Adhesion Of Ovarian Cancer Cells Research Articles

A new 3D organotypic model of ovarian cancer to help evaluate the antimetastatic activity of RAPTA-C conjugated micelles.

Ovarian cancer is often diagnosed at a late stage, when disease has spread to extra-pelvic regions such as the omentum. There are limited treatment options available for women with extensive disease and tumours often relapse after current chemotherapy regimens. Therefore, novel drugs should be investigated for the treatment of ovarian cancer. A 3D organotypic model of ovarian cancer can provide a specific platform for the evaluation of nano-drugs. Using patient derived primary cells, the 3D model mimics the ovarian metastatic microenvironment allowing efficient and reproducible testing of many nanoparticles. Dichlororuthenium(ii) (p-cymene) (1,3,5-triaza-7-phosphaadamantane) (RAPTA-C) conjugated fructose-micelles have been used as the promising nano-drug for the treatment of metastatic cancer. Therefore we aimed to investigate the anti-metastatic properties of RAPTA-C conjugated micelles in ovarian cancer metastasis. Ovarian cancer cell adhesion and invasion into a model of omentum were analyzed with and without RAPTA-C conjugated micelles in a range of conditions. We observed that RAPTA-C showed low general toxicity to both primary healthy and cancer cell lines. RAPTA-C loaded micelles significantly enhance the internalization of ruthenium inside the cells compared to free drugs. RAPTA-C did not affect adhesion of OVCAR4 ovarian cancer cells; however, it significantly inhibited invasion of these cells within the omentum model, either in its free form or as cargos inside the micelles. However, when OVCAR4 were treated prior to implantation, invasion was not inhibited. A 3D organotypic model provides a clinically relevant and simple method to evaluate the efficiency of nano-drug treatment of ovarian cancer. The ability to inhibit metastasis of RAPTA-C delivered in fructose coated nanoparticles was investigated for the first time via this model. These results provide a good basis to continue the development of this nano-drug in vivo.

Abstract A38: Modeling ascites-induced changes in peritoneal mechanobiology and ovarian cancer metastatic success

Abstract Ovarian cancer (OvCa) is frequently accompanied by accumulation of intraperitoneal ascites fluid early in disease progression. This fluid is rich in soluble and cellular components including tumor cells and multicellular aggregates (MCAs) of 150-300 um diameter shed from the primary tumor. In addition to chemical cues, ascites fluid buildup can also alter the force environment in the peritoneal cavity, thereby impacting the primary tumor, disseminating cells and MCAs, and host peritoneal tissues. Whereas the intraperitoneal pressure (IPP) of the normal peritoneal cavity is subatmospheric (-5 mmHg), the IPP measured in ovarian cancer patients with tense ascites is reported to be 24 mmHg. The potential effect of ascites-induced changes in peritoneal mechanobiology on tumor cells and host structures has not been investigated due to a lack of appropriate model systems. As a first approximation, we have begun preliminary investigations into the response of tumor and host structures to compressive and strain (stretching) forces. Our initial experiments used MCAs sealed in nonadherent cell culture bags placed into a temperature-controlled stainless-steel pressure vessel and subjected to a compressive force of 22-24 mmHg using an Instron system. While this approach is feasible for short-term experiments, longer-term compression experiments require a system with gas exchange to maintain cell viability. Thus, we fabricated a mold designed to fit within a Flexcell-400C Compression system Biopress+ Bioflex 6-well plate. This mold was used to produce porous hydrogels containing defined void areas so as to encapsulate MCAs within the hydrogel carrier and thereby ensure a more uniform encounter with the Flexcell compression plate. Our initial experiments investigated the effects of MCA compression on gene expression associated with epithelial-to-mesenchymal transition (EMT). Data indicate that short-term static compression (6h) downregulates CDH2 (N-cadherin, Ncad) with cell line-dependent inhibition of EMT regulators including SNAI1, SNAI2, and TWIST. In contrast, long-term compression (24h) upregulated expression of mesenchymal genes including CDH2, MMP14, Wnt5a, ROR1, and ROR2. To examine the impact of strain on receptivity of host peritoneal tissues to metastatic implantation, we used control or strained ex vivo explants of murine peritoneal tissue immobilized on silastic resin. Strained peritoneal tissue exhibited a 3-fold increase in stiffness as determined by atomic force microscopy. Concomitantly, adhesion of ovarian cancer cells to strained peritoneum increased by 4.5-fold. Together these data provide support for a more detailed investigation of the complex role of peritoneal mechanobiology as an important microenvironmental regulator of ovarian cancer metastatic success. Citation Format: Yuliya Klymenko, Rebecca Wates, Yueying LIu, Rachel Lombard, Holly Weiss-Bilka, Leigh Campbell, Diane Wagner, Matthew J. Ravosa, M. Sharon Stack. Modeling ascites-induced changes in peritoneal mechanobiology and ovarian cancer metastatic success. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A38.

