Exploring a new approach to target KRAS oncogene

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Targeting mutant KRAS in human colon cancer cells through G-quadruplex ligands

Publication . Brito, Hugo Miguel de Sousa e; Borralho, Pedro M.; Rodrigues, Cecília M. P.

KRAS mutations occur in 35 to 45% of colon tumors, being an important target for cancer treatment, for which there are no targeted therapeutics available. The objectives of this thesis were to downregulate transcription of mutant KRAS using DNA G-quadruplex ligands, and to evaluate their impact on proliferation and death of colon cancer (CC) cells. Ligands evaluated included ten indoloquinolines derivative compounds, seven iQc (3e, 3g, 3h, 3m, 13a, 14a, 3c) and three iQb (2a, 1a and 2d). These test compounds were synthesized and evaluated for preferential selectivity to the G-quadruplex region in human KRAS oncogene promoter (KRas21R), compared to a double-stranded hairpin loop sequence (T-Loop), by fluorescence resonance energy transfer (FRET) melting experiments. Effects on cell viability and death were evaluated using mutant KRAS CC cell lines (HCT116 and SW620) and non-malignant cells with wild-type KRAS (HEK293T and CCD18co). For most test compounds, we obtained IC50 and IC65 lower than those for 5-Fluorouracil (5-FU). Interestingly, in CC cell lines, particularly in HCT116, test compounds increased cell death, mainly by apoptosis, up to ~ 60% when compared to vehicle control, accompanied by increased p53 protein steady-state levels. Finally, to evaluate the effect of test compounds on KRAS repression, we evaluated KRAS protein and mRNA steady-state levels, obtaining a significant reduction of both KRAS protein and mRNA. Importantly, we next used a Dual-Luciferase Reporter Assay specific for KRAS promoter, to validate repression of KRAS promoter by the DNA G-quadruplex ligand test compounds. Our results showed that all test compounds interact with the KRAS promoter, inducing significant promoter repression. Collectively, our results suggest a higher dependence on KRAS signaling by tumor cells, particularly in HCT116, suggesting that inhibition of KRAS by these ligands could provide a promising novel therapeutic approach for the treatment of tumors with mutant KRAS, warranting additional investigation.

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Entidade financiadora

Fundação para a Ciência e a Tecnologia

Programa de financiamento

3599-PPCDT

Número da atribuição

EXPL/QEQ-MED/0502/2012