RESUMO
SWI/SNF chromatin remodeling enzymes are multisubunit complexes that contain one of two catalytic subunits, BRG1 or BRM and 9-11 additional subunits called BRG1 or BRM-associated factors (BAFs). BRG1 interacts with the microphthalmia-associated transcription factor (MITF) and is required for melanocyte development in vitro and in vivo. The subunits of SWI/SNF that mediate interactions between BRG1 and MITF have not been elucidated. Three mutually exclusive isoforms of a 60-kDa subunit (BAF60A, B, or C) often facilitate interactions with transcription factors during lineage specification. We tested the hypothesis that a BAF60 subunit promotes interactions between MITF and the BRG1-containing SWI/SNF complex. We found that MITF can physically interact with BAF60A, BAF60B, and BAF60C. The interaction between MITF and BAF60A required the basic helix-loop-helix domain of MITF. Recombinant BAF60A pulled down recombinant MITF, suggesting that the interaction can occur in the absence of other SWI/SNF subunits and other transcriptional regulators of the melanocyte lineage. Depletion of BAF60A in differentiating melanoblasts inhibited melanin synthesis and expression of MITF target genes. MITF promoted BAF60A recruitment to melanocyte-specific promoters, and BAF60A was required to promote BRG1 recruitment and chromatin remodeling. Thus, BAF60A promotes interactions between MITF and the SWI/SNF complex and is required for melanocyte differentiation.
Assuntos
Diferenciação Celular , Proteínas Cromossômicas não Histona/metabolismo , DNA Helicases/metabolismo , Melanócitos/citologia , Melanócitos/metabolismo , Fator de Transcrição Associado à Microftalmia/metabolismo , Proteínas Nucleares/metabolismo , Fatores de Transcrição/metabolismo , Animais , Ciclo Celular , Diferenciação Celular/genética , Regulação da Expressão Gênica , Células HEK293 , Humanos , Melaninas/biossíntese , Glicoproteínas de Membrana/genética , Glicoproteínas de Membrana/metabolismo , Camundongos , Fator de Transcrição Associado à Microftalmia/química , Modelos Biológicos , Oxirredutases/genética , Oxirredutases/metabolismo , Regiões Promotoras Genéticas/genética , Ligação Proteica , Subunidades Proteicas/metabolismoRESUMO
Mutations in SOX10 cause neurocristopathies which display varying degrees of hypopigmentation. Using a sensitized mutagenesis screen, we identified Smarca4 as a modifier gene that exacerbates the phenotypic severity of Sox10 haplo-insufficient mice. Conditional deletion of Smarca4 in SOX10 expressing cells resulted in reduced numbers of cranial and ventral trunk melanoblasts. To define the requirement for the Smarca4 -encoded BRG1 subunit of the SWI/SNF chromatin remodeling complex, we employed in vitro models of melanocyte differentiation in which induction of melanocyte-specific gene expression is closely linked to chromatin alterations. We found that BRG1 was required for expression of Dct, Tyrp1 and Tyr, genes that are regulated by SOX10 and MITF and for chromatin remodeling at distal and proximal regulatory sites. SOX10 was found to physically interact with BRG1 in differentiating melanocytes and binding of SOX10 to the Tyrp1 distal enhancer temporally coincided with recruitment of BRG1. Our data show that SOX10 cooperates with MITF to facilitate BRG1 binding to distal enhancers of melanocyte-specific genes. Thus, BRG1 is a SOX10 co-activator, required to establish the melanocyte lineage and promote expression of genes important for melanocyte function.
Assuntos
Diferenciação Celular , DNA Helicases/metabolismo , Melanócitos/fisiologia , Proteínas Nucleares/metabolismo , Fatores de Transcrição SOXE/metabolismo , Fatores de Transcrição/metabolismo , Animais , Linhagem Celular , Elementos Facilitadores Genéticos , Expressão Gênica , Regulação da Expressão Gênica , Melaninas/biossíntese , Glicoproteínas de Membrana/genética , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Oxirredutases/genéticaRESUMO
Epigenetics refers to mitotically/meiotically heritable mechanisms that regulate gene transcription without a need for changes in the DNA code. Covalent modifications of DNA, in the form of methylation, and histone post-translational modifications, in the form of acetylation and methylation, constitute the epigenetic code of a cell. Both DNA and histone modifications are highly dynamic and often work in unison to define the epigenetic state of a cell. Most epigenetic mechanisms regulate gene transcription by affecting localized/genome-wide transitions between heterochromatin and euchromatin states, thereby altering the accessibility of the transcriptional machinery and in turn, reduce/increase transcriptional output. Altered chromatin structure is associated with cancer progression, and epigenetic plasticity primarily governs the resistance of cancer cells to therapeutic agents. In this chapter, we specifically focus on regulators of histone methylation and acetylation, the two well-studied chromatin post-translational modifications, in the context of prostate cancer.
