Fluid Of Ovarian Cancer Patients Research Articles

Abstract 31: Metabolic reprogramming in ovarian cancer

Abstract Ovarian cancer is currently the most lethal gynecologic malignancy, with no new significant changes in treatment options for these patients in the last 30 years. Importantly, ovarian cancer patients have increased levels of lysophosphatidic acid (LPA), a bioactive phospholipid in ascites and serum that has been linked to driving oncogenesis and progression of ovarian cancer. Accumulating evidence has implicated Hypoxia-inducible factor-1α (HIF-1α) as a critical mediator of the glycolytic shift observed in cancer cells. However, the precise biological role of LPA in regulating HIF-1α-mediated glycolytic shift is largely unknown. Therefore, we were interested in examining the potential role of LPA in promoting metabolic reprogramming in ovarian cancer via HIF-1α signaling. In this study, we identified a novel mechanism by which LPA upregulates HIF-1α expression via Gαi2 (the gip2 oncogene) in human ovarian cancer cells. This study demonstrates that LPA induces a glycolytic shift via LPA-mediated activation of Gαi2, which causes a subsequent upregulation of HIF-1α in ovarian cancer cells. Additionally, we show that LPA-signaling via Gαi2 induces an increase in the expression of Hexokinase-2 (HK2) and Glucose transporter 1 (GLUT1), which are known targets of HIF-1α. We also demonstrate that LPA induces an increase in extracellular acidification rate (ECAR) in a dose dependent manner in both ovarian cancer cell lines and in patient-derived cells taken from the ascites fluid of ovarian cancer patients using an XFe96 analyzer. Furthermore, we found that inhibition of Rac signaling caused a reduction in LPA-induced ECAR, identifying Rac as a critical downstream component of Gαi2-medidated increase of ECAR. Moreover, using NAC, an inhibitor of redox signaling, we found that this caused a decrease in LPA-induced ECAR in SKOV3-ip cells, indicating that inhibition of redox signaling abolishes LPA-induced ECAR in ovarian cancer cells. Similarly, the use of EUK-134, a scavenger of superoxide and H2O2, also decreased LPA-induced increase in ECAR. Altogether, these results indicate that LPA regulates glycolysis through redox signaling via a Gαi2-Rac-HIF1α-dependent signaling pathway. Most importantly, the Gαi2-Rac-dependent pathway identified in this study more than likely serves as a potential driver of HIF-1α-mediated metabolic changes in ovarian cancer cells and represents a potential target for therapy in these patients. Citation Format: JI HEE HA, Rangasudhagar Radhakrishnan, Jeremy D. Ward, Muralidharan Jayaraman, Danny N. Dhanasekaran. Metabolic reprogramming in ovarian cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 31.

Abstract B82: Exosomes derived from ascitic fluid of patients with ovarian carcinoma induce IL-4 gene expression in T lymphocytes

