Targeting Epigenetic Regulation of miR-34a for Treatment of Pancreatic Cancer by Inhibition of Pancreatic Cancer Stem Cells

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-2'-deoxycytidine (5-Aza-dC) and HDAC inhibitor Vorinostat (SAHA).Re-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, p21(WAF1), p27(KIP1) 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.The 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

Deletion of the mitochondrial flavoprotein apoptosis inducing factor (AIF) induces beta-cell apoptosis and impairs beta-cell mass.

BACKGROUND:Apoptosis is a hallmark of beta-cell death in both type 1 and type 2 diabetes mellitus. Understanding how apoptosis contributes to beta-cell turnover may lead to strategies to prevent progression of diabetes. A key mediator of apoptosis, mitochondrial function, and cell survival is apoptosis inducing factor (AIF). In the present study, we investigated the role of AIF on beta-cell mass and survival using the Harlequin (Hq) mutant mice, which are hypomorphic for AIF. METHODOLOGY/PRINCIPAL FINDINGS:Immunohistochemical evaluation of pancreata from Hq mutant mice displayed much smaller islets compared to wild-type mice (WT). Analysis of beta-cell mass in these mice revealed a greater than 4-fold reduction in beta-cell mass together with an 8-fold increase in beta-cell apoptosis. Analysis of cell cycle dynamics, using BrdU pulse as a marker for cells in S-phase, did not detect significant differences in the frequency of beta-cells in S-phase. In contrast, double staining for phosphorylated Histone H3 and insulin showed a 3-fold increase in beta-cells in the G2 phase in Hq mutant mice, but no differences in M-phase compared to WT mice. This suggests that the beta-cells from Hq mutant mice are arrested in the G2 phase and are unlikely to complete the cell cycle. beta-cells from Hq mutant mice display increased sensitivity to hydrogen peroxide-induced apoptosis, which was confirmed in human islets in which AIF was depleted by siRNA. AIF deficiency had no effect on glucose stimulated insulin secretion, but the impaired effect of hydrogen peroxide on beta-cell function was potentiated. CONCLUSIONS/SIGNIFICANCE:Our results indicate that AIF is essential for maintaining beta-cell mass and for oxidative stress response. A decrease in the oxidative phosphorylation capacity may counteract the development of diabetes, despite its deleterious effects on beta-cell survival

Decreased β-cell mass in Hq mutant mice.

<p>(A). Representative Western blots (panel 1,3) and PCR analyzes (panel 2,4) of AIF expression in isolated mouse (panel 1,2) and human (panel 3,4) islets. Actin was used as loading control/ house keeping gene. Western blots/ PCRs are representatives of three independent experiments from three mice or from 3 organ donors, respectively. (B) Histological analysis by insulin staining in green and glucagon staining in red show a normal islet cellular composition and smaller islets in 2-week-old <i>Hq</i> mutant mice. (C,D) Analysis of β-cell mass (C) or β-cell mass divided by body weight (D) of WT and <i>Hq</i> mutant mice at 2 and 9 weeks of age. Values are representative of 5 slides spanning the whole pancreas of each mouse and 4 mice for each group at each age (magnification x125). (E) Cell cycle characteristics of β-cells from WT mice and <i>Hq</i> mutant mice as measured by BrdU and pHH3 staining. BrdU⁺insulin⁺ cells are counted as β-cells at S phase (see example in F, upper panel). pHH3⁺ (with punctuated pattern) insulin+ cells are counted as β-cells at G2 phase (see example in F, Hq mice lower right panel). pHH3⁺ (with strong nuclear expression) insulin+ cells were counted as β-cells at M phase (see example in F, WT mice lower left panel). Data are shown as mean±SE. *<i>P</i><0.05 in <i>Hq</i> mutant mice <i>vs</i>. WT mice.</p

AIF depletion leads to increased β-cell apoptosis in human islets without affecting insulin secretion.

<p>(A) Isolated human pancreatic islets were exposed to siRNA to AIF (siAIF) or scrambled control siRNA (siScr) for 3 days. The knockdown efficiency was determined by Western blot analysis. Actin was used as a loading control on the same membrane after stripping. This Western blot is representative of three independent experiments from three different organ donors. The density of expression levels were quantified after scanning and normalised to actin levels. (B–D) 3 days after transfection, islets were exposed to 50 µM H₂O₂ for 2 h. (B) Islet sections were prepared for analysis of β-cell apoptosis by the TUNEL assay. Islets were double-stained for insulin in green and counterstained for DAPI in blue. Results are means±SE of the percentage of TUNEL-positive β-cells. The mean number of β-cells counted was 3400 for each treatment condition. (C,D) GSIS: after the H₂O₂ treatment, islets were washed and basal and stimulated insulin secretion analyzed during successive 1-h incubations at 2.8 mM (basal) and 16.7 mM (stimulated) glucose. Data are normalized to insulin content. (D) Stimulatory index denotes the ratio between stimulated and basal values of insulin secretion. (B–D) All assays were performed in triplicate or quadruplicate in three independent experiments from 3 different organ donors, respectively. *p<0.05 to siScr control, **<i>p</i><0.05 in H₂O₂ treated vs. untreated control.</p