RAS specific protease induces irreversible growth arrest via p27 in several KRAS mutant colorectal cancer cell lines

Caleb K Stubbs,Vania Vidimar,Marco Biancucci,Karla J F Satchell

doi:10.1038/s41598-021-97422-0

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. Yet, our understanding of the mechanisms by which RRSP drives growth inhibition are unknown. Here, we 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. To investigate 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 apoptosis, RRSP treatment of GP5d and SW620 cells induces G1 cell cycle arrest. In some cell lines, growth effects were dictated by rescued expression of the tumor suppressor protein p27 (Kip1). The ability of RRSP to irreversibly inhibit cancer cell growth highlights the antitumor potential of RRSP, and further warrants investigation as a potential anti-tumor therapeutic.

Highlights

Ras-specific proteases to degrade Rat sarcoma GTPase (RAS) within cancer cells are under active development as an innovative strategy to treat tumorigenesis
RAS/related protein 1 (RAP1)-specific endopeptidase (RRSP) was previously shown to cleave HRAS, NRAS, and KRAS when the proteins were ectopically expressed in HeLa cells and recombinant RRSP was shown to process purified KRAS G12D, G13D, and Q61R in biochemical assays[26]
To get an even broader sense of RRSP effectiveness across different isoforms and mutants of RAS, we tested RRSP against the ‘RAS-less’ mouse embryonic fibroblast (MEF) cell line panel developed by Drosten et al.[7]

Summary

Introduction

Ras-specific proteases to degrade RAS within cancer cells are under active development as an innovative strategy to treat tumorigenesis. Small molecules targeting KRAS G12C have been developed and are undergoing clinical trials[14,15,16] Many of these agents have shown clinical success with one molecule receiving FDA accelerated approval earlier this year for treatment of KRAS G12C tumors in non-small cell lung carcinoma[17]. Despite this success, the strategy of selective inhibition has problems of being applicable to only a limited range of cancers integrated with personalized medicine and cannot be used to treat cancers that lack the specific mutation. Approaches are being developed to more broadly target RAS either with proteases that directly cleave R AS18,19 or with linkers that target RAS for cellular degradation[20,21,22,23]

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