Abstract A21: Computational methods and novel in vitro model elucidate a therapeutic target against ovarian cancer metastasis

Abstract High-grade serous ovarian cancer (HGSOC) is the most lethal gynecologic cancer, and patients with HGSOC are often subject to extensive metastasis. The primary mode of metastasis in HGSOC is transcoelomic, where tumor cells detach and float in the peritoneal fluid before reattaching to the mesothelial-lined peritoneum. Levels of alternatively activated macrophages (AAMs) in ascites in the peritoneal cavity are correlated with disease stage, but it is unclear if AAMs play a role in transcoelomic spread. We hypothesized that AAM-secreted ligands enhance adhesion of ovarian cancer to mesothelial cells, which would support tumor progression. To address this hypothesis, we adapted a novel coculture device from our lab to create an in vitro model of the metastatic microenvironment and partial-least squares regression (PLSR) modeling to identify AAM-secreted ligands that enhanced adhesion. Methods: The device consists of two parallel culture surfaces (a well within a 24-well tissue culture plate and a glass coverslip) that are separated by a PDMS ring such that only paracrine interactions can occur between cells cultured on the surfaces. LP-9 or LP-3 mesothelial cells were seeded to confluency onto adsorbed collagen I within the PDMS ring and primary monocytes isolated from whole blood were differentiated to the M2 phenotype on the coverslip. Mesothelial cells were then cultured for 24 hours with a primary AAM coverslip, mimicking paracrine signaling between macrophages and the peritoneal mesothelium. As a control, mesothelial cells were cultured with a cell-free coverslip for 24 hours. Cell-Tracker Green-labeled ovarian cancer cells (OVCAR5, CaOV3, or OV-90) were seeded on top of the mesothelial cells and allowed to adhere for 3 hours. Nonadherent cells were washed off and the adherent cells were quantified. Media were collected from the devices and screened for 36 cytokines and matrix metalloproteinases using Bioplex assays. The data were analyzed using PLSR to identify secreted factors that correlated to the observed changes in cell adhesion. To validate the model, adhesion experiments were repeated using blocking antibodies and recombinant proteins. To examine the presence of these factors in patients, ascites fluid from patients with benign conditions or HGSOC were obtained under protocols approved by the Institutional Review Board at UW-Madison. Results and Conclusions: Coculture of primary AAMs with mesothelial cells increased ovarian cancer cell adhesion by at least 40% in all three cell lines. Bioplex analysis revealed that cytokines such as MIP-1β, MMP-7, and IL-13 were present in the media of AAM cultures. A PLSR model constructed with this data indicated a strong correlation between the ligands measured in the cultures and cell adhesion (R2Y= 0.95, Q2Y= 0.84). MIP-1β correlated strongly with adhesion and contributed highly to the model’s predictive capacity. To validate this model-predicted relationship, we experimentally tested the impact of inhibiting MIP-1β and found that while the baseline adhesion was unaffected, the ability of AAMs to enhance adhesion was lost with all three tumor cell lines. Similarly, treatment of mesothelial cells with MIP-1β alone was sufficient to increase tumor cell adhesion. Additionally, analysis of benign and HGSOC ascites showed elevated levels of MIP-1β in HGSOC patients. Overall, our findings suggest a role of AAMs in the multicellular process of transcoelomic spread of HGSOC, and identify MIP-1β as a potential target in the peritoneal cavity. Ongoing efforts are decoding the mechanisms by which MIP-1β increases adhesion between mesothelial and tumor cells. Citation Format: Molly J. Carroll, Kaitlin C. Fogg, Harin A. Patel, Anne-Sophie Mancha, Manish S. Patankar, Pamela K. Kreeger. Computational methods and novel in vitro model elucidate a therapeutic target against ovarian cancer metastasis. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A21.

Abstract A66: The tissue transglutaminase 2–fibronectin protein complex: A new target in ovarian cancer

