Invasion of ovarian cancer cells is induced by PITX2-mediated activation of TGF-β and Activin-A

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

Studies on The Role of Pitx2 Homeodomain Transcription Factor to Maintain Ovarian Structure and Function

The bicoid-related homeobox gene Pitx2 is a member of a multi gene family with multiple functional aspects. Pitx2, have been first implicated in pituitary development and transcriptional regulation (Gaje PJ et al., 1997, 1999). Later on, extensive studies demonstrated that Pitx2-deficient mice are embryonic lethal and showed severe defects in heart, eye, pituitary and tooth organogenesis (Ai D et al., 2007; Lin CR et al., 1999). Three different isoforms of PITX2 (PITX2A/B/C) differ only in their amino terminus and differentially regulate transcription of their target genes (Ganga M et al., 2003; Vadlamudi U et al., 2005). The Pitx isoforms are members of bicoid class of homeodomain transcription factors and besides Plod2 gene, they regulate genes like LHβ, FSHβ, GH, PRL and GnRH receptor (Quentien MH et al, 2002; Tremblay JJ et al, 2000, 1998) by interacting with cell-restricted factors such as Steroidogenic Factor1 (SF-1), Early Growth Response-1 (Egr-1), the heterodimeric NeuroD1/ Pan1, Tpit and Pit1 (Quentien MH et al, 2002). Pitx2 controls cell cycle regulating genes, such as cyclin D1 and -D2 as well as it regulates Wnt dependent mRNA turn over (Briata P et al, 2003). Pitx isoforms regulate the expression of other transcription factors, like Hesx1, Prop1, Lhx3 and Gata2 (Charles MA et al, 2005). Upon finding of the expression of PITX2 in ovary (Ghosh P et al., 2007), we speculated its significant involvement in maintaining the ovarian structural integrity. The extracellular matrix (ECM) proteins, especially collagens play very critical role in maintaining the normal function of ovary. Procollagen Lysine 2-oxoglutarate 5-dioxygenase (Plod) is the key enzyme responsible for hydroxylation of lysines in collagens that plays a role in specifying the ECM and provides a foundation for the morphogenesis of tissues and organs (Cox CJ et al, 2002). Three Plod isoforms have been characterized in human, mouse and in rats. The ECM has an important role in the formation and maintenance of follicles and corpora lutea (Vogel W et al, 1997; Luck MR,1994; Rodgers HF et al, 1998, 1999; Belkin AM & Stepp MA, 2000). PLOD, a peripheral membrane protein (within the endoplasmic reticulum), catalyses lysine hydroxylation in collagens and more than 15 other proteins (Rautavuoma K et al, 2002). The Plod2 is under the regulation of Pitx2 in tissues like mice brain and the proximal promoter region of Plod2 harbors multiple bicoid elements that bind to Pitx2 (Hjalt et al., 2001)

ETS-1 Protein Regulates Vascular Endothelial Growth Factor-induced Matrix Metalloproteinase-9 and Matrix Metalloproteinase-13 Expression in Human Ovarian Carcinoma Cell Line SKOV-3

The mechanism of vascular endothelial growth factor (VEGF)-regulated expression of MMPs followed by cancer cell scattering/invasion is poorly understood. VEGF induces MMP-9, MMP-13, and ETS-1 through PI3K/AKT and p38 MAPK pathways in SKOV-3 cells. VEGF induces ETS-1, which activates specific MMPS, leading to the invasion/scattering in SKOV-3 cells. This study provides useful information that reveals the molecular mechanism of ovarian cancer metastasis

Exploring the major cross-talking edges of competitive endogenous RNA networks in human Chronic and Acute Myeloid Leukemia

