Epithelial-to-mesenchymal Transition In Ovarian Cancer Research Articles

Machine learning-assisted analysis of epithelial mesenchymal transition pathway for prognostic stratification and immune infiltration assessment in ovarian cancer.

Ovarian cancer is the most lethal gynaecological malignancy, and serous ovarian cancer (SOC) is one of the more important pathological subtypes. Previous studies have reported a significant association of epithelial tomesenchymal transition (EMT) with invasive metastasis and immune modulation of SOC, however, there is a lack of prognostic and immune infiltration biomarkers reported for SOC based on EMT. Gene expression data for ovarian cancer and corresponding patient clinical data were collected from the TCGA database and the GEO database, and cell type annotation and spatial expression analysis were performed on single cell sequencing data from the GEO database. To understand the cell type distribution of EMT-related genes in SOC single-cell data and the enrichment relationships of biological pathways and tumour functions. In addition, GO functional annotation analysis and KEGG pathway enrichment analysis were performed on mRNAs predominantly expressed with EMT to predict the biological function of EMT in ovarian cancer. The major differential genes of EMT were screened to construct a prognostic risk prediction model for SOC patients. Data from 173 SOC patient samples obtained from the GSE53963 database were used to validate the prognostic risk prediction model for ovarian cancer. Here we also analysed the direct association between SOC immune infiltration and immune cell modulation and EMT risk score. and calculate drug sensitivity scores in the GDSC database.In addition, we assessed the specific relationship between GAS1 gene and SOC cell lines. Single cell transcriptome analysis in the GEO database annotated the major cell types of SOC samples, including: T cell, Myeloid, Epithelial cell, Fibroblast, Endothelial cell, and Bcell. cellchat revealed several cell type interactions that were shown to be associated with EMT-mediated SOC invasion and metastasis. A prognostic stratification model for SOC was constructed based on EMT-related differential genes, and the Kapan-Meier test showed that this biomarker had significant prognostic stratification value for several independent SOC databases. The EMT risk score has good stratification and identification properties for drug sensitivity in the GDSC database. This study constructed a prognostic stratification biomarker based on EMT-related risk genes for immune infiltration mechanisms and drug sensitivity analysis studies in SOC. This lays the foundation for in-depth clinical studies on the role of EMT in immune regulation and related pathway alterations in SOC. It is also hoped to provide effective potential solutions for early diagnosis and clinical treatment of ovarian cancer.

A Luminacin D Analog HL142 Inhibits Ovarian Tumor Growth and Metastasis by Reversing EMT and Attenuating the TGFβ and FAK Pathways.

Epithelial to mesenchymal transition (EMT) is known to contribute to tumor metastasis and chemoresistance. Reversing EMT using small molecule inhibitors to target EMT associated gene expression represents an effective strategy for cancer treatment. The purpose of this study is to test whether a new luminacin D analog HL142 reverses EMT in ovarian cancer (OC) and has the therapeutic potential for OC. We chemically synthesized HL142 and tested its functions in OC cells in vitro and its efficacy in inhibiting ovarian tumor growth and metastasis in vivo using orthotopic OC mouse models. We first demonstrate that ASAP1 is co-amplified and interacts with the focal adhesion kinase (FAK) protein in serous ovarian carcinoma. HL142 inhibits ASAP1 and its interaction protein FAK in highly invasive OVCAR8 and moderately invasive OVCAR3 cells. HL142 inhibits EMT phenotypic switch, accompanied by upregulating epithelial marker E-cadherin and cytokeratin-7 and downregulating mesenchymal markers vimentin, β-catenin, and snail2 in both cell lines. Functionally, HL142 inhibits proliferation, colony formation, migration, and invasion. HL142 also sensitizes cell responses to chemotherapy drug paclitaxel treatment and inhibits ovarian tumor growth and metastasis in orthotopic OC mouse models. We further show that HL142 attenuates the TGFβ and FAK pathways in vitro using OC cells and in vivo using orthotopic mouse models.

