A triple action CDK4/6-PI3K-BET inhibitor with augmented cancer cell cytotoxicity

National Institutes of Health GM125195, GM135671, CA192656, FD00511

Regulation of Cell Proliferation and Migration by miR-203 via GAS41/miR-10b Axis in Human Glioblastoma Cells

<div><p>Glioma amplified sequence 41(GAS41) is a potent transcription factor that play a crucial role in cell proliferation and survival. In glioblastoma, the expression of GAS41 at both transcriptional and post transcriptional level needs to be tightly maintained in response to cellular signals. Micro RNAs (miRNA) are small non coding RNA that act as important regulators for modulating the expression of various target genes. Studies have shown that several miRNAs play role in the post-transcriptional regulation of GAS41. Here we identified GAS41 as a novel target for endogenous miR-203 and demonstrate an inverse correlation of miR-203 expression with GAS41 in glioma cell lines (HNGC2 and U87). Over expression of miR-203 negatively regulates GAS41 expression in U87 and HNGC2 cell lines. Moreover, miR-203 restrained miR-10b action by suppressing GAS41. GAS41 is essential for repressing p53 in tumor suppressor pathway during cell proliferation. Enforced expression of GAS41 produced contradictory effect on miR-203 but was able to enhance p53 tumor suppressor pathway associated protein. It was also found that miR-203 maintains the stability of p53 as knock down of p53 expression using siRNA resulted in down regulation of pri-miR and mature miR-203 expression. Conversely reconstitution of miR-203 expression induced apoptosis and inhibited migratory property of glioma cells. Taken together, we show that miR-203 is a key negative regulator of GAS41 and acts as tumor suppressor microRNA in glioma.</p></div

miR-203 dispensable for p53 stabilization.

<p><b>(A)</b> Co immunoprecipitation studies using antip53 antibody in HNGC2 and U87 cells transfected with empty vector and miR-203 construct for 24 hr.The p53/MDM2 complex and the input was detected by westernblot. <b>(B)</b> Real time PCR analysis of p53 mRNA expression in HNGC2 and U87 cells transfected with p53 siRNA (p53si) and scrambled siRNA(scrsi) as control. GAPDH used for normalization. Bars represent RQ (2^-ddct) of p53 expression normalized with GAPDH. ***represent the significant lable with p value <0.001 vs control.All experiments were conducted for at least three times. <b>(C)</b>Western blot analysis showing the level of p53 expression in HNGC2 and U87 cells transiently transfected with scrambled siRNA and p53 siRNA. β-actin <b>is</b> used as gel loading control <b>(D)</b> Quantitative PCR analysis of pri-miR-203 and mature miR-203 in cells introduced with scrambled siRNA (scrsi) and p53 siRNA (p53 si). Bars represent RQ (2^-ddct) of pri-miR-203 and mature miR-203 expression normalized with U6. *** represents p value <0.001,**represent p value< 0.01,*represent p value <0.05. All the above experiments were performed in triplicates</p

miR-203 regulate p21 promoter activity.

<p><b>(A)</b> Luciferase assay showing p21FL promoter activity in HNGC2 and U87 cell line transfected with PGL3vector, p21FL promoter and p21FL (full length) plus miR-203. Bars represent relative luciferase activity normalized to β-gal.* represents p value <0.05, ** represents p value <0.01, *** represents p value <0.001 vs. PGL3 and p21FL. <b>(B)</b> Luciferase assay showing p21 promoter activity in HNGC2 and U87 cells transfected with miR-203 plus GAS41 and miR-203 plus GAS41 siRNA (GAS41si). Bars represent relative luciferase activity normalized to β-gal.* represent p value <0.05, ** represents p value <0.01,***p<0.001 vs. PGL3 and p21FL.ns represent non significant. <b>(C)</b> Luciferase assay showing p21 promoter activityin HNGC2 and U87 cells transiently transfected with -2500 to -1400 region containing p53 RES with miR-203. Bars determine relative luciferase activity normalized toβ-gal from three independent experiment.* represents p value <0.05,** represents p value <0.01. ns denotes non significant. <b>(D)</b> Luciferase assay showing p21 promoter activityin HNGC2 and U87 cells transiently transfected with p53RES2 (-1400 to +100) region along with miR-203. Bars represent relative luciferase activity normalized to β-gal from three separate experiments. ** represent p value <0.01 vs. PGL3 vector. <b>(E)</b> Schematic representation of p21FL (full length) promoter (spanning +1 to -2500kb) and two deletion construct from (-2500bp to -1400bp and -1400 to +100) cloned in PGL3 basic vector.</p

miR-203 induces apoptosis in glioblastoma cells.

