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

Basu M, Bhattacharya R, Ray U, Mukhopadhyay S, Chatterjee U, Roy SS - Mol. Cancer (2015)

Bottom Line: The physiological effect of PITX2 on invasion/motility was checked by matrigel invasion and wound healing assay.We have also identified the previously unknown involvement of activin-A in promoting EMT.The extension of this study have the potential for therapeutic applications in future.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Physiology Division, CSIR-Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, 4 Raja S. C. Mullick Road, Kolkata, 700032, India. moitrri_basu@yahoo.co.in.

ABSTRACT

Background: Most 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.

Methods: The 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.

Results: Human 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.

Conclusions: Overall, 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.

No MeSH data available.


Related in: MedlinePlus

PITX2 acts through Activin-A to affect the cellular invasion and EMT. a Matrigel transwell assay was performed in OAW-42 cells which were treated either with rhActivin-A or transfected with INHBA-siRNA in presence or absence of PITX2A-overexpression and the percentage of cell invasion was calculated. Scale bar 100 μm. b Cells at three independent fields for each well were counted and plotted with error bar. c-f RNA was isolated from cells tarnsfected with INHBA-siRNA in presence or absence of PITX2A- overexpression and the expression of MMP9, ZEB1, SNAI1, VIM, CLDN7 was quantified by Q- PCR with specific primers. Relative gene expression is indicated as ‘fold’ change in the Y-axis (mean ± SEM). The statistical analysis is done as described previously.*represents p < 0.05
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Fig7: PITX2 acts through Activin-A to affect the cellular invasion and EMT. a Matrigel transwell assay was performed in OAW-42 cells which were treated either with rhActivin-A or transfected with INHBA-siRNA in presence or absence of PITX2A-overexpression and the percentage of cell invasion was calculated. Scale bar 100 μm. b Cells at three independent fields for each well were counted and plotted with error bar. c-f RNA was isolated from cells tarnsfected with INHBA-siRNA in presence or absence of PITX2A- overexpression and the expression of MMP9, ZEB1, SNAI1, VIM, CLDN7 was quantified by Q- PCR with specific primers. Relative gene expression is indicated as ‘fold’ change in the Y-axis (mean ± SEM). The statistical analysis is done as described previously.*represents p < 0.05

Mentions: We next attempted to investigate the possible contribution of activin-A in PITX2-induced invasion and EMT in ovarian cancer cells. rhActivin-A treatment resulted in ~4 fold increase in invasion of OAW-42 cells compared to controls as shown by matrigel invasion assay (Fig. 7a-b). In contrast, PITX2A-enhanced cell invasiveness was reduced by ~ 50 % on transfection with INHBA-siRNA (Fig. 7a-b). PITX2A increased mRNA levels of MMP9 (Fig. 7c), SNAI1 and ZEB1 (Fig. 7d) that were significantly reduced by transfection with activin-A siRNA. Similarly, the mRNA levels of the mesenchymal marker VIM was up-regulated by ~ 2.5 folds on PITX2A over-expression, whereas it decreased drastically upon activin-A knockdown in presence of PITX2A over-expression (Fig. 7e). Knockdown of INHBA rescued the PITX2A-mediated suppression of the epithelial marker CLDN7 (Fig. 7f).Fig. 7


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

Basu M, Bhattacharya R, Ray U, Mukhopadhyay S, Chatterjee U, Roy SS - Mol. Cancer (2015)

PITX2 acts through Activin-A to affect the cellular invasion and EMT. a Matrigel transwell assay was performed in OAW-42 cells which were treated either with rhActivin-A or transfected with INHBA-siRNA in presence or absence of PITX2A-overexpression and the percentage of cell invasion was calculated. Scale bar 100 μm. b Cells at three independent fields for each well were counted and plotted with error bar. c-f RNA was isolated from cells tarnsfected with INHBA-siRNA in presence or absence of PITX2A- overexpression and the expression of MMP9, ZEB1, SNAI1, VIM, CLDN7 was quantified by Q- PCR with specific primers. Relative gene expression is indicated as ‘fold’ change in the Y-axis (mean ± SEM). The statistical analysis is done as described previously.*represents p < 0.05
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4546816&req=5

Fig7: PITX2 acts through Activin-A to affect the cellular invasion and EMT. a Matrigel transwell assay was performed in OAW-42 cells which were treated either with rhActivin-A or transfected with INHBA-siRNA in presence or absence of PITX2A-overexpression and the percentage of cell invasion was calculated. Scale bar 100 μm. b Cells at three independent fields for each well were counted and plotted with error bar. c-f RNA was isolated from cells tarnsfected with INHBA-siRNA in presence or absence of PITX2A- overexpression and the expression of MMP9, ZEB1, SNAI1, VIM, CLDN7 was quantified by Q- PCR with specific primers. Relative gene expression is indicated as ‘fold’ change in the Y-axis (mean ± SEM). The statistical analysis is done as described previously.*represents p < 0.05
Mentions: We next attempted to investigate the possible contribution of activin-A in PITX2-induced invasion and EMT in ovarian cancer cells. rhActivin-A treatment resulted in ~4 fold increase in invasion of OAW-42 cells compared to controls as shown by matrigel invasion assay (Fig. 7a-b). In contrast, PITX2A-enhanced cell invasiveness was reduced by ~ 50 % on transfection with INHBA-siRNA (Fig. 7a-b). PITX2A increased mRNA levels of MMP9 (Fig. 7c), SNAI1 and ZEB1 (Fig. 7d) that were significantly reduced by transfection with activin-A siRNA. Similarly, the mRNA levels of the mesenchymal marker VIM was up-regulated by ~ 2.5 folds on PITX2A over-expression, whereas it decreased drastically upon activin-A knockdown in presence of PITX2A over-expression (Fig. 7e). Knockdown of INHBA rescued the PITX2A-mediated suppression of the epithelial marker CLDN7 (Fig. 7f).Fig. 7

Bottom Line: The physiological effect of PITX2 on invasion/motility was checked by matrigel invasion and wound healing assay.We have also identified the previously unknown involvement of activin-A in promoting EMT.The extension of this study have the potential for therapeutic applications in future.

View Article: PubMed Central - PubMed

Affiliation: Cell Biology and Physiology Division, CSIR-Indian Institute of Chemical Biology, Council of Scientific and Industrial Research, 4 Raja S. C. Mullick Road, Kolkata, 700032, India. moitrri_basu@yahoo.co.in.

ABSTRACT

Background: Most 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.

Methods: The 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.

Results: Human 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.

Conclusions: Overall, 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.

No MeSH data available.


Related in: MedlinePlus