Abstract
IntroductionBasal phenotype breast cancers (BPBC) are often associated with apparent epithelial to mesenchymal transition (EMT). The role of progesterone (P4) in regulating EMT of BPBC has not been reported.MethodsThe EMT relevant biology was investigated in vitro using human BPBC cell models (MDA-MB468 and MDA-MB231) with P4, PR agonist (RU486), and PR antagonist (R5020) treatments. The essential role of membrane progesterone receptor α (mPRα) in the P4-regulated EMT was demonstrated by knocking down the endogenous gene and/or stably transfecting exogenous mPRα gene in the BPBC cell models.ResultsThe expression of snail and down-stream EMT proteins such as occludin, fibronectin, and E-cadherin was significantly regulated by P4 incubation, which was accompanied by cell morphological reversion from mesenchymal to epithelial phenotypes. In searching for the cell mediator of P4' action in the MDA-MB468 (MB468) cells, it was found that mPRα but not the nuclear PR has an essential role in the P4 mediated EMT inhibition. Knocking down the expression of mPRα with specific siRNA blocked the P4's effects on expression of the EMT proteins. In another BPBC cell line - MDA-MB231 (MB231), which is mPRα negative by Western blotting, P4 treatment did not alter cell proliferation and EMT protein expressions. Introduction of the exogenous mPRα cDNA into these cells caused cell proliferation, but not EMT, to become responsive to P4 treatment. In further studies, it was found that activation of the PI3K/Akt pathway is necessary for the P4-induced EMT reversion. To define the potential inter-mediate steps between mPRα and PI3K, we demonstrated that mPRα, caveolin-1 (Cav-1), and epidermal growth factor receptor (EGFR) are colocalized in the membrane of caveolar vesicle and the P4-repressed EMT in MB468 cells can be blocked by EGFR inhibitor (AG1478) and PI3K inhibitor (wortmannin).ConclusionsOur data suggest that the signaling cascade of P4 induced mesenchymal repression is mediated through mPRα and other caveolae bound signaling molecules namely Cav-1, EGFR, and PI3K. This novel finding may have great impact on fully understanding the pathogenesis of BPBC and provide an essential clue for developing a targeted therapeutic strategy for treatment of BPBC.
Highlights
Basal phenotype breast cancers (BPBC) are often associated with apparent epithelial to mesenchymal transition (EMT)
Anti-snail antibody was from Abcam (1:1000, Cambridge, MA, USA); anti-E-cadherin and antifibronectin antibodies were obtained from EPITMICS (1:1000, Burlingame, CA, USA); anti-membrane progesterone receptor α (mPRα), antiGAPDH (1:500) and secondary antibodies (1:2000) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA); anti-occludin antibody was from BD transduction (1:500, San Jose, CA, USA); and anti-α-tubulin antibody was from Sigma
P4 regulates expression of snail and other EMT-relevant proteins in MB468 but not in MB231 cells In this study, we focused on the effects of P4 on expression of snail and other EMT marker proteins
Summary
Basal phenotype breast cancers (BPBC) are often associated with apparent epithelial to mesenchymal transition (EMT). The actions of P4 on cancer cells are attributed to the binding of nuclear progesterone receptor (PR), translocation of P4/PR complex into the nucleus and subsequent activation of target genes over the course of several hours These mechanisms, are not applicable to BPBC due to a lack or very low level of PR expression in these cancers. The binding of progestin to mPRα alters the secondary messenger pathways through activation of the pertussis toxin-sensitive inhibitory Gproteins and activates the mitogen activated protein kinases (MAPK)/Erk 1/2 pathway [6,7,11,12] This theory has been debated because others failed to demonstrate mPRs on the cell surface or mediate P4dependent signaling events, such as coupling to G proteins [13].
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