RESUMO
The Ubiquitin-Specific Protease 22 (USP22) is a deubiquitinating subunit of the mammalian SAGA transcriptional co-activating complex. USP22 was identified as a member of the so-called "death-from-cancer" signature predicting therapy failure in cancer patients. However, the importance and functional role of USP22 in different types and subtypes of cancer remain largely unknown. In the present study, we leveraged human cell lines and genetic mouse models to investigate the role of USP22 in HER2-driven breast cancer (HER2+-BC) and demonstrate for the first time that USP22 is required for the tumorigenic properties in murine and human HER2+-BC models. To get insight into the underlying mechanisms, we performed transcriptome-wide gene expression analyses and identified the Unfolded Protein Response (UPR) as a pathway deregulated upon USP22 loss. The UPR is normally induced upon extrinsic or intrinsic stresses that can promote cell survival and recovery if shortly activated or programmed cell death if activated for an extended period. Strikingly, we found that USP22 actively suppresses UPR induction in HER2+-BC cells by stabilizing the major endoplasmic reticulum (ER) chaperone HSPA5. Consistently, loss of USP22 renders tumor cells more sensitive to apoptosis and significantly increases the efficiency of therapies targeting the ER folding capacity. Together, our data suggest that therapeutic strategies targeting USP22 activity may sensitize tumor cells to UPR induction and could provide a novel, effective approach to treat HER2+-BC.
Assuntos
Neoplasias da Mama/metabolismo , Receptor ErbB-2/metabolismo , Ubiquitina Tiolesterase/metabolismo , Resposta a Proteínas não Dobradas , Animais , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Linhagem Celular Tumoral , Bases de Dados Genéticas , Modelos Animais de Doenças , Estresse do Retículo Endoplasmático , Feminino , Humanos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Prognóstico , Receptor ErbB-2/genética , Taxa de Sobrevida , Ubiquitina Tiolesterase/genéticaRESUMO
As a member of the 11-gene "death-from-cancer" gene expression signature, overexpression of the Ubiquitin-Specific Protease 22 (USP22) was associated with poor prognosis in various human malignancies. To investigate the function of USP22 in cancer development and progression, we sought to detect common USP22-dependent molecular mechanisms in human colorectal and breast cancer cell lines. We performed mRNA-seq to compare gene expression profiles of various colorectal (SW837, SW480, HCT116) and mammary (HCC1954 and MCF10A) cell lines upon siRNA-mediated knockdown of USP22. Intriguingly, while USP22 depletion had highly heterogeneous effects across the cell lines, all cell lines displayed a common reduction in the expression of Heat Shock Protein 90 Alpha Family Class B Member 1 (HSP90AB1). The downregulation of HSP90AB1 was confirmed at the protein level in these cell lines as well as in colorectal and mammary tumors in mice with tissue-specific Usp22 deletions. Mechanistically, we detected a significant reduction of H3K9ac on the HSP90AB1 gene in USP22-deficient cells. Interestingly, USP22-deficient cells displayed a high dependence on HSP90AB1 expression and diminishing HSP90 activity further using the HSP90 inhibitor Ganetespib resulted in increased therapeutic vulnerability in both colorectal and breast cancer cells in vitro. Accordingly, subcutaneously transplanted CRC cells deficient in USP22 expression displayed increased sensitivity towards Ganetespib treatment in vivo. Together, we discovered that HSP90AB1 is USP22-dependent and that cooperative targeting of USP22 and HSP90 may provide an effective approach to the treatment of colorectal and breast cancer.
