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"Mechanisms of Cellular Signaling and Tumorigenesis
by Crk proto-oncogene"

Sushil Kumar
Molecular Biology, Genetics & Cancer Program
M.Sc., 2009, Bangalore University, India
B.Sc., 2007, Bangalore University, India

Thesis Advisor: Raymond B. Birge, Ph.D.
Department of Microbiology, Biochemistry and Molecular Genetics

Tuesday, March 28, 2017
1:00 P.M., Cancer Center, G-1196


Crk is the prototypical member of a class of Src homology 2 (SH2) and Src homology 3 (SH3) domain-containing adaptor proteins that positively regulate cell motility via the activation of Rac1 and, in certain tumor types such as glioblastoma multiformae and breast cancer, can promote cell invasion and metastasis by mechanisms that are not well understood. To interrogate the role of Crk in tumor progression and metastasis, we studied the tyrosine phosphorylation of Crk on Tyrosine 251 and Tyrosine 239 in the carboxyl-terminal SH3 domain. Our results demonstrate that Crk, via its phosphorylation at Tyr251, promotes invasive behavior of tumor cells, is a prominent feature in GBM, and correlates with aggressive glioma grade IV staging and overall poor survival outcomes. In addition, Crk via its phosphorylation on Tyr239, regulates the Csk/Src axis. Using SH2 domain arrays we show that Crk pTyr239 binds selectively to the CSK SH2 domain and indirectly alter Src activity and function. Functionally, expression of Y239F Crk in Crk (-/-) MEFs or in HS683 cells suppressed PDGF-b-inducible Src phosphorylation on Tyr416, suggesting that Crk pTyr239-CSK-SH2 protein assemblage negatively regulate Src activation. Results obtained through loss-of-function and epistasis experiments using CRISPR-Cas9 engineered 4T1 murine breast cancer cells, implicate a role for Crk Tyr239 phosphorylation in breast cancer tumor growth and metastasis. These results provide a potential molecular explanation for a long-standing question as to how Crk activates Src. Finally, we investigated the tumor cell extrinsic role of Crk proto-oncogene in modulation of host anti-tumor immune response. Our results show an unexpected role for Crk oncogene (that is usually upregulated in many solid cancers) in immune cell infiltration into tumor in immunocompetent murine breast adenocarcinoma model. W demonstrate that genetic ablation of Crk in tumor cells leads to suppression of tumor suppresive immune cell populations in primary tumor. Pathologically, this leads to a significant reduction in tumor growth and lung metastasis. Mechanistically, we show that Crk KO suppresses EMT and PD-L1 expression on tumor cells and hence does not synergies with anti-PD1 therapy for tumor growth and metastasis outcomes. These studies provide a basis for a role of Crk proto-oncogene in regulation of tumor immune microenvironment.

Significance: Taken together, studies in this dissertation provide mechanistic insights into poorly understood Crk SH3C domain in regulation of Abl and Src kinase activation which consequently lead to tumor promoting cell biological phenotypes. Additionally, our results provide new evidence for role of Crk in regulation of host anti-tumor immune response.

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