טכניון מכון טכנולוגי לישראל
הטכניון מכון טכנולוגי לישראל - בית הספר ללימודי מוסמכים  
Ph.D Thesis
Ph.D StudentTocci Stefania
SubjectNovel PRMT5 Targets in Melanoma and Pancreatic Cancer
DepartmentDepartment of Medicine
Supervisors Professor Amir )Oryan )Orian
Professor Zeev Ronai


Abstract

Regulation of post-translational modifications, including protein methylation, is central for the normal maintenance of cellular homeostasis, often deregulated in cancer. Protein Arginine Methyl-Transferase 5 (PRMT5), which catalyzes the arginine mono and symmetric di-methylation, has been implicated in the regulation of diverse processes in oncogenesis. To further understand mechanisms underlying PRMT5 function in cancer, we have undertaken three complementing routes to identify and characterize novel substrates that are subject to PRMT5 regulation either via its adaptor protein SHARPIN or in a SHARPIN-independent manner.

Using an unbiased proteomic approach, we identified new putative PRMT5 targets and regulatory pathways not yet associated with PRMT5 activity. Among them is PROSER2, a proline and serine-rich 2 protein, previously proposed as a biomarker in cancer. The knockdown of PROSER2 decreased 2D and 3D growth of human melanoma and pancreatic cancer cells, whereas its ectopic expression mediated the opposite effect. The pro-tumorigenic function for PROSER2 is supported by transcriptomic PDAC patient specimens’ datasets, in which PROSER2 expression significantly associates with poor prognosis. Furthermore, a mutant form of PROSER2, which may no longer be methylated by PRMT5, loses its tumorigenic potential. Mechanistically, PROSER2 controls p38 MAPK phosphorylation under basal growth conditions and following UV light radiation. Initial assessment of kinases and phosphatases known to affect p38 signaling identified changes in transcriptional level of Wip1, a phosphatase responsible for the inactivation of the p38 signal, whose expression inversely correlates with PROSER2 expression in PDAC patients.

Given the hypothesis that SHARPIN may guide PRMT5 to select substrates, we performed analyses of human melanoma samples, which revealed that both SHARPIN and PRMT5 expression inversely coincided with the immune activation in melanoma. We discovered that PRMT5 methylates IFI16, component of the cGAS/STING complex, and a SHARPIN-bound protein. Complementary to the regulation of IFI16 was PRMT5 effect on NLRC5, the master regulator of antigen presentation pathways, including the major histocompatibility complex class I (MHCI). Correspondingly, through the induction of IFN, cytokines, and MHCI in melanoma, the inhibition of PRMT5 resulted in increased immune recognition of murine melanoma, and the suppression of these tumors when combined with anti-PD1 therapy, providing a rationale for clinical evaluation of PRMT5 inhibitors together with immune-checkpoint therapy for cancer.

We finally tested the possibility that SHARPIN-interacting proteins may serve as PRMT5 substrates. We identified the RNA binding protein, RBM10 as a SHARPIN-bound protein in melanoma cells, which expression was altered upon SHARPIN manipulation. The inhibition of RBM10 dramatically reduced the cell viability in vitro settings, suggesting its critical role in melanoma growth. We found that PRMT5 interacts to and regulates RBM10 expression, pointing to the possible SHARPIN-PRMT5-RBM10 regulatory axis in cancer. Taken together, our findings identify PRMT5 and SHARPIN as important regulators contributing to tumor intrinsic functions and implicated in tumor microenvironment response.