Basal-type triple-negative breast cancers (TNBC) are aggressive and difficult to treat relative to luminal type breast cancers. TNBC often express abundant Met receptors and are enriched for transcriptional targets regulated by hypoxia inducible factor 1-alpha (HIF-1α), which independently predicts cancer relapse and increased risk of metastasis. Brk/PTK6 is a critical downstream effector of Met signaling and required for HGF-induced cell migration. Herein, we examined the regulation of Brk by HIFs in TNBC in vitro and in vivo. Brk mRNA and protein levels are upregulated strongly in vitro by hypoxia, low glucose and reactive oxygen species. In HIF-silenced cells, Brk expression relied upon both HIF-1α and HIF-2α, which we found to regulate BRK transcription directly. HIF-1α/2α silencing in MDA-MB-231 cells diminished xenograft growth and Brk re-expression reversed this effect. These findings were pursued in vivo by crossing WAP-Brk (FVB) transgenic mice into the METmut knock-in (FVB) model. In this setting, Brk expression augmented METmut-induced mammary tumor formation and metastasis. Unexpectedly, tumors arising in either METmut or WAP-Brk X METmut mice expressed abundant levels of Sik, the mouse homolog of Brk, which conferred increased tumor formation and decreased survival. Taken together, our results identify HIF-1α/2α as novel regulators of Brk expression and suggest that Brk is a key mediator of hypoxia-induced breast cancer progression. Targeting Brk expression or activity may provide an effective means to block the progression of aggressive breast cancers.

Cancer cells use stress response pathways to sustain their pathogenic behavior. In breast cancer, stress response-associated phenotypes are mediated by the breast tumor kinase, Brk (PTK6), via the hypoxia-inducible factors HIF-1α and HIF-2α. Given that glucocorticoid receptor (GR) is highly expressed in triple negative breast cancer (TNBC), 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, indicating that Brk is a direct GR/HIF target. Notably, HIF-2α, not HIF-1α, expression was induced by GR signaling and the important steroid receptor coactivator PELP1 was also found to be induced in a HIF-dependent manner. Mechanistic investigations showed how PELP1 interacted with GR to activate Brk expression and demonstrated that physiologic cell stress, including hypoxia, promoted phosphorylation of GR serine 134, initiating a feed-forward signaling loop that contributed significantly to Brk upregulation. Collectively, 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.

Tamoxifen (Tam) is the only FDA-approved chemoprevention agent for pre-menopausal women at high risk for developing breast cancer. While Tam reduces a woman's risk of developing estrogen receptor positive (ER+) breast cancer, the molecular mechanisms associated with risk reduction are poorly understood. Prior studies have shown that cytoplasmic proline, glutamic acid and leucine rich protein 1 (PELP1) promotes Tam resistance in breast cancer cell lines. Herein, we tested for PELP1 localization in breast epithelial cells from women at high risk for developing breast cancer and found that PELP1 was localized to the cytoplasm in 36% of samples. In vitro, immortalized HMECs expressing a nuclear localization signal (NLS) mutant of PELP1 (PELP1-cyto) were resistant to Tam-induced death. Furthermore, PELP1-cyto signaling through estrogen-related receptor gamma (ERRγ) promoted cell survival in the presence of Tam. Overexpression of ERRγ in immortalized HMECs protected cells from Tam-induced death, while knockdown of ERRγ sensitized PELP1-cyto expressing HMECs to Tam. Moreover, Tam-induced HMEC cell death was independent of apoptosis and involved accumulation of the autophagy marker LC3-II. Expression of PELP1-cyto and ERRγ reduced Tam-induced LC3-II accumulation, and knockdown of ERRγ increased LC3-II levels in response to Tam. Additionally, PELP1-cyto expression led to the upregulation of MMP-3 and MAOB, known PELP1 and ERRγ target genes, respectively. Our data indicate that cytoplasmic PELP1 induces signaling pathways that converge on ERRγ to promote cell survival in the presence of Tam. These data suggest that PELP1 localization and/or ERRγ activation could be developed as tissue biomarkers for Tam responsiveness.

The metastatic cascade is a complex process that requires cancer cells to survive despite conditions of high physiologic stress. Previously, cooperation between the glucocorticoid receptor (GR) and hypoxia-inducible factors (HIF) was reported as a point of convergence for host and cellular stress signaling. These studies indicated p38 MAPK-dependent phosphorylation of GR on Ser134 and subsequent p-GR/HIF-dependent induction of breast tumor kinase (PTK6/Brk), as a mediator of aggressive cancer phenotypes. Herein, p-Ser134 GR was quantified in human primary breast tumors ( = 281) and the levels of p-GR were increased in triple-negative breast cancer (TNBC) relative to luminal breast cancer. Brk was robustly induced following exposure of TNBC model systems to chemotherapeutic agents (Taxol or 5-fluorouracil) and growth in suspension [ultra-low attachment (ULA)]. Notably, both Taxol and ULA resulted in upregulation of the Aryl hydrocarbon receptor (AhR), a known mediator of cancer prosurvival phenotypes. Mechanistically, AhR and GR copurified and following chemotherapy and ULA, these factors assembled at the Brk promoter and induced Brk expression in an HIF-dependent manner. Furthermore, Brk expression was upregulated in Taxol-resistant breast cancer (MCF-7) models. Ultimately, Brk was critical for TNBC cell proliferation and survival during Taxol treatment and in the context of ULA as well as for basal cancer cell migration, acquired biological phenotypes that enable cancer cells to successfully complete the metastatic cascade. These studies nominate AhR as a p-GR binding partner and reveal ways to target epigenetic events such as adaptive and stress-induced acquisition of cancer skill sets required for metastatic cancer spread. Breast cancer cells enlist intracellular stress response pathways that evade chemotherapy by increasing cancer cell survival and promoting migratory phenotypes. .