Background: Human Chronic and Acute Myeloid Leukemia are myeloproliferative disorders in myeloid lineage of blood cells characterized by accumulation of aberrant white blood cells. In cancer, the anomalous transcriptome includes deregulated expression of non-coding RNAs in conjunction with protein-coding mRNAs in human genome. The coding or non-coding RNA transcripts harboring miRNA-binding sites can converse with and regulate each other by explicitly contending for a limited pool of shared miRNAs and act as competitive endogenous RNAs (ceRNAs). An unifying hypothesis attributing modulation of expression of transcripts in this fashion had been defined as competitive endogenous RNA hypothesis. Network built with ceRNAs evidently offers a platform to elucidate complex regulatory interactions at post-transcriptional level in human cancers. Methods: Contemplating cancers of human myeloid lineage we constructed ceRNA networks for CML and AML coding and non-coding repertoire utilizing patient sample data. Through functional enrichment analysis we selected the significant functional modules for transcripts being differentially expressed in Blastic phases of each cancer types with respect to Normal. After retrieving free energy of binding and duplex formation of shared miRNAs on ceRNAs, we performed statistical averaging of energy values over the ensemble of populations considering cellular system as in canonical (Iso-thermal) situation. Results and conclusions: We aimed to shed light on Sibling Rivalry in ceRNA partners from the perspective of statistical thermodynamics, identified major cross-talking tracks and ceRNAs influencing transcripts concerned in myeloid cancer systems. General significance: Insights into ceRNA-regulation will shed light on progression and prognosis of human Chronic and Acute Myeloid Leukemia.Funding Agencies|Science and Engineering Research Board, Department of Science and Technology, Govt. of India [YSS/2015/000115]</p

Additional file 1: Figure S1. of Invasion of ovarian cancer cells is induced byPITX2-mediated activation of TGF-β and Activin-A

The expression of PITX2 is up-regulated in human ovarian cancer. (A) The level of PITX2 was observed by IHC in human ovarian tissue-sections with specific antibody followed by Alexa Fluor-488 (green) of low malignant potential (i; n = 20) and high malignant potential metastatic adenocarcinoma patients (ii; n = 20). The DAPI-stained nuclei and the merged images were also shown. Scale bar, 10 μm. (TIFF 28495 kb

Dual histone reader ZMYND8 inhibits cancer cell invasion by positively regulating epithelial genes

Enhanced migratory potential and invasiveness of cancer cells contribute crucially to cancer progression. These phenotypes are achieved by precise alteration of invasionassociated genes through local epigenetic modifications which are recognized by a class of proteins termed a chromatin reader. ZMYND8 [zinc finger MYND (myeloid, Nervy and DEAF-1)-type containing 8], a key component of the transcription regulatory network, has recently been shown to be a novel reader of H3.1K36Me2/H4K16Ac marks. Through differential gene expression analysis upon silencing this chromatin reader, we identified a subset of genes involved in cell proliferation and invasion/migration regulated by ZMYND8. Detailed analysis uncovered its antiproliferative activity through BrdU incorporation, alteration in the expression of proliferation markers, and cell cycle regulating genes and cell viability assays. In addition, performing wound healing and invasion/migration assays, its anti-invasive nature is evident. Interestingly, epithelial–mesenchymal transition (EMT), a key mechanism of cellular invasion, is regulated by ZMYND8 where we identified its selective enrichment on promoters of CLDN1/CDH1 genes, rich in H3K36Me2/ H4K16Ac marks, leading to their up-regulation. Thus, the presence of ZMYND8 could be implicated in maintaining the epithelial phenotype of cells. Furthermore, syngeneic mice, injected with ZMYND8-overexpressed invasive breast cancer cells, showed reduction in tumor volume and weight. In concert with this, we observed a significant down-regulation of ZMYND8 in invasive ductal and lobular breast cancer tissues compared with normal tissue. Taken together, our study elucidates a novel function of ZMYND8 in regulating EMT and invasion of cancer cells, possibly through its chromatin reader function

Wnt/�-Catenin Pathway Is Regulated by PITX2 Homeodomain Protein and Thus Contributes to the Proliferation of Human Ovarian Adenocarcinoma Cell, SKOV-3

Wnt pathway and homeodomain proteins are associated with cancer, but their interaction in ovarian cancer cells has not been studied.PITX2 itself and through inducing Wnt ligands activates the canonical Wnt pathway and cell proliferation. Downregulation of Frizzled receptors limits further Wnt activation.PITX2 enhances proliferation of SKOV-3 cells by inducing canonical Wnt signaling. This study will help understand the mechanism of proliferation in ovarian cancer cells