ABSTRACT
RAS is one of the most frequently mutated oncogenes in cancer with ~30% of all human tumors harboring a mutation in either HRAS, NRAS, or KRAS isoforms. Despite countless efforts for development of small molecule inhibitors for RAS, it remains an elusive target in the clinic. Here we demonstrated that the pan-RAS biological inhibitor RAS/RAP1-specific endopeptidase (RRSP) has proteolytic activity in ‘Ras-less’ mouse embryonic fibroblasts expressing human RAS isoforms (H/N/KRAS) or major oncogenic KRAS mutants (G12C, G12V, G12D, G13D, and Q61R). The cleavage of RAS inhibited phosphorylation of ERK and cell proliferation. To investigate how RAS processing affects colon cancer cells, we tested RRSP against KRAS-dependent (SW620 and GP5d) and KRAS-independent (HCT-116, SW1463, and HT-29) cell lines and found that RRSP inhibited growth. The cleavage of RAS was cytotoxic in some cell lines and induced either irreversible cell cycle arrest or uncharacterized growth inhibition in others. The G1 cell cycle arrest in some colon cancer cells was mediated through rescue of p27 (Kip1) protein expression resulting in reduced phosphorylation of retinoblastoma protein. Together, this work demonstrated that complete ablation of RAS in cells induces growth inhibition, but the mechanism of inhibition can vary in different tumor cell lines. This ability of RAS processing to halt cell proliferation by multiple strategies highlights RRSP both as a potential anti-tumor therapy and as a tool for studying RAS signaling across tumor types.
Competing Interest Statement
K.J.F.S. has been granted a patent (US 10,829,752 B2) on use of RRSP to treat cancer. K.J.F.S. is a consultant for Buoy Health on topics unrelated to this manuscript. K.J.F.S. has a significant financial interest in Situ Biosciences, LLC, a contract research organization that pursues research unrelated to cancer. C.K.S. is an intern as a scientific and financial advisor for Aspire Capital Partners, LLC, which invests in oncotherapies.