Targeting replication stress response using polypurine reverse hoogsteen hairpins directed against WEE1 and CHK1 genes in human cancer cells

Abstract

In response to DNA damage, cell cycle checkpoints produce cell cycle arrest to repair and maintain genomic integrity. Due to the high rates of replication and genetic abnormalities, cancer cells are dependent on replication stress response (RSR) and inhibitors of this pathway are being studied as an anticancer approach. In this direction, we investigated the inhibition of CHK1 and WEE1, key components of RSR, using Polypurine Reverse Hoogsteen hairpins (PPRHs) as gene silencing tool. PPRHs designed against WEE1 or CHK1 reduced the viability of different cancer cell lines and showed an increase of apoptosis in HeLa cells. The effect of the PPRHs on cell viability were dose- and time-dependent in HeLa cells. Both the levels of mRNA and protein for each gene were decreased after treatment with the PPRHs. When analyzing the levels of the two CHK1 mRNA splicing variants, CHK1 and CHK1-S, there was a proportional decrease of the two forms, thus maintaining the same expression ratio. PPRHs targeting WEE1 and CHK1 also proved to disrupt cell cycle after 15 h of treatment. Moreover, PPRHs showed a synergy effect when combined with DNA damaging agents, such as methotrexate or 5-Fluorouracil, widely used in clinical practice. This work validates in vitro the usage of PPRHs as a silencing tool against the RSR genes WEE1 and CHK1 and corroborates the potential of inhibiting these targets as a single agent therapy or in combination with other chemotherapy agents in cancer research.

Keywords: CHK1; Cell cycle checkpoint; DNA damage; PPRH; Replication stress; WEE1.