BackgroundHypoxia-inducible factors (HIFs) are well-established mediators of tumor growth, the epithelial to mesenchymal transition (EMT) and metastasis. In several types of solid tumors, including breast cancers, the HIFs play a critical role in maintaining cancer stem cell (CSC) activity. Thus, we hypothesized that HIFs may also regulate transcription of markers of breast CSC activity. One approach to enrich for breast cells with stem-like phenotypes is FACS sorting, in which sub-populations of live cells are gated based on the expression of cell surface antigens, including various integrin subunits. Integrin alpha 6 (ITGA6; CD49f) is routinely used in combination with other integrin subunits to enrich for breast stem cells by FACS. Integrins not only mediate interactions with the extracellular matrix (ECM), but also drive intracellular signaling events that communicate from the tumor microenvironment to inside of the tumor cell to alter phenotypes including migration and invasion.MethodsWe used two models of metastatic breast cancer (MBC), polyoma middle T (MMTV-PyMT) and MDA-MB-231 cells, to compare the expression of ITGA6 in wild type and knockout (KO) or knockdown cells. Chromatin immunoprecipitation (ChIP) and luciferase reporter assays verified that ITGA6 is a direct HIF transcriptional target. We also used FACS sorting to enrich for CD49f + cells to compare tumorsphere formation, tumor initiating cell activity, invasion and HIF activity relative to CD49fneg or low cells. Knockdown of ITGA6 significantly reduced invasion, whereas re-expression of ITGA6 in the context of HIF knockdown partially rescued invasion. A search of public databases also revealed that ITGA6 expression is an independent prognostic factor of survival in breast cancer patients.ResultsWe report that ITGA6 is a HIF-dependent target gene and that high ITGA6 expression enhances invasion and tumor-initiating cell activities in models of MBC. Moreover, cells that express high levels of ITGA6 are enriched for HIF-1α expression and the expression of HIF-dependent target genes.ConclusionsOur data suggest that HIF-dependent regulation of ITGA6 is one mechanism by which sorting for CD49f + cells enhances CSC and metastatic phenotypes in breast cancers. Our results are particularly relevant to basal-like breast cancers which express higher levels of the HIFα subunits, core HIF-dependent target genes and ITGA6 relative to other molecular subtypes.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-016-0510-x) contains supplementary material, which is available to authorized users.

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.

A crucial step in the cellular adaptation to oxygen deficiency is the binding of hypoxia-inducible factors (HIFs) to hypoxia response elements (HREs) of oxygen-regulated genes. Genome-wide HIF-1α/2α/β DNA-binding studies revealed that the majority of HREs reside distant to the promoter regions, but the function of these distal HREs has only been marginally studied in the genomic context. We used chromatin immunoprecipitation (ChIP), gene editing (TALEN) and chromosome conformation capture (3C) to localize and functionally characterize a 82 kb upstream HRE that solely drives oxygen-regulated expression of the newly identified HIF target gene PAG1. PAG1, a transmembrane adaptor protein involved in Src signalling, was hypoxically induced in various cell lines and mouse tissues. ChIP and reporter gene assays demonstrated that the −82 kb HRE regulates PAG1, but not an equally distant gene further upstream, by direct interaction with HIF. Ablation of the consensus HRE motif abolished the hypoxic induction of PAG1 but not general oxygen signalling. 3C assays revealed that the −82 kb HRE physically associates with the PAG1 promoter region, independent of HIF-DNA interaction. These results demonstrate a constitutive interaction between the −82 kb HRE and the PAG1 promoter, suggesting a physiologically important rapid response to hypoxia.

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<p>PDF file, 2022K, Western and mRNA data downing retroviral transduction of Brk gene and subsequent over expression, as well as maintenance of HIF1a/2a knockdown.</p>

<p>PDF file, 5252K, Table detailing patient information from HCI tumor samples used.</p>

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 Schörg, 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

<p>PDF file, 4524K, Regulation of Brk protein and mRNA expression in MCF7 cells.</p>