Abstract Here we describe a small-molecule inhibitor (SMI) that disrupts the tissue transglutaminase 2 (TG2)-fibronectin (FN) interaction. TG2 is a multifunctional protein, with enzymatic (transglutaminase, isopeptidase, protein disulfide isomerase), GTPase, and extracellular matrix (ECM) scaffold activities. TG2 directly interacts with FN through its N-terminus domain, playing an important role in stabilizing ternary TG2/FN/integrin complexes, which regulate cell adhesion to the matrix. Our group has previously shown that TG2 overexpressed in ovarian cancer (OC) promotes epithelial-to-mesenchymal transition (EMT) contributing to OC cell invasiveness and metastatic behavior. We showed that the interaction with FN is critical to these functions. Proximity ligation assay was used to measure TG2/integrin beta 1 interaction in OC tumors and identified TG2/integrin beta 1 complex in >50% of ovarian tumors, irrespective of histologic type. Based on those findings, we conducted the high-throughput screening and identified TG53 as the lead TG2/FN inhibitory compound. Structure-activity relationship-based approach was used to optimize the physicochemical properties of TG53, leading to design and chemical synthesis of 6 analogues (MT1-6). All analogues blocked TG2/FN interaction, as measured by an ELISA assay, and OC cell adhesion to FN, as measured by a solid phase assay, MT4 being the most potent inhibitor. MT4 downregulates integrin-initiated signaling in OC cells, leading to downstream inhibition of FAK and ERK. Immunofluorescence staining demonstrated that the actin cytoskeleton adopts a cortical localization pattern in MT4-treated cells. MT4 caused membrane ruffling delaying the formation of stable focal contacts and mature adhesions points. Pretreatment with MT4 sensitized OC cells to paclitaxel, as measured by colony-forming assay. In an intraperitoneal OC mouse model, MT4 caused decreased OC cell adhesion to the peritoneum, inhibiting peritoneal dissemination. Hence, MT4 is a potential candidate to be developed and used in combination with current chemotherapeutics to target OC tumor progression. Further studies will continue to optimize this new class of TG2/FN SMIs for potential clinical use in OC. Citation Format: Livia Elena Sima, Bakhtiyor Yakubov, Salvatore Condello, Horacio Cardenas, Zhong Yin Zhang, Lan Chen, Daniela Elena Matei. The tissue transglutaminase 2–fibronectin protein complex: A new target in ovarian cancer. [abstract]. In: Proceedings of the AACR Conference: Addressing Critical Questions in Ovarian Cancer Research and Treatment; Oct 1-4, 2017; Pittsburgh, PA. Philadelphia (PA): AACR; Clin Cancer Res 2018;24(15_Suppl):Abstract nr A66.

Alternatively-Activated Macrophages Upregulate Mesothelial Expression of P-Selectin to Enhance Adhesion of Ovarian Cancer Cells.

Peritoneal metastasis of high-grade serous ovarian cancer (HGSOC) occurs when tumor cells suspended in ascites adhere to mesothelial cells. Despite the strong relationship between metastatic burden and prognosis in HGSOC, there are currently no therapies specifically targeting the metastatic process. We utilized a coculture model and multivariate analysis to examine how interactions between tumor cells, mesothelial cells, and alternatively-activated macrophages (AAM) influence the adhesion of tumor cells to mesothelial cells. We found that AAM-secreted MIP-1β activates CCR5/PI3K signaling in mesothelial cells, resulting in expression of P-selectin on the mesothelial cell surface. Tumor cells attached to this de novo P-selectin through CD24, resulting in increased tumor cell adhesion in static conditions and rolling underflow. C57/BL6 mice treated with MIP-1β exhibited increased P-selectin expression on mesothelial cells lining peritoneal tissues, which enhanced CaOV3 adhesion ex vivo and ID8 adhesion in vivo Analysis of samples from patients with HGSOC confirmed increased MIP-1β and P-selectin, suggesting that this novel multicellular mechanism could be targeted to slow or stop metastasis in HGSOC by repurposing anti-CCR5 and P-selectin therapies developed for other indications.Significance: This study reports novel insights on the peritoneal dissemination occurring during progression of ovarian cancer and has potential for therapeutic intervention.Graphical Abstract: http://cancerres.aacrjournals.org/content/canres/78/13/3560/F1.large.jpg Cancer Res; 78(13); 3560-73. ©2018 AACR.

PO-176 Hepatocyte growth factor activator inhibitor-2 suppresses human prostate and lung cancer cell invasion and metastasis

IntroductionIntegrins are glycosylated transmembrane receptor family proteins, mediate cancer cell to extracellular matrix (ECM) interaction. Integrins are essential for adhesion and migration of cancer cells and frequently associate with poor disease outcome. In addition, glycosylation of integrins modulates receptor and ligand binding affinity. However, the physiological role of glycosylation of integrins remain unclear. Based on our recent findings that disruption of glycosphingolipids triggering loss of α2,6-sialylation Alam et al. 2017 here, we identified integrin α2 as candidate and investigated its potential role in facilitating ovarian cancer metastasis.Material and methodsHypersialylated proteins were identified by lectin-enrichment proteomic analysis. CRISPR-Cas9 mediated site-specific deletion of integrin α2 together with a lentiviral-mediated rescue system were established in four ovarian cancer cell lines (IGROV1, SKOV3, SKOV3ip, and OVCAR4). ECM- and mesothelial cell- adhesion assays were applied to evaluate integrin-mediated in vitro cell adhesion, while a zebrafish transplant model was used to study in vivo cell invasion ability. Finally, integrin expression and sialylation profile were determined in patient-derived matched primary and metastatic ovarian cancer tumour specimens.Results and discussionsProteomic analysis identified integrin α2 as a hypersialylated protein in ovarian cancer cells. Loss of sialylation on N-glycoproteins of cancer cells reduced cell adhesion to collagen type I, fibronectin, and laminin. Furthermore, integrin α2 knockout cancer cells abolished cell adhesion to collagen whereas rescuing integrin α2 expression in those knockout cells fully restored the adhesion ability. We also identified an elevated level of intergin α2 in patient-derived metastatic tumour compared to primary site and tumor-free omentum, indicating a niche for ovarian cancer cells toward omental metastasis.ConclusionOur data demonstrate that α2,6 sialylation on integrin α2 triggers ovarian cancer cell adhesion to metastatic sites. Therefore, blocking sialylation and integrin α2 may be a therapeutic target for preventing ovarian cancer metastasis in the future.

