miRNA-143 and miRNA-145 modulation in colon cancer : from anticancer activity to drug sensitization

Tese de doutoramento, Farmácia (Biologia Celular e Molecular), Universidade de Lisboa, Faculdade de Farmácia, 2017Overall survival of patients with colon cancer has slightly increased over the past 15 years due to the introduction of novel therapies and screening tests in clinical practice. However, mortality from colon cancer remains high, being the fourth most common cause of cancer-related death worldwide. Although most patients are initially responsive to several chemotherapy regimens, the effectiveness of the current systemic therapies in patients with advanced colon cancer remains limited by drug resistance. Therefore, increasing drug sensitivity is a key step towards more effective therapeutic strategies to treat cancer patients. Recent discoveries have revealed that microRNAs (miRNAs/miRs) hold great promise to render tumours responsive to chemotherapy and targeted therapy. In fact, miRNA expression profiles in tumour cells have been shown to promote anti-cancer therapy resistance, by abnormally regulating the expression of genes involved in cellular processes, such as apoptotic responses and redox-mediated mechanisms. Therefore, the studies included in the present thesis were conducted with the purpose of further characterizing the molecular mechanisms by which the tumour suppressors miR-143 and miR-145 regulate anticancer activity and drug sensitization of colon cancer cells. In initial studies, we evaluated the signalling pathways and players modulated by miR-143 and miR-145 expression in colon cancer cells. Functional proteomic analysis revealed that miR-143 and miR-145 modulate several proteins associated with regulation of oxidative stress, cell death and protein folding. In particular, the antioxidant enzyme SOD1 was among the identified proteins whose steady state levels were downregulated by either miR-143 or miR-145. In addition, forced miR-143 expression strongly increased endogenous production of reactive oxygen species (ROS) production, which was completely abrogated by re-introduction of SOD1. Importantly, miR-143 overexpression increased oxaliplatin-induced apoptosis associated with ROS generation, which was abrogated by genetic and pharmacological inhibition of oxidative stress. Therefore, our data provide evidence to support a role for miR-143-induced ROS in mediating cell sensitivity to oxaliplatin. Next, considering the involvement of miR-143 and miR-145 in the EGFR/RAS/MAPK signalling network, we hypothesized that miR-143 and miR-145 play a role in the regulation of colon cancer cellular sensitivity to the anti-EGFR antibody, cetuximab. Our data show that overexpression of miR-143 and miR-145 reduced cell proliferation and migration, sensitized both mutant and wild-type KRAS cells to cetuximab, and strongly potentiated the antitumour effects of cetuximab-mediated antibody-dependent cellular cytotoxicity (ADCC). These changes were associated with increased apoptosis and caspase-3/7 activity, and reduced Bcl-2 protein expression. Interestingly, we observed that the mechanism underlying cetuximab-mediated ADCC proceeds through a granzyme Bdependent pathway, as granzyme B inhibition abrogated cell death induced by cetuximabmediated ADCC, as well as caspase-3/7 activity. These results further suggest that miR-143 and miR-145 re-sensitize colon cancer cells to cetuximab by stimulating cetuximabdependent ADCC to induce cell death. Finally, we suggest that miR-145 inhibits cancer stem-like cell characteristics, including colon sphere formation and propagation, as well as the expression of specific stem cell markers. Notably, augmented levels of miR-145 in colon cancer stem-like cells increased a panel of well-established differentiation markers, suggesting that miR-145 may contribute to the induction of colon cancer stem-like cell differentiation. In addition, miR-145 reversed chemoresistance by reducing cell viability of colon spheres exposed to 5- fluorouracil-based chemotherapeutic regimens, and undergoing efficient apoptosis elicited by chemotherapy. Overall, our findings underscore the importance of miR-143 and miR-145 in colon cancer, expanding their roles as pro-apoptotic and chemosensitizer components. miR-145 additionally regulates colon cancer stem cells. Importantly, replenishing such tumour suppressor miRNAs in combination with anti-cancer therapy should be regarded and further studied as potential therapeutic options for colon cancer.Fundação para a Ciência e a Tecnologia (FCT), SAICTPAC/0019/201