Cancer cells use stress response pathways to sustain their pathogenic behavior. In breast cancer, stress response-associated phenotypes are mediated by breast tumor kinase, Brk, a driver of breast cancer cell migration and survival. Triple negative (basal type) breast cancers (TNBC) are aggressive and difficult to treat. These tumors are frequently enriched for transcriptional targets regulated by hypoxia inducible factor 1-alpha (HIF-1a), a principal mediator of physiologic cell stress that independently predicts cancer relapse and increased risk of metastasis. Hormone stress signaling also impacts breast tumor biology and is mediated by the glucocorticoid receptor (GR). High levels of GR expression in TNBC are predictive of decreased overall survival and increased risk of metastasis. Herein, we examined the regulation of Brk by HIFs and GR in TNBC cell lines and mouse models. Brk mRNA and protein levels are upregulated by physiologic stress stimuli in vitro, dependent upon expression of both HIF-1a and HIF-2a Chromatin-immunoprecipitation (ChIP) assays revealed that BRK is a direct transcriptional target of HIF-1a/2a. Notably, knockdown of HIF-1a/2a in MDA-MB-231 cells significantly diminished xenograft growth; Brk re-expression reversed this effect. We investigated crosstalk between stress hormone-driven GR signaling and HIF-regulated physiologic stress. Primary TNBC tumor explants or cell lines treated with the GR ligand dexamethasone (dex) exhibited robust induction of Brk mRNA and protein that was HIF1/2-dependent. HIF and GR co-assembled on the BRK promoter in response to either hypoxia or dex. Notably, HIF-2a, not HIF-1a, expression was induced by GR signaling and the important steroid receptor coactivator PELP1 was also induced in a HIF-dependent manner. Physiologic cell stress, including hypoxia, promoted phosphorylation of GR serine 134, initiating a feed-forward signaling loop that contributed significantly to Brk upregulation. Finally, we crossed WAP-Brk (FVB) transgenic mice into the METmut knock-in (FVB) model and found expression of the Brk transgene augmented METmut-induced mammary tumor formation and metastasis. These results identify HIF-1a/2a, GR, and PELP1 as novel regulators of Brk expression. Ultimately, our findings linked cellular stress (HIF) and stress hormone (cortisol) signaling in TNBC, identifying the phospho-GR/HIF/PELP1 complex as a potential therapeutic target to limit Brk-driven progression and metastasis in TNBC patients.
University of Minnesota Ph.D. dissertation. August 2016. Major: Microbiology, Immunology and Cancer Biology. Advisor: Carol Lange. 1 computer file (PDF); viii, 119 pages.
Regan Anderson, Tarah.
Integration of physiologic (HIF) and hormone (GR) stress signaling pathways in the regulation of breast tumor kinase (Brk/PTK6) expression in triple-negative breast cancer..
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