Dysregulation of fibulin-5 and matrix metalloproteases in epithelial ovarian cancer.

Fibulin 5 (FBLN5) is an extracellular matrix glycoprotein that suppresses matrix metalloprotease 9 (MMP-9), angiogenesis and epithelial cell motility. Here, we investigated the regulation and function of FBLN5 in epithelial ovarian cancer (EOC). FBLN5 mRNA was down-regulated 5-fold in EOC relative to benign ovary. Not surprisingly, MMP9 mRNA and enzyme activity were increased significantly, and inversely correlated with FBLN5 gene expression. FBLN5 degradation products of 52.8 and 41.3 kDa were increased substantially in EOC. We identified two candidate proteases (serine elastase and MMP-7, but not MMP-9) that cleave FBLN5. MMP-7, but not neutrophil elastase, gene expression was increased dramatically in EOC. Recombinant FBLN5 significantly inhibited adhesion of EOC cells to both laminin and collagen I. Finally, using immunohistochemistry, we found immunoreactive FBLN5 within tumor macrophages throughout human EOC tumors. This work indicates that FBLN5 is degraded in EOC most likely by proteases enriched in macrophages of the tumor microenvironment. Proteolysis of FBLN5 serves as a mechanism to promote cell adhesion and local metastasis of ovarian cancer cells. Promotion of a stable ECM with intact FBLN5 in the tumor matrix may serve as a novel therapeutic adjunct to prevent spread of ovarian cancer.

Human Epididymis Protein 4 Promotes Events Associated with Metastatic Ovarian Cancer via Regulation of the Extracelluar Matrix.

Human epididymis protein 4 (HE4) has received much attention recently due to its diagnostic and prognostic abilities for epithelial ovarian cancer. Since its inclusion in the Risk of Ovarian Malignancy Algorithm (ROMA), studies have focused on its functional effects in ovarian cancer. Here, we aimed to investigate the role of HE4 in invasion, haptotaxis, and adhesion of ovarian cancer cells. Furthermore, we sought to gain an understanding of relevant transcriptional profiles and protein kinase signaling pathways mediated by this multifunctional protein. Exposure of OVCAR8 ovarian cancer cells to recombinant HE4 (rHE4) promoted invasion, haptotaxis toward a fibronectin substrate, and adhesion onto fibronectin. Overexpression of HE4 or treatment with rHE4 led to upregulation of several transcripts coding for extracellular matrix proteins, including SERPINB2, GREM1, LAMC2, and LAMB3. Gene ontology indicated an enrichment of terms related to extracellular matrix, cell migration, adhesion, growth, and kinase phosphorylation. LAMC2 and LAMB3 protein levels were constitutively elevated in cells overexpressing HE4 and were upregulated in a time-dependent manner in cells exposed to rHE4 in the media. Deposition of laminin-332, the heterotrimer comprising LAMC2 and LAMB3 proteins, was increased in OVCAR8 cells treated with rHE4 or conditioned media from HE4-overexpressing cells. Enzymatic activity of matriptase, a serine protease that cleaves laminin-332 and contributes to its pro-migratory functional activity, was enhanced by rHE4 treatment in vitro. Proteomic analysis revealed activation of focal adhesion kinase signaling in OVCAR8 cells treated with conditioned media from HE4-overexpressing cells. Focal adhesions were increased in cells treated with rHE4 in the presence of fibronectin. These results indicate a direct role for HE4 in mediating malignant properties of ovarian cancer cells and validate the need for HE4-targeted therapies that will suppress activation of oncogenic transcriptional activation and signaling cascades.

Effects of CD44 and E-cadherin overexpression on the proliferation, adhesion and invasion of ovarian cancer cells.

