Regulation of Brk Expression via Stress-activated Signaling in Breast Cancer

Objectives:
Breast tumors experience high levels of physiologic stress due to limited access to oxygen and nutrients, and utilize stress response pathways to sustain their pathogenic behavior. Adaptation to cellular and host stress is mediated by hypoxia inducible factors HIF-1 and HIF-2 and glucocorticoid receptor (GR) signaling, respectively. We previously identified a novel signaling complex including HIFs/GR that is critical for inducing breast tumor kinase (Brk/PTK6) expression in breast tumors. Brk is overexpressed in 85% of breast cancers and is a driver of aggressive phenotypes, including cell survival and migration.
Aryl hydrocarbon receptor (AhR) is a transcription factor activated in response to environmental toxins. Recent literature suggests that AhR is also involved in tumor progression. We hypothesize that crosstalk between AhR, GR, and HIF’s results in induction of Brk expression, allowing cells to adapt during stress, including altered growth conditions.

Methods: Techniques utilized herein include immunoblotting assays and quantitative real-time polymerase chain reactions (qRT-PCR) in cells grown in attachment or in suspension (using ultra-low attachment plates). Cell lines included MD-MBA-231, BT-549, and T47D-co.

Results: Results revealed that AhR, HIF2a, and Brk were upregulated in suspension (compared to attached plates) in all three cell lines. This increase was seen at the level of mRNA and protein expression. Additionally, a multitude of AhR target genes including TDO2, NFE2L2, CYP1A1, CYP1B1, and KYNU were highly expressed in suspension relative to adherent conditions as positive controls.
Conclusions: These data suggest that AhR activation in suspension upregulates Brk expression, thus allowing the cancer cell to survive in non-adherent conditions, reminiscent of multiple steps in the metastatic process. A better understanding of the crosstalk that occurs between AhR and GR will provide important insights into mechanisms used by cancer cells to adapt to stress, survive when not attached to the basement membrane, and metastasize.