysis of c-Myc activity has been hampered by the extreme difficulty in obtaining a soluble form of the purified full-length protein. The chapter also discusses cell proliferation and c-Myc as a transcription factor. The majority of studies concerning c-Myc expression in adult tissues or embryos are related to the production of mRNA, although gene expression does not always strictly correlate with the temporal pattern and level of protein expression during development. While proliferating cells exhibit stable c-Myc RNA levels and abrupt changes in c-Myc expression are often observed during exit from the cell cycle, because cells undergo terminal differentiation. During the past decade it has become increasingly apparent that programmed cell death, or apoptosis, is a fundamental process affecting both growth and development.
The protooncogene c-myc regulates cell growth, differentiation, and apoptosis, and its aberrant expression is frequently observed in human cancer. However, the consequences of activating c-Myc in an adult tissue, in which these cellular processes are part of normal homeostasis, remain unknown. In order to achieve this, we have targeted expression of a switchable form of the c-Myc protein to the skin epidermis, a well characterized homeostatic tissue. We show that activation of c-MycERTM in adult suprabasal epidermis rapidly triggers proliferation and disrupts differentiation of postmitotic keratinocytes. Sustained activation of c-Myc is sufficient to induce papillomatosis together with angiogenesis—changes that resemble hyperplastic actinic keratosis, a commonly observed human precancerous epithelial lesion. All these premalignant changes spontaneously regress upon deactivation of c-MycERTM.
Recent evidence indicates that the genetic alterations of the multistage process of malignant transformation appear to activate tumor neovascularization by altering the balance between stimulators and inhibitors of angiogenesis. In the present study, we have attempted to define the effect of enhanced MYCN oncogene expression on the profile of endothelial cell growth modulators in neuroblastoma cells. We report here that conditioned medium of human neuroblastoma cells with normal MYCN expression contains three inhibitors of endothelial cell proliferation, which appear to be novel proteins as judged by their physicochemical, immunological and biological properties. All three inhibitors are diminished or become undetectable upon experimental increase of MYCN expression. Our results suggest that enhanced MYCN expression in human neuroblastoma cells alters the angiogenic balance by down-regulating endothelial cell growth inhibitors but leaving the expression of the stimulators unaffected. These data shed light on the molecular mechanisms linking the genetic changes of malignant transformation with initiation of tumor angiogenesis. Moreover, our observations might explain the poor prognosis of human neuroblastomas following MYCN oncogene amplification through initiation of angiogenesis and subsequent tumor growth and spread.
The c-Myc protein functions as a transcription factor to facilitate oncogenic transformation; however, the biochemical and
genetic pathways leading to transformation remain undefined. We demonstrate here that the recently described c-Myc cofactor
TRRAP recruits histone acetylase activity, which is catalyzed by the human GCN5 protein. Since c-Myc function is inhibited
by recruitment of histone deacetylase activity through Mad family proteins, these opposing biochemical activities are likely
to be responsible for the antagonistic biological effects of c-Myc and Mad on target genes and ultimately on cellular transformation.
The bcl-2 proto-oncogene is activated by translocation in a variety of B-lymphoid tumours and synergizes with the c-myc oncogene in tumour progression. The mechanism of synergy is unclear but bcl-2 expression inhibits apoptosis, a property presumably pertinent to its proto-oncogenic mode of action. We have shown that the c-myc gene is a potent inducer of apoptosis, in addition to its established role in mitogenesis. Here we show that expression of the bcl-2 protein, Bcl-2, specifically abrogates c-myc-induced apoptosis without affecting the c-myc mitogenic function. This provides a novel mechanism for oncogene cooperation, of potential importance both in carcinogenesis and in the evolution of drug resistance in tumours.
