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Abstract

Cancer is a leading cause of death globally and most cancer-related deaths are attributable to cancer metastasis, a process describing the movement of cells from primary tumours to distant sites within the body. As metastasis is the primary cause of cancer patient death, understanding the process of metastasis is critical for the development of new therapeutics to treat or prevent metastasis. Gamma-aminobutyric acid (GABA), a prominent neurotransmitter, has been linked to processes modulating tumour cell proliferation and metastasis. GABA is expressed at elevated levels in many tumour types including triple negative breast cancers (TNBC), where it is associated with tumour progression and poor prognosis. However, the precise molecular mechanism underlying GABA-induced pro-metastatic phenotypes in tumour cells is largely undescribed. Recently, our lab described a role for GABA in promoting tumour cell extravasation into GABA-rich environments, with early evidence suggesting a role for GABA in promoting invadopodia formation. Invadopodia, are actin-rich membrane protrusions that have been observed in vitro and in vivo in many metastatic cell lines and are critical to the progression of cancer metastasis. Here, we demonstrate GABA’s role in invadopodia formation and function in three TNBC cell lines. We evaluated endogenous GABA levels in a panel of TNBC cell lines through dot-blot analysis; incubated cells with exogenous GABA and the GABA receptor (GABAR) inhibitors, gabazine and 2-OH-saclofen, then assessed their effects on invadopodia formation. We observed that stimulation of cells with exogenous GABA significantly increased invadopodia formation and inhibition of GABAARs using gabazine significantly reduced invadopodia formation. Conversely, our control had no significant differences relative to cells treated with the GABABR antagonist, 2-OH-Saclofen. Collectively, our findings indicate GABA is important for invadopodia formation and function; the reduction in invadopodia formation in response to gabazine suggests GABA signals through GABAARs to mediate invadopodia formation. Lastly, our dot-blots confirmed endogenous GABA expression in our TNBC cells, providing further evidence to suggest TNBC cells utilize GABA to promote invadopodia formation, local-tumour invasion and increase extravasation into GABA-rich microenvironments. Understanding how GABA mediates tumor metastasis in TNBC has the potential to identify novel therapeutic targets with clinical utility for TNBC treatment.