|dc.description.abstract||The human ether-a-go-go-related gene (HERG) potassium channel, a known regulator of cell proliferation, is overexpressed in several cancer cell lines. Despite its importance, there have not been many studies regarding the mechanism by which it contributes to aberrant proliferation of cancer cells. In this study, we identified a novel estrogen signalling pathway that plays a role in regulating cell proliferation in estrogen receptor positive breast cancer cells. We provide the initial characterization of this signal transduction pathway which results in up-regulation of HERG channels and increased proliferation of estrogen receptor positive breast cancer cell lines.
Using biochemical and confocal microscopy imaging, we revealed that there is a protein-protein interaction between HERG and Signal Transducers and Activators of Transcription 1 (STAT1) in breast cancer cell lines that express estrogen receptors (ER+) (MCF-7, T47D) and in those that lack ERs (ER-) (MDA-MB-231, BT-20). After estrogen treatment (E2, 10µM), only the ER+ human breast cancer cell lines showed increased co-precipitation of HERG and STAT1 and higher levels of subcellular colocalization, and these effects were prevented by pharmacological blockers of ERs (ICI 182 780, 5nM) or STAT1 (fludarabine, 50µM). Furthermore, we demonstrated that the enhanced STAT1 and HERG interaction induced by estrogen is important for upregulation of the HERG channel surface expression. Consistent with our prediction that HERG surface expression plays a critical role in cancer cell proliferation, proliferation assays revealed a marked upregulation of ER+ cancer cell proliferation after E2 stimulation, and this was prevented by ICI, fludarabine and the HERG blocker E4031 (10µM). Together, these results suggest that there is an estrogen receptor mediated signalling pathway involving a physical complex between STAT1 and HERG channels, and this proposed pathway may be an attractive strategy for anti-cancer therapeutic targeting of ER+ human breast tumors. Next, since it is known that HERG contains multiple tyrosine residues whose phosphorylation could be increased after estrogen receptor stimulation, we hypothesized that these phosphorylated tyrosine residues could serve as binding sites of the SH2 domain of STAT1. We have used FR-peptide, a 28 amino acid peptide mimetic of STAT1 SH2 domain, to disrupt interaction between HERG and STAT1. Our biochemistry and imaging data demonstrated that FR-peptide did disrupt the HERG-STAT1 interaction, and this cell-permeable peptide did prevent the estrogen-induced upregulation of HERG channel and cancer cell proliferation of ER+ breast cancer cells. Finally, we also demonstrated that the HERG-STAT1 interaction was specific to breast cancer cells, as similar biochemical studies failed to show physical interactions in normal brain and cardiac tissue.
Together, this study reveals a novel estrogen receptor signalling pathway which enhances the HERG channel surface expression and its contribution to cancer cell proliferation in ER+ human breast cancer cell lines. The identification of the FR-peptide to occlude HERG-STAT1 interaction and prevent HERG surface expression and cancer cell proliferation, may form the basis for a rational design of anti-cancer therapeutics. The regulatory factors involved in the regulation of endogenous HERG-STAT1 interaction in ER- cell line remains to be established.||