RESUMO
Ovarian cancer is the most lethal cancer of the female reproductive system. In that regard, several epidemiological studies suggest that long-term exposure to estrogen could increase ovarian cancer risk, although its precise role remains controversial. To decipher a mechanism for this, we previously generated a mathematical model of how estrogen-mediated upregulation of the transcription factor, E2F6, upregulates the ovarian cancer stem/initiating cell marker, c-Kit, by epigenetic silencing the tumor suppressor miR-193a, and a competing endogenous (ceRNA) mechanism. In this study, we tested that previous mathematical model, showing that estrogen treatment of immortalized ovarian surface epithelial cells upregulated both E2F6 and c-KIT, but downregulated miR-193a. Luciferase assays further confirmed that microRNA-193a targets both E2F6 and c-Kit. Interestingly, ChIP-PCR and bisulphite pyrosequencing showed that E2F6 also epigenetically suppresses miR-193a, through recruitment of EZH2, and by a complex ceRNA mechanism in ovarian cancer cell lines. Importantly, cell line and animal experiments both confirmed that E2F6 promotes ovarian cancer stemness, whereas E2F6 or EZH2 depletion derepressed miR-193a, which opposes cancer stemness, by alleviating DNA methylation and repressive chromatin. Finally, 118 ovarian cancer patients with miR-193a promoter hypermethylation had poorer survival than those without hypermethylation. These results suggest that an estrogen-mediated E2F6 ceRNA network epigenetically and competitively inhibits microRNA-193a activity, promoting ovarian cancer stemness and tumorigenesis.
Assuntos
Fator de Transcrição E2F6/genética , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/genética , RNA/genética , Transcrição Gênica/genética , Animais , Linhagem Celular Tumoral , Metilação de DNA/efeitos dos fármacos , Metilação de DNA/genética , Epigênese Genética/efeitos dos fármacos , Epigênese Genética/genética , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/patologia , Estrogênios/efeitos adversos , Feminino , Genes Supressores de Tumor/fisiologia , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Camundongos SCID , MicroRNAs/genética , Células-Tronco Neoplásicas/efeitos dos fármacos , Neoplasias Ovarianas/etiologia , Transcrição Gênica/efeitos dos fármacos , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
In this study, we aim to determine the relationship between methylation level of an inflammatory-related gene, SOCS-1 in serum samples of patients with ankylosing spondylitis (AS) and their degree of inflammation as well as serum cytokine level. Quantitative real time methylation specific PCR was performed to examine the promoter methylation of SOCS-1 in serum samples of 43 HLA-B27+ AS patients and 6 B27+ healthy controls. Degree of inflammation was accessed by spondylopathy, sacroiliitis as well as acute phase reactant, erythrocyte sedimentation rate and C-reactive protein (CRP). Serum IL-6 and TNF-α level was determined by ELISA assay. SOCS-1 methylation can only be found in serums samples from patients but not normal control. Methylation of SOCS-1 significantly associated with severity of patient's spondylopathy (P < 0.005), sacroiliitis (P < 0.005) and acute phase reactant CRP (P = 0.0278). AS patients also exhibited higher serum IL-6 (P < 0.001) and TNF-α level (P < 0.001). Importantly, patients with high serum IL-6 or TNF-α level demonstrated a significantly higher SOCS-1 methylation (P < 0.001). In conclusion, this proof-of-principle study suggested that methylation of SOCS-1 can be detected in serum of HLA-B27+ AS patients but not in B27+ controls. The pathogenic potential of SOCS-1 methylation in AS deserves further investigation.
Assuntos
Metilação de DNA/genética , Interleucina-6/sangue , Espondilite Anquilosante/genética , Proteínas Supressoras da Sinalização de Citocina/genética , Fator de Necrose Tumoral alfa/sangue , Adulto , Idoso , Idoso de 80 Anos ou mais , Epigênese Genética/genética , Feminino , Estudos de Associação Genética , Humanos , Inflamação/sangue , Inflamação/genética , Masculino , Pessoa de Meia-Idade , Regiões Promotoras Genéticas , Espondilite Anquilosante/sangue , Espondilite Anquilosante/patologia , Proteína 1 Supressora da Sinalização de CitocinaRESUMO
Accumulating data indicate that cancer stem cells contribute to tumor chemoresistance and their persistence alters clinical outcome. Our previous study has shown that ovarian cancer may be initiated by ovarian cancer initiating cells (OCIC) characterized by surface antigen CD44 and c-KIT (CD117). It has been experimentally demonstrated that a microRNA, namely miR-193a, targets c-KIT mRNA for degradation and could play a crucial role in ovarian cancer development. How miR-193a is regulated is poorly understood and the emerging picture is complex. To unravel this complexity, we propose a mathematical model to explore how estrogen-mediated up-regulation of another target of miR-193a, namely E2F6, can attenuate the function of miR-193a in two ways, one through a competition of E2F6 and c-KIT transcripts for miR-193a, and second by binding of E2F6 protein, in association with a polycomb complex, to the promoter of miR-193a to down-regulate its transcription. Our model predicts that this bimodal control increases the expression of c-KIT and that the second mode of epigenetic regulation is required to generate a switching behavior in c-KIT and E2F6 expressions. Additional analysis of the TCGA ovarian cancer dataset demonstrates that ovarian cancer patients with low expression of EZH2, a polycomb-group family protein, show positive correlation between E2F6 and c-KIT. We conjecture that a simultaneous EZH2 inhibition and anti-estrogen therapy can constitute an effective combined therapeutic strategy against ovarian cancer.
