Epithelial-to-mesenchymal Transition In Ovarian Cancer Cells Research Articles

Ovarian Primary and Metastatic Tumors Suppressed by Survivin Knockout or a Novel Survivin Inhibitor.

Survivin, a member of the inhibitor of apoptosis family, is upregulated in multiple cancers including ovarian cancer, but is rarely detectable in normal tissues. We previously reported that survivin promoted epithelial-to-mesenchymal transition (EMT) in ovarian cancer cells, suggesting that survivin may contribute to ovarian tumor metastasis and chemoresistance. In this study, we tested whether knockout or pharmacologic inhibition of survivin overcomes chemoresistance and suppresses tumor metastasis. The genetic loss of survivin suppressed tumor metastasis in an orthotopic ovarian cancer mouse model. To pharmacologically test the role of survivin on ovarian tumor metastasis, we treated chemo-resistant ovarian cancer cells with a selective survivin inhibitor, MX106, and found that MX106 effectively overcame chemoresistance in vitro MX106 inhibited cell migration and invasion by attenuating the TGFβ pathway and inhibiting EMT in ovarian cancer cells. To evaluate the efficacy of MX106 in inhibiting ovarian tumor metastasis, we treated an orthotopic ovarian cancer mouse model with MX106, and found that MX106 efficiently inhibited primary tumor growth in ovaries and metastasis in multiple peritoneal organs as compared with vehicle-treated control mice. Our data demonstrate that inhibition of survivin using either genetic knockout or a novel inhibitor MX106 suppresses primary ovarian tumor growth and metastasis, supporting that targeting survivin could be an effective therapeutic approach in ovarian cancer.

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.

Abstract NT-091: TGF-β1-INDUCED CHEMORESISTANCE IS REVERSED BY BG-4 PEPTIDE

Abstract Transforming growth factor-β (TGF-β) superfamily plays a significant role in ovarian physiology but dysregulated TGF-β signaling is associated with promotion of ovarian carcinogenesis. TGF-β signaling in ovarian cancer favors cancer invasion and metastasis and controls ovarian cancer cell proliferation. The objective of this study is to determine the ability of a food-derived peptide, called BG-4, to suppress TGF-β1 signaling in ovarian cancer cells. BG-4 is a novel bioactive peptide isolated from the seeds of Momordica charantia with demonstrated anticancer and strong trypsin inhibitory properties. Different ovarian cancer cells (A2780-1A9, BR and PEO4) were exposed to 10 ng/mL TGF-β1 with or without non-cytotoxic dose of BG-4 (100 µg/mL) and response to chemotherapeutic reagents paclitaxel and cisplatin was evaluated. In addition, markers of epithelial-to-mesenchymal transition (EMT) and apoptosis were evaluated using western blot and immunofluorescence microscopy. TGF-β1 treatment resulted in cisplatin resistance in three cell lines as evidenced by increase in ED50 values from 42.9 µM to 88.5 µM, 53.7 µM to 108.3 µM and 44.8 µM to 67.3 µM for BR, PEO4 and A2780-1A9 cells, respectively. In the presence of BG-4, ED50 values of 35.6 µM, 56.4 µM and 46.6 µM were obtained for BR, PEO4 and A2780-1A9 cells, respectively. These values are similar to non-TGF-β1-treated cells indicating the ability of BG-4 to reverse TGF-β1-induced chemoresistance in ovarian cancer cell lines. Mechanistic studies showed that TGF-β1 signaling induces EMT in ovarian cancer cells as shown by differential expression of epithelial marker E-cadherin and mesenchymal markers N-cadherin, vimentin and transcription factors Snail, Slug and Twist1. In addition, the expression of apoptosis markers were affected by TGF-β1 signaling. On the other hand, treatment of TGF-β1 in the presence of 100 µg/mL BG-4 led to reversal of the EMT process. These results showed that the ability of BG-4 to reverse TGF-β1-induced chemoresistance in ovarian cancer cell lines may be associated with inhibition of the EMT process. Citation Format: Vermont P Dia. TGF-β1-INDUCED CHEMORESISTANCE IS REVERSED BY BG-4 PEPTIDE [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-091.

