Tarah M. Regan Anderson

Breast Tumor Kinase (Brk/PTK6) Is a Mediator of Hypoxia- Associated Breast Cancer Progression

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-1α (HIF-1α), which independently predict cancer relapse and increased risk of metastasis. Brk/PTK6 is a critical downstream effector of Met signaling and is required for hepatocyte growth factor (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 reexpression reversed this effect. These findings were pursued in vivo by crossing WAP-Brk (FVB) transgenic mice into the MET(Mut) knockin (FVB) model. In this setting, Brk expression augmented MET(Mut)-induced mammary tumor formation and metastasis. Unexpectedly, tumors arising in either MET(Mut) or WAP-Brk × MET(Mut) 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.

Cytoplasmic PELP1 and ERRgamma Protect Human Mammary Epithelial Cells from Tam-Induced Cell Death

Tamoxifen (Tam) is the only FDA-approved chemoprevention agent for pre-menopausal women at high risk for developing breast cancer. While Tam reduces a womans 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.

Breast Tumor Kinase (Brk/PTK6) Is Induced by HIF, Glucocorticoid Receptor, and PELP1-Mediated Stress Signaling in Triple-Negative Breast Cancer.

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 cross-talk between stress hormone-driven GR signaling and HIF-regulated physiologic stress. Primary TNBC tumor explants or cell lines treated with the GR ligand dexamethasone exhibited robust induction of Brk mRNA and protein that was HIF1/2-dependent. HIF and GR coassembled on the BRK promoter in response to either hypoxia or dexamethasone, 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.

Taxol Induces Brk-dependent Prosurvival Phenotypes in TNBC Cells through an AhR/GR/HIF–driven Signaling Axis

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 (n = 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. Implication: Breast cancer cells enlist intracellular stress response pathways that evade chemotherapy by increasing cancer cell survival and promoting migratory phenotypes. Mol Cancer Res; 16(11); 1761–72. ©2018 AACR.

Abstract 1331: TGFβ1 induces breast tumor kinase overexpression in triple negative breast cancer via p38 MAPK signaling to glucocorticoid receptors

Triple negative breast cancer (TNBC) is the deadliest breast cancer (BC) subtype, accounting for 20-30% of all BCs. It has a heterogeneous pathology and pathogenicity, but it is defined by the lack of estrogen receptor, progesterone receptor, and Her2 epidermal growth factor receptor expression. Because targeted therapy through these receptors is not possible, treatment relies on chemotherapy and surgery, which are often inadequate. Thus, the identification of possible molecular targets is critically important in TNBC. Breast tumor kinase (Brk) is a soluble tyrosine kinase that is overexpressed in 85% of BCs and a driver of aggressive and metastatic phenotypes. Overexpression of Brk mRNA and protein occurs in TNBC by unknown mechanisms. The glucocorticoid receptor (GR), a very potent modulator of cytokine mediated actions of the immune system, is emerging as a mediator of chemoresistance and recurrence in TNBC. We previously demonstrated that GR signaling cooperates with physiologic stress signaling mediated by hypoxia inducible factors HIF-1a and HIF-2 to modulate the expression of Brk mRNA and protein in TNBC cells. Moreover, phosphorylation of GR at ser134 by p38 MAPK is essential for GR regulation of Brk expression. P38 is an essential Ser/Thr kinase that regulates cellular transduction of growth factors, such as Hepatocyte Growth Factor (HGF), and cytokines (e.g. TGFβ1) in TNBC cells, and was previously shown to be co-expressed with Brk in human breast tumors. Herein, we further probed mechanisms of crosstalk between key cytokines, GR, and p38 signaling in the regulation of Brk overexpression. We hypothesize that TGFβ1 signaling modulates EMT and metastasis in part by increasing the expression of Brk in TNBC. Treatment of MDA-MB-231 cells with TGFβ1 for 1, 2, 24 and 48 hours increased Brk protein expression relative to vehicle controls. Additionally, TGFβ1 increased both HIF1 and HIF2 protein levels (at 24 and 48 hours respectively). TGFβ1 regulated Brk expression at the level of mRNA, as measured using RT-PCR. Moreover, TGFβ1 synergized with activated GR to further increase Brk mRNA levels. In contrast, mRNA levels of HIF1 and HIF2 were not modulated by TGFβ1, suggesting that the observed protein increases are due to stabilization of HIFs. Finally, TGFβ1 robustly induced p38-dependent phosphorylation of GR at serine 134. This phosphorylation event promoted ligand-independent GR transcriptional activity at the Brk promoter. Human breast tumors significantly co-express active p38 MAPK and Brk. Our molecular model implicates TGFβ1 signaling (via p38 MAPKs) to phospho-GR in the aberrant overexpression of Brk in TNBC. We conclude that blocking of the TGFβ1 pathway may provide a strategy to inhibit Brk mediated TNBC tumor progression. This work was supported by NIH/NCI R01 CA192178 (to CAL) and T32 GM008244-24. Citation Format: Carlos J. Santos Perez, Tarah Regan Anderson, Carol A. Lange. TGFβ1 induces breast tumor kinase overexpression in triple negative breast cancer via p38 MAPK signaling to glucocorticoid receptors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1331. doi:10.1158/1538-7445.AM2017-1331

Abstract A56: Regulation of breast tumor kinase (Brk) expression in triple-negative breast cancer integrates cellular (HIF-2alpha) and hormonal (cortisol) stress signaling

Abstracts: AACR Special Conference: Advances in Breast Cancer; October 17-20, 2015; Bellevue, WA 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.