Although Rat-1 fibroblasts expressing c-myc constitutively are unable to arrest growth in low serum, their numbers do not increase in culture because of substantial cell death. We show this cell death to be dependent upon expression of c-myc protein and to occur by apoptosis. Regions of the c-myc protein required for induction of apoptosis overlap with regions necessary for cotransformation, autoregulation, and inhibition of differentiation, suggesting that the apoptotic function of c-myc protein is related to its other functions. Moreover, cells with higher levels of c-myc protein are more prone to cell death upon serum deprivation. Finally, we demonstrate that deregulated c-myc expression induces apoptosis in cells growth arrested by a variety of means and at various points in the cell cycle.
A gene-transfer approach was used to explore the function of the BCL2 (B-cell lymphoma/leukemia 2) gene in a human T-cell line, Jurkat. Though stable introduction of a BCL2 expression plasmid into Jurkat T cells was by itself insufficient, the combined transfer of BCL2 and MYC genes markedly enhanced the tumorigenicity of these cells in athymic mice. Moreover, a BCL2 antisense expression plasmid ablated tumor formation by Jurkat cells, providing further evidence that this oncogene contributes to the regulation of the in vivo growth of these human T lymphocytes. In addition to their influence on tumor formation, BCL2 sense and antisense expression plasmids increased and decreased, respectively, the in vitro survival of Jurkat T cells in serum-free medium. These observations extend to T cells the finding of synergy of BCL2 with MYC previously reported for B cells and provide evidence that BCL2 can regulate the growth of human T cells.
In the murine interleukin 3 (IL-3)-dependent myeloid cell line 32D, down-regulation of c-myc and ornithine decarboxylase (ODC) expression is an immediate response to IL-3 deprivation. This is followed by an accumulation of cells in the G1 phase of the cell cycle, and eventual cell death. However, clones of 32D cells harboring an expression vector which constitutively expresses murine c-myc did not down-regulate ODC transcripts in response to IL-3 withdrawal, and they failed to G1 arrest. Moreover, in contrast to control cultures in which the majority of death occurred following G1 arrest, c-myc clones rapidly initiated a program of cell death characteristic of apoptosis following IL-3 deprivation, and their subsequent loss of viability occurred with accelerated kinetics. The premature induction of apoptosis in cells harboring a deregulated c-myc gene suggests that apoptosis may be an important mechanism in the elimination of hematopoietic cells harboring mutations, such as constitutive c-myc expression, which imbalance normal cell cycle regulatory controls.
The myc protooncogene family has been implicated in cell proliferation, differentiation, and neoplasia, but its mechanism
of function at the molecular level is unknown. The carboxyl terminus of Myc family proteins contains a basic region helix-loop-helix
leucine zipper motif (bHLH-Zip), which has DNA-binding activity and has been predicted to mediate protein-protein interactions.
The bHLH-Zip region of c-Myc was used to screen a complementary DNA (cDNA) expression library, and a bHLH-Zip protein, termed
Max, was identified. Max specifically associated with c-Myc, N-Myc, and L-Myc proteins, but not with a number of other bHLH,
bZip, or bHLH-Zip proteins. The interaction between Max and c-Myc was dependent on the integrity of the c-Myc HLH-Zip domain,
but not on the basic region or other sequences outside the domain. Furthermore, the Myc-Max complex bound to DNA in a sequence-specific
manner under conditions where neither Max nor Myc exhibited appreciable binding. The DNA-binding activity of the complex was
dependent on both the dimerization domain and the basic region of c-Myc. These results suggest that Myc family proteins undergo
a restricted set of interactions in the cell and may belong to the more general class of eukaryotic DNA-binding transcription
factors.
This chapter discusses normal c-myc gene regulation and abnormal c-myc regulation in cancer cells. All normal c-myc transcription units are composed of three exons: the second two encoding the major c-myc proteins. These two exons have from 70% to over 90% sequence identity between species. All c-myc genes contain a long untranslated exon 1, suggesting an important function for this feature. Other members of the myc oncogene family, N-myc and L-myc, share the three-exon gene organization with exons 2 and 3 providing the major coding regions that exhibit highly conserved stretches of amino acids. A long untranslated exon 1 is present in both N-myc and L-myc genes. These exons have little homology to each other or to exon 1 of c-myc, lending further support to the notion of an important structural or regulatory role for c-myc leader regions via a sequence-independent mechanism. Ample direct and circumstantial evidence exists to implicate c-myc in neoplastic transformation. Indirect evidence is provided by the presence of the c-myc gene at various DNA rearrangements that characteristically accompany tumors, such as leukemias, lymphomas, and small-cell lung carcinomas. These rearrangements may lead to one or more of increased levels, constitutive synthesis, or alterations in ratios between the c-myc products. Mutations in c-myc protein coding regions occur, but are not characteristic of rearranged c-myc in tumor cells.