CD44 is a prognostic indicator of shorter survival time in ovarian cancer. E-cadherin fragmentation promotes the progression of ovarian cancer. However, the effects of CD44 and E-cadherin overexpression on ovarian cancer cells have remained elusive. The present study aimed to investigate the effects of overexpression of CD44 and E-cadherin on cell proliferation, adhesion and invasion of SKOV-3 and OVCAR-3 ovarian cancer cells. Overexpression of CD44 and E-cadherin was achieved by transfecting SKOV-3 and OVCAR-3 cells with viruses carrying the CD44 or E-cadherin gene, respectively. Expression of CD44 and E-cadherin was detected by western blot analysis. The proliferation of SKOV-3 and OVCAR-3 cells was measured by a Cell Counting Kit-8 at 0, 24 and 48 h after viral transfection. The adhesion ability of SKOV-3 and OVCAR-3 cells to the endothelial layer was detected. A Transwell invasion assay was utilized to assess the invasion ability of the cells. Overexpression of CD44 and E-cadherin in SKOV-3 and OVCAR-3 cells was confirmed by western blot. Compared with the blank or negative control groups, the CD44 overexpression groups of SKOV-3 and OVCAR-3 cells exhibited an increased cell proliferation rate at 24 and 48 h, whereas overexpression of E-cadherin did not alter the proliferation of these cells. Furthermore, compared with the blank and negative control groups, the cell adhesion and invasion ability in the CD44 overexpression groups of SKOV-3 and OVCAR-3 cells was markedly higher. There were no significant differences in adhesion ability between the E-cadherin overexpression group and the blank/negative control group. Of note, overexpression of E-cadherin decreased the invasive ability of SKOV-3 and OVCAR-3 cells. In conclusion, Overexpression of CD44 increased the proliferation, adhesion and invasion of ovarian cancer cells, while overexpression of E-cadherin decreased the invasion of ovarian cancer cells.

Abstract 5885: Sialyl Lewis X-P-selectin connection between ovarian tumor-mesothelium in early stage metastasis

Abstract Metastasis is one of the major challenges for the treatment of ovarian cancer. Unlike other solid tumors, ovarian cancer primarily disseminates within the peritoneal cavity. A crucial step in metastasis formation is the adhesion of ovarian cancer cells onto the peritoneal mesothelium under ascitic shear flow. However, the adhesion mechanisms engaged in this tumor-mesothelium interaction remain elusive due to a lack of physiologically relevant model to manipulate and investigate this dynamic process. In this study, using a 3D microfluidic platform, we found that the metastatic population of cancer stem cells (M-CSCs) exhibited slower rolling velocity and higher binding ability to the peritoneal mesothelium than non-metastatic (NM)-CSCs under flow condition. This adhesion cascade was mediated by P-selectin which expressed on the peritoneal mesothelium. The key carbohydrate determinant on M-CSCs was a glycoprotein, but not a glycolipid, with its recognition as sialyl-Lewis X (sLeX) in a sialic acid- and fucose-dependent manner. Moreover, several glycosyltransferase genes including B4GALT4, ST3GAL3, ST3GAL4 and FUT-5 involved the synthesis of sLeX were upregulated in M-CSCs. Knocking down FUT-5 significantly inhibited ovarian tumor cell adhesion on the mesothelium in vitro and reduced the metastatic potential in vivo. Taken together, our findings revealed that a distinct sLeX-P-selectin axis of ovarian tumor-mesothelium interaction in early metastasis and may offer the possibility of new therapeutic targets. (This work is supported by RGC grant 17122014) Citation Format: Shan-Shan Li, Carman K. M. Ip, Ayon A. Hassan, Matthew Y. H. Tang, Susan Yung, Tak-Mao Chan, Ho Cheung Shum, Alice S. T. Wong. Sialyl Lewis X-P-selectin connection between ovarian tumor-mesothelium in early stage metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5885. doi:10.1158/1538-7445.AM2017-5885

Oxidative stress contributes to hepatocyte growth factor-dependent pro-senescence activity of ovarian cancer cells

The cancer-promoting activity of senescent peritoneal mesothelial cells (HPMCs) has already been well evidenced both in vitro and in vivo. Here we sought to determine if ovarian cancer cells may activate senescence in HPMCs. The study showed that conditioned medium (CM) from ovarian cancer cells (OVCAR-3, SKOV-3, A2780) inhibited growth and promoted the development of senescence phenotype (increased SA-β-Gal, γ-H2A.X, 53BP1, and decreased Cx43) in HPMCs. An analysis of tumors isolated from the peritoneum of patients with ovarian cancer revealed an abundance of senescent HPMCs in proximity to cancerous tissue. The presence of senescent HPMCs was incidental when fragments of peritoneum free from cancer were evaluated. An analysis of the cells' secretome followed by intervention studies with exogenous proteins and neutralizing antibodies revealed hepatocyte growth factor (HGF) as the mediator of the pro-senescence impact of the cancer cells. The activity of cancerous CM and HGF was associated with an induction of mitochondrial oxidative stress. Signaling pathways involved in the senescence of HPMCs elicited by the cancer-derived CM and HGF included p38 MAPK, AKT and NF-κB. HPMCs that senesced prematurely in response to the cancer-derived CM promoted adhesion of ovarian cancer cells, however this effect was effectively prevented by the cell protection against oxidative stress. Collectively, our findings indicate that ovarian cancer cells can elicit HGF-dependent senescence in HPMCs, which may contribute to the formation of a metastatic niche for these cells within the peritoneal cavity.

