Identification of microRNAs regulated by oncogenic KRAS in lung and pancreatic cancer

Author(s)

Mateus Nóbrega Aoki

• Document type: Doctoral Thesis
• Press: São Paulo.
• Institution: Universidade de São Paulo (USP). Conjunto das Químicas (IQ e FCF) (CQ/DBDCQ)
• Defense date: 2014-07-04

Examining board members

Daniela Sanchez Bassères; Roger Chammas; Silvya Stuchi Maria Engler; Eduardo Moraes Rego Reis; Deborah Schechtman

• Advisor: Daniela Sanchez Bassères

Abstract

KRAS-induced lung cancer is a very common disease, for which there are currently no effective therapies. Direct targeting of KRAS has failed in clinical trials and intense efforts are underway to identify KRAS targets that play a crucial role in oncogenesis. One promising KRAS-regulated pathway that has so far been overlooked is the micro RNA (miRNA) pathway. Our goal was to identify miRNAs regulated by oncogenic KRAS in lung and pancreatic cells that could contribute to the oncogenic phenotype. In order to achieve this goal we used two different approaches: (1) We investigated miRNA 486-5p as a KRAS target in lung and pancreatic cancer. The expression of this miRNA had been correlated to the presence of KRAS mutations in colon cancer patients; (2) we used a microarray platform to identify differentially expressed miRNAs between immortalized human primary pulmonary or pancreatic epithelial cell lines and their isogenic K-Ras-transformed counterparts. In our first approach, we were able to show that mi486-5p expression correlates with KRAS status in lung primary cells, but not in pancreatic primary cells. Furthermore, we generated lung cancer cells with either gain-of-function or loss-of-function of KRAS and demonstrated that KRAS regulates miRNA 486-5p in these cells. We also found, in all lung cell models analyzed, a negative correlation between expression of KRAS and expression of miR-486-5p target FoxO1, a tumor suppressor. In order to evaluate how miR-486-5p affects KRAS-induced oncogenic properties, we transfected miR-486-5p inhibitor oligonucleotides into KRAS-positive lung cancer cell lines. Inhibition of miR-486-5p expression leads to reduced clonogenic growth and viability. This reduction is not associated with increased cell death, but with decreased cell proliferation. Interestingly, transfection of miR-486-5p double-stranded RNA mimic oligonucleotides in to KRAS negative lung cancer cell lines or into cells with loss-of-function of KRAS by RNAi leads to enhanced proliferation and clonogenicity. These results indicate, not only that miR-486-5p is a KRAS target in lung cancer, but also that miR-486-5p acts as an oncomiR contributing to KRAS-induced cell proliferation. In our second approach, we identified 17 upregulated microRNAs and 3 downregulated microRNAs in the primary pancreatic cell line expressing KRAS. Of these, 9 miRNAs were also identified by a metanalysis of published microarray datasets comparing pancreatic cancer patient samples to non-cancerous pancreatic tissues. Even though our array experiment in the primary pulmonary cells did not produce statistically significant results after FDR correction, differential expression trends were seen for many miRNAs and we validated miRNAs 720 and 139-3p as differentially expressed. In conclusion we were able to identify miRNAs regulated by KRAS both in lung and pancreatic cancer cells. Further understanding of their biological function, as well as the targets they regulate in these settings, could uncover novel pathways for therapy design. (AU)

• FAPESP's process: 11/06709-6 - Identification of differentially expressed microRNAs in pulmonary and pancreatic cells transformed by the K-Ras oncogene
• Grantee: Mateus Nóbrega Aoki
• Support Opportunities: Scholarships in Brazil - Doctorate