Lentiviral vector mediated-ASAP1 expression promotes epithelial to mesenchymal transition in ovarian cancer cells.

Ovarian cancer is one of the most common malignancies in women and has a high mortality rate due to metastatic progression and tumor recurrence. ASAP1 (ArfGAP with SH3 Domain, Ankyrin Repeat and PH Domain 1) is an ADP-ribosylation factor GTPase-activating protein, which is involved in tumor metastasis. However, the role of ASAP1 in ovarian cancer is completely unknown. The present study reported that ASAP1 was highly expressed in ovarian carcinoma, and expression positively-correlated with overall poor survival and prognosis of patients. Lentiviral vector mediated ASAP1 expression promoted cell migration and invasion in ovarian cancer cell lines SKOV3 and OVCAR3. In addition, ASAP1 promoted cell proliferation, survival and inhibited chemotherapy drug paclitaxel-induced cell apoptosis. Furthermore, ASAP1 expression promoted epithelial to mesenchymal transition (EMT) by upregulating the mesenchymal cell markers N-cadherin and vimentin, and downregulating epithelial cell marker E-cadherin in the ovarian cancer cell lines. The data indicate for the first time that ASAP1 exhibits an oncogenic role by promoting EMT in ovarian cancer cells.

Linc-ROR induces epithelial-to-mesenchymal transition in ovarian cancer by increasing Wnt/β-catenin signaling.

Long intergenic non-protein coding RNA, regulator of reprogramming (linc-ROR) is an intergenic long non-coding RNA (lncRNA) previously shown to contribute to tumorigenesis in several malignancies. However, little is known about whether linc-ROR has a role in ovarian cancer progression. In this study, we found that linc-ROR expression was increased in high-grade ovarian serous cancer tissues compared with normal ovarian tissues or normal fallopian tube tissues. Furthermore, the level of linc-ROR expression was associated with ovarian cancer International Federation of Gynecology and Obstetrics stage and lymph node metastasis. Linc-ROR promoted ovarian cancer cell proliferation both in vitro and in vivo, and contributed to cell migration and invasion. Linc-ROR knockdown in ovarian cancer cell lines inhibited the epithelial-to-mesenchymal transition (EMT) program, which led to ovarian cancer cell metastasis through the repression of canonical Wnt/β-catenin signaling. Together, our results indicated that linc-ROR induces EMT in ovarian cancer cells and may be an important molecule in the invasion and metastasis of ovarian cancer.

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.

Lentiviral CRISPR/Cas9 vector mediated miR-21 gene editing inhibits the epithelial to mesenchymal transition in ovarian cancer cells.

CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats) mediated genome editing is a powerful approach for loss of function studies. Here we report that lentiviral CRISPR/Cas9 vectors are highly efficient in introducing mutations in the precursor miRNA sequence, thus leading to the loss of miRNA expression and function. We constructed four different lentiviral CRISPR/Cas9 vectors that target different regions of the precursor miR-21 sequence and found that these lentiviral CRISPR/Cas9 miR-21 gRNA vectors induced mutations in the precursor sequences as shown by DNA surveyor mutation assay and Sanger sequencing. Two miR-21 lentiviral CRISPR/Cas9 gRNA vectors were selected to probe miR-21 function in ovarian cancer SKOV3 and OVCAR3 cell lines. Our data demonstrate that disruption of pre-miR-21 sequences leads to reduced cell proliferation, migration and invasion. Moreover, CRISPR/Cas9-mediated miR-21 gene editing sensitizes both SKOV3 and OVCAR3 cells to chemotherapeutic drug treatment. Disruption of miR-21 leads to the inhibition of epithelial to mesenchymal transition (EMT) in both SKOV3 and OVCAR3 cells as evidenced by the upregulation of epithelial cell marker E-cadherin and downregulation of mesenchymal marker genes, vimentin and Snai2. The miR-21 target genes PDCD4 and SPRY2 were upregulated in cells transduced with miR-21gRNAs compared to controls. Our study indicates that lentiviral CRISPR/Cas9-mediated miRNA gene editing is an effective approach to address miRNA function, and disruption of miR-21 inhibits EMT in ovarian cancer cells.