Assuntos
Neoplasias da Mama/genética , Neoplasias Colorretais/genética , Resistencia a Medicamentos Antineoplásicos/genética , Regulação Neoplásica da Expressão Gênica , Proteínas de Choque Térmico HSP90/antagonistas & inibidores , Ubiquitina Tiolesterase/metabolismo , Animais , Linhagem Celular Tumoral , Regulação para Baixo/genética , Feminino , Proteínas de Choque Térmico HSP90/genética , Proteínas de Choque Térmico HSP90/metabolismo , Humanos , Camundongos Endogâmicos C57BL , Camundongos SCID , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Ubiquitina Tiolesterase/deficiência , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
Bromodomain-containing protein 4 (BRD4) is a member of the bromo- and extraterminal (BET) domain-containing family of epigenetic readers which is under intensive investigation as a target for anti-tumor therapy. BRD4 plays a central role in promoting the expression of select subsets of genes including many driven by oncogenic transcription factors and signaling pathways. However, the role of BRD4 and the effects of BET inhibitors in non-transformed cells remain mostly unclear. We demonstrate that BRD4 is required for the maintenance of a basal epithelial phenotype by regulating the expression of epithelial-specific genes including TP63 and Grainy Head-like transcription factor-3 (GRHL3) in non-transformed basal-like mammary epithelial cells. Moreover, BRD4 occupancy correlates with enhancer activity and enhancer RNA (eRNA) transcription. Motif analyses of cell context-specific BRD4-enriched regions predicted the involvement of FOXO transcription factors. Consistently, activation of FOXO1 function via inhibition of EGFR-AKT signaling promoted the expression of TP63 and GRHL3. Moreover, activation of Src kinase signaling and FOXO1 inhibition decreased the expression of FOXO/BRD4 target genes. Together, our findings support a function for BRD4 in promoting basal mammary cell epithelial differentiation, at least in part, by regulating FOXO factor function on enhancers to activate TP63 and GRHL3 expression.
Assuntos
Mama/metabolismo , Proteínas de Ligação a DNA/genética , Células Epiteliais/metabolismo , Regulação da Expressão Gênica , Proteínas Nucleares/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Mama/citologia , Proteínas de Ciclo Celular , Linhagem Celular , Proteínas de Ligação a DNA/biossíntese , Elementos Facilitadores Genéticos , Fatores de Transcrição Forkhead/metabolismo , Humanos , Proteínas Proto-Oncogênicas c-akt/metabolismo , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , RNA Polimerase II/metabolismo , Transdução de Sinais , Fatores de Transcrição/biossíntese , Transcrição Gênica , Proteínas Supressoras de Tumor/biossínteseRESUMO
Proper temporal epigenetic regulation of gene expression is essential for cell fate determination and tissue development. The Bromodomain-containing Protein-4 (BRD4) was previously shown to control the transcription of defined subsets of genes in various cell systems. In this study we examined the role of BRD4 in promoting lineage-specific gene expression and show that BRD4 is essential for osteoblast differentiation. Genome-wide analyses demonstrate that BRD4 is recruited to the transcriptional start site of differentiation-induced genes. Unexpectedly, while promoter-proximal BRD4 occupancy correlated with gene expression, genes which displayed moderate expression and promoter-proximal BRD4 occupancy were most highly regulated and sensitive to BRD4 inhibition. Therefore, we examined distal BRD4 occupancy and uncovered a specific co-localization of BRD4 with the transcription factors C/EBPb, TEAD1, FOSL2 and JUND at putative osteoblast-specific enhancers. These findings reveal the intricacies of lineage specification and provide new insight into the context-dependent functions of BRD4.