Assuntos
Cromatina/genética , Epigênese Genética , Neoplasias da Próstata/genética , Acetilação , Metilação de DNA , Histonas/química , Histonas/metabolismo , Humanos , Masculino , Processamento de Proteína Pós-TraducionalRESUMO
Melanocytes are pigment producing cells in the skin that give rise to cutaneous malignant melanoma, which is a highly aggressive and the deadliest form of skin cancer. Studying melanocytes in vivo is often difficult due to their small proportion in the skin and the lack of specific cell surface markers. Several genetically-engineered mouse models (GEMMs) have been created to specifically label the melanocyte compartment. These models give both spatial and temporal control over the expression of a cellular 'beacon' that has an added benefit of inducible expression that can be activated on demand. Two powerful models that are discussed in this review include the melanocyte-specific, tetracycline-inducible green fluorescent protein expression system (iDct-GFP), and the fluorescent ubiquitination-based cell cycle indicator (FUCCI) model that allows for the monitoring of the cell-cycle. These two systems are powerful tools in studying melanocyte and melanoma biology. We discuss their current uses and how they could be employed to help answer unresolved questions in the fields of melanocyte and melanoma biology.
Assuntos
Ciclo Celular , Proteínas de Fluorescência Verde/genética , Melanócitos/citologia , Camundongos Transgênicos/genética , Animais , Linhagem da Célula , Proteínas de Fluorescência Verde/metabolismo , Melanócitos/metabolismo , Melanoma/patologia , Camundongos , Camundongos Transgênicos/metabolismo , Microscopia de Fluorescência/métodosRESUMO
Macrophages are conventionally classified into M1 and M2 subtypes according to their differentiation status and functional role in the immune system. However, accumulating evidence suggests that this binary classification system is insufficient to account for the remarkable plasticity of macrophages that gives rise to an immense diversity of subtypes. This diverse spectrum of macrophage subtypes play critical roles in various homeostatic and immune functions, but remain far from being fully characterised. In addition to their roles in normal physiological conditions, macrophages also play crucial roles in disease conditions such as cancer. In this review, we discuss the roles tumour-associated macrophages (TAMs) play in regulating different steps of tumour progression and metastasis, and the opportunities to target them in the quest for cancer prevention and treatment.
Assuntos
Macrófagos/fisiologia , Metástase Neoplásica , Neoplasias/imunologia , Animais , Progressão da Doença , Humanos , Tolerância Imunológica , Invasividade Neoplásica , Células-Tronco Neoplásicas/fisiologia , Neovascularização Patológica/etiologia , Microambiente TumoralRESUMO
Brahma (BRM) and Brahma-related gene 1(BRG1) are catalytic subunits of SWItch/sucrose non-fermentable (SWI/SNF) chromatin remodeling complexes. BRM is epigenetically silenced in a wide-range of tumors. Mutations in the v-raf murine sarcoma viral oncogene homolog B1 (BRAF) gene occur frequently in melanoma and lead to constitutive activation of the mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase (ERK1/2) pathway. We tested the hypothesis that BRM expression is modulated by oncogenic BRAF and phosphorylation of ERK1/2 in melanocytes and melanoma cells. Expression of oncogenic BRAF in melanocytes and melanoma cells that are wild-type for BRAF decreased BRM expression and increased BRG1 expression. Inhibition of mitogen-activated protein/extracellular signal-regulated kinase kinase (MEK) or selective inhibition of BRAF in melanoma cells that harbor oncogenic BRAF increased BRM expression and decreased BRG1 expression. Increased BRM expression was associated with increased histone acetylation on the BRM promoter. Over-expression of BRM in melanoma cells that harbor oncogenic BRAF promoted changes in cell cycle progression and apoptosis consistent with a tumor suppressive role. Upon inhibition of BRAF(V600E) with PLX4032, BRM promoted survival. PLX4032 induced changes in BRM function were correlated with increased acetylation of the BRM protein. This study provides insights into the epigenetic consequences of inhibiting oncogenic BRAF in melanoma through modulation of SWI/SNF subunit expression and function.
