SUN-007 Discovery of Novel Signaling Pathways Downstream of PTK6 in TNBC Models

Abstract

Triple negative breast cancer (TNBC) patients have a worse prognosis relative to other breast cancer subtypes. The glucocorticoid receptor (GR) is a ubiquitous steroid receptor that mediates homeostatic responses to life-induced stressors via glucocorticoids synthesized in the adrenal cortex. High GR expression predicts poor outcome in TNBC. The molecular mechanisms for how stress and GR contribute to TNBC progression are largely unknown. We previously described overexpression of protein tyrosine kinase 6 (PTK6) in response to both cellular and endocrine stress, coordinated by GR/HIF complexes in TNBC models. PTK6-induced signaling further amplified phosphorylation of GR on Ser134. PTK6 knock-down impaired TNBC motility, and blocked outgrowth of cancer stem-like cells (CSC) as measured by tumorsphere assays. Furthermore, PTK6 expression was integral for cancer cell survival upon chemotherapy treatment (Taxol, 5-FU). Interestingly, kinase activity of PTK6 was not required for TNBC cell motility or CSC outgrowth. This finding is of critical importance for drug design, given that previous efforts have primarily focused on targeting the PTK6 kinase domain. To probe the mechanisms of PTK6 signaling, we have created kinase-intact domain structure mutants of PTK6 via in frame deletions of the N-terminal SH3 or SH2 domain. MDA-MB-231 cells expressing PTK6 which lacks an SH2 domain (SH2-del PTK6) exhibited increased formation of tumorspheres relative to wt or kinase-dead (KM) full-length PTK6. Surprisingly, SH2-del PTK6+ cells were strikingly less responsive to both serum- and growth factor-stimulated cell motility relative to wt or KM controls. To track signal transduction pathways activated in TNBC cells harboring PTK6 domain mutants (wt, KM, SH2-del PTK6, PTK6 null), we used Reverse Phase Protein Array (RPPA). Our data suggests that the SH2 domain of PTK6 mediates TNBC motility via activation of the Rho and/or AhR signaling pathways, while CSC outgrowth primarily occurs via activation of the p38 and/or canonical Wnt signaling pathways. Together, these studies nominate PTK6 as an effector of advanced cancer phenotypes in GR+ TNBC cells and identify novel therapeutic targets (Rho, AhR) for treatment of TNBC tumors. Additionally, our work defines PTK6 domain-specific functions and reveals new opportunities for pharmacologically targeting the PTK6 SH2-domain in TNBC.