Abstract Exosomes are endosome derived secreted membrane vesicles within sizes ranging from 50nm to 100nm. They are secreted from viable cells varying from hematopoietic cells such as B lymphocytes and dendritic cells to non-hematopoietic cells such as intestinal epithelial cells, cortical neurons and tumor cells. Exosomes consist of a great number of membrane associated and cytosolic proteins; they are rich in membrane rafts and contains nucleic acid types such as mRNA, miRNA and DNA. This molecular complexity suggests that exosomes may carry out a variety of physiological functions via mediating intercellular communications. With the findings that cancer cells secrete great amount of exosomes compared to normal cells, it has become apparent that tumor exosomes may have functional implications especially on the tumor microenvironment. In this context, understanding the roles of tumor exosomes on cancer pathogenesis in large measure depends on revealing the interactions between the tumor exosomes and the immune system. Considering the host anti-tumor immunity, T lymphocytes constitute the major part of the adaptive immune responses generated against tumors. In this study it was aimed to identify the gene expression profiles of T lymphocytes under the condition of tumor exosome treatments. For this aim Jurkat T cells, CD4+ T cell leukemia cells, was used as the T lymphocyte model. As the tumor exosome sources, ascitic fluids of patients with ovarian cancer were used. Tumor exosomes was isolated from ascitic fluids of ovarian cancer patients using gel filtration method. 106 Jurkat T cells were treated with 50µg of ascites derived tumor exosomes for one hour and three hour time periods. Control groups included untreated cells and cells treated with control exosomes. After tumor exosome treatment of Jurkat T cells, total RNA isolation and cDNA synthesis were performed. Expression of total of 84 genes involved in JAK-STAT signaling pathway was evaluated with real-time PCR using SYBR green method. ΔCt values were calculated and the fold changes of gene expression between experimental and control groups were determined. As a result it was found that among the evaluated 84 genes, interleukin four (IL-4) gene expression was significantly up-regulated in tumor exosome treated Jurkat T cells in one hour but not three hour treatment time periods with compare to control groups. Observing tumor exosome induced IL-4 gene expression in Jurkat T cells as early as one hour treatment time period suggests that tumor exosomes may up regulate IL-4 gene expression in Jurkat T cells via activation of receptor(s) expressed at the cell surface. Based on this cytokine-induction profile, tumor exosomes derived from ovarian cancer patients appears to lead to a Th2 biased response that has tumor promoting effects. Citation Format: Ergin Sahin, Douglas D. Taylor, Cicek Gercel Taylor. Exosomes derived from ascitic fluid of patients with ovarian carcinoma induce IL-4 gene expression in T lymphocytes. [abstract]. In: Proceedings of the AACR Special Conference: Tumor Immunology and Immunotherapy: A New Chapter; December 1-4, 2014; Orlando, FL. Philadelphia (PA): AACR; Cancer Immunol Res 2015;3(10 Suppl):Abstract nr B82.

M-Trap: Exosome-Based Capture of Tumor Cells as a New Technology in Peritoneal Metastasis.

Remodeling targeted tissues for reception of tumor cells metastasizing from primary lesions is a consequence of communication between the tumor and the environment that governs metastasis. This study describes a novel approach that aims to disrupt the process of metastasis by interfering with this intense dialogue. Proteomics and adhesion assays identified exosomes purified from the ascitic fluid of ovarian cancer patients (n = 9) as intermediaries of tumor cell attachment. A novel tumor cell capture device was fabricated by embedding exosomes onto a 3D scaffold (metastatic trap [M-Trap]). Murine models of ovarian metastasis (n = 3 to 34 mice per group) were used to demonstrate the efficacy of M-Trap to capture metastatic cells disseminating in the peritoneal cavity. Kaplan-Meier survival curves were used to estimate cumulative survival probabilities. All statistical tests were two-sided. The exosome-based M-Trap device promoted tumor cell adhesion with a nonpharmacological mode of action. M-Trap served as a preferential site for metastasis formation and completely remodeled the pattern of peritoneal metastasis in clinically relevant models of ovarian cancer. Most importantly, M-Trap demonstrated a statistically significant benefit in survival outcomes, with mean survival increasing from 117.5 to 198.8 days in the presence of M-Trap; removal of the device upon tumor cell capture further improved survival to a mean of 309.4 days (P < .001). A potent artificial premetastatic niche based on exosomes is an effective approach to impair the crosstalk between metastatic cells and their environment. In the clinical setting, the capacity to modulate the pattern of dissemination represents an opportunity to control the process of metastasis. In summary, M-Trap transforms a systemic, fatal disease into a focalized disease where proven therapeutic approaches such as surgery can extend survival.

Regulated delivery of molecular cargo to invasive tumour-derived microvesicles.