The putative oncogene bcl-2 is juxtaposed to the immunoglobulin heavy chain (Igh) locus by the t(14;18) chromosomal translocation typical of human follicular B-cell lymphomas. The bcl-2 gene product is not altered by the translocation, but its expression is deregulated, presumably by the Igh enhancer E mu. Constitutive bcl-2 expression seems to augment cell survival, as infection with a bcl-2 retrovirus enables certain growth factor-dependent mouse cell lines to maintain viability when deprived of factor. Furthermore, high levels of the bcl-2 product can protect human B and T lymphoblasts under stress and thereby confer a growth advantage. Mice expressing a bcl-2 transgene controlled by the Igh enhancer accumulate small non-cycling B cells which survive unusually well in vitro but do not show a propensity for spontaneous tumorigenesis. In contrast, an analogous myc transgene, designed to mimic the myc-Igh translocation product typical of Burkitt's lymphoma and rodent plasmacytoma, promotes B lymphoid cell proliferation and predisposes mice to malignancy in pre-B and B lymphoid cells. Previous experiments have suggested that bcl-2 can cooperate with deregulated myc to improve in vitro growth of pre-B and B cells. Here we describe a marked synergy between bcl-2 and myc in doubly transgenic mice. E mu-bcl-2/myc mice show hyperproliferation of pre-B and B cells and develop tumours much faster than E mu-myc mice. Suprisingly, the tumours derive from a cell with the hallmarks of a primitive haemopoietic cell, perhaps a lymphoid-committed stem cell.
We have analysed c-myc, N-myc and L-myc gene expression in developing human fetal brain by Northern hybridization, RNAase protection and in situ hybridization. The unique zonal organization of the developing fetal brain allows a particularly good assessment of the coupling of myc gene expression to cell proliferation and differentiation in vivo. By Northern and in situ hybridization, L-myc as well as c-myc and N-myc transcripts in the brain were found in the post-mitotic cortical and intermediate layers, as well as in the mitotically active layers containing the neuroepithelial precursor cells. Consistent results were also obtained for L-myc using RNAase protection analysis. Both the 3.6 and 3.8kb forms of the L-myc mRNA, resulting from alternative splicing of intron I, were detected in layers of neuroectodermal origin, but not in the meninges or choroid plexus. We also extended L-myc expression and splicing analyses to other developing human fetal tissues. L-myc mRNA was expressed in several other fetal tissues, particularly in fetal skin. Predominantly intron I containing L-myc mRNA was observed in fetal striated and cardiac muscle. Thus, L-myc is expressed in a wider spectrum of developing tissues than previously known. Our findings also, show that L-myc as well as N-myc and c-myc expression is uncoupled from cell division in developing brain.
The tyrosine kinase P210 is the gene product of the rearranged BCR-ABL locus on the Philadelphia chromosome (Ph1), which is
found in leukemic cells of patients with chronic myelogenous leukemia. It has a weakly oncogenic effect in immature murine
hematopoietic cells and does not transform NIH 3T3 cells. We have found that P210 has a strikingly different effect in Rat-1
cells, another line of established rodent fibroblasts. Stable expression of P210 in Rat-1 cells caused a distinct morphological
change and conferred both tumorigenicity and capacity for anchorage-independent growth. The introduction of v-myc into Rat-1
cells expressing P210 led to complete morphological transformation and enhanced tumorigenicity. No such interaction took place
in NIH 3T3 cells. Thus, Rat-1 cells can be used to detect cooperation between BCR-ABL and other oncogenes and may prove useful
for the identification of secondary oncogenic events in chronic myelogenous leukemia.