Abstract TMIM-084: PKCα INDUCES TWIST1 PHOSPHORYLATION AT SERINE 144 AND PROMOTES EMT IN OVARIAN CANCER CELLS

Abstract PURPOSE: Metastasis and carcinomatosis remains a major problem in ovarian cancer. Metastasis requires a process known as Epithelial-to-Mesenchymal Transition (EMT), where by epithelial cancer cells undergo dynamic changes to acquire a mesenchymal and invasive phenotype. Twist1 is a transcription factor with a central role in EMT. We previously showed that in epithelial ovarian cancer (EOC), Twist1 is constitutively ubiquitinated and targeted for proteasomal degradation and inhibition of Twist degradation is associated with mesenchymal phenotype and chemoresistance. Accordingly, identification of mechanisms that promote EMT by inhibiting the active degradation of Twist1 is critical for prevention of carcinomatosis. In this study, we report the identification of PKCα as a central regulator of Twist1. We identified 11 possible PKCα phosphorylation sites on Twist1. We show that Twist1 is a novel substrate of the kinase PKCα and that PKCα-induced Twist1 phosphorylation abrogates Twist1 ubiquitination leading to its stabilization and consequently EMT. METHODS: The following cell lines were used: (1) ovarian cancer cells lines OVCAR3 and OVCA432; (2) in-house developed cultures of ovarian cancer cells, R182 and R2615; and (3) HEK293T cells. The effect of constitutively active (PKCαCAT), dominant negative (PKCαDN), and wild-type (wt-PKCα) PKCα on levels of Twist1 were determined by transient transfection using qRT-PCT and western blot analysis. Levels of phosphorylated Twist1 were measured in Twist1 immunoprecipitate-complex using anti-phospho-serine/threonine/tyrosine antibody. The phospho-deficient mutant Twist1 S144A was constructed using QuickChange Site-directed Mutagenesis Kit. PKCα was knocked-out in ovarian cancer cells using CRISPR/Cas9. EMT was induced by treatment with 10 ng/ml TGFβ1 and confirmed molecularly (i.e. loss of epithelial markers E-cadherin, Ck18, Claudin 3 and gain of mesenchymal markers Twist1, N-cadherin, and vimentin). Intra-peritoneal tumors were established by injecting 10 million ovarian cancer cells in athymic nude mice. RESULTS: Transfection with PKCαCAT(active) resulted in a significant increase in Twist1 protein levels compared to PKCαDN(inactive) or empty vector control in EOC and HEK293T cells. This was not a transcriptional effect (no increase in Twist1 mRNA) but due to increase in the levels of phospho-serine/threonine/tyrosine on Twist1. We identified S144 as the putative PKCα phosphorylation site on Twist1 protein. Whereas wt-Twist1 was readily ubiquitinated when transfected in ovarian cancer cells and HEK293T, the phosphomimic S144D Twist1 demonstrated significantly less ubiquitination. Furthermore, we identified TGFβ1 as an activator of the endogenous PKCα-Twist1 axis in ovarian cancer cells. TGFβ1 is able to: (1) activate PKCα; (2) increase Twist1 protein levels; (3) and induce EMT (spheroid formation). Knock-out of PKCα in ovarian cancer cells abrogated TGFβ1-induced EMT in vitro and inhibit carcinomatosis in vivo in athymic nude mice model. CONCLUSION: We demonstrate for the first time a TGFβ1-PKCα-Twist1 signaling pathway that specifically targets Twist1 protein for phosphorylation and stabilization. This is a non-classical pathway of TGFβ1 induced EMT. Moreover, we identify S144 on Twist1 as novel and direct PKCα-phosphorylation site that can control Twist1 stability. Given the pleiotropic nature of TGFβ1 signaling, the identification of PKCα as a novel target may aid in the development of better therapeutic modalities that can prevent EMT and curtail metastasis formation in ovarian cancer. Citation Format: Roslyn Tedja, Cai Roberts, Carlos Cardenas, Ayesha B. Alvero, Mary Pitruzzello, Yang Yang-Hartwich, Carlotta Glackin and Gil G. Mor. PKCα INDUCES TWIST1 PHOSPHORYLATION AT SERINE 144 AND PROMOTES EMT IN OVARIAN CANCER CELLS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr TMIM-084.