Assuntos
Epigênese Genética , MicroRNAs/genética , Modelos Genéticos , Células-Tronco Neoplásicas/metabolismo , Células-Tronco Neoplásicas/patologia , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Sequência de Bases , Linhagem Celular Tumoral , Bases de Dados Genéticas , Fator de Transcrição E2F6/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Redes Reguladoras de Genes , Humanos , MicroRNAs/metabolismo , Dados de Sequência Molecular , Proteínas Proto-Oncogênicas c-kit/metabolismo , Reprodutibilidade dos TestesRESUMO
Aberrant TGFß signaling pathway may alter the expression of down-stream targets and promotes ovarian carcinogenesis. However, the mechanism of this impairment is not fully understood. Our previous study has identified RunX1T1 as a putative SMAD4 target in an immortalized ovarian surface epithelial cell line, IOSE. In this study, we report that transcription of RunX1T1 was confirmed to be positively regulated by SMAD4 in IOSE cells and epigenetically silenced in a panel of ovarian cancer cell lines by promoter hypermethylation and histone methylation at H3 lysine 9. SMAD4 depletion increased repressive histone modifications of RunX1T1 promoter without affecting promoter methylation in IOSE cells. Epigenetic treatment can restore RunX1T1 expression by reversing its epigenetic status in MCP3 ovarian cancer cells. When transiently treated with a demethylating agent, the expression of RunX1T1 was partially restored in MCP3 cells, but gradual re-silencing through promoter re-methylation was observed after the treatment. Interestingly, SMAD4 knockdown accelerated this re-silencing process, suggesting that normal TGF-beta signaling is essential for the maintenance of RunX1T1 expression. In vivo analysis confirmed that hypermethylation of RunX1T1 was detected in 35.7% (34/95) of ovarian tumors with high clinical stages (P=0.035) and in 83% (5/6) of primary ovarian cancer-initiating cells. Additionally, concurrent methylation of RunX1T1 and another SMAD4 target, FBXO32 which was previously found to be hypermethylated in ovarian cancer was observed in this same sample cohort (P< 0.05). Restoration of RunX1T1 inhibited cancer cell growth. Taken together, dysregulated TGFß/SMAD4 signaling may lead to epigenetic silencing of a putative tumor suppressor, RunX1T1, during ovarian carcinogenesis.
Assuntos
Epigênese Genética , Neoplasias Ovarianas/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Transdução de Sinais , Fatores de Transcrição/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Linhagem Celular Tumoral , Metilação de DNA , Feminino , Inativação Gênica , Histonas/metabolismo , Humanos , Estadiamento de Neoplasias , Neoplasias Ovarianas/genética , Neoplasias Ovarianas/patologia , Regiões Promotoras Genéticas , Proteínas Proto-Oncogênicas/genética , Proteína 1 Parceira de Translocação de RUNX1 , Fatores de Transcrição/genética , Fator de Crescimento Transformador beta/genéticaRESUMO
BACKGROUND: Bladder cancer is the sixth most common cancer in the world and the incidence is particularly high in southwestern Taiwan. Previous studies have identified several tumor-related genes that are hypermethylated in bladder cancer; however the DNA methylation profile of bladder cancer in Taiwan is not fully understood. METHODS: In this study, we compared the DNA methylation profile of multiple tumor suppressor genes (APC, DAPK, E-cadherin, hMLH1, IRF8, p14, p15, RASSF1A, SFRP1 and SOCS-1) in bladder cancer patients from different Chinese sub-populations including Taiwan (104 cases), Hong Kong (82 cases) and China (24 cases) by MSP. Two normal human urothelium were also included as control. To investigate the diagnostic potential of using DNA methylation in non-invasive detection of bladder cancer, degree of methylation of DAPK, IRF8, p14, RASSF1A and SFRP1 was also accessed by quantitative MSP in urine samples from thirty bladder cancer patients and nineteen non-cancer controls. RESULTS: There were distinct DNA methylation epigenotypes among the different sub-populations. Further, samples from Taiwan and China demonstrated a bimodal distribution suggesting that CpG island methylator phentotype (CIMP) is presented in bladder cancer. Moreover, the number of methylated genes in samples from Taiwan and Hong Kong were significantly correlated with histological grade (P < 0.01) and pathological stage (P < 0.01). Regarding the samples from Taiwan, methylation of SFRP1, IRF8, APC and RASSF1A were significantly associated with increased tumor grade, stage. Methylation of RASSF1A was associated with tumor recurrence. Patients with methylation of APC or RASSF1A were also significantly associated with shorter recurrence-free survival. For methylation detection in voided urine samples of cancer patients, the sensitivity and specificity of using any of the methylated genes (IRF8, p14 or sFRP1) by qMSP was 86.7% and 94.7%. CONCLUSIONS: Our results indicate that there are distinct methylation epigenotypes among different Chinese sub-populations. These profiles demonstrate gradual increases with cancer progression. Finally, detection of gene methylation in voided urine with these distinct DNA methylation markers is more sensitive than urine cytology.