Cells of the established REF52 line completely resist stable transformation by activated ras oncogenes, apparently because expression of ras p21 above a low threshold level inhibits cell proliferation. Adenovirus E1A and simian virus 40 (SV40) large T antigen enable ras oncogenes to transform REF52 cells and therefore protect REF52 cells from ras-induced growth arrest. The present study investigated the role of c-myc in regulating the responses of REF52 cells to ras oncogenes. We report that transcriptionally activated c-myc oncogenes enabled ras to transform REF52 cells but the efficiency of transformation was 20- to 30-fold lower than with E1A. In contrast, myc and E1A were similarly active as ras collaborators when assayed on primary baby rat kidney (BRK) cells. Relative difficulties transforming REF52 celis by myc and ras did not result from a requirement to express either gene at higher levels in REF52 as compared with BRK transformants. Steady state levels of endogenous c-myc RNA were unaltered in REF52 cells transformed by ras together with c-myc, E1A or SV40 large T antigen. Furthermore, ras-induced growth arrest was not accompanied by a decline in c-myc RNA levels. These results suggest that transcriptional control of c-myc is not affected either by the anti-proliferative effects of ras or by the collaborating activities of E1A and SV40 large T antigen.
The c-myc protooncogene in mouse embryos was shown by RNA in situ hybridization to be preferentially expressed in tissues
of endodermal and mesodermal origin. Most organs developing from the ectoderm, such as skin, brain, and spinal cord, displayed
low levels of c-myc RNA. The thymus represented the only hematopoietic organ with high c-myc expression. In organs and structures
strongly hybridizing to c-myc probes, for example the fetal part of the placenta, gut, liver, kidney, pancreas, submandibular
glands, enamel organs of the molars, and skeletal cartilage, the level of expression depended on the stage of development.
Expression was observed to be correlated with proliferation, particularly during expansion and folding of partially differentiated
epithelial cells.
The requirements for transformation of rat embryo fibroblasts (REFs) by transfected ras and myc oncogenes were explored. Under
conditions of dense monolayer culture, neither oncogene was able to transform REFs on its own. However, the introduction of
a ras oncogene together with a selectable neomycin resistance marker into REFs allowed killing of the normal nontransfected
cells and the outgrowth of colonies of ras transformants, 10% of which survived crisis and became tumorigenic. These cells
expressed greater than 10-fold-higher levels of ras p21 than tumorigenic cells cotransfected with ras and myc oncogenes. The
myc oncogene similarly was unable to induce tumorigenic conversion of REFs unless especially refractile colonies of oncogene-bearing
cells, produced by use of a cotransfected selectable marker, were picked and subcultured. Tumorigenic conversion of REFs by
single transfected oncogenes appears to require special culture conditions and high levels of gene expression.
Transgenic mice bearing the cellular myc oncogene coupled to the immunoglobulin mu or kappa enhancer frequently develop a fatal lymphoma within a few months of birth. Since the tumours represent represent both immature and mature B lymphocytes, constitutive c-myc expression appears to be highly leukaemogenic at several stages of B-cell maturation. These myc mice should aid study of lymphoma development, B-cell ontogeny and immunoglobulin regulation.
The chicken genome contains nucleotide sequences homologous to transforming genes (oncogenes) of a number of avian retroviruses. We have isolated chicken DNA (c-myc) that is homologous to the oncogene (v-myc) of the avian myelocytomatosis virus MC29 and have compared the structures of the cellular and viral genes. Results from restriction endonuclease mapping of c-myc and from analysis of heteroduplexes between the DNAs of the cellular and viral genes show that c-myc is homologous to 1,500 nucleotides in v-myc DNA. This homologous region is interrupted in c-myc by an intron-like sequence of 1,100 nucleotides which is absent from v-myc. Nuclear RNA from normal chicken cells contains at least five species of transcripts from c-myc ranging from 2.5 to 6.5 kilobases in length. By contrast, cytoplasm contains only the 2.5-kilobase c-myc RNA. These features of the c-myc gene and its nuclear transcripts are characteristic of normal cellular genes and suggest that the myc gene is of cellular rather than viral origin. The exons in c-myc may define two functional domains in the gene and may therefore facilitate the dissection of the different oncogenic potentials of the MC29 virus.