Cells release multiple, distinct, forms of extracellular vesicles including structures known as microvesicles which are known to alter the extracellular environment. Despite growing understanding of microvesicle biogenesis, function, and contents, mechanisms regulating cargo delivery and enrichment remain largely unknown. Here we demonstrate that in amoeboid-like invasive tumor cell lines, the v-SNARE, VAMP3, regulates delivery of microvesicle cargo such as the membrane-type 1 matrix metalloprotease (MT1-MMP) to shedding microvesicles. MT1-MMP delivery to nascent microvesicles depends on the association of VAMP3 with the tetraspanin CD9 and facilitates the maintenance of amoeboid cell invasion. VAMP3-shRNA expression depletes shed vesicles of MT1-MMP and decreases cell invasiveness when embedded in cross-linked collagen matrices. Finally, we describe functionally similar microvesicles isolated from bodily fluids of ovarian cancer patients. Together these studies demonstrate the importance of microvesicle cargo sorting in matrix degradation and disease progression.

Abstract 1984: Role of nectin-4 in the progression of ovarian cancer

Abstract The mechanisms by which ovarian cancer cells are released from the primary tumor, seed throughout the peritoneal cavity, attach to local organs, and then invade these organs, are not understood. In this study, the cell adhesion molecule, Nectin-4, was investigated as a potential mediator of several steps involved in ovarian cancer progression. In particular, due to the ability of Nectin-4 to promote cell-cell adhesions, we were interested in determining its role in mediating the formation of cell-cell aggregates (spheroids) which are commonly seen in the peritoneal fluid of ovarian cancer patients. Furthermore, since spheroids are known to have a decreased rate of cell proliferation and are chemoresistant, we were also interested in the role of Nectin-4 in ovarian cancer cell proliferation and invasion. In our previous studies, we performed gene microarray analysis and identified Nectin-4 overexpression in ovarian cancer tissues compared to normal ovaries and other normal tissues. By ELISA, we found soluble Nectin-4 in the sera of 50% of women with ovarian cancer. In this study, we performed a series of experiments in which two human ovarian cancer cells lines were transfected with Nectin-4 or shRNA targeting Nectin-4, and then the resultant cells were tested in functional assays. In the first case, MA148 cells, which do not express Nectin-4, were transfected with full-length Nectin-4. The resultant MA148 cells which expressed high levels of Nectin-4 showed an increased rate of proliferation compared to control cells which did not express Nectin-4. They also formed the initial cell-cell adhesions involved in spheroid formation faster than control cells. Furthermore, the spheroids which expressed Nectin-4 were slower to disaggregate and invade through monolayers of mesothelial cells. In the second case, NIH:OVCAR5 cells, which express moderate levels of Nectin-4, were transfected with shRNA targeting Nectin-4, resulting in the partial knockdown of Nectin-4 expression. We found that the NIH:OVCAR5 cells that express lower levels of Nectin-4 on their surface required a substantially longer time to form spheroids and migrated more slowly in a wound healing assay. In both cases, we verified the expression of Nectin-4 in the transfected cells by Western immunoblotting and flow cytometry. These results suggest that Nectin-4: (a) is important in the early events of cell-cell adhesion, (b) plays a role in multicellular aggregation within the ascites of ovarian cancer patients, and (c) causes tight spheroid formations that may impede disaggregation. Additional functional studies leading to a better understanding of the role of Nectin-4 overexpression and shedding in ovarian cancer are currently underway. Understanding the role of Nectin-4 in ovarian cancer progression is critical in order to facilitate its development as a novel therapeutic target for ovarian cancer. Citation Format: Kristin L. Boylan, Adam Meyer, Petra C. Buchanan, Melissa S. DeRycke, Amy P. Skubitz. Role of nectin-4 in the progression of ovarian cancer. [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 1984. doi:10.1158/1538-7445.AM2014-1984

Abstract A21: Proteomic analysis of residual Pap test fluid: Novel source for detection of ovarian cancer biomarkers