Assuntos
Linhagem da Célula/genética , Epigênese Genética , Células Epiteliais/metabolismo , Células-Tronco Mesenquimais/metabolismo , Proteínas Nucleares/genética , Osteoblastos/metabolismo , Osteócitos/metabolismo , Fatores de Transcrição/genética , Proteína beta Intensificadora de Ligação a CCAAT/genética , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Proteínas de Ciclo Celular , Diferenciação Celular , Linhagem Celular , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/citologia , Antígeno 2 Relacionado a Fos/genética , Antígeno 2 Relacionado a Fos/metabolismo , Perfilação da Expressão Gênica , Humanos , Células-Tronco Mesenquimais/citologia , Proteínas Nucleares/metabolismo , Especificidade de Órgãos , Osteoblastos/citologia , Osteócitos/citologia , Regiões Promotoras Genéticas , Ligação Proteica , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais , Fatores de Transcrição de Domínio TEA , Fatores de Transcrição/metabolismo , Sítio de Iniciação de TranscriçãoRESUMO
UNLABELLED: Regulatory pathways that drive early hematogenous dissemination of tumor cells are insufficiently defined. Here, we used the presence of disseminated tumor cells (DTC) in the bone marrow to define patients with early disseminated breast cancer and identified low retinoic acid-induced 2 (RAI2) expression to be significantly associated with DTC status. Low RAI2 expression was also shown to be an independent poor prognostic factor in 10 different cancer datasets. Depletion of RAI2 protein in luminal breast cancer cell lines resulted in dedifferentiation marked by downregulation of ERα, FOXA1, and GATA3, together with increased invasiveness and activation of AKT signaling. Functional analysis of the previously uncharacterized RAI2 protein revealed molecular interaction with CtBP transcriptional regulators and an overlapping function in controlling the expression of a number of key target genes involved in breast cancer. These results suggest that RAI2 is a new metastasis-associated protein that sustains differentiation of luminal breast epithelial cells. SIGNIFICANCE: We identified downregulation of RAI2 as a novel metastasis-associated genetic alteration especially associated with early occurring bone metastasis in ERα-positive breast tumors. We specified the role of the RAI2 protein to function as a transcriptional regulator that controls the expression of several key regulators of breast epithelial integrity and cancer.
Assuntos
Medula Óssea/patologia , Neoplasias da Mama/genética , Neoplasias da Mama/patologia , Células Neoplásicas Circulantes/patologia , Proteínas/genética , Oxirredutases do Álcool/metabolismo , Sequência de Aminoácidos , Neoplasias da Mama/mortalidade , Linhagem Celular Tumoral , Análise por Conglomerados , Proteínas de Ligação a DNA/metabolismo , Feminino , Expressão Gênica , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Peptídeos e Proteínas de Sinalização Intercelular , Dados de Sequência Molecular , Prognóstico , Ligação Proteica , Domínios e Motivos de Interação entre Proteínas , Proteínas/química , Proteínas/metabolismo , Reprodutibilidade dos Testes , Alinhamento de Sequência , TranscriptomaRESUMO
The estrogen receptor α (ERα) controls cell proliferation and tumorigenesis by recruiting various cofactors to estrogen response elements (EREs) to control gene transcription. A deeper understanding of these transcriptional mechanisms may uncover therapeutic targets for ERα-dependent cancers. We show that BRD4 regulates ERα-induced gene expression by affecting elongation-associated phosphorylation of RNA polymerase II (RNAPII) and histone H2B monoubiquitination. Consistently, BRD4 activity is required for proliferation of ER(+) breast and endometrial cancer cells and uterine growth in mice. Genome-wide studies revealed an enrichment of BRD4 on transcriptional start sites of active genes and a requirement of BRD4 for H2B monoubiquitination in the transcribed region of estrogen-responsive genes. Importantly, we demonstrate that BRD4 occupancy on distal EREs enriched for H3K27ac is required for recruitment and elongation of RNAPII on EREs and the production of ERα-dependent enhancer RNAs. These results uncover BRD4 as a central regulator of ERα function and potential therapeutic target.
Assuntos
Neoplasias da Mama/metabolismo , Neoplasias do Endométrio/metabolismo , Proteínas Nucleares/metabolismo , Elementos de Resposta , Fatores de Transcrição/metabolismo , Ativação Transcricional , Animais , Proteínas de Ciclo Celular , Proliferação de Células , Receptor alfa de Estrogênio/genética , Receptor alfa de Estrogênio/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Histonas/metabolismo , Células MCF-7 , Camundongos , Camundongos Endogâmicos C57BL , Proteínas Nucleares/genética , RNA Polimerase II/metabolismo , Fatores de Transcrição/genéticaRESUMO
Tumor metastasis is the major cause of mortality and morbidity in most solid cancers. A growing body of evidence suggests that the epithelial-to-mesenchymal transition (EMT) plays a central role during tumor metastasis and frequently imparts a stem cell-like phenotype and therapeutic resistance to tumor cells. The induction of EMT is accompanied by a dynamic reprogramming of the epigenome involving changes in DNA methylation and several post-translational histone modifications. These changes in turn promote the expression of mesenchymal genes or repress those associated with an epithelial phenotype. Importantly, in order for metastatic colonization and the formation of macrometastases to occur, tumor cells frequently undergo a reversal of EMT referred to as the mesenchymal-to-epithelial transition (MET). Thus, a high degree of epigenetic plasticity is required in order to induce and reverse EMT during tumor progression. In this review, we describe various epigenetic regulatory mechanisms employed by tumor cells during EMT and elaborate on the importance of the histone code in controlling both the expression and activity of EMT-associated transcription factors. We propose that a more thorough understanding of the epigenetic mechanisms controlling EMT may provide new opportunities which may be harnessed for improved and individualized cancer therapy based on defined molecular mechanisms.
