Abstract
Cancer cells are able to survive under conditions that cause endoplasmic reticulum stress (ER-stress), and can adapt to this stress by upregulating cell-survival signalling pathways and down-regulating apoptotic pathways. The cellular response to ER-stress is controlled by the unfolded protein response (UPR). Small Rho family GTPases are linked to many cell responses including cell growth and apoptosis. In this study, we investigate the function of small GTPases in cell survival under ER-stress. Using siRNA screening we identify that RAC1 promotes cell survival under ER-stress in cells with an oncogenic N92I RAC1 mutation. We uncover a novel connection between the UPR and N92I RAC1, whereby RAC1 attenuates phosphorylation of EIF2S1 under ER-stress and drives over-expression of ATF4 in basal conditions. Interestingly, the UPR connection does not drive resistance to ER-stress, as knockdown of ATF4 did not affect this. We further investigate cancer-associated kinase signalling pathways and show that RAC1 knockdown reduces the activity of AKT and ERK, and using a panel of clinically important kinase inhibitors, we uncover a role for MEK/ERK, but not AKT, in cell viability under ER-stress. A known major activator of ERK phosphorylation in cancer is oncogenic NRAS and we show that knockdown of NRAS in cells, which bear a Q61 NRAS mutation, sensitises to ER-stress. These findings highlight a novel mechanism for resistance to ER-stress through oncogenic activation of MEK/ERK signalling by small GTPases.
Highlights
Oncogenesis and uncontrolled cancer cell division often lead to conditions that perturb protein folding and induce endoplasmic reticulum stress (ER-stress) [1]
In HT-1080 cells (N92I related C3 botulinum toxin substrate 1 (RAC1)), while pools of siRNA against RHOA and RHOQ had a small but significant effect on sensitivity to ER-stress, siRNA against RAC1 had the strongest effect and was comparable to the ATF6 positive control (Fig. 1B). These results suggest that RHOA, RHOC, RHOQ and RAC1 may be involved in cell survival under ERstress in wild-type cells, while oncogenic RAC1 mutation may overcome this in HT-1080 cells where RAC1 is the predominant Rho GTPase involved in ER-stress resistance
The cell signalling response that controls the balance between cell survival and cell death upon ER-stress is the unfolded protein response (UPR) [9] and previous studies have shown that this plays an important role in cancer cell adaption to stress and cancer development [30]
Summary
Oncogenesis and uncontrolled cancer cell division often lead to conditions that perturb protein folding and induce endoplasmic reticulum stress (ER-stress) [1]. There are three ubiquitously expressed controlling arms to the UPR: endoplasmic reticulum to nucleus signalling 1 (ERN1, hereafter referred to by its common name, IRE1); eukaryotic translation initiation factor 2-alpha kinase 3 (EIF2AK3, hereafter referred to by its common name, PERK); and cyclic AMP-dependent transcription factor ATF-6 alpha (ATF6). PERK is a kinase that dimerizes upon activation by ER-stress [5] and phosphorylates its major target, eukaryotic translation initiation factor 2 subunit alpha (EIF2S1) This phosphorylation event attenuates protein translation relieving the protein folding burden within the ER and promoting cell survival [6]. Under acute ER-stress, the UPR promotes cell survival and adaptation This function is superseded by pro-apoptotic signalling pathways (e.g. ATF4 and DDIT3) under prolonged ER-stress [9]
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