Abstract NT-120: KNOCKOUT OF MTF1 RESULTS IN THE INHIBITION OF EMT IN OVARIAN CANCER CELLS

Abstract Due to peritoneal metastasis and frequent recurrence, ovarian cancer has the highest mortality among gynecological cancers. Epithelial to mesenchymal transition (EMT) contributes to ovarian tumor metastasis. In this study, we report for the first time that metal regulatory transcription factor 1 (MTF1) was upregulated in ovarian cancer, and its high expression was associated with poor patient survival and disease relapse. Knockout of MTF1 using lentiviral CRISPR/Cas9 nickase vector-mediated gene editing inhibited EMT by upregulating epithelial cell markers E-cadherin and cytokeratin 7, and downregulating mesenchymal markers Snai2 and β-catenin in ovarian cancer SKOV3 and OVCAR3 cells. Loss of MTF1 reduced cell proliferation, migration, and invasion in both SKOV3 and OVCAR3 cells. Knockout of MTF1 upregulated the expression of the KLF4 transcription factor, and attenuated two cellular survival pathways, ERK1/2 and AKT. Our studies demonstrated that MTF1 plays an oncogenic role and contributes to ovarian tumor metastasis by promoting EMT. MTF1 may be a novel biomarker for early diagnosis as well as a drug target for clinical therapy. Citation Format: Guannan Zhao, Lawrence M Pfeffer Junming Yue. KNOCKOUT OF MTF1 RESULTS IN THE INHIBITION OF EMT IN OVARIAN CANCER CELLS [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-120.

GPR30 signaling to regulate epithelial-mesenchymal transition and predict survival in ovarian cancer.

e17041 Background: G protein-coupled receptor 30 (GPR30) is a 7-transmembrane estrogen receptor that functions alongside traditional estrogen receptors to regulate the cellular responses to estrogen. Recent studies suggest that the high expression of GPR30 is associated with a poor prognosis in breast cancer or endometrial cancer. Although the role of GPR30 in ovarian cancer was unclear, we revealed that GPR30 is associated with poor prognosis in ovarian cancer. On the other hand, Epithelial-to-Mesenchymal Transition (EMT) is involved in cancer metastasis. The purpose of this study is to reveal how GPR30 was associated with poor prognosis and whether associated with EMT in ovarian cancer. Methods: We examined whether GPR30 signaling activates the EGFR-Akt pathway in an ovarian cancer cell line (Caov-3) by a Western blotting analysis. We also examined the effect of GPR30 on EMT were evaluated in Caov-3, which were cultured both in two-dimensional (2D) culture and three-dimensional (3D) culture model. GPR30 agonist, G1, was used to confirm the regulatory effects of GPR30 on the change of phenotypic modulation and EMT markers expression. Results: The phosphorylation of the EGFR and Akt could be significantly enhanced by G1 (p < 0.05) and inhibited by a Src family kinase inhibitor. In 3D culture, the stimulation of GPR30 leads the floating and sphere formation in Caov-3. G1-induced EMT was observed with related regulation of EMT markers expression at both mRNA and protein level. G1 induced the decrease of E-cadherin level and the increase of Snail and Vimentin in RT-PCR and Western blotting. Knockdown of GPR30, using siRNA, blocked G1-induced EMT. Conclusions: GPR30 increases the phosphorylation of Akt via the EGFR in ovarian cancer cells and changes ovarian cancer cells to the EMT state.GPR30 might be an important molecule related to metastasis process in ovarian cancer.

