Defining the ER-associated degradation factors required for cancer cell survival

Thesis

Constitutively engaging the pro-survival outcomes 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 to mitigate ER stress. ERAD capacity is supplemented by the UPR’s pro-survival outcomes through transcriptional upregulation of HRD1 and key cofactors in the complex. To assess whether attenuating ERAD could compromise viability of stressed cancer cells, components of the HRD1 complex were knocked down in multiple myeloma (AMO-1/L363) and TNBC (MDA-MB-231/MDA-MB-436) cell lines. Both HRD1 and its cofactor HERP were found to be required for cells to grow in 3D as spheroids. We next turned to a temporally controlled model of ER stress, inducing expression of unpaired IgM heavy chain (µS) with mifepristone in HeLa cells (HeLa_µS). In this model, cells were dependent on HRD1 and select individual cofactors for viability, but only when ER stress was elicited by µS. To delineate the contribution made by ERAD (via the HRD1 complex) to viability during ER stress resolution, we generated HeLa_µS cell lines where canonical XBP1S/ATF6p50 transcription factor binding ERSE sequences of HRD1, HERP and Derlin3 were disrupted by CRISPR-Cas9. ERSE sequence disruption effectively decoupled each factor from UPR regulation, reflected by blunted transcriptional responses during ER stress. Uncoupling UPR regulation of HRD1, HERP and Derlin3 attenuated HeLa_µS growth during µS induction, but none were as severe as complete knockouts. Lastly, we investigated HRD1 complex heterogeneity and remodelling during stress by Size-Exclusion Chromatography (SEC). Fractions representing HRD1 complexes were enriched with different cofactors and co-exist within the ER. The contributions of these different HRD1 complexes to ER stress resolution, remains to be established. These data underscore the key roles played by HRD1 complexes constitutively expressed to maintain homeostasis and those added by the UPR to supplement ERAD and restore homeostasis. These will provide insight toward identifying key targets for small-molecule inhibitors aimed at selectively disrupting the HRD1 complex, as new therapeutic avenues in cancer.

Authors: Gaeta, G

• DOI: 10.5287/ora-7ryj5grjj
• Type of award: DPhil
• Level of award: Doctoral
• Awarding institution: University of Oxford
• Language: English
• Keywords: ERAD; protein degradation; ER; HRD1
• Subjects: Endoplasmic reticulum; Protein degradation