Assuntos
Sistema de Sinalização das MAP Quinases , Melanoma/genética , Melanoma/metabolismo , Fatores de Transcrição/genética , Substituição de Aminoácidos , Pontos de Checagem do Ciclo Celular , Linhagem Celular Tumoral , Proliferação de Células , Sobrevivência Celular , Células Cultivadas , DNA Helicases/genética , Epigênese Genética , Regulação Neoplásica da Expressão Gênica , Técnicas de Silenciamento de Genes , Histonas/metabolismo , Humanos , Melanócitos/citologia , Melanócitos/metabolismo , Melanoma/patologia , Mutação , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas B-raf/genética , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , RNA Interferente Pequeno/genética , Proteínas Recombinantes/genética , Proteína do Retinoblastoma/metabolismo , Fatores de Transcrição/antagonistas & inibidoresRESUMO
Trastuzumab improves overall survival for HER2+ breast cancer, but its short half-life in the cerebrospinal fluid (~2-4 days) and delivery limitations restrict the ability to target HER2+ central nervous system (CNS) disease. We developed an adeno-associated virus (AAV) vector expressing a codon-optimized, ubiquitin C (UbC)-promoter-driven trastuzumab sequence (AAV9.UbC.trastuzumab) for intrathecal administration. Transgene expression was evaluated in adult Rag1 knockout mice and rhesus nonhuman primates (NHPs) after a single intracerebroventricular (ICV) or intra-cisterna magna (ICM) AAV9.UbC.trastuzumab injection, respectively, using real-time PCR, ELISA, Western blot, in situ hybridization, single-nucleus RNA sequencing, and liquid chromatography-mass spectrometry; antitumor efficacy was evaluated in brain xenografts using HER2+ breast cancer cell lines (BT-474, MDA-MB-453). Transgene expression was detected in brain homogenates of Rag1 knockout mice following a single ICV injection of AAV9.UbC.trastuzumab (1 × 1011 vector genome copies [GC]/mouse) and tumor progression was inhibited in xenograft models of breast-to-brain metastasis. In NHPs, ICM delivery of AAV9.UbC.trastuzumab (3 × 1013 GC/animal) was well tolerated (36-37 days in-life) and resulted in transgene expression in CNS tissues and cerebrospinal fluid at levels sufficient to induce complete tumor remission in MDA-MB-453 brain xenografts. With AAV9's proven clinical safety record, this gene therapy may represent a viable approach for targeting HER2 + CNS malignancies.
Assuntos
Neoplasias Encefálicas , Sistema Nervoso Central , Dependovirus , Sistemas de Liberação de Medicamentos , Receptor ErbB-2 , Trastuzumab , Animais , Humanos , Camundongos , Antineoplásicos Imunológicos/farmacologia , Antineoplásicos Imunológicos/uso terapêutico , Antineoplásicos Imunológicos/administração & dosagem , Neoplasias Encefálicas/terapia , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/secundário , Neoplasias Encefálicas/patologia , Neoplasias da Mama/patologia , Neoplasias da Mama/terapia , Neoplasias da Mama/genética , Neoplasias da Mama/tratamento farmacológico , Linhagem Celular Tumoral , Sistema Nervoso Central/efeitos dos fármacos , Sistema Nervoso Central/metabolismo , Vetores Genéticos/administração & dosagem , Vetores Genéticos/genética , Macaca mulatta , Camundongos Knockout , Receptor ErbB-2/metabolismo , Receptor ErbB-2/genética , Trastuzumab/administração & dosagem , Trastuzumab/farmacologia , Trastuzumab/uso terapêutico , Ensaios Antitumorais Modelo de Xenoenxerto , Sistemas de Liberação de Medicamentos/métodosRESUMO
The androgen receptor (AR) is a ligand-responsive transcription factor that binds at enhancers to drive terminal differentiation of the prostatic luminal epithelia. By contrast, in tumors originating from these cells, AR chromatin occupancy is extensively reprogrammed to drive hyper-proliferative, metastatic, or therapy-resistant phenotypes, the molecular mechanisms of which remain poorly understood. Here, we show that the tumor-specific enhancer circuitry of AR is critically reliant on the activity of Nuclear Receptor Binding SET Domain Protein 2 (NSD2), a histone 3 lysine 36 di-methyltransferase. NSD2 expression is abnormally gained in prostate cancer cells and its functional inhibition impairs AR trans-activation potential through partial off-loading from over 40,000 genomic sites, which is greater than 65% of the AR tumor cistrome. The NSD2-dependent AR sites distinctly harbor a chimeric AR-half motif juxtaposed to a FOXA1 element. Similar chimeric motifs of AR are absent at the NSD2-independent AR enhancers and instead contain the canonical palindromic motifs. Meta-analyses of AR cistromes from patient tumors uncovered chimeric AR motifs to exclusively participate in tumor-specific enhancer circuitries, with a minimal role in the physiological activity of AR. Accordingly, NSD2 inactivation attenuated hallmark cancer phenotypes that were fully reinstated upon exogenous NSD2 re-expression. Inactivation of NSD2 also engendered increased dependency on its paralog NSD1, which independently maintained AR and MYC hyper-transcriptional programs in cancer cells. Concordantly, a dual NSD1/2 PROTAC degrader, called LLC0150, was preferentially cytotoxic in AR-dependent prostate cancer as well as NSD2-altered hematologic malignancies. Altogether, we identify NSD2 as a novel subunit of the AR neo-enhanceosome that wires prostate cancer gene expression programs, positioning NSD1/2 as viable paralog co-targets in advanced prostate cancer.