Resveratrol Inhibits Cisplatin-Induced Epithelial-to-Mesenchymal Transition in Ovarian Cancer Cell Lines

BackgroundMany patients diagnosed with ovarian cancer experience recurrence and metastasis, two aspects that will often cause their demise. Epithelial-to-mesenchymal transition (EMT) is a key process involved in cancer progression. With increasing evidence linking Cisplatin and EMT, we wanted to identify a compound able to counter EMT progression when cancer cells are treated with Cisplatin.Methodology/Principal FindingsCell death was evaluated by cytometry with Annexin V/PI staining in A2780 and A2780CP cells. Ovarian cancer cell lines were treated with Cisplatin (24 h, 10 µM) and different concentrations of Resveratrol to evaluate its effect on Cisplatin-induced EMT using Western Blot and RT-PCR analysis. Morphological studies and wound healing assay to evaluate cell motility were performed using 72 h Cisplatin treatment with A2780 and A2780CP cells. Densitometry was done on Western Blot and PCR results, and statistical significance was determined using One-Way ANOVA followed by Tukey post-hoc test. Our results show that Cisplatin induced EMT-associated morphological changes in the A2780 ovarian cancer cell line and to a lesser extent in its Cisplatin-resistant counterpart A2780CP. Resveratrol caused cell death in A2780 and A2780CP cell lines in an apoptotic-independent manner. Resveratrol inhibited Cisplatin-induced Snail expression by reducing the Erk pathway activation, reverted morphological changes induced by Cisplatin and decreased cell migration.ConclusionsThese results indicate that Resveratrol has interesting potential to prevent Cisplatin-induced EMT in ovarian cancer cells. By increasing cell death, it also represents an inviting approach as adjuvant therapy to be used with chemotherapy. Using Erk pathway inhibitors could also prove helpful in ovarian cancer treatment to reduce the risk of metastasis.

Gab2 regulates the migratory behaviors and E-cadherin expression via activation of the PI3K pathway in ovarian cancer cells

Ovarian cancer, the most deadly gynecologic malignancy, is often diagnosed late and at the advanced stage when the cancer cells have already migrated and invaded into other tissues and organs. Better understanding of the mechanism of metastasis in ovarian cancer cells is essential to the design of effective therapy. In this study, we investigated the function of scaffolding adaptor protein Gab2 in ovarian cancer cells. Gab2 is found to be overexpressed in a subset of ovarian tumors and cancer cell lines. Gab2 expression mainly regulates the migratory behaviors of ovarian cancer cells. Overexpression of Gab2 promotes the migration and invasion, and down-regulates E-cadherin expression in ovarian cancer cells with low-Gab2 expression. Conversely, knockdown of Gab2 expression inhibits the migration and invasion, and promotes E-cadherin expression in ovarian cancer cells with high-Gab2 expression. By expressing Gab2 wild type and Gab2 mutants that are defective in activation the PI3K and Shp2-Erk pathways, we find that Gab2 inhibits E-cadherin expression and enhances the expression of Zeb1, a transcription factor involved in epithelial-to-mesenchymal transition (EMT), and cell migration and invasion through the activation of the PI3K pathway. Knockdown of Zeb1 expression blocks Gab2-induced suppression of E-cadherin expression and increase in cell invasion. LY294002 and GDC-0941, inhibitors of PI3K, or Rapamycin, an inhibitor of PI3K downstream target mTOR, can reverse the effects of Gab2 on migration and invasion. Overall, our studies reveal that Gab2 overexpression, via activation of the PI3K-Zeb1 pathway, promotes characteristics of EMT in ovarian cancer cells.

