Perez Kerkvliet, Carlos2020-09-222020-09-222020-07https://hdl.handle.net/11299/216350University of Minnesota Ph.D. dissertation. July 2020. Major: Microbiology, Immunology and Cancer Biology. Advisor: Carol Lange. 1 computer file (PDF); xii, 168 pages.Triple negative breast cancer (TNBC) is the most metastatic and deadly breast cancer subtype, accounting for 20-30% of all breast cancer cases. There is a critical need to identify molecular targets that could be exploited as new biomarkers of TNBC prognosis and for improving therapies. Although TNBC lacks estrogen and progesterone receptors, 15-40% of TNBC patients express the glucocorticoid receptor (GR). Women with TNBC that express high levels of GR have poor outcomes. We hypothesize that GR is a key mediator of advanced cancer phenotypes in TNBC. Altered signaling pathways typify breast cancer and serve as direct inputs to steroid hormone receptor “sensors.” We previously reported that phospho-Ser134-GR (pS134-GR) species are elevated in TNBC and cooperate with hypoxia-inducible factors, providing a novel avenue for activation of GR in response to local or cellular stress. Specifically, we propose that GR acts as a “sensor” for stress signaling pathways commonly activated by soluble factors that are abundant within the tumor microenvironment (TME) of TNBC. Herein, we show that in the absence of GR ligands, GR is transcriptionally activated via p38-dependent phosphorylation of Ser134 as a mechanism of homeostatic stress-sensing and regulated upon exposure of TNBC cells to TME-derived agents. The ligand-independent pS134-GR transcriptome encompasses Transforming Growth Factor β1 (TGFβ1) and Mitogen Activated Protein Kinase (MAPK) signaling gene sets associated with TNBC cell survival and migration/invasion. Accordingly, pS134-GR was essential for TNBC cell anchorage-independent growth in soft-agar, migration, invasion, and tumorsphere formation, an in vitro readout of cancer stemness properties. Both pS134-GR and expression of the MAPK-scaffolding molecule 14-3-3ζ were essential for a functionally intact p38 MAPK signaling pathway downstream of MAP3K5/ASK1, indicative of a feed-forward signaling loop wherein self-perpetuated GR phosphorylation enables cancer cell autonomy. A 24-gene pS134-GR-dependent signature induced by TGFβ1 predicts shortened overall survival in breast cancer patients. Additionally, GR is known to induce the expression of PTK6, a soluble protein tyrosine kinase important in mediating signaling in response to cellular stress. PTK6 is overexpressed in 86% of breast cancer patients, regardless of subtypes. Although GR is known to modulate PTK6 expression in TNBC, PTK6-driven signaling events in the context of TNBC are largely undefined. We sought to delineate the functions of downstream of PTK6. To do this, 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 the SH2 domain of PTK6 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. The combination of AhR and Rho inhibitors enhanced paclitaxel cytotoxicity in TNBC cells, including a taxane-refractory TNBC model. In conclusion, our results identified pS134-GR as a critical downstream effector of p38 MAPK signaling and TNBC migration/invasion, survival, and stemness properties. Our studies define a ligand-independent role for GR as a homeostatic “sensor” of intrinsic stimuli as well as extrinsic factors rich within the TME (TGFβ1) that enables potent activation of the p38 MAPK stress-sensing pathway and nominate pS134-GR as a therapeutic target in aggressive TNBC. Additionally, we identified that PTK6 (SH2-domain), a downstream gene of GR, is a potent effector of advanced cancer phenotypes in TNBC. We identified both RhoA and AhR as novel therapeutic targets in PTK6+ breast tumors since these two proteins are downstream effectors of PTK6 functions in TNBC.enBreast CancerGlucocorticoid ReceptorphosphorylationThesis or Dissertation