Invasion of ovarian cancer cells is induced byPITX2-mediated activation of TGF-β and Activin-A

Abstract

BackgroundMost ovarian cancers are highly invasive in nature and the high burden of metastatic disease make them a leading cause of mortality among all gynaecological malignancies. The homeodomain transcription factor, PITX2 is associated with cancer in different tissues. Our previous studies demonstrated increased PITX2 expression in human ovarian tumours. Growing evidence linking activation of TGF-β pathway by homeodomain proteins prompted us to look for the possible involvement of this signalling pathway in PITX2-mediated progression of ovarian cancer.MethodsThe status of TGF-β signalling in human ovarian tissues was assessed by immunohistochemistry. The expression level of TGFB/INHBA and other invasion-associated genes was measured by quantitative-PCR (Q-PCR) and Western Blot after transfection/treatments with clones/reagents in normal/cancer cells. The physiological effect of PITX2 on invasion/motility was checked by matrigel invasion and wound healing assay. The PITX2- and activin-induced epithelial-mesenchymal transition (EMT) was evaluated by Q-PCR of respective markers and confocal/phase-contrast imaging of cells.ResultsHuman ovarian tumours showed enhanced TGF-β signalling. Our study uncovers the PITX2-induced expression of TGFB1/2/3 as well as INHBA genes (p < 0.01) followed by SMAD2/3-dependent TGF-β signalling pathway. PITX2-induced TGF-β pathway regulated the expression of invasion-associated genes, SNAI1, CDH1 and MMP9 (p < 0.01) that accounted for enhanced motility/invasion of ovarian cancers. Snail and MMP9 acted as important mediators of PITX2-induced invasiveness of ovarian cancer cells. PITX2 over-expression resulted in loss of epithelial markers (p < 0.01) and gain of mesenchymal markers (p < 0.01) that contributed significantly to ovarian oncogenesis. PITX2-induced INHBA expression (p < 0.01) contributed to EMT in both normal and ovarian cancer cells.ConclusionsOverall, our findings suggest a significant contributory role of PITX2 in promoting invasive behaviour of ovarian cancer cells through up-regulation of TGFB/INHBA. We have also identified the previously unknown involvement of activin-A in promoting EMT. Our work provides novel mechanistic insights into the invasive behavior of ovarian cancer cells. The extension of this study have the potential for therapeutic applications in future.Electronic supplementary materialThe online version of this article (doi:10.1186/s12943-015-0433-y) contains supplementary material, which is available to authorized users.

Co-Activation of TGFβ and Wnt Signalling Pathways Abrogates EMT in Ovarian Cancer Cells

“…Immunofluorescence was carried out as described previously [30] using the antibodies Claudin 7, Vimentin, Snail and β-catenin.…”

“…Previous reports suggest that Wnt and TGFβ promote EMT [27-29]. Considering background references regarding Wnt and TGFβ signalling cascades in EMT and ovarian cancer [28-30], we wanted to study the effect of this co-activation in these processes to mimick a part of the signalling cascade functioning in the tumour microenvironment. In this report, we have shown the effect of the functioning of Wnt and TGFβ in unison in abrogating EMT.…”

Lysophosphatidic Acid Promotes Epithelial to Mesenchymal Transition in Ovarian Cancer Cells by Repressing SIRT1

“…Immunofluorescence staining with E-cadherin and N-cadherin (1: 100) antibodies followed by Alexafluor 488-conjugated secondary antibody was performed as described previously [22]. For phalloidin staining 10 5 cells/well were treated and the cellular morphology was observed as mentioned previously [23].…”

Activin A stimulates migration of the fallopian tube epithelium, an origin of high-grade serous ovarian cancer, through non-canonical signaling

“…The role of activin A in epithelial ovarian cancer is less clear. Basu et al observed higher phospho-Smad2 immunostaining in ovarian tumors compared to normal ovaries [32], and Do et al found that ovarian cystadenocarinoma patients with high immunostaining for INHBA tended to have shorter survival times than patients with low immunostaining [33]. Activin A stimulates migration of OCC1 and SKOV3 cells [34].…”