Metastatic melanoma remains a mostly incurable disease. Although newly approved targeted therapies are efficacious in a subset of patients, resistance and relapse rapidly ensue. Alternative therapeutic strategies to manipulate epigenetic regulators and disrupt the transcriptional program that maintains tumor cell identity are emerging. Bromodomain and extraterminal domain (BET) proteins are epigenome readers known to exert key roles at the interface between chromatin remodeling and transcriptional regulation. Here, we report that BRD4, a BET family member, is significantly upregulated in primary and metastatic melanoma tissues compared with melanocytes and nevi. Treatment with BET inhibitors impaired melanoma cell proliferation in vitro and tumor growth and metastatic behavior in vivo, effects that were mostly recapitulated by individual silencing of BRD4. RNA sequencing of BET inhibitor–treated cells followed by Gene Ontology analysis showed a striking impact on transcriptional programs controlling cell growth, proliferation, cell-cycle regulation, and differentiation. In particular, we found that, rapidly after BET displacement, key cell-cycle genes (SKP2, ERK1, and c-MYC) were downregulated concomitantly with the accumulation of cyclin-dependent kinase (CDK) inhibitors (p21 and p27), followed by cell-cycle arrest. Importantly, BET inhibitor efficacy was not influenced by BRAF or NRAS mutational status, opening the possibility of using these small-molecule compounds to treat patients for whom no effective targeted therapy exists. Collectively, our study reveals a critical role for BRD4 in melanoma tumor maintenance and renders it a legitimate and novel target for epigenetic therapy directed against the core transcriptional program of melanoma.
Neuroblastoma (NB) is a neoplasm of the sympathetic nervous system, and is the most common solid tumor of infancy. NBs are very heterogeneous, with a clinical course ranging from spontaneous regression to resistance to all current forms of treatment. High-risk patients need intense chemotherapy, and only 30-40% will be cured. Relapsed or metastatic tumors acquire multi-drug resistance, raising the need for alternative treatments. Owing to the diverse mechanisms that are responsible of NB chemoresistance, we aimed to target epigenetic factors that control multiple pathways to bypass therapy resistance. We found that the SWI/SNF-related, matrix-associated, actin-dependent regulator of chromatin, subfamily a, member 4 (SMARCA4/BRG1) was consistently upregulated in advanced stages of NB, with high BRG1 levels being indicative of poor outcome. Loss-of-function experiments in vitro and in vivo showed that BRG1 is essential for the proliferation of NB cells. Furthermore, whole-genome transcriptome analysis revealed that BRG1 controls the expression of key elements of oncogenic pathways such as PI3K/AKT and BCL2, which offers a promising new combination therapy for high-risk NB.
Lung cancer is one of the most aggressive tumours with very low life expectancy. Altered microRNA expression is found in human tumours because it is involved in tumour growth, progression and metastasis. In this study, we analysed microRNA expression in 47 lung cancer biopsies. Among the most downregulated microRNAs we focussed on the miR-99a characterisation. In vitro experiments showed that miR-99a expression decreases the proliferation of H1650, H1975 and H1299 lung cancer cells causing cell cycle arrest and apoptosis. We identified two novel proteins, E2F2 (E2F transcription factor 2) and EMR2 (EGF-like module-containing, mucin-like, hormone receptor-like 2), downregulated by miR-99a by its direct binding to their 3′-UTR. Moreover, miR-99a expression prevented cancer cell epithelial-to-mesenchymal transition (EMT) and repressed the tumourigenic potential of the cancer stem cell (CSC) population in both these cell lines and mice tumours originated from H1975 cells. The expression of E2F2 and EMR2 at protein level was studied in 119 lung cancer biopsies. E2F2 and EMR2 are preferentially expressed in adenocarcinomas subtypes versus other tumour types (squamous and others). Interestingly, the expression of E2F2 correlates with the presence of vimentin and both E2F2 and EMR2 correlate with the presence of β-catenin. Moreover, miR-99a expression correlates inversely with E2F2 and directly with β-catenin expression in lung cancer biopsies. In conclusion, miR-99a reveals two novel targets E2F2 and EMR2 that play a key role in lung tumourigenesis. By inhibiting E2F2 and EMR2, miR-99a represses in vivo the transition of epithelial cells through an EMT process concomitantly with the inhibition of stemness features and consequently decreasing the CSC population.
