Abstract A56: Regulation of breast tumor kinase (Brk) expression in triple-negative breast cancer integrates cellular (HIF-2alpha) and hormonal (cortisol) stress signaling
Authors: Anderson Tarah M. Regan , Ma Shihong , Raj Ganesh V. , Lange Carol A. 
Published: May 2016

Triple-negative breast cancers (TNBC) have a worse prognosis relative to other breast cancer subtypes, underscoring the urgent need for identification of driver molecules or pathways for targeted therapies. Breast tumor kinase (Brk) is a soluble tyrosine kinase that is aberrantly elevated and active in 86% of breast cancers. Our lab has shown Brk to be a potent driver of basal-type mammary tumors. Mechanisms through which Brk overexpression is acquired in breast cancer cells are largely unknown. We recently reported that Brk is a direct target gene of hypoxia-inducible factor 1 alpha (HIF-1alpha) and HIF-2alpha, activated in response to cellular stresses such as hypoxia, low glucose, or nutrient starvation. It is becoming increasingly evident that the stress sensing hormone, cortisol, via activation of the glucocorticoid receptor (GR), leads to cell survival and chemoresistance in tumors of epithelial origin, such as breast cancer. In fact, GR expression in TNBC predicts poor outcome. Herein, we sought to investigate crosstalk between cell stress pathways and GR signaling that may influence expression of Brk in TNBC. An explant model of primary human TNBC demonstrated robust induction of Brk mRNA and protein with the GR ligand, dexamethasone (dex). Brk mRNA and protein were also induced in response to dex in TNBC cell line models. MDA-MB-231 cells with HIF-1a/2a knockdown (DKD), failed to induce Brk expression following dex treatment, suggesting that GR regulation of Brk requires HIF-1a/2a. Chromatin immunoprecipitation (ChIP) assays showed HIF and GR co-recruitment to the Brk promoter in response to either hypoxia or dex, indicating that Brk is a direct GR/HIF target gene. HIF-2a mRNA and protein were also directly regulated by GR in response to dex treatment. Notably, expression of Proline, glutamate and leucine rich protein 1 (PELP1), an important steroid receptor coactivator, was significantly induced by hypoxic cell stress, while DKD cells (lacking HIFs) exhibited markedly reduced PELP1 protein levels relative to control cells. Co-immunoprecipitation (co-IP) assays showed that PELP1 and GR interact basally and in response to dex treatment in multiple TNBC cell lines. Moreover, PELP1 was recruited to the Brk promoter with HIF2a and GR following dex treatment. Inhibition of PELP1 with the peptidomemtic, D2, blocked dex induction of Brk mRNA. Physiologic cell stress resulted in phosphorylation of GR at serine 134 (S134) and this event was required for the GR and PELP1 interaction. Enhanced phosphorylation at this site via H2O2 treatment increased GR recruitment to the Brk promoter, while blockade of this site via the p38 MAPK inhibitor SB203580 diminished GR recruitment to the Brk promoter and blocked Brk induction. Notably, mutant GR in which S134 was mutated to an alanine (S134A) was not recruited to the Brk promoter basally or in response to dex treatment, highlighting the importance of this phosphorylation event in the GR regulation of Brk expression. Our data show that GR initiates a feed-forward signaling loop leading to upregulation of Brk in TNBC and reveal molecular linkage between cell stress and stress hormone signaling in driving aggressive phenotypes in breast cancer. Collectively, our studies suggest that GR, HIF, PELP1 cross talk may promote aggressive tumor behavior, in part via upregulation of Brk. Breast cancer patients are routinely given high doses of dex to alleviate the inflammatory side effects of chemotherapy. This treatment may inadvertently promote chemoresistance and tumor progression via robust induction of Brk expression. Targeting the GR/HIF/PELP1 complex may provide a means of blocking Brk-dependent tumor progression and metastasis in patients with TNBC.

Citation Format: Tarah M. Regan Anderson, Shihong Ma, Ganesh V. Raj, Carol A. Lange. Regulation of breast tumor kinase (Brk) expression in triple-negative breast cancer integrates cellular (HIF-2alpha) and hormonal (cortisol) stress signaling. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Breast Cancer Research; Oct 17-20, 2015; Bellevue, WA. Philadelphia (PA): AACR; Mol Cancer Res 2016;14(2_Suppl):Abstract nr A56.