Upregulation of USP11 promotes epithelial‑to‑mesenchymal transition by deubiquitinating Snail in ovarian cancer.

Ubiquitin specific peptidase11 (USP11) is a deubiquitinating enzyme (DUB) and has been observed to promote mammary tumor initiation and progression. Several reports have shown that USP11 plays an important role in cancer development. Previous research has shown that USP11 is upregulated in breast cancer, but the function of USP11 in ovarian cancer has not been determined to date. In the present study, we observed that USP11 was upregulated in ovarian cancer tissues and cell lines. To determine the role of USP11 in ovarian cancer, we performed several functional experiments, including Transwell migration and invasion analysis, in vitro and in vivo deubiquitination assays, colony formation assays, and CCK‑8 assays. Notably, we observed that USP11 facilitated the epithelial‑to‑mesenchymal transition (EMT), which is an important process in cancer cell invasion and metastasis. Furthermore, we observed that USP11 regulated EMT through the regulation of Snail. Our results revealed that USP11 promoted EMT in ovarian cancer by deubiquitinating Snail, and USP11 may be a novel therapeutic target for ovarian cancer treatment.

Decoding critical long non-coding RNA in ovarian cancer epithelial-to-mesenchymal transition

Long non-coding RNA (lncRNA) are emerging as contributors to malignancies. Little is understood about the contribution of lncRNA to epithelial-to-mesenchymal transition (EMT), which correlates with metastasis. Ovarian cancer is usually diagnosed after metastasis. Here we report an integrated analysis of >700 ovarian cancer molecular profiles, including genomic data sets, from four patient cohorts identifying lncRNA DNM3OS, MEG3, and MIAT overexpression and their reproducible gene regulation in ovarian cancer EMT. Genome-wide mapping shows 73% of MEG3-regulated EMT-linked pathway genes contain MEG3 binding sites. DNM3OS overexpression, but not MEG3 or MIAT, significantly correlates to worse overall patient survival. DNM3OS knockdown results in altered EMT-linked genes/pathways, mesenchymal-to-epithelial transition, and reduced cell migration and invasion. Proteotranscriptomic characterization further supports the DNM3OS and ovarian cancer EMT connection. TWIST1 overexpression and DNM3OS amplification provides an explanation for increased DNM3OS levels. Therefore, our results elucidate lncRNA that regulate EMT and demonstrate DNM3OS specifically contributes to EMT in ovarian cancer.

Abstract TMEM-022: LYSOPHOSPHATIDIC ACID IN OVARIAN CANCER EPITHELIAL–TO–MESENCHYMAL TRANSITION

Abstract Lysophosphatidic acid (LPA) is a bioactive phospholipid found in elevated concentrations in ovarian cancer (OVCA) patients. The objective of this study was to define the mechanism of action by which LPA contributes to ovarian carcinogenesis focusing on epithelial-to-mesenchymal transition (EMT) and role of human macrophages. THP-1 human macrophages and A27801AP human OVCA cells were used and treated with LPA up to 40 µM for 24 h. In THP-1 human macrophages, LPA treatment led to a dose-dependent increase in IL-1β secretion from 333.5 pg/mL (untreated) to 581.9, 661.8 and 1270.1 pg/mL for 10, 20 and 40 µM LPA treatment. We then used the conditioned medium (CM) from THP-1 macrophages to determine the effect on A27801AP OVCA cells. LPA-treated THP-1 CM increased the expression of EMT transcription factors p65, Slug and Snail as compared to non-LPA-treated THP-1 CM and resting OVCA cells. When A27801AP OVCA cells were treated with LPA alone, 20 µM LPA led to the same significant increase in the expression of EMT transcription factors p65, Slug and Snail. In addition, LPA treatment reduced the expression of the epithelial marker E-cadherin while increasing the expression of mesenchymal markers N-cadherin and vimentin which might be associated with increased phosphorylation of extracellular regulated kinase 1/2. The data show the role of LPA in modifying not only OVCA cells but also macrophages that can be found in the tumor microenvironment to promote progression of OVCA by promoting EMT. Citation Format: Vermont P Dia, Ph D. LYSOPHOSPHATIDIC ACID IN OVARIAN CANCER EPITHELIAL–TO–MESENCHYMAL TRANSITION [abstract]. In: Proceedings of the 11th Biennial Ovarian Cancer Research Symposium; Sep 12-13, 2016; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2017;23(11 Suppl):Abstract nr TMEM-022.