RESUMO
Metastatic castration-resistant prostate cancer (CRPC) is a fatal disease, primarily resulting from the transcriptional addiction driven by androgen receptor (AR). First-line CRPC treatments typically target AR signaling, but are rapidly bypassed, resulting in only a modest survival benefit with antiandrogens. Therapeutic approaches that more effectively block the AR-transcriptional axis are urgently needed. Here, we investigated the molecular mechanism underlying the association between the transcriptional coactivator MED1 and AR as a vulnerability in AR-driven CRPC. MED1 undergoes CDK7-dependent phosphorylation at T1457 and physically engages AR at superenhancer sites, and is essential for AR-mediated transcription. In addition, a CDK7-specific inhibitor, THZ1, blunts AR-dependent neoplastic growth by blocking AR/MED1 corecruitment genome-wide, as well as reverses the hyperphosphorylated MED1-associated enzalutamide-resistant phenotype. In vivo, THZ1 induces tumor regression of AR-amplified human CRPC in a xenograft mouse model. Together, we demonstrate that CDK7 inhibition selectively targets MED1-mediated, AR-dependent oncogenic transcriptional amplification, thus representing a potential new approach for the treatment of CRPC. SIGNIFICANCE: Potent inhibition of AR signaling is critical to treat CRPC. This study uncovers a driver role for CDK7 in regulating AR-mediated transcription through phosphorylation of MED1, thus revealing a therapeutically targetable potential vulnerability in AR-addicted CRPC.See related commentary by Russo et al., p. 1490.This article is highlighted in the In This Issue feature, p. 1469.
Assuntos
Subunidade 1 do Complexo Mediador/metabolismo , Fenilenodiaminas/administração & dosagem , Neoplasias de Próstata Resistentes à Castração/tratamento farmacológico , Pirimidinas/administração & dosagem , Receptores Androgênicos/genética , Animais , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Elementos Facilitadores Genéticos , Amplificação de Genes , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Humanos , Masculino , Camundongos , Células PC-3 , Fenilenodiaminas/farmacologia , Fosforilação/efeitos dos fármacos , Neoplasias de Próstata Resistentes à Castração/genética , Neoplasias de Próstata Resistentes à Castração/metabolismo , Pirimidinas/farmacologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Present: Due to an error made by the authors while submitting a revision, Dr. Tuan Zea Tan was omitted from the list of authors.Corrected: Correct author list can be found below. Authors sincerely apologize for this oversight. Ila Datar1, Xiaoliang Qiu1, Hong Zhi Ma1, Miranda Yeung1, Shweta Aras1, Ivana de la Serna1, Fahd Al-Mulla2, Tuan Zea Tan3, Jean Paul Thiery3, Robert Trumbly1, Xuan Fan4, Hongjuan Cui4 and Kam C. Yeung1 1 Department of Biochemistry and Cancer Biology, University of Toledo, College of Medicine, Health Science Campus, Toledo, OH, USA 2 Kuwait University, Faculty of Medicine. P.O. Box 24923, Safat, Kuwait 3 Department of Biochemistry, Yong Loo Lin School of Medicine, National University of Singapore, Singapore 4 State Key Laboratory Of Silkworm Genome Biology, Chongqing, China Original article: Oncotarget. 2015; 6(36): 39050-61. doi: 10.18632/oncotarget.5176.
