The RalB small GTPase-SEC5 effector complex regulates survival of RAS mutant colorectal cancer by regulating trafficking of DR5


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Hajrah Awais Khawaja1,Emma Evergren1,Jamie Roberts1,Daniel Longley1,Paul O'Reilly1,Darragh McArt1,Patrick Johntson1,Timothy Harrison1,Sandra Van Schaeybroeck1
1Queen's University Belfast

Abstract

Background

Oncogenic mutations in RAS most commonly involve amino acid substitution at codons 12, 13 or 61 and occur in > 50% of colorectal cancer (CRC). These mutations results in constitutive activation of RAS effector pathways, have been associated with poor outcome and resistance to anti-EGFR therapies in CRC. Combined MAPK/PI3K inhibition (i) has shown no activity in RASMT CRC. The RALGDS/RAL pathway constitutes a third RAS effector pathway that is essential in the transformation of murine fibroblasts, downstream of RAS. The aim of this study is to evaluate the roles of the small GTPases, RALA and RALB, as potential novel targets for RAS mutant (MT) CRC.

Method

RNAi against RALA/B was used in isogenic/non-isogenic RASMT/WT CRC cells, which have been stratified according to the key consensus molecular subtypes (CMS) of CRC. MTT, Flow Cytometry, Western blotting and activity assays were used to measure cell survival/death. DR5 cell localization was performed using flow cytometry and immunofluorescence.

Results

We found that silencing of RALB but not RALA, led to the greatest amount of cell death in CMS 1 and 3 RASMT, but not RASWT CRC cells. In addition, significant increases in cell death were observed when siRALB was combined with MEKi in RASMT CRC. Furthermore, cell death induced by siRALB was found to be dependent on caspase-8 activation and on up-regulation of the death receptor 5 (DR5). Further data indicates that RalB regulates the trafficking and stability of DR5 and that this occurs through its effector SEC5.

Conclusion

The RALB-SEC5 effector complex regulates survival and MEKi sensitivity of RASMT CRC cells, stratified to CMS1 and 3. We have elucidated a novel mechanism of RALB-induced cell survival, which involves SEC5 and DR5. Thus, the development of novel RALB/SEC5-specific therapies may lead to new treatment strategies for RASMT CRC.