ReviewModifications to glucocorticoid and progesterone receptors AbstractSteroid hormone receptors (SRs) are heavily posttranslationally modified by the reversible addition of a variety of molecular moieties, including phosphorylation, acetylation, methylation, SUMOylation, and ubiquitination. These rapid and dynamic modifications may be combinatorial and interact (i.e. may be sequential, complement, or oppose each other), creating a vast array of uniquely modified receptor subspecies that allow for diverse receptor behaviors that enable highly sensitive and context-dependent hormone action. For example, in response to hormone or growth factor membrane-initiated signaling events, posttranslational modifications (PTMs) to SRs alter protein-protein interactions that govern the complex process of promoter or gene-set selection coupled to transcriptional repression or activation. Unique phosphorylation events allow SRs to associate or disassociate with specific cofactors that may include pioneer factors and other tethering partners, which specify the resulting transcriptome and ultimately change cell fate. The impact of PTMs on SR action is particularly profound in the context of breast tumorigenesis, in which frequent alterations in growth factor-initiated signaling pathways occur early and act as drivers of breast cancer progression toward endocrine resistance. In this article, with primary focus on breast cancer relevance, we review the mechanisms by which PTMs, including reversible phosphorylation events, regulate the closely related SRs, glucocorticoid receptor and progesterone receptor, allowing for precise biological responses to ever-changing hormonal stimuli.

Breast cancer is the number one diagnosed cancer in women. Luminal breast cancers express steroid hormone receptors (SR) and these cases can be effectively treated with endocrine therapies that block estrogen receptor (ER) activity or estrogen synthesis. Unfortunately, at least 40% of women develop resistance to anti-estrogen treatments and progress to metastatic disease. One of the important transitions from hormone responsive (ER+/PR+ luminal A) to refractory (ER+ luminal B) disease involves the loss of progesterone receptor (PR) protein expression. However, data from large clinical trials showed that exposure to a synthetic progestin (MPA) and estrogen, but not estrogen alone, increased breast cancer incidence and tumor grade. These data, while somewhat controversial, implicate PR action in breast cancer development and progression. PR isoforms (A and B) are ligand-activated transcription factors, though they can be activated in the absence of progestin via signaling pathways commonly elicited downstream of growth factor receptors. We have shown that growth factor signaling (EGF or heregulin) through PR-B can initiate rapid proliferation and survival of breast cancer cells growing in soft-agar. PR-B is phosphorylated and hyperactived by mitogenic protein kinases, including MAPK, CDK2, and CK2. Notably, over-expression and activation of Aurora A kinase (downstream of MAPKs) is associated with invasive breast cancer and poor prognosis. We hypothesize that activation of protein kinases commonly overexpressed in breast cancer provide a context for aberrant PR activity, resulting in upregulation of Her2/erbB2 signaling, EMT, and increased expression of mammary stem-cell mediators (stemness). To model this process, we over-expressed oncogenic Raf1 in ER+/PR+ MCF-7 breast cancer cells. MCF-7-Raf1 cells exhibited constitutive activation of the MAPK pathway and upregulation of both ER and PR mRNA and protein relative to parental controls. Addition of progestin to MCF-7-Raf1 cells increased PR phosphorylation and soft agar growth that was blocked by MLN8237, an Aurora kinase inhibitor. Interestingly, passage of MCF-7-Raf1 cells as mouse xenografts (1GX) led to rapid tumor progression that was associated with increased Aurora A phosphorylation, loss of PR mRNA and protein, expression of epithelial-to-mesenchymal (EMT) markers, increased stemness, and tamoxifen resistance. These data suggest that Aurora A kinase and PR cross-talk may drive early breast cancer progression in response to growth promoting signals. Targeting this cross-talk with Aurora kinase inhibitors and anti-progestins, in conjunction with conventional estrogen-blocking therapies, may improve survival outcomes by preventing progression to endocrine failure. MCF-7-Raf1 and MCF-7-Raf-1 (1GX) cells may provide a useful model for the study of SR-loss during luminal A to luminal B transition. Citation Format: Katherine A. Leehy, Tarah M. Regan Anderson, Andrea R. Daniel, Antonino B. D'Assoro, Carol A. Lange. Aurora A kinase and progesterone receptor cross talk in breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2107. doi:10.1158/1538-7445.AM2014-2107

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<p>PDF file, 3449K, ChIP data showing recruitment of HIF-1a, HIF-2a, and RNA pol II to HREs 2-5 in the Brk promoter.</p>