Transfection of embryo fibroblasts by a human ras oncogene does not convert them into tumour cells unless the fibroblasts are established and immortalized before transfection. The embryo fibroblasts become tumorigenic if a second oncogene such as a viral or cellular myc gene or the gene for the polyoma large-T antigen is introduced together with the ras gene.
Growth factors such as platelet-derived growth factor (PDGF) elicit the transcriptional activation of a large number of immediate early genes (many of which encode transcription factors), and ultimately DNA synthesis. Both AP1 and Myc are activated in fibroblasts in response to growth factor stimulation, and various experiments suggest their importance in proliferation. Src family kinases are required for PDGF (and other growth factors) to induce DNA synthesis. We have examined which transcription factors, when constitutively expressed, 'rescue' the block elicited by dominant negative Src. We report here that Myc, but not Fos and/or Jun, was able to rescue the block. In contrast, Fos and Jun, but not Myc, rescued the block induced by dominant negative Ras. Our data suggest that Src kinases control the transcriptional activation of Myc.
A number of properties of the cancer-related genes c-myc and p53 suggest that they might collaborate to induce tumorigenesis. To test this notion, we produced doubly heterozygotic mice bearing disrupted p53 alleles and a fusion transgene consisting of the mouse mammary tumor virus (MMTV) LTR and the oncogene c-myc. Mice bearing both the MMT/c-myc transgene and a single p53- allele develop very aggressive pre-T- and T-cell lymphomas with a significantly shorter latency than mice carrying either the p53- allele or the c-myc transgene alone. Moreover, every lymphoma occurring in these animals has lost or suffers an inactivation of its wild type p53 allele indicating that loss of p53 activity is necessary for this c-myc-accelerated lymphomagenesis. Nonetheless, p53 inactivation and expression of the MMTV/c-myc transgene are not sufficient for lymphoid transformation. Tumors that arise in homozygous p53- mice carrying the c-myc transgene are monoclonal, suggesting that at least one additional event is necessary for their transformation. Moreover, since mice bearing only the MMTV/c-myc transgene predominantly develop mammary carcinomas, it was surprising that the p53- allele failed to accelerate the incidence of mammary carcinomas. Further, in contrast to the lymphomas, only one in four mammary tumors that arose in the double heterozygotic mice had lost its wild type p53 allele. Apparently cell context influences the ability of c-myc and p53- to cooperate in inducing oncogenesis.
Activation of the c-myc oncogene and functional loss of the p53 tumour suppressor gene are among the most frequently recorded genetic lesions in neoplasia but their combined effect has not previously been investigated. By breeding together mice transgenic for human c-myc (CD2-myc) and mice carrying an inactive p53 allele (p53-/-) we found that these genetic lesions act synergistically in vivo. Offspring carrying the CD2-myc transgene and the homozygous p53 null mutation (p53-/-/CD2-myc) were viable but developed thymic lymphomas with dramatically increased frequency and reduced latency compared to both parental groups. The tumour phenotype was similar to that previously recorded for CD2-myc mice (predominantly CD3+, CD4+8+) but tumour clonal complexity and metastasis was significantly greater in the p53-/-/CD2-myc mice. In contrast, no significant increase in tumour incidence was seen in p53+/-/CD2-myc vs p53+/+/CD2-myc mice over a 6 month observation period. However, the loss of wild type p53 in a proportion of tumour cells in p53+/-/CD2-myc lymphomas suggests that wild type allele loss can occur as a late progression step rather than an initiating step in these tumours. We suggest that p53 loss of function may collaborate with the CD2-myc transgene at more than one stage in thymic lymphoma development.