KDM5A promotes proliferation and EMT in ovarian cancer and closely correlates with PTX resistance.

The authors initially performed reverse transcription‑quantitative polymerase chain reaction to determine the expression profile of KDM5A in ovarian cancer tissues and adjacent normal tissue. Compared with adjacent normal tissue, it was identified that KDM5A was highly expressed in ovarian cancer tissues. Moreover, human ovarian cell lines also confirmed that KDM5A was highly expressed in ovarian cancer. KDM5A was especially highly expressed in SKOV3/paclitaxel (PTX) cells, which are resistant to PTX. Previous studies demonstrated that chemoresistance in cancer cells facilitates epithelial‑to‑mesenchymal transition (EMT). Following this, whether KDM5A influenced EMT and metastasis was investigated. The expression of KDM5A and N‑cadherin were obviously higher in SKOV3/PTX cells than in SKOV3 cells. The expression of E‑cadherin was decreased and the expression of N‑cadherin was increased following ectopic expression of KDM5A, while the expression of E‑cadherin was increased and the expression of N‑cadherin was decreased following KDM5A depletion. Transwell and wound healing assays were used to explore the function of KMD5A in metastasis. The present results indicated that KDM5A facilitated EMT and metastasis in ovarian cells. Moreover, it was identified that P‑glycoprotein was increased while KDM5A was expressed ectopically in SKOV3 cells. Following fluorescence‑activated cell sorting flow cytometry analysis and CCK‑8 assay all revealed that KDM5A regulated the PTX sensitivity in SKOV3 and SKOV3/PTX cells. In brief, KDM5A is a crucial oncogene that is significantly upregulated in ovarian cancer. Its expression is closely correlated with cancer cell proliferation, EMT and metastasis. KDM5A suppresses ovarian cancer cell apoptosis under PTX treatment.

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

Background/Aims: Epithelial-to-mesenchymal transition (EMT) plays an essential role in the transition from early to invasive phenotype, however the underlying mechanisms still remain elusive. Herein, we propose a mechanism through which the class-III deacetylase SIRT1 regulates EMT in ovarian cancer (OC) cells. Methods: Expression analysis was performed using Q-PCR, western blot, immunofluorescence and fluorescence-IHC study. Matrigel invasion assay was used for the invasion study. Morphological alterations were observed by phalloidin-staining. Co-immunoprecipitation study was performed to analyze protein-protein interaction. Results: Overexpression of SIRT1-WT as well as Resveratrol-mediated SIRT1 activation antagonized the invasion of OC cells by suppressing EMT. SIRT1 deacetylates HIF1α, to inactivate its transcriptional activity. To further validate HIF1α inactivation, its target gene, i.e. ZEB1, an EMT-inducing factor was found to attenuate upon SIRT1 activation. To uncover the regulatory factor governing SIRT1 expression, lysophosphatidic acid (LPA), a highly enriched oncolipid in ascites/serum of OC patients, was found to down-regulate SIRT1 expression. Importantly, LPA was found to induce the mesenchymal switch in OC cells through suppression of SIRT1. Decreased level of SIRT1 was further validated in ovarian tissue samples of OC patients. Conclusion: We have identified a mechanism that relates SIRT1 down-regulation to LPA-induced EMT in OC cells and may open new arenas on developing novel anti-cancer therapeutics.

