Abstract
BackgroundMicroRNA-34a (miR-34a) is a transcriptional target of p53 and is down-regulated in pancreatic cancer. This study aimed to investigate the functional significance of miR-34a in pancreatic cancer progression through its epigenetic restoration with chromatin modulators, demethylating agent 5-Aza-2′-deoxycytidine (5-Aza-dC) and HDAC inhibitor Vorinostat (SAHA).Methodology/Principal FindingsRe-expression of miR-34a in human pancreatic cancer stem cells (CSCs) and in human pancreatic cancer cell lines upon treatment with 5-Aza-dC and SAHA strongly inhibited the cell proliferation, cell cycle progression, self-renewal, epithelial to mesenchymal transition (EMT) and invasion. In pancreatic CSCs, modulation of miR-34a induced apoptosis by activating caspase-3/7. Treatment of pancreatic CSCs with the chromatin-modulating agents resulted in the inhibition of Bcl-2, CDK6 and SIRT1, which are the putative targets of miR-34a. MiR-34a upregulation by these agents also induced acetylated p53, p21WAF1, p27KIP1 and PUMA in pancreatic CSCs. Inhibition of miR-34a by antagomiR abrogates the effects of 5-Aza-dC and SAHA, suggesting that 5-Aza-dC and SAHA regulate stem cell characteristics through miR-34a. In CSCs, SAHA inhibited Notch pathway, suggesting its suppression may contribute to inhibition of the self-renewal capacity and induction of apoptosis. Interestingly, treatment of pancreatic CSCs with SAHA resulted in the inhibition of EMT with the transcriptional up-regulation of E-Cadherin and down-regulation of N-Cadherin. Expression of EMT inducers (Zeb-1, Snail and Slug) was inhibited in CSCs upon treatment with SAHA. 5-Aza-dC and SAHA also retard in vitro migration and invasion of CSCs.ConclusionsThe present study thus demonstrates the role of miR-34a as a critical regulator of pancreatic cancer progression by the regulating CSC characteristics. The restoration of its expression by 5-Aza-dC and SAHA in CSCs will not only provide mechanistic insight and therapeutic targets for pancreatic cancer but also promising reagents to boost patient response to existing chemotherapies or as a standalone cancer drug by eliminating the CSC characteristics.
Highlights
Pancreatic cancer is the fourth leading cause of cancer death in United States and is a devastating invasive disease and one of the most aggressive cancers [1]
The present study demonstrates the role of miR-34a as a critical regulator of pancreatic cancer progression by the regulating cancer stem cells (CSCs) characteristics
We first measured the expression of miR-34a in human pancreatic CSCs derived from primary tumors and pancreatic cancer cell lines [ASPC-1 (p53wt) and MiaPaCa-2 (p53mutant)] and compared with normal pancreatic ductal epithelial cells using quantitative reverse transcriptase polymerase chain reaction (RTPCR-Taqman) and Taqman Real Time Assays
Summary
Pancreatic cancer is the fourth leading cause of cancer death in United States and is a devastating invasive disease and one of the most aggressive cancers [1]. MicroRNAs (miRNAs) are endogenous, noncoding small RNAs 19–25 nucleotides in length, which are recognized as crucial post transcriptional regulators of gene expression [2,3,4]. The ability of miRNAs to regulate multiple genes conform them to play important roles in biological processes that effect tumor progression including migration, invasion, epithelial to mesenchymal transition (EMT) and metastasis [5,6,7,8,9,10]. MiRNAs are very promising as early biomarkers, prognostic indicators and mechanism based therapeutic targets for anticancer treatments [11,12,13,14,15] because their aberrant expressions are linked to cancer stem cell (CSC) deregulation and oncogenesis [16]. MicroRNA-34a (miR-34a) is a transcriptional target of p53 and is down-regulated in pancreatic cancer. This study aimed to investigate the functional significance of miR-34a in pancreatic cancer progression through its epigenetic restoration with chromatin modulators, demethylating agent 5-Aza-29-deoxycytidine (5-Aza-dC) and HDAC inhibitor Vorinostat (SAHA)
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