Pituitary tumor transforming gene induces epithelial to mesenchymal transition by regulation of Twist, Snail, Slug, and E-cadherin

Pituitary tumor-transforming gene (PTTG) is an oncogene with its expression levels correlating with tumor development and metastasis. Epithelial to mesenchymal transition (EMT) is a crucial step in tumor progression and metastasis. Using ovarian epithelial tumor cell line (A2780) for loss-of-function or gain-of-function of PTTG in our experiments, we observed up regulation of TGF-β, Twist, Snail, Slug, vimentin and down regulation of E-cadherin on infection of cells with Ad-PTTG cDNA. In contrast reverse phenomena was observed on depletion of PTTG on infection of cells with Ad-PTTG siRNA, suggesting an important role of PTTG in induction of EMT in ovarian cancer cells.

The Epidermal Growth Factor Receptor Responsive miR-125a Represses Mesenchymal Morphology in Ovarian Cancer Cells

The epithelial-to-mesenchymal transition (EMT) that occurs during embryonic development is recapitulated during tumor metastasis. Important regulators of this process include growth factors, transcription factors, and adhesion molecules. New evidence suggests that microRNA (miRNA) activity contributes to metastatic progression and EMT; however, the mechanisms leading to altered miRNA expression during cancer progression remain poorly understood. Importantly, overexpression of the epidermal growth factor receptor (EGFR) in ovarian cancer correlates with poor disease outcome and induces EMT in ovarian cancer cells. We report that EGFR signaling leads to transcriptional repression of the miRNA miR-125a through the ETS family transcription factor PEA3. Overexpression of miR-125a induces conversion of highly invasive ovarian cancer cells from a mesenchymal to an epithelial morphology, suggesting miR-125a is a negative regulator of EMT. We identify AT-rich interactive domain 3B (ARID3B) as a target of miR-125a and demonstrate that ARID3B is overexpressed in human ovarian cancer. Repression of miR-125a through growth factor signaling represents a novel mechanism for regulating ovarian cancer invasive behavior.

P70 S6 Kinase Promotes Epithelial to Mesenchymal Transition through Snail Induction in Ovarian Cancer Cells

p70 S6 kinase (p70(S6K)) is a downstream effector of phosphatidylinositol 3-kinase and is frequently activated in human ovarian cancer. Here we show that p70(S6K) functions in epithelial to mesenchymal transition (EMT) responsible for the acquisition of invasiveness during tumor progression. This tumorigenic activity is associated with the ability of p70(S6K) to repress E-cadherin through the up-regulation of Snail. p70(S6K) activation induced phenotypic changes consistent with EMT in ovarian cancer cells: The cells lost epithelial cell morphology, acquired fibroblast-like properties, and showed reduced intercellular adhesion. Western blot showed that p70(S6K) activation led to decreased expression of the epithelial marker E-cadherin and increased expression of mesenchymal markers N-cadherin and vimentin. Inhibition of p70(S6K) by a specific inhibitor or small interfering RNA reversed the shift of EMT markers. Importantly, p70(S6K) activation also stimulated the expression of Snail, a repressor of E-cadherin and an inducer of EMT, but not other family members such as Slug. This induction of Snail was regulated at multiple levels by increasing transcription, inhibiting protein degradation, and enhancing nuclear localization of Snail. RNA interference-mediated knockdown of Snail suppressed p70(S6K)-induced EMT, confirming that the effect was Snail specific. Furthermore, phospho (active)-p70(S6K) staining correlated with higher tumor grade. We also showed a significant positive correlation between p70(S6K) activation and Snail expression in ovarian cancer tissues. These results indicate that p70(S6K) may play a critical role in tumor progression in ovarian cancer through the induction of EMT. Targeting p70(S6K) may thus be a useful strategy to impede cancer cell invasion and metastasis.