Cell cycle arrest following pan-RAS inhibition can occur through upregulation of p27 in both KRAS-dependent and KRAS-independent colon cancer cell lines

Abstract

Ras-specific proteases to degrade RAS within cancer cells are under active development as an innovative strategy to treat tumorigenesis. The naturally occurring biological toxin effector called RAS/RAP1-specific endopeptidase (RRSP) is known to cleave all RAS within a cell, including HRAS, KRAS, NRAS and mutant KRAS G13D. In the course of studies developing RRSP as an anti-cancer therapeutic, it was shown that cleavage of total RAS by RRSP results in a range of cell fates from cytotoxicity to moderate growth inhibition. Despite the considerable amount of evidence demonstrating RRSP anti-tumor effects in vivo, our understanding of the mechanisms involved are unknown. Here, we first demonstrate, using isogenic mouse fibroblasts expressing a single isoform of RAS or mutant KRAS, that RRSP equally inactivates all isoforms of RAS as well as the major oncogenic KRAS mutants. The cleavage of RAS inhibited phosphorylation of ERK and cell proliferation regardless of the RAS isoform. To investigate further how RAS processing might lead to varying outcomes in cell fate within cancer cells, we tested RRSP against four colorectal cancer cell lines with a range of cell fates. While cell lines highly susceptible to RRSP (HCT116 and SW1463) undergo cytotoxic death, RRSP treatment of GP5d cells induces G1 cell cycle arrest, and SW620 cells instead induces growth inhibition through cell senescence. In three of four cell lines tested, growth effects were dictated by rescued expression of the tumor suppressor protein p27 (Kip1). The ability of RRSP to inactivate all RAS and inhibit cancer cell growth through a variety of mechanisms highlights the antitumor potential of RRSP, and further warrants investigation as a potential anti-tumor therapeutic.