Protein tyrosine kinase 6 (PTK6; also called Brk) is overexpressed in 86% of patients with breast cancer; high PTK6 expression predicts poor outcome. We reported PTK6 induction by HIF/GR complexes in response to either cellular or host stress. However, PTK6-driven signaling events in the context of triple-negative breast cancer (TNBC) remain undefined. In a mouse model of TNBC, manipulation of PTK6 levels (i.e., via knock-out or add-back) had little effect on primary tumor volume, but altered lung metastasis. To delineate the mechanisms of PTK6 downstream signaling, we created kinase-dead (KM) and kinase-intact domain structure mutants of PTK6 via in-frame deletions of the N-terminal SH3 or SH2 domains. While the PTK6 kinase domain contributed to soft-agar colony formation, PTK6 kinase activity was entirely dispensable for cell migration. Specifically, TNBC models expressing a PTK6 variant lacking the SH2 domain (SH2-del PTK6) were unresponsive to growth factor-stimulated cell motility relative to SH3-del, KM, or wild-type PTK6 controls. Reverse-phase protein array revealed that while intact PTK6 mediates spheroid formation via p38 MAPK signaling, the SH2 domain of PTK6 limits this biology, and instead mediates TNBC cell motility via activation of the RhoA and/or AhR signaling pathways. Inhibition of RhoA and/or AhR blocked TNBC cell migration as well as the branching/invasive morphology of PTK6 +/AhR + primary breast tumor tissue organoids. Inhibition of RhoA also enhanced paclitaxel cytotoxicity in TNBC cells, including in a taxane-refractory TNBC model. IMPLICATIONS: The SH2-domain of PTK6 is a potent effector of advanced cancer phenotypes in TNBC via RhoA and AhR, identified herein as novel therapeutic targets in PTK6 + breast tumors.
Bibliographical noteFunding Information:
We are grateful to MD Anderson RPPA Core Facility. We thank Dr. Thu H. Truong for her critical reading of this manuscript. We also thank Alana Welm for sharing her PDX models and for graciously performing gene expression analysis of cHCI-10 cells to authenticate their origin. All paraffin-embedded tissue sections were prepared in the UTHSC Research Histology Core. We thank Dr. Radhika Sekhri for digitally scanning stained tissue slides and for providing access to the quantification software housed in the Department of Pathology at UTHSC. The UTHSC Center for Cancer Research provided the IncuCyte S3 shared instrumentation. This research received patient specimen procurement assistance from the University of Minnesota’s Biorepository and Laboratory Services program and was supported by the NIH’s National Center for Advancing Translational Services, grant UL1TR002494. This work was supported by NIH grants R01 CA159712 (to C.A. Lange), R01 CA138488 and Dept. of Defense BC150640 (to T.N. Seagroves), F30CA228261, T32CA009138 (to C.P. Kerkvliet), and NIH’s National Center for Advancing Translational Sciences, grant UL1TR002494 (to A.R. Dwyer and C.P. Kerkvliet). B.A. Smeester was previously supported by an NIH NIAMS T32AR050938 Musculoskeletal Training Grant and is currently supported by a doctoral dissertation fellowship through the graduate school at the University of Minnesota. Some reagents used in this study were also supported by the Sobiech Osteosarcoma Fund Award and Children’s Cancer Research Fund grants (to B.S. Moriarity). The Tickle Family Land Grant Endowed Chair in Breast Cancer Research (to C.A. Lange) and the West Cancer Center Research award (to T.N. Seagroves) also supported this work.
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