The Heat Shock Transcription Factor HSF1 Induces Ovarian Cancer Epithelial-Mesenchymal Transition in a 3D Spheroid Growth Model.

Ovarian cancer is the most lethal gynecological cancer, with over 200,000 women diagnosed each year and over half of those cases leading to death. The proteotoxic stress-responsive transcription factor HSF1 is frequently overexpressed in a variety of cancers and is vital to cellular proliferation and invasion in some cancers. Upon analysis of various patient data sets, we find that HSF1 is frequently overexpressed in ovarian tumor samples. In order to determine the role of HSF1 in ovarian cancer, inducible HSF1 knockdown cell lines were created. Knockdown of HSF1 in SKOV3 and HEY ovarian cancer cell lines attenuates the epithelial-to-mesenchymal transition (EMT) in cells treated with TGFβ, as determined by western blot and quantitative RT-PCR analysis of multiple EMT markers. To further explore the role of HSF1 in ovarian cancer EMT, we cultured multicellular spheroids in a non-adherent environment to simulate early avascular tumors. In the spheroid model, cells more readily undergo EMT; however, EMT inhibition by HSF1 becomes more pronounced in the spheroid model. These findings suggest that HSF1 is important in the ovarian cancer TGFβ response and in EMT.

EZH2 inhibition promotes epithelial-to-mesenchymal transition in ovarian cancer cells.

Cancer cells acquire essential characteristics for metastatic dissemination through the process of epithelial-to-mesenchymal transition (EMT), which is regulated by gene expression and chromatin remodeling changes. The enhancer of zeste homolog 2 (EZH2), the catalytic subunit of the polycomb repressive complex 2 (PRC2), catalyzes trimethylation of lysine 27 of histone H3 (H3K27me3) to repress gene transcription. Here we report the functional roles of EZH2-catalyzed H3K27me3 during EMT in ovarian cancer (OC) cells. TGF-β-induced EMT in SKOV3 OC cells was associated with decreased levels of EZH2 and H3K27me3 (P<0.05). These effects were delayed (~72 h relative to EMT initiation) and coincided with increased (>15-fold) expression of EMT-associated transcription factors ZEB2 and SNAI2. EZH2 knockdown (using siRNA) or enzymatic inhibition (by GSK126) induced EMT-like changes in OC cells. The EMT regulator ZEB2 was upregulated in cells treated with either approach. Furthermore, TGF-β enhanced expression of ZEB2 in EZH2 siRNA- or GSK126-treated cells (P<0.01), suggesting that H3K27me3 plays a role in TGF-β-stimulated ZEB2 induction. Chromatin immunoprecipitation assays confirmed that TGF-β treatment decreased binding of EZH2 and H3K27me3 to the ZEB2 promoter (P<0.05). In all, these results demonstrate that EZH2, by repressing ZEB2, is required for the maintenance of an epithelial phenotype in OC cells.

Abstract 1057: LPA stimulates EMT of ovarian cancer cells via gip2 and gep oncogenes

Abstract Ovarian cancer is currently the most fatal gynecological cancer with a 5-year survival rate of only 45%. Recent studies have identified a critical role for lysophosphatidic acid (LPA) in the genesis and the progression of ovarian cancer. In order to identify transcription factors that are induced by LPA, a commercial pathway reporter array was used. Hypoxia induced factor 1 (HIF-1α) induction was the most prominent response showing more than 150 fold transcriptional activation compared with control. LPA treatment under normoxic and hypoxic (1% O2) conditions increased HIF-1α protein level, which leads to epithelial-to-mesenchymal transition (EMT) in the ovarian cancer cell line OVCA432. We also found that stimulation of the OVCA432 cells with LPA at normoxic and hypoxic conditions led to a significant increase of the mRNA and protein expression of the EMT transcription factor Twist. Immunofluorescent imaging confirmed that Twist levels were significantly up-regulated and that Twist was translocated to the nucleus as early as 4 hours after stimulation with LPA in both a normoxic and hypoxic environment. Additionally, immunofluorescent imaging showed that expression of E-cadherin on the periphery of the cell as well as in the whole cell was greatly reduced when cells were stimulated with LPA in normoxic and hypoxic conditions. Finally, our study demonstrated that LPA-mediated induction of EMT required both gip2 and gep oncogenes. Overall, our study indicated that the downstream components regulated by oncogenic G-proteins could potentially be targeted in the future to prevent EMT in ovarian cancer. Citation Format: Ji Hee Ha, Jeremy Ward, Danny N. Dhanasekaran. LPA stimulates EMT of ovarian cancer cells via gip2 and gep oncogenes. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1057. doi:10.1158/1538-7445.AM2014-1057