Highlights d ATRX IFFs are redistributed genome wide and are enriched at active promoters d The neuronal silencing transcription factor REST is an ATRX IFF target gene d REST and EZH2 silence neuronal gene programs in ATRX IFF NB d REST loss or EZH2 inhibition induces neuronal gene expression programs and NB cell death
Approximately 15,000 new cases of pediatric cancer are diagnosed yearly in Europe, with 8–10% corresponding to neuroblastoma, a rare disease with an incidence of 8–9 cases per million children <15 years of age. Although the survival rate for low-risk and intermediate-risk patients is excellent, half of children with high-risk, refractory, or relapsed tumors will be cured, and two-thirds of the other half will suffer major side effects and life-long disabilities. Epigenetic therapies aimed at reversing the oncogenic alterations in chromatin structure and function are an emerging alternative against aggressive tumors that are or will become resistant to conventional treatments. This approach proposes targeting epigenetic regulators, which are proteins that are involved in the creation, detection, and interpretation of epigenetic signals, such as methylation or histone post-translational modifications. In this review, we focused on the most promising epigenetic regulators for targeting and current drugs that have already reached clinical trials.
Despite multimodal therapies, a high percentage of high-risk neuroblastoma (NB) become refractory to current treatments, most of which interfere with cell cycle and DNA synthesis or function, activating the DNA damage response (DDR). In cancer, this process is frequently altered by deregulated expression or function of several genes which contribute to multidrug resistance (MDR). MicroRNAs are outstanding candidates for therapy since a single microRNA can modulate the expression of multiple genes of the same or different pathways, thus hindering the development of resistance mechanisms by the tumor. We found several genes implicated in the MDR to be overexpressed in high-risk NB which could be targeted by microRNAs simultaneously. Our functional screening identified several of those microRNAs that reduced proliferation of chemoresistant NB cell lines, the best of which was miR-497. Low expression of miR-497 correlated with poor patient outcome. The overexpression of miR-497 reduced the proliferation of multiple chemoresistant NB cell lines and induced apoptosis in MYCN-amplified cell lines. Moreover, the conditional expression of miR-497 in NB xenografts reduced tumor growth and inhibited vascular permeabilization. MiR-497 targets multiple genes related to the DDR, cell cycle, survival and angiogenesis, which renders this molecule a promising candidate for NB therapy.
MicroRNAs (miRNAs) are a class of endogenous non-coding small RNAs that post-transcriptionally control the translation and stability of target mRNAs in a sequence-dependent manner. MiRNAs are essential for key cellular processes including proliferation, differentiation, cell death and metabolism, among others. Consequently, alterations of miRNA expression contribute to developmental defects and a myriad of diseases.The expression of miRNAs can be altered by several mechanisms including gene copy number alterations, aberrant DNA methylation, defects of the miRNA processing machinery or unscheduled expression of transcription factors. In this work, we sought to analyze the regulation of the miR-182 cluster, located at the 7q32 locus, which encodes three different miRNAs that are abundantly expressed in human embryonic stem cells and de-regulated in cancer. We have found that the Krüppel-like factor 4 (KLF4) directly regulates miR-182 cluster expression in human embryonic stem cells (hESCs) and in melanoma tumors, in which the miR-182 cluster is highly expressed and has a pro-metastatic role. Furthermore, higher KLF4 expression was found to be associated with metastatic progression and poor patient outcome. Loss of function experiments revealed that KLF4 is required for melanoma cell maintenance. These findings provide new insights into the regulation of the miR-182 cluster expression and new opportunities for therapeutic intervention in tumors in which the KLF4-miR-182 cluster axis is deregulated.
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