RESUMO
Accumulating evidence suggests that presence of macrophages in the tumor microenvironment add to the invasive and tumor-promoting hallmarks of cancer cells by secreting angiogenic and growth factors. RKIP is a known metastasis suppressor and interferes with several steps of metastasis. However, the mechanistic underpinnings of its function as a broad metastasis suppressor remain poorly understood. Here, we establish a novel pathway for RKIP regulation of metastasis inhibition through the negative regulation of RANTES/CCL5 thereby limiting tumor macrophage infiltration and inhibition of angiogenesis. Using a combination of loss- and gain-of- function approaches, we show that RKIP hinders breast cancer cell invasion by inhibiting expression of the CC chemokine CCL5 in vitro. We also show that the expression levels of RKIP and CCL5 are inversely correlated among clinical human breast cancer samples. Using a mouse allograft breast cancer transplantation model, we highlight that ectopic expression of RKIP significantly decreases tumor vasculature, macrophage infiltration and lung metastases. Mechanistically, we demonstrate that the inhibition of the CCL5 expression is the cause of the observed effects resulting from RKIP expression. Taken together, our results underscore the significance of RKIP as important negative regulator of tumor microenvironment.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Quimiocina CCL5/biossíntese , Macrófagos/patologia , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Animais , Neoplasias da Mama/irrigação sanguínea , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Quimiocina CCL5/genética , Quimiocina CCL5/metabolismo , Feminino , Xenoenxertos , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Invasividade Neoplásica , Metástase Neoplásica , Neovascularização Patológica/metabolismo , Neovascularização Patológica/patologia , Proteína de Ligação a Fosfatidiletanolamina/genética , Microambiente TumoralRESUMO
Raf Kinase Inhibitory Protein or RKIP was initially identified as a Raf-1 binding protein using the yeast 2-hybrid screen. RKIP inhibits the activation phosphorylation of MEK by Raf-1 by competitively inhibiting the binding of MEK to Raf-1 and thus exerting an inhibitory effect on the Raf-MEK-Erk pathway. RKIP has been identified as a metastasis suppressor gene. Expression of RKIP is low in cancer metastases. Although primary tumor growth remains unaffected, re- expression of RKIP inhibits cancer metastasis. Mechanistically, RKIP constrains metastasis by inhibiting angiogenesis, local invasion, intravasation, and colonization. The molecular mechanism of how RKIP inhibits these individual steps remains undefined. In our present study, using an unbiased PCR based screening and by analyzing DNA microarray expression datasets we observe that the expression of multiple metalloproteases (MMPs) including MMP1, MMP3, MMP10 and MMP13 are negatively correlated with RKIP expression in breast cancer cell lines and clinical samples. Since expression of MMPs by cancer cells is important for cancer metastasis, we hypothesize that RKIP may mediate suppression of breast cancer metastasis by inhibiting multiple MMPs. We show that the expression signature of RKIP and MMPs is better at predicting high metastatic risk than the individual gene. Using a combination of loss- and gain-of-function approaches, we find that MMP13 is the cause of RKIP-mediated inhibition of local cancer invasion. Interestingly expression of MMP13 alone is not sufficient to reverse the inhibition of breast cancer cell metastasis to the lung due to the expression of RKIP. We find that RKIP negatively regulates MMP13 through the Erk2 signaling pathway and the repression of MMP13 by RKIP is transcription factor AP-1 independent. Together, our findings indicate that RKIP inhibits cancer cell invasion, in part, via MMP13 inhibition. These data also implicate RKIP in the regulation of MMP transcription, suggesting a potential mechanism by which RKIP inhibits tumor progression and metastasis.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Metaloproteinase 13 da Matriz/genética , Proteína de Ligação a Fosfatidiletanolamina/metabolismo , Ativação Transcricional , Animais , Neoplasias da Mama/genética , Linhagem Celular Tumoral , Transformação Celular Neoplásica , Intervalo Livre de Doença , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Invasividade Neoplásica , Metástase Neoplásica , Transdução de SinaisRESUMO
Microphthalmia-associated transcription factor (MITF) is a survival factor in melanocytes and melanoma cells. MITF regulates expression of antiapoptotic genes and promotes lineage-specific survival in response to ultraviolet (UV) radiation and to chemotherapeutics. SWI/SNF chromatin-remodeling enzymes interact with MITF to regulate MITF target gene expression. We determined that the catalytic subunit, BRG1, of the SWI/SNF complex protects melanoma cells against UV-induced death. BRG1 prevents apoptosis in UV-irradiated melanoma cells by activating expression of the melanoma inhibitor of apoptosis (ML-IAP). Down-regulation of ML-IAP compromises BRG1-mediated survival of melanoma cells in response to UV radiation. BRG1 regulates ML-IAP expression by cooperating with MITF to promote transcriptionally permissive chromatin structure on the ML-IAP promoter. The alternative catalytic subunit, BRM, and the BRG1-associated factor, BAF180, were found to be dispensable for elevated expression of ML-IAP in melanoma cells. Thus, we illuminate a lineage-specific mechanism by which a specific SWI/SNF subunit, BRG1, modulates the cellular response to DNA damage by regulating an antiapoptotic gene and implicate this subunit of the SWI/SNF complex in mediating the prosurvival function of MITF.