Overexpression of miR-429 induces mesenchymal-to-epithelial transition (MET) in metastatic ovarian cancer cells

ObjectiveOvarian cancer (OC) is the most lethal of all gynecological malignancies primarily due to the sloughing-off of highly metastatic cells from primary tumors and their subsequent spread throughout the peritoneal cavity. Since the epithelial-to-mesenchymal transition (EMT) of OC cells located at the periphery of primary tumors is essential to this process, molecular interventions that can block EMT are of potential clinical significance. Members of the miR200 family of microRNAs have been implicated in EMT in other cancers. Our purpose was to determine if miR200 family microRNAs may be involved in EMT in OC and of potential therapeutic value in reducing OC metastasis. MethodsGene expression profiles of two OC cell lines with different metastatic potentials were monitored using qRT-PCR (quantitative reverse transcription polymerase chain reaction). The effect of over-expression of a miR-200 family microRNA (miR-429) in metastatic OC cells was monitored on molecular (qRT-PCR and microarray) and functional (morphology, migration, invasiveness and anchorage independence assays) levels. ResultsMolecular profiling of two OC cell lines with differing metastatic potentials identified significant differences in previously established epithelial and mesenchymal cell biomarkers including E-cadherin, ZEB1, ZEB2, miR-205 and miR-200 family microRNAs. Ectopic overexpression of miR-429, a member of the miR-200 family of microRNAs, in mesenchymal-like OC cells resulted in reversal of the mesenchymal phenotype (mesenchymal–epithelial transition, MET). ConclusionsOur results indicate that miR-429 may not only be a useful biomarker of EMT in ovarian cancer, but also of potential therapeutic value in abating OC metastasis.

Loss of MKK4 expression in ovarian cancer: A potential role for the epithelial to mesenchymal transition

In the current study, we investigated the mechanism relating downregulation of mitogen-activated protein kinase kinase 4 (MKK4) expression to development of ovarian cancer. Over-expression of the MKK4 gene in TOV-21 G cells, a line with homozygous deletion of MKK4, resulted in morphologic changes in which cells growing in a scattered, fibroblast-like pattern formed tightly packed colonies. Based on a wound healing assay and a Matrigel invasion assay, we determined that both motility and invasiveness of MKK4-transfected TOV-21G cells were significantly reduced compared to control vector-transfected cells. To confirm that MKK4 expression related to tumor invasion resulted from an epithelial to mesenchymal transition (EMT)-like morphological change, we used 2 independent but complementary approaches. MKK4 gene knockdown in MDAH 2774 cells over-expressing MKK4 increased invasion activity. Additionally, engineered expression of MKK4 in SKOV3 cells, a line with low endogenous MKK4 expression, produced a phenotype similar to that of TOVG-21G. Interestingly, we found that MKK4 upregulation caused downregulation of phosphorylated NF-κB and Twist, as well as upregulation of E-cadherin, in TOVG-21G and SKOV3 cells. Reciprocal results were obtained in MDAH 2774 cells with MKK4 knockdown. Our results suggest that MKK4 downregulation causes increased phosphorylation NF-κB. This promotes Twist over-expression, resulting in E-cadherin downregulation that induces EMT in ovarian cancer.