Assuntos
Epigênese Genética/fisiologia , Transição Epitelial-Mesenquimal/genética , Regulação Neoplásica da Expressão Gênica/fisiologia , Neoplasias/genética , Animais , HumanosRESUMO
BACKGROUND: In conjunction with posttranslational chromatin modifications, proper arrangement of higher order chromatin structure appears to be important for controlling transcription in the nucleus. Recent genome-wide studies have shown that the Estrogen Receptor-alpha (ERα), encoded by the ESR1 gene, nucleates tissue-specific long-range chromosomal interactions in collaboration with the cohesin complex. Furthermore, the Mediator complex not only regulates ERα activity, but also interacts with the cohesin complex to facilitate long-range chromosomal interactions. However, whether the cohesin and Mediator complexes function together to contribute to estrogen-regulated gene transcription remains unknown. RESULTS: In this study we show that depletion of the cohesin subunit SMC3 or the Mediator subunit MED12 significantly impairs the ERα-regulated transcriptome. Surprisingly, SMC3 depletion appears to elicit this effect indirectly by rapidly decreasing ESR1 transcription and ERα protein levels. Moreover, we provide evidence that both SMC3 and MED12 colocalize on the ESR1 gene and are mutually required for their own occupancy as well as for RNAPII occupancy across the ESR1 gene. Finally, we show that extended proteasome inhibition decreases the mRNA expression of cohesin subunits which accompanies a decrease in ESR1 mRNA and ERα protein levels as well as estrogen-regulated transcription. CONCLUSIONS: These results identify the ESR1 gene as a cohesin/Mediator-dependent gene and indicate that this regulation may potentially be exploited for the treatment of estrogen-dependent breast cancer.
RESUMO
Murine embryonic stem cells (mESCs) have the potential to differentiate into almost any type of cell, and hence, represent a useful biological resource for tissue engineering. The differentiation of mESCs into osteoblasts in vitro is usually dampened by simultaneous differentiation of adipocytes. Insulin exerts a profound effect on bone development through increased differentiation of osteoblasts and concurrent formation of adipocytes. Comparatively, Sirt1, which plays a crucial role in osteoblast differentiation, has been reported to down regulate adipocyte formation during osteoblast differentiation. This study analyzed the combined effects of insulin and Sirt1 on the differentiation of osteoblasts. Osteoblast differentiation was quantified by estimating the accumulation of mineralized matrix and expression of osteogenic genes. The present data show that the simultaneous action of the insulin and Sirt1-mediated pathways increased the efficiency of osteoblast differentiation. When the cells were tested for ALP activity and Alizarin red staining, there was a respective increase of ~180% and ~166% (P<0.05) compared to the control. Furthermore, the mRNA expression patterns of osteoprotegerin, osterix, runx2, and osteopontin were increased by 3.6, 2.3, 1.8, and 1.7-fold, respectively, with a concomitant decrease in the mRNA expression levels of adipocyte marker genes. Interestingly, blocking the effects of both Sirt1 and insulin resulted in decreased osteoblastogenesis (60%) and subsequent increased adipocyte differentiation (195%) (P<0.05). Moreover, immunoblotting analysis demonstrated that this activation was via an Akt-dependent pathway. In conclusion, the present data suggests an enhanced process of osteoblast differentiation that can be exploited further to improve mESC differentiation.