RESUMO
Membrane type-1 matrix metalloproteinase (MT1-MMP) is often activated and expressed in tumor cells with significant invasive properties, and is associated with poor prognosis of patients. This could partly be due to deregulated expression of microRNAs (miRNAs) which regulates the expression of MT1-MMP and PTEN (phosphatase and tensin homolog) contributing to tumor invasion and metastasis. We initially compared the expression profile of miR-200 family, PTEN and MT1-MMP expression in six pancreatic cancer (PC) cell lines by qRT-PCR and western blot analysis. We found loss of expression of miR-200a, b and c in chemo-resistant PC cell lines, which was correlated with loss of PTEN and over-expression of MT1-MMP. Based on our initial findings, we chose BxPC-3, MIAPaCa-2 and MIAPaCa-2-GR cells for further mechanistic studies We assessed the effect of two separate novel agents CDF (a synthetic analog of curcumin) and BR-DIM (a natural agent) on PC cells. The expression of miR-200 family and PTEN was significantly re-expressed whereas the expression of MT1-MMP was down-regulated by CDF and BR-DIM treatment. Forced over-expression or silencing of miR-200c, followed by either CDF or BR-DIM treatment of MIAPaCa-2 cells, altered the morphology of cells, wound-healing capacity, colony formation and the expression of MT1-MMP and PTEN. These results provide strong experimental evidence showing that the loss of miR-200 family and PTEN expression and increased level of MT1-MMP leads to aggressive behavior of PC cells, which could be attenuated through re-expression of miR-200c by CDF and/or BR-DIM treatment, suggesting that these agents could be useful for PC treatment.
Assuntos
Metaloproteinase 1 da Matriz/metabolismo , MicroRNAs/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias Pancreáticas/metabolismo , Humanos , Neoplasias Pancreáticas/enzimologia , Neoplasias Pancreáticas/patologiaRESUMO
Human pancreatic cancer (PC) is an aggressive disease, which has been recapitulated in transgenic animal model that provides unique opportunity for mechanistic understanding of disease progression and also for testing the efficacy of novel therapeutics. Emerging evidence suggests deregulated expression of microRNAs (miRNAs) in human PC, and thus we investigated the expression of miRNAs in pancreas tissues obtained from transgenic mouse models of K-Ras (K), Pdx1-Cre (C), K-Ras;Pdx1-Cre (KC), and K-Ras;Pdx1-Cre;INK4a/Arf (KCI), initially from pooled RNA samples using miRNA profiling, and further confirmed in individual specimens by quantitative RT-PCR. We found over-expression of miR-21, miR-221, miR-27a, miR-27b, and miR-155, and down-regulation of miR-216a, miR-216b, miR-217, and miR-146a expression in tumors derived from KC and KCI mouse model, which was consistent with data from KCI-derived RInk-1 cells. Mechanistic investigations revealed a significant induction of EGFR, K-Ras, and MT1-MMP protein expression in tissues from both KC and KCI mouse compared to tissues from K or C, and these results were consistent with similar findings in RInk-1 cells compared to human MIAPaCa-2 cells. Furthermore, miR-155 knock-down in RInk-1 cells resulted in the inhibition of cell growth and colony formation consistent with down-regulation of EGFR, MT1-MMP, and K-Ras expression. In addition, miR-216b which target Ras, and forced re-expression of miR-216b in RInk-1 cells showed inhibition of cell proliferation and colony formation, which was correlated with reduced expression of Ras, EGFR, and MT1-MMP. These findings suggest that these models would be useful for preclinical evaluation of novel miRNA-targeted agents for designing personalized therapy for PC.
Assuntos
Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Regulação para Baixo/genética , Genes ras , MicroRNAs/genética , Neoplasias Pancreáticas/genética , Animais , Linhagem Celular , Proliferação de Células , Inibidor p16 de Quinase Dependente de Ciclina/genética , Receptores ErbB/genética , Receptores ErbB/metabolismo , Regulação Neoplásica da Expressão Gênica/genética , Humanos , Metaloproteinase 14 da Matriz/genética , Metaloproteinase 14 da Matriz/metabolismo , Camundongos , Camundongos Transgênicos/genética , Camundongos Transgênicos/metabolismo , MicroRNAs/metabolismo , Pâncreas/metabolismo , Neoplasias Pancreáticas/metabolismo , Células-Tronco/metabolismoRESUMO
The histone methyltransferase EZH2 is a central epigenetic regulator of cell survival, proliferation, and cancer stem cell (CSC) function. EZH2 expression is increased in various human cancers, including highly aggressive pancreatic cancers, but the mechanisms underlying for its biologic effects are not yet well understood. In this study, we probed EZH2 function in pancreatic cancer using diflourinated-curcumin (CDF), a novel analogue of the turmeric spice component curcumin that has antioxidant properties. CDF decreased pancreatic cancer cell survival, clonogenicity, formation of pancreatospheres, invasive cell migration, and CSC function in human pancreatic cancer cells. These effects were associated with decreased expression of EZH2 and increased expression of a panel of tumor-suppressive microRNAs (miRNA), including let-7a, b, c, d, miR-26a, miR-101, miR-146a, andmiR-200b, c that are typically lost in pancreatic cancer. Mechanistic investigations revealed that reexpression of miR-101 was sufficient to limit the expression of EZH2 and the proinvasive cell surface adhesion molecule EpCAM. In an orthotopic xenograft model of human pancreatic cancer, administration of CDF inhibited tumor growth in a manner associated with reduced expression of EZH2, Notch-1, CD44, EpCAM, and Nanog and increased expression of let-7, miR-26a, and miR-101. Taken together, our results indicated that CDF inhibited pancreatic cancer tumor growth and aggressiveness by targeting an EZH2-miRNA regulatory circuit for epigenetically controlled gene expression.