<p><b>(A)</b>Western blot analysis showing relative expression of proteins related to apoptosis (Bax, Bcl-2, Cyto-C, Caspase-3) in HNGC2 and U87 cells transfected with either empty vector or miR-203 over expressed plasmid.Beta actin served as housekeeping gel loading control. Bars represent the average of three independent experiments with SD. <b>(B)</b> Tunnel assay performed in HNGC2 and U87 cells transfected with miR-203 or empty vector showing a clear occurrence of apoptosis. <b>(C)</b> Caspase 3 activity in HNGC2 and U87 cells transfected with empty vector or miR-203 over expressing plasmid. Bars represent the average of three independent experiments with SD.</p

miR-203 down regulates GAS41 expression in glioblastoma cell.

<p><b>(A)</b> Real time PCR studies showing the expression of GAS41 in HNGC2 and U87 cells transfected with miR-203. GAPDH is used as loading control in the experiment.Transfection of both the cell lines with empty vector (EV) served as control. Bars represent relative GAS41 mRNA expression normalized to GAPDH from same sample. ** represents p value<0.01 vs empty vector transfected control. <b>(B)</b> Western blots showing expression of GAS41 protein in HNGC2 and U87 cells transfected with miR-203 mimics. <b>(C)</b> Immunofluorescence studies showing the expression and localization of GAS41 in HNGC2 and U87 cells transfected with miR-203. Scale bars represent 5μm. <b>(D)</b> Quantitative RT-PCR studies showing expression of miR-203 in HNGC2 and U87 cells transfected with antimiR-203. *** represents p value <0.001, ** represents p value <0.01vs control. <b>(E)</b> Quantitative RT-PCR studies showing expression of GAS41 after exposure to anti-miR-203 in HNGC2 and U87 cells. ** represents p value <b><</b>0.01vs control <b>(F)</b> Quantitative Real time PCR studies showing expression of miR-203 in HNGC2 and U87 cells upon over expression of GAS41.** represents p value <0.01 and *** represents p value <0.001 vs EV (cells transfected with pCMVTag1 vector). All the above experiments were performed in triplicates.</p

GAS41 mediates suppression of miR-10b through miR-203.

<p><b>(A)</b> Quantitative Real time PCR analysis showing the expression of miR-10b in HNGC2 and U87 cells transfected with miR-203 plasmid. Bars represent relative miR-10b expression normalized toU6 snRNA. ** represents p value <0.01, *** represents p value <0.001 vs empty vector (EV). <b>(B)</b> Quantitative RT-PCR analysis showing the expression of miR-10b in HNGC2 and U87 cells transfected with scrambled siRNA and GAS41 siRNA (GAS41si). U6sn RNA is used for normalization. ** represents p value <0.01, *** represents p value <0.001vs scrambled siRNA. <b>(C)</b> qRT-PCR analysis showing expression of miR-10b in HNGC2 and U87 cells transfected with constructs carrying over expressed GAS41.* represents p value <0.05, ** represents p value <0.01 vs. EV (empty vector). All the above experiments were performed in triplicates</p

miR-203 mediated GAS41 Regulation.

<p><b>(A)</b> Schematic representation depicting the cloning scheme of 3’-UTR of GAS41 (wild type and mutant) into luciferase ORF of psiCHECK2 vector. <b>(B & C)</b> Reporter Luciferase expression in HNGC2 and U87 cells co transfected with miR-203 and 3’UTR of wild type (WT) GAS41 compared to cells co transfected with miR-203 and 3’UTR of mutant GAS41 (MT). U87 and HNGC2 cells co transfected with blank vector or negative control miRNA (NC) and 3’-UTR of wild type or mutant GAS41 served as control. *** represents p value <0.001 vs. control.</p

miR-203 induces p53 pathway by targeting GAS41.

<p><b>(A)</b> Western blots analysis showing the expression of proteins related to p53pathway (p53, P-p53, p21, p14, Mdm2) in HNGC2 and U87 cells transfected with mir-203. Bars represent relative protein expression normalized to β-actin (housekeeping gene as gel loading control) from same sample with SD. <b>(B)</b> Western blots analysis showing the expression of proteins related to p53pathway (p53, P-p53, p21, p14, Mdm2) in HNGC2 and U87 cells transfected withscrambled siRNA and GAS41 siRNA (GAS41si). Bars represent relative protein expression normalized to β-actin (housekeeping gene as gel loading control) from same sample with SD. <b>(C)</b> Western blots analysis showing the expression of proteins related to p53pathway (p53, P-p53, p21, p14, Mdm2) in HNGC2 and U87 cells transfected with empty vector (EV) and upon over expression of GAS41 (GAS41 OVX). Bars represent relative protein expression normalized to β-actin (housekeeping gene as gel loading control) from same sample with SD.</p