MiR-10a Regulation of Drug Response and Cancer Stem Cell Populations in Non-Small Cell Lung Carcinomas
DeSevo, Christopher Gerard
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Phosphatidylinositol 3-kinases (PI3Ks) are enzymes involved in diverse cellular functions including cell growth, proliferation, differentiation, motility, survival and apoptosis. Many of these functions relate to class I PI3Ks, heterodimers composed of regulatory and catalytic subunits that convert extracellular cues to intracellular responses upon activation. Overall, this signaling pathway is under tight regulation and even slight perturbations can lead to aberrant pathway activation. In NSCLC cell lines, we found that manipulation of miR-10a results in significant changes to both mRNA and protein levels of PI3K. In the context of cellular response to front-line chemotherapeutic agents used to treat NSCLC, I uncovered that miR-10a mimic decreases cell viability10-fold in the presence of paclitaxel relative to drug alone, while inhibiting miR-10a results in a 10-fold increase, suggesting that high levels of miR-10a may be predictive of response to such agents. To assess its prognostic value, we interrogated miR-10a expression in NSCLC tumors and found that high miR-10a levels correlate with longer overall patient survival. miRNAs can target hundreds of genes, meaning that miR-10a may regulate PIK3CA expression both directly and indirectly. We identified the transcription factor GATA6 as both a target of miR-10a with a predicted miR-10a target site in its 3’UTR and a regulator of PI3K expression, with several conserved binding sites in the promoter of PIK3CA. These findings demonstrate that miR-10a regulates the PI3K pathway at two distinct levels. Microarray expression profiling of NSCLC cells treated with miR-10a mimic had significant down regulation of ALDH1A3, a marker of cancer stem cells. This relationship was confirmed through functional validation of ALDH activity. Multiple miRNA target prediction algorithms showed that ALDH1A3 is not a direct target of miR-10a. To uncover the direct target of miR-10a we used a targeted siRNA screen containing genes implicated in stem cell maintenance to reveal that the WNT and Notch pathway are important for cell survival. Both pathways are down-regulated when cells are treated with a miR-10a mimic. Bioinformatic analysis identified DVL3 as a miR-10a target gene. Manipulation of miR-10a levels resulted in significant changes in both mRNA and protein levels of DVL3. Finally, loss of DVL3 expression significantly decreased ALDH1A3 protein levels and the population of ALDH+ cells. Collectively, my work has uncovered miR-10a as mediator of the potent PI3K oncogenic pathway through both direct and indirect mechanisms, a modulator of cellular response to paclitaxel and finally its identification in NSCLC stem cell maintenance through regulation of the WNT and NOTCH pathways.