Documented interactions among members of the Myc superfamily support a yin-yang model for the regulation of Myc-responsive genes in which transactivation-competent Myc-Max heterodimers are opposed by repressive Mxi1-Max or Mad-Max complexes. Analysis of mouse mxi1 has led to the identification of two mxi1 transcript forms possessing open reading frames that differ in their capacity to encode a short amino-terminal alpha-helical domain. The presence of this segment dramatically augments the suppressive potential of Mxi1 and allows for association with a mammalian protein that is structurally homologous to the yeast transcriptional repressor SIN3. These findings provide a mechanistic basis for the antagonistic actions of Mxi1 on Myc activity that appears to be mediated in part through the recruitment of a putative transcriptional repressor.
The bHLH-ZIP protein Mad heterodimerizes with Max as a sequence-specific transcriptional repressor. Mad is rapidly induced upon differentiation, and the associated switch from Myc-Max to Mad-Max heterocomplexes seem to repress genes normally activated by Myc-Max. We have identified two related mammalian cDNAs that encode Mad-binding proteins. Both possess sequence homology with the yeast transcription repressor Sin3, including four conserved paired amphipathic helix (PAH) domains. mSin3A and mSin3B bind specifically to Mad and the related protein Mxi1. Mad-Max and mSin3 form ternary complexes in solution that specifically recognize the Mad-Max E box-binding site. Mad-mSin3 association requires PAH2 of mSin3A/mSin3B and the first 25 residues of Mad, which contains a putative amphipathic alpha-helical region. Point mutations in this region eliminate interaction with mSin3 proteins and block Mad transcriptional repression. We suggest that Mad-Max represses transcription by tethering mSin3 to DNA as corepressors and that a transcriptional repression mechanism is conserved from yeast to mammals.
Transgenic mice expressing the simian virus-40 large T-antigen (Tag) under the control of the insulin gene regulatory region offer a useful model for tumorigenesis. All the islets of Langerhans express Tag, although there is at first no aberrant proliferation. Over half of the islets become hyperplastic, however, and neovascularization of a further subset (about 10%)3 leads eventually to formation of highly vascularized solid tumours in 1-2% of islets by about 14 weeks of age. Here we show that the initial proliferative switch is correlated with focal activation of insulin-like growth factor II (IGF-II). Transfection with an antisense oligonucleotide to the IGF-II messenger RNA interferes with tumour cell proliferation in vitro, and transgenic mice homozygous for a disruption of the IGF-II gene develop tumours with reduced malignancy and a higher incidence of apoptosis. Several signals, in this case including an oncoprotein and a growth/survival factor, thus appear to be needed to elicit hyperproliferation.
Tumor necrosis factor-alpha (TNF) is a multifunctional cytokine which is cytotoxic for some tumor cells and transformed cells. The molecular mechanisms which render transformed and tumor cells sensitive to the cytotoxic action of TNF are unclear. We show here that an increased expression of the c-Myc oncoprotein strongly increases cellular sensitivity to TNF cytotoxicity. In Rat1A fibroblasts, which are resistant to TNF, the addition of TNF with a concomitant activation of a hormone-inducible c-Myc-estrogen receptor chimera (MycER) resulted in apoptotic cell death. Similarly, c-Myc overexpression enhanced the sensitivity of NIH3T3 fibroblasts to TNF-induced death. The c-Myc and TNF-induced apoptosis was inhibited by ectopic expression of the Bcl2 oncoprotein and by the free oxygen radical scavenging enzyme Mn superoxide dismutase. Furthermore, in highly TNF-sensitive fibrosarcoma cells, antisense c-myc oligodeoxynucleotides caused a specific inhibition of TNF cytotoxicity. Our results suggest that the deregulation of c-Myc, which is common in human tumors and tumor cell lines is one reason why these cells are TNF sensitive.
E2F-1, the first gene product identified among a family of E2F transcription factors, is thought to play a critical role in
G1/S progression of the cell cycle. Transcriptional activities of E2F are modulated during the cell cycle, mainly by the formation
of complexes between E2F and several key regulators of cell cycle such as the retinoblastoma protein and related proteins.