Abstract 2100: Regulation of breast tumor kinase (Brk) expression in triple negative breast cancer integrates cell stress and cortisol signaling pathways

Triple negative breast cancers (TNBC) lack expression of the molecules currently exploited for targeted therapy (estrogen receptor, progesterone receptor, and HER2). These tumors frequently respond well to chemotherapy, but emergence of chemotherapy-resistance is a common clinical problem. Thus, identification of drivers of TNBC that may be exploited for targeted therapy is of great importance. Breast tumor kinase (Brk), also known as protein tyrosine kinase 6 (PTK6), is a soluble tyrosine kinase that is absent from normal mammary epithelial cells but inappropriately expressed in 85% of breast cancers. Previous studies from our lab have shown Brk to be a critical regulator of breast cancer cell migration in vitro and a potent driver of basal-type mammary tumors in vivo. Notably, we recently identified a novel mechanism of upregulation of Brk expression in TNBC cells. Brk expression is induced following exposure to cell stress, including hypoxia, oxidative damage, and nutrient starvation. Moreover, we found that Brk is a direct target gene of both hypoxia-inducible factor 1 alpha (HIF-1α) and HIF-2α. It is becoming increasingly evident that cortisol signaling via activation of the glucocorticoid receptor (GR) leads to enhanced 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 (HIFs) and cortisol signaling (GR) that may influence expression of Brk in TNBC. Brk mRNA and protein were induced in response to the GR ligand, dexamethasone (dex), in both normoxia and hypoxia, and blocked following treatment with RU486, a GR antagonist. Brk mRNA and protein failed to be induced following dex treatment in cells lacking HIF-1α and HIF-2α, suggesting that even in normoxia, GR regulation of Brk requires HIF-1α/2α. Chromatin immunoprecipitation (ChIP) assays showed HIF and GR recruitment to multiple regions of the Brk promoter in response to either hypoxia (a ligand-independent GR action) or treatment with dex, indicating that Brk is a direct GR/HIF target gene. These data show that cortisol signaling via GR is a novel mechanism of upregulation of Brk expression in TNBC. Additionally, GR upregulation of Brk is HIF-dependent, revealing molecular linkage between cell stress pathways and stress hormone signaling. Collectively, our studies suggest that GR and HIF cross talk may promote aggressive tumor behavior, in part via upregulation of Brk. Breast cancer patients are routinely given dex (a GR agonist) to alleviate the inflammatory side effects of chemotherapy. This treatment may inadvertently promote tumor progression via induction of Brk expression. Citation Format: Tarah M. Regan Anderson, Andrea R. Daniel, Carol A. Lange. Regulation of breast tumor kinase (Brk) expression in triple negative breast cancer integrates cell stress and cortisol signaling pathways. [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 2100. doi:10.1158/1538-7445.AM2014-2100

Abstract 2927: Breast tumor kinase (Brk/PTK6) mediates hypoxia/HIF-1α-associated breast cancer progression.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Triple negative (basal type) breast cancers are more aggressive and difficult to treat relative to HER2/ER/PR positive (luminal type) breast cancers; these tumors frequently express abundant Met receptors and contain high constitutive levels of hypoxia inducible factor alpha (HIF-1α), a principal mediator of cellular stress that predicts cancer relapse and increased risk of metastasis. Breast tumor kinase (Brk/PTK6; Sik in mice) is a soluble tyrosine kinase that is not found in normal breast tissue, but is aberrantly expressed in up to 86% of all breast cancers. We recently showed that Brk is a critical downstream effector of Met signaling and required for HGF-induced cell migration. Herein, we show that Brk and HIF-1α are co-expressed in human triple negative cell lines and tumors. Brk mRNA and protein levels are strongly upregulated during conditions that induce cellular stress in vitro, including low glucose, H2O2, and hypoxia. Chromatin-immunoprecipitation (ChIP) assays revealed that Brk is a direct early transcriptional target of HIF-1α; HIF knockdown demonstrated that Brk expression is dependent upon both HIF-1α and HIF-2α. Notably, Brk expression rescued the growth of MDA-MB-231 xenografts knocked-down for both HIF1α and HIF2α in vivo. Finally, we crossed WAP-Brk (FVB) transgenic mice into the MMTV-METmut model (FVB mice engineered to express MMTV-driven constitutively active mutant Met receptors); the addition of the human wt Brk transgene decreased survival in this model. Notably, mouse mammary tumors that arose in MMTV-METmut mice expressed abundant Sik, the mouse homolog of Brk; high Sik expression conferred markedly decreased latency to tumor formation independently of the presence of the Brk transgene. These results identify HIF-1α as a novel regulator of Brk mRNA expression and suggest that Brk is a key mediator of HIF-1α-induced breast cancer progression. Targeting Brk expression or activity (downstream of the HGF/Met receptor signaling pathway) may provide an effective means to block the progression of deadly triple-negative breast cancers. Citation Format: Tarah Regan Anderson, Gregory Hubbard, Andrea R. Daniel, Danielle Peacock, Roland H. Wenger, Alexandra Schorg, David Hoogewijs, George Vande Woude, Tiffany N. Seagroves, Carol A. Lange. Breast tumor kinase (Brk/PTK6) mediates hypoxia/HIF-1α-associated breast cancer progression. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2927. doi:10.1158/1538-7445.AM2013-2927