Elucidating the Role of C16orf72 in the Cellular Stress Response Network

PROJECT SUMMARY A hallmark of cancer is the ability of malignant cells to maintain viability in the face of stressors such as aneuploidy, nutrient scarcity, and xenobiotic compounds. This multi-stress-resilience phenotype enables tumor formation, metastasis, and therapy resistance. Individual molecular pathways co-opted by cancer cells to promote stress resilience have been thoroughly characterized, but how these specialized pathways are integrated in complex physiological stress states remains poorly understood. Recently, we used genome-scale fitness screening data to estimate the intrinsic stress phenotype of 689 diverse cancer cell and identify genes which are selectively essential in cancer cells highly reliant on multiple distinct stress response pathways. Through our integrative analysis, we discovered C16orf72, a previously uncharacterized protein which is broadly stress-inducible and promotes resilience to mechanistically diverse stress insults. We have determined that C16orf72 physically interacts with HUWE1, an E3 ligase known to ubiquitinate proteins involved in stress response pathways. Moreover, loss of either C16orf72 or HUWE1 produces highly similar transcriptomic and ubiquitination phenotypes while not affecting expression of the other factor. Thus, our overarching hypothesis is that C16orf72 mediates stress resilience by physically interacting with HUWE1 to promote HUWE1-mediated ubiquitination of proteins with critical roles in diverse stress response pathways. In Aim 1 of this proposal, we will define the binding interface of C16orf72 and HUWE1 and test several non-exclusive models by which C16orf72 regulates HUWE1 enzymatic activity. In Aim 2, we will determine the substrates and specific modifications which underly the role of C16orf72 and HUWE1 in cellular stress resilience and canonical stress response signaling. In Aim 3, we will determine the extent to which C16orf72 is required for in vivo tumorigenesis and the development of therapy resistance in breast cancer. The long-term goals of the proposed work are to determine the molecular mechanism by which C16orf72 promotes cellular stress resilience and investigate the therapeutic potential of blocking the C16orf72/HUWE1 axis in cancer cells.