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The Contribution of ERAD to ER Stress Resolution
DEFINING ER-ASSOCIATED DEGRADATION FACTORS REQUIRED FOR CANCER CELL SURVIVAL
G. Gaeta, S. Picaud, F. Lari, E. van Anken, and J.C. Christianson
Botnar Research Centre, University of Oxford, United Kingdom
Constitutively engaging the pro-survival branches of the unfolded protein response (UPR) permits cancer cells experiencing lethal proteotoxic stress levels in the endoplasmic reticulum (ER) to remain viable. Disabling UPR-mediated adaptation represents a potential strategy for cancer therapies to break this form of non-oncogenic addiction. The Hrd1 ubiquitin ligase complex is the principal executor of ER-Associated Degradation (ERAD) - clearing proteotoxic burden and mitigating ER stress. ERAD capacity through Hrd1 is a key aspect supplemented by the UPR’s pro-survival Ire1-XBP1 and ATF6 branches through transcriptional upregulation of key genes in the Hrd1 complex. To assess whether attenuating ERAD could compromise viability of stressed cancer cells, individual Hrd1 complex components were knocked down by shRNAs in both multiple myeloma (AMO-1) and triple negative breast cancer (MBA-MD-231) cell lines. Using both 2D growth and 3D spheroid assays, Hrd1 and important cofactors (e.g. SEL1L, Ube2j1) were found to be required for cell growth and viability. We next modelled ER stress in cancer cells using a HeLa cell line expressing a mifepristone-induced IgM heavy chain (µS). Hrd1 and its cofactors were dispensable for unstressed cells while ER stress elicited by µS induction revealed dependence on the Hrd1 complex and its ubiquitylating activity. To establish the contribution made by ERAD to ER stress resolution and viability made by the Hrd1 complex added during the UPR, we generated µS-expressing HeLa cell lines in which canonical XBP1/ATF6p50 transcription factor (TF)-binding ERSE sequences of Hrd1 and Herp were mutated by CRISPR/Cas9. Disrupting TF binding sites in Hrd1 (Hrd1∆ERSE) and Herp (Herp∆ERSE) was sufficient to decouple each factor from UPR regulation, indicated by blunted responsiveness of either gene to ER stress induced by tunicamycin or µS. Decoupling UPR regulation for Hrd1 or Herp only attenuated growth upon µS induction but neither were as severe as complete knockouts (∆Hrd1, ∆Herp). These data indicate the key roles played by the Hrd1 complexes constitutively expressed and those supplementing ERAD activity during the UPR. Together with ongoing studies of composition changes in Hrd1 complexes, these studies will provide insight toward identifying the key targets for small-molecule inhibitors aimed specifically at selectively disrupting the Hrd1 complex, as new avenues for therapeutic benefit in cancer.