Correction: Pinin interacts with C-terminal binding proteins for RNA alternative splicing and epithelial cell identity of human ovarian cancer cells.

// Yanli Zhang 1 , Jamie Sui-Lam Kwok 2 , Pui-Wah Choi 1 , Minghua Liu 2 , Junzheng Yang 1 , Margit Singh 1 , Shu-Kay Ng 4 , William R. Welch 5 , Michael G. Muto 1 , Stephen KW Tsui 2 , Stephen P. Sugrue 3 , Ross S. Berkowitz 1 , Shu-Wing Ng 1 1 Laboratory of Gynecologic Oncology, Division of Gynecologic Oncology, Department of Obstetrics, Gynecology and Reproductive Biology, Harvard Medical School, Boston, MA, USA 2 School of Biomedical Sciences, The Chinese University of Hong Kong, Hong Kong 3 Department of Anatomy and Cell Biology, University of Florida College of Medicine, Gainesville, FL, USA 4 School of Medicine and Menzies Health Institute Queensland, Griffith University, Meadowbrook, Australia 5 Department of Pathology, Brigham and Women’s Hospital, Harvard Medical School, Boston, MA, USA Correspondence to: Shu-Wing Ng, e-mail: sng@partners.org Keywords: ovarian cancer, tumorigenesis, RNA metabolism, cell adhesion, RNA sequencing Received: October 21, 2015 Accepted: January 24, 2016 Published: February 08, 2016 ABSTRACT Unlike many other human solid tumors, ovarian tumors express many epithelial markers at a high level for cell growth and local invasion. The phosphoprotein Pinin plays a key role in epithelial cell identity. We showed that clinical ovarian tumors and ovarian cancer cell lines express a high level of Pinin when compared with normal ovarian tissues and immortalized normal ovarian surface epithelial cell lines. Pinin co-localized and physically interacted with transcriptional corepressor C-terminal binding proteins, CtBP1 and CtBP2, in the nuclei of cancer cells. Knockdown of Pinin in ovarian cancer cells resulted in specific reduction of CtBP1 protein expression, cell adhesion, anchorage-independent growth, and increased drug sensitivity. Whole transcriptomic comparison of next-generation RNA sequencing data between control ovarian cancer cell lines and cancer cell lines with respective knockdown of Pinin, CtBP1, and CtBP2 expression also showed reduced expression of CtBP1 mRNA in the Pinin knockdown cell lines. The Pinin knockdown cell lines shared significant overlap of differentially expressed genes and RNA splicing aberrations with CtBP1 knockdown and in a lesser degree with CtBP2 knockdown cancer cells. Hence, Pinin and CtBP are oncotargets that closely interact with each other to regulate transcription and pre-mRNA alternative splicing and promote cell adhesion and other epithelial characteristics of ovarian cancer cells.

The expression of Nectin-4 on the surface of ovarian cancer cells alters their ability to adhere, migrate, aggregate, and proliferate.

The cell adhesion molecule Nectin-4 is overexpressed in epithelial cancers, including ovarian cancer. The objective of this study was to determine the biological significance of Nectin-4 in the adhesion, aggregation, migration, and proliferation of ovarian cancer cells. Nectin-4 and its binding partner Nectin-1 were detected in patients’ primary tumors, omental metastases, and ascites cells. The human cell lines NIH:OVCAR5 and CAOV3 were genetically modified to alter Nectin-4 expression. Cells that overexpressed Nectin-4 adhered to Nectin-1 in a concentration and time-dependent manner, and adhesion was inhibited by antibodies to Nectin-4 and Nectin-1, as well as synthetic Nectin peptides. In functional assays, CAOV3 cells with Nectin-4 knock-down were unable to form spheroids and migrated more slowly than CAOV3 parental cells expressing Nectin-4. NIH:OVCAR5 parental cells proliferated more rapidly, migrated faster, and formed larger spheroids than either the Nectin-4 knock-down or over-expressing cells. Parental cell lines expressed higher levels of epithelial markers and lower levels of mesenchymal markers compared to Nectin-4 knock-down cells, suggesting a role for Nectin-4 in epithelial-mesenchymal transition. Our results demonstrate that Nectin-4 promotes cell-cell adhesion, migration, and proliferation. Understanding the biology of Nectin-4 in ovarian cancer progression is critical to facilitate its development as a novel therapeutic target.