Abstract Biomarkers that can be used to screen women in the general population for ovarian cancer have yet to be identified. In contrast, screening for cervical cancer by Pap tests has been routinely performed for over 50 years. The liquid-based Pap test consists of collecting cervical cells from the external cervical os and placing them into an alcohol-based fixative. The cells are stained and examined by a pathologist to identify the presence of premalignant and malignant cells. It has been reported that ovarian cancer cells can be identified in the liquid-based Pap test fluid. We hypothesize that proteins shed by ovarian cancer cells can be detected in the Pap test fluid by mass spectrometry (MS)-based proteomic techniques. This biospecimen source is ideal for the discovery phase of biomarker development, since it is readily available at the time of a Pap test, derived from a site near the ovarian cancer, and may not contain the high abundance proteins found in blood that could mask a potential biomarker. In this study, we developed a sample preparation and MS methodology to document the protein profile in residual Pap test fluid. The residual fluid from discarded Pap tests from women over the age of 55 with normal cytology was used to optimize the methodology for sample preparation prior to analysis by MS. The cells were removed by centrifugation and the protein concentration of the cell-free supernatant was determined. The protein composition of individual samples was visualized by separating the proteins on SDS-PAGE gels followed by silver staining. The presence of serum proteins in the residual Pap test fluid was examined by western immunoblot using an antibody against human serum albumin. In order to determine the number of peptides (and proteins) present in the Pap test fluid, samples were precipitated with acetone. The proteins were trypsinized and the resulting peptides were fractionated followed by analysis with tandem MS. The average volume of the residual Pap tests was 1.5 ml and the average protein concentration was 160 ug/ml. By western blot, we showed that the amount of albumin in each sample was significantly reduced compared to normal serum. The number of peptides and proteins present in residual Pap fluid was determined by searching the MS data against the Human Unipro database. Over 300 proteins were identified in samples pooled from 40 patients with normal cytology. When the proteins were classified according to cellular localization and biological function, we found over half of the proteins identified were extracellular or plasma membrane proteins. The largest category of biological function identified was for proteins involved in immunity and defense; including several mucin proteins, such as MUC16 (CA125) which are characteristic of the healthy cervico-vaginal proteome. Approximately 10% of the proteins identified were components of the blood. Using a modification of this method, we have also analyzed the proteins from individual residual Pap test samples by tandem MS, and were able to identify over 1,000 proteins. From these preliminary studies, we conclude that residual Pap test fluid contains a sufficient amount of protein for analysis by MS. Studies to perform MS-based proteomic analysis of proteins from residual Pap test fluid of ovarian cancer patients are in progress. Citation Format: Kristin L.M. Boylan, Melissa A. Geller, Timothy J. Griffin, Stefan E. Pambuccian, Somi Afiuni, Amy P.N. Skubitz. Proteomic analysis of residual Pap test fluid: Novel source for detection of ovarian cancer biomarkers. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr A21.

Abstract B31: Aryl hydrocarbon receptor translocation, CYP1A1 induction and ROS formation: pharmacodynamic markers of sensitivity to antitumor Benzothiazole 5F203 in ovarian cancer