To further understand the roles of E2F in the cell cycle progression, we have overexpressed exogenous E2F-1 by using a tetracycline-controlled
expression system. We have found that the induced expression of E2F-1 in Rat-2 fibroblasts promotes S-phase entry and subsequently
leads to apoptosis. The apoptosis occurs in an E2F-1 dose-dependent manner. Cells resistant to the induction of apoptosis
have lost the ability to express exogenous E2F-1. Cells growing in low serum are more sensitive to the E2F-1-mediated cell
death. Overexpression of E2F-1 mutants that impair DNA binding or transactivation does not alter cell cycle progression or
induce apoptosis. These results define a novel pathway to apoptosis and demonstrate that premature S-phase entry is associated
with apoptotic cell death.
The c-Myc oncoprotein is a basic-helix-loop-helix-leucine zipper (b-HLH-LZ) transcription factor involved in regulating cell proliferation and differentiation. We have used retrovirus-mediated gene transfer to investigate the effect of ectopic c-Myc expression on the spontaneous differentiation of primary quail myoblasts in vitro. Unlike normal myoblasts, c-Myc-expressing myoblasts are unable to form myotubes or express muscle-specific genes, such as myosin, and show severely reduced expression of the myogenic regulatory factors myoD, myogenin, and myf5. The c-Myc leucine zipper (LZ) motif is essential for the differentiation block since myoblasts expressing a mutant with a partial deletion of this region, c-Myc delta 7, differentiate and express myoD family regulators and muscle-specific genes normally. Remarkably, c-Myc delta 7, like wild-type c-Myc, retains the capacity to transform the growth phenotype of myoblasts, and associates with the b-HLH-LZ Myc partner protein Max in transformed cells. We conclude that the block to myogenic differentiation induced by c-Myc can be dissociated from cell transformation per se, and that this attribute correlates more closely with down-regulation of myoD family gene expression. These findings are discussed in the light of current models of Myc function.
E2F-1 is a transcription factor suspected of activating genes required for S phase and a known target for the action of RB, the retinoblastoma gene product. Its induction in quiescent fibroblasts led to S-phase entry followed by apoptosis. E2F-1-mediated apoptosis was suppressed by coexpression of wild-type RB or a transdominant negative mutant species of p53. In contrast, coexpression of a naturally occurring loss-of-function RB mutant or wild-type p53 did not suppress the induction of apoptosis under these conditions. Thus, deregulated E2F-1 activity gives rise to proliferative and apoptotic signals. p53 appears to participate in the execution of the latter.
The tumor-suppressor protein p53 appears to function at the G1 phase of the cell cycle as a checkpoint in response to DNA damage. Mutations in the p53 gene lead to an increased rate of genomic instability and tumorigenesis. The E2F-1 transcription factor is a protein partner of the retinoblastoma-susceptibility gene product, RB. E2F-1 appears to function as a positive regulator or signal for entry into S phase. To explore possible interactions of p53 and E2F-1 in the cell cycle, a human E2F-1 expression plasmid was introduced into a murine cell line containing a temperature-sensitive p53 allele which produces a p53 protein in the wild-type conformation at 32 degrees C and the mutant form at 37.5 degrees C. Coexpression of the wild-type p53 protein and E2F-1 in these cells resulted in a rapid loss of cell viability through a process of apoptosis. Thus, the cell cycle utilizes an interacting or communicative pathway between RB-E2F-1 and p53.
The myc family of cellular oncogenes (c-, N- and L-myc) encode for nuclear phosphoproteins that appear to regulate cellular proliferation and differentiation during normal vertebrate development. In addition, their overexpression has been correlated with malignant transformation and apoptotic cell death. The transgenic mouse technology has been utilized to study the unique and overlapping functions of the myc family in these cellular processes in the context of the developing animal. These studies suggest that Myc oncoproteins serve important roles during normal development and that their deregulated expression is causal in the development of many malignancies. Furthermore, these experiments have provided insight into the cell type specific activities of each gene and into the unique and overlapping roles of the genes during normal development.