MiR-17 inhibits ovarian cancer cell peritoneal metastasis by targeting ITGA5 and ITGB1.

An essential step in the peritoneal spread of ovarian cancer is the adhesion and implantation of tumor cells to the mesothelium layer. Integrinα5 and β1 have been reported to mediate the initial adhesion process and to correlate with disease survival in ovarian cancer. However, the molecular mechanism of integrinα5β1 dysregulation in tumorigenesis and metastasis remained enigmatic. In the present study, using the USNCI60 database, we identified miR-17 as a candidate regulator targeting both integrinα5 and β1. The level of miR-17 was evidently inversely correlated with that of α5 and β1 in ovarian cancer cell lines. Specifically, miR-17 bound directly to the 3'untranslated region (3'UTR) of α5 and β1 and suppressed their expression. Forced expression of miR-17 led to markedly diminished adhesion and invasion of ovarian cancer cells invitro, and notably reduced metastatic nodules inside the peritoneal cavity in invivo SKOV3 xenografts model. Moreover, ectopic expression of miR-17 in ovarian cancer cells resulted in repressed ILK phosphorylation as well as decreased production of active matrix metalloproteinase-2 (MMP-2). Our results indicated that miR-17 hampered ovarian cancer peritoneal propagation by targeting integrinα5 andβ1. These findings supported the utility of miR-17/α5β1 to be considered as valuable marker for metastatic potential of ovarian cancer cells, or a therapeutic target in ovarian cancer treatment.

Short-form Ron is a novel determinant of ovarian cancer initiation and progression.

Short-form Ron (sfRon) is an understudied, alternative isoform of the full-length Ron receptor tyrosine kinase. In contrast to Ron, which has been shown to be an important player in many cancers, little is known about the role of sfRon in cancer pathogenesis. Here we report the striking discovery that sfRon expression is required for development of carcinogen-induced malignant ovarian tumors in mice. We also show that sfRon is expressed in several subtypes of human ovarian cancer including high-grade serous carcinomas, which is in contrast to no detectable expression in healthy ovaries. In addition, we report that introduction of sfRon into OVCAR3 cells resulted in epithelial-to-mesenchymal transition, activation of the PI3K and PDK1 pathway, and inhibition of the MAPK pathway. We demonstrated that sfRon confers an aggressive cancer phenotype in vitro characterized by increased proliferation and migration, and decreased adhesion of ovarian cancer cells. Moreover, the in vivo studies show that OVCAR3 tumors expressing sfRon exhibit significantly more robust growth and spreading to the abdominal cavity when compared with the parental sfRon negative OVCAR3 cells. These data suggest that sfRon plays a significant role in ovarian cancer initiation and progression, and may represent a promising therapeutic target for ovarian cancer treatment.

Sex-determining region Y-box3 (SOX3) functions as an oncogene in promoting epithelial ovarian cancer by targeting Src kinase.

Ovarian cancer is one of the most common cancers which cause female mortality. The knowledge of ovarian cancer initiation and progression is critical to develop new therapeutic strategies to treat and prevent it. Recently, SOX3 has been reported to play a pivotal role in tumor progression. However, the clinical significance of SOX3 in human ovarian cancer remains elusive, and the identity of SOX3 in ovarian cancer initiation, progression, and the related underlying mechanism is unknown. In this study, we showed that SOX3 expression increased from benign and borderline to malignant ovarian tumors. Subsequently, we found that overexpression of SOX3 in EOC cells promoted proliferation, migration, and invasion, while restrained apoptosis and adhesion of ovarian cancer cells. In contrast, silencing of SOX3 gained the opposite results. Finally, we discovered SOX3 targeted Src kinase in EOC cells. These data imply that SOX3, acting as an oncogene in EOC, is not only a crucial factor in the carcinogenesis but also a promising therapeutic target for EOC.

Extracellular matrix protein ITGBL1 promotes ovarian cancer cell migration and adhesion through Wnt/PCP signaling and FAK/SRC pathway.

Despite the advances in cancer treatment and the progresses in tumor biological, ovarian cancer remains a bad situation. In current study, we found a novel extracellular matrix protein, ITGBL1, which is highly expressed in ovarian cancer tissues by immunohistochemistry examination. The expression pattern of ITGBL1 in malignant tissues inspired us to investigate its role in ovarian cancer progression. Both loss- and gain-function assays revealed that ITGBL1 could promote ovarian cancer cell migration and adhesion. As it's a secreted protein, we further used recombinant ITGBL1 protein treated cancer cells and found that ITGBL1 promotes cell migration and adhesion in a concentration dependent manner. Furthermore, we found that ITGBL1 not only influences the activity of Wnt/PCP signaling but also affects FAK/src pathway in vitro. Taken together, our results suggest that highly expressed ITGBL1 could promotes cancer cell migration and adhesion in ovarian cancer and as a secreted protein, ITGBL1 might be a novel biomarker for ovarian cancer diagnosis.

Pinin interacts with C-terminal binding proteins for RNA alternative splicing and epithelial cell identity of human ovarian cancer cells

Unlike many other human solid tumors, ovarian tumors express many epithelial markers at a high level for cell growth and local invasion. The phosphoprotein Pinin plays a key role in epithelial cell identity. We showed that clinical ovarian tumors and ovarian cancer cell lines express a high level of Pinin when compared with normal ovarian tissues and immortalized normal ovarian surface epithelial cell lines. Pinin co-localized and physically interacted with transcriptional corepressor C-terminal binding proteins, CtBP1 and CtBP2, in the nuclei of cancer cells. Knockdown of Pinin in ovarian cancer cells resulted in specific reduction of CtBP1 protein expression, cell adhesion, anchorage-independent growth, and increased drug sensitivity. Whole transcriptomic comparison of next-generation RNA sequencing data between control ovarian cancer cell lines and cancer cell lines with respective knockdown of Pinin, CtBP1, and CtBP2 expression also showed reduced expression of CtBP1 mRNA in the Pinin knockdown cell lines. The Pinin knockdown cell lines shared significant overlap of differentially expressed genes and RNA splicing aberrations with CtBP1 knockdown and in a lesser degree with CtBP2 knockdown cancer cells. Hence, Pinin and CtBP are oncotargets that closely interact with each other to regulate transcription and pre-mRNA alternative splicing and promote cell adhesion and other epithelial characteristics of ovarian cancer cells.

Connective tissue growth factor as a novel therapeutic target in high grade serous ovarian cancer.

Ovarian cancer is the most common cause of death among women with gynecologic cancer. We examined molecular profiles of fibroblasts from normal ovary and high-grade serous ovarian tumors to identify novel therapeutic targets involved in tumor progression. We identified 2,300 genes that are significantly differentially expressed in tumor-associated fibroblasts. Fibroblast expression of one of these genes, connective tissue growth factor (CTGF), was confirmed by immunohistochemistry. CTGF protein expression in ovarian tumor fibroblasts significantly correlated with gene expression levels. CTGF is a secreted component of the tumor microenvironment and is being pursued as a therapeutic target in pancreatic cancer. We examined its effect in in vitro and ex vivo ovarian cancer models, and examined associations between CTGF expression and clinico-pathologic characteristics in patients. CTGF promotes migration and peritoneal adhesion of ovarian cancer cells. These effects are abrogated by FG-3019, a human monoclonal antibody against CTGF, currently under clinical investigation as a therapeutic agent. Immunohistochemical analyses of high-grade serous ovarian tumors reveal that the highest level of tumor stromal CTGF expression was correlated with the poorest prognosis. Our findings identify CTGF as a promoter of peritoneal adhesion, likely to mediate metastasis, and a potential therapeutic target in high-grade serous ovarian cancer. These results warrant further studies into the therapeutic efficacy of FG-3019 in high-grade serous ovarian cancer.

Phage display screening identifies a novel peptide to suppress ovarian cancer cells in vitro and in vivo in mouse models.

BackgroundOvarian cancer is a possibly lethal gynecological malignancy and this study utilized phage display technology to screen and identify peptides that specifically bind to ovarian cancer cells and explored the effects of these peptides on ovarian cancer cells in vitro and in vivo.MethodsThe phage displayed peptide library was used to isolate the peptides binding to and internalizing into the ovarian carcinoma cells. Positive phage clones were characterized with DNA sequencing and bioinformatics analysis and then validated with immunofluorescence. Subsequently, the selected peptides were investigated for their cancer-related functions, including cell adhesion, spreading, motility, and invasion in vitro and in vivo.ResultsPeptide1 read as SWQIGGNwas the positive peptide and showed preferential binding to the target cells. Peptide 1 also inhibited cell proliferation, migration, invasion and adhesion of ovarian cancer HO8910 cells in vitro. In vivo, Peptide 1 led to a lower tumorigenicity of HO8910 cells, which was characterized by the inhibitory effect on tumor growth and metastasis of ovarian cells.ConclusionThese studies demonstrate that the phage display-identified tumor cell-binding peptide was able to control ovarian cancer cell viability, migration, invasion, and adhesion capacity in vitro as well as tumor growth and metastasis in vivo. Future studies will be aimed at evaluating the clinical efficacy of the peptide SWQIGGN in ovarian cancer patients.