Poster Session 2 - R8
1,2Justin Rondeau, 1Lubna Nadeem, 1,2,3Stephen Lye
1 LTRI, Sinai Health System, Toronto; 2 Dept. of Physiology, University of Toronto; 3 Dept. of Obstetrics and Gynaecology, University of Toronto, Toronto, ON, Canada
Objectives: Progesterone (P4) imparts distinct effects in the breast via its progesterone receptors (PRs) PRA and PRB. We have recently found that the gap junction protein Connexin 43 (Cx43) is differentially regulated by P4 via PRA/B in human myometrial cells, where P4/PRA upregulates Cx43 expression and trafficking to the plasma membrane (PM) and P4/PRB inhibits these processes. Since Cx43-mediated gap junction intercellular connectivity (GJIC) has the potential to suppress tumorigenesis via the spread of pro-apoptotic signals between adjacent cells, we sought to delineate the roles of PRA/PRB on Cx43 expression and trafficking in breast cancer tumorigenesis. Methods: Overexpression of PRA/PRB and Cx43-GFP in the PR-ve cell line MDA231 and the PRB+ve cell line MFM223 was examined via immunofluorescence after 24 hour treatment with P4 (100nM) or its vehicle control (70% ethanol). Actin staining was also executed using immunofluorescence, while surface area and Cx43 travelling distance was quantified using Volocity. Live cell imaging of MDA231 and MFM223 cells were imaged via IN Cell Analyzer 2000. To verify our results endogenously, CRISPR was performed on PRA+ve/PRB+ve and Cx43+ve MCF7 cells to selectively knock out (KO) PRB. Cx43 expression analysis via Western Blotting and qPCR was performed on wildtype (WT) versus PRB KO cells treated with P4 or its vehicle control. Results: Immunofluorescence analysis of MDA231 and MFM223 revealed PRA and PRB differentially regulate Cx43 trafficking towards the PM. Actin quantification showed PRA cells exhibit mesenchymal-like morphology while PRB expressing cells retain their roundness, and Cx43 travels farther in PRA expressing cells. Live cell imaging revealed Cx43 is actively trafficked towards the PM in PRA expressing cells after 24 hours, while Cx43 is retained in the perinuclear space in PRB expressing cells. Furthermore, Cx43 is differentially expressed in MCF7 WT versus MCF7 PRB KO cells, where P4 increases Cx43 protein expression in PRB KO cells whereas P4 decreases Cx43 expression in WT cells. Conclusions: Our data suggests that in endocrine responsive tumours, PR isoform profiling may be critical to the assessment of hormonal therapy options. In patients with PRA-rich tumours, P4 treatment may prove beneficial due to its restorative action on Cx43 expression and trafficking, thereby enhancing its tumour-suppressive properties. Funding: Foundation Grant/CIHR