Abstract Ovarian malignancies, the second cause of gynaecological cancer death in developed countries possess poor prognoses. Detected at advanced stages of disease (III and IV), the standard of care is cytoreductive surgery followed by platin- and taxane-based chemotherapy. Intrinsic- or acquired resistance to cytotoxic drugs result in significant risk of recurrence; therefore, the discovery of new molecular targets and more effective therapies remain vital. 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F203, NSC 703786) lysylamide belongs to a novel mechanistic class of antitumor agents. It elicits activity against ovarian, breast, kidney and colorectal cancer models. We have previously showed 5F203 activates aryl hydrocarbon receptor (AhR) signaling: enhanced CYP1A1 expression and AhR nuclear translocation in sensitive IGROV-1 ovarian cancer cells. This AhR modulation triggers an antiproliferative/pro-apoptotic activity evoking ROS formation, DNA damage and cell cycle arrest prior to apoptosis. In contrast, 5F203 failed to induce CYP1A1 expression, AhR translocation or oxidative stress in 5F203-resistant SKOV-3 cells. In this work we further investigated 5F 203 antitumor mechanism of action. We found that 5F203 ROS formation was accompanied by JNK, ERK and P38MAPK phosphorylation only in IGROV-1 sensitive cells. In addition, cancer cells have been isolated from ascites fluid of ovarian cancer patients; sensitivity to 5F203 and concurrent AhR signal transduction has been examined. 5F203 induced enhanced CYP1A1 expression, AhR translocation and ROS formation in ascites-isolated ovarian cancer cells that were sensitive to 5F203. We propose that AhR may represent a new molecular target in the treatment of ovarian tumors and 5F203 may exemplify a potential novel treatment. Finally, our studies demonstrate theranostic pharmacodynamic markers of 5F203 activity and provide additional insight into molecular targets of this novel anticancer agent, which may benefit ovarian cancer patients with limited therapeutic options. Note: This abstract was not presented at the conference. Citation Format: Mariana Alejandra Callero, Gabriela Luzzani, Diana De Dios, Bradshaw Tracey, Andrea Loaiza Perez. Aryl hydrocarbon receptor translocation, CYP1A1 induction and ROS formation: pharmacodynamic markers of sensitivity to antitumor Benzothiazole 5F203 in ovarian cancer. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research: From Concept to Clinic; Sep 18-21, 2013; Miami, FL. Philadelphia (PA): AACR; Clin Cancer Res 2013;19(19 Suppl):Abstract nr B31.

Biomarkers of sensitivity to potent and selective antitumor 2-(4-amino-3-methylphenyl)-5-fluorobenzothiazole (5F203) in ovarian cancer

2-(4-Amino-3-methylphenyl)-5-fluorobenzothiazole (5F203, NSC 703786) lysylamide belongs to a novel mechanistic class of antitumor agents. It elicits activity against ovarian, breast, kidney and colorectal cancer models. In sensitive breast cancer cells, 5F203 activates aryl hydrocarbon receptor (AhR) signaling. Herein, we evaluate the role of AhR in 5F203 activity in two ovarian cancer cell lines: IGROV-1 (sensitive to 5F203), SKOV-3 (resistant to this agent). In addition, cancer cells have been isolated from ascites fluid of ovarian cancer patients; sensitivity to 5F203 and concurrent AhR signal transduction has been examined in ascites-isolated ovarian cancer patients' cells. 5F203 induced enhanced CYP1A1 expression, AhR translocation and ROS formation in IGROV-1 cells and ascites-isolated ovarian cancer cells that were sensitive to 5F203. In IGROV-1 cells 5F203-induced ROS formation was accompanied by JNK, ERK and P38MAPK phosphorylation, DNA damage and cell cycle arrest prior to apoptosis. In contrast, 5F203 failed to induce CYP1A1 expression, AhR translocation or oxidative stress in 5F203-resistant SKOV-3 cells, or in ovarian cancer ascites cells inherently resistant to this agent. We propose that AhR may represent a new molecular target in the treatment of ovarian tumors and 5F203 may exemplify a potential novel treatment. Furthermore, putative biomarkers of sensitivity to this agent have been identified.

Characterization of disease progression in ovarian cancer by utilizing ‘chemograms’ of ovarian cancer stem cells

Introduction: Ovarian cancer is one of the leading causes of death in women with cancer. First-line chemotherapy with platinum compounds and taxane compounds has been effective, but most patients develop a relapse of the disease due to drug resistance. There is growing evidence that this resistance may be due to the presence of ovarian cancer stem cells.Discussion: Cells with properties of cancer stem cells have been isolated from the ascitic fluid of ovarian cancer patients. This subset of cells is highly tumourigenic compared to the rest of the cells in the ascitic fluid. They are known to exude harmful chemicals from their cytoplasm and have been found to be resistant to chemotherapeutic agents. This property has been utilized to purify them by fluorescence assisted cytometry to yield a subset of cells which are called ‘side population’. These cells exhibit the properties of cancer stem cells and their role in disease progression is being currently investigated. The course of the disease can be potentially characterized at the cellular level by closely studying this cell population. They can also be cultured in different combinations of chemotherapeutic agents at varying concentrations to obtain ‘chemograms’ which are sensitivity charts. Chemotherapeutic agents which produce the most effective kill curves can then be rationally used as a second-line chemotherapy if the disease relapses. These sensitivity charts can provide insight into emerging patterns of chemoresistance and also help discover surface markers that accurately identify ovarian cancer stem cells.Conclusion: The high rate of disease relapse in patients with ovarian cancer requires a new and different approach utilizing the sensitivity of cancer stem cells. Isolating and characterizing the resistance patterns of ovarian cancer stem cells may provide a rational approach towards an effective and individualized chemotherapeutic regimen.

Ovarian cancer stem cells: elusive targets for chemotherapy

Ovarian cancer is one of the major causes of death in women with gynecologic malignancy. Most patients respond favorably to platinum therapy, but relapses are common. There is emerging evidence that a special subset of cells that is highly tumorigenic is responsible for recurrence of the disease. This subset of cells has been characterized by several groups and has been found to have the properties of cancer stem cells. They have been isolated from tumor samples obtained during surgical cytoreduction and also from the ascitic fluid of ovarian cancer patients. Currently, there are no known unique markers to define these cells, but several groups have used different approaches to purify them. Although some heterogeneity has been observed in these cells, most of them satisfy the functional definition of a stem cell. Advances in characterization of ovarian cancer stem cells are instrumental in developing therapies that specifically target them. This review describes the advances made in characterization of these cells, basis of their resistance to conventional chemotherapeutic agents and the prognostic implications of utilizing mechanisms specific to ovarian cancer stem cell for therapeutic interventions. Eliminating ovarian cancer stem cells could possibly lead to a prolongation of the disease-free survival period and hopefully a definitive cure.

Abstract 433: Expression of stem cell and EMT markers on circulating tumor cells

Abstract Tumor metastasis contributes significantly to cancer mortality especially in breast, colon, lung and prostate cancers and is believed to be associated with distant seeding of circulating tumor cells (CTC) present in the peripheral blood that arise from the primary tumor. Various studies on breast, colon and prostate cancer suggest that CTC numbers are good prognostic indicators. Although there are different technologies available to detect CTCs from peripheral blood, the CellSearchTM (Veridex, NJ, USA) technology is the only system approved by the US FDA for clinical utilization in metastatic breast, colorectal and prostate cancer. This system is based on a semiautomated immunomagnetic enrichment in combination with image cytometry and uses ferrofluid-conjugated antibodies for epithelial cell adhesion molecule (EpCAM), a cell surface antigen expressed on most normal epithelial cells and carcinomas to capture CTCs and cytokeratin (CK) 8, 18 &amp; 19 to image. Additionally, by definition, CTCs are negative for CD45 (leukocyte common antigen). One major limitation of the CellSearchTM system is the sole dependence on anti-EpCAM antibody-based enrichment. Recent studies have demonstrated that epithelial cancer cells transition into mesenchymal-like tumor cells to gain migratory capacity, and therefore, CTC enumeration using the EpCAM-based system may be misleading due to the lower expression of this antigen. Based on this observation, we hypothesize that CTCs might express stem cell and EMT-associated markers, both hallmarks of the metastatic properties of these cells. To test this concept, we determined the expression of the stem/progenitor (Doublecortin like Kinase-I [DCLK-1]; CD133) and EMT (β-Catenin, Fibronectin and Vimentin) markers on a panel of lung cancer cells (A549: epithelial lung carcinoma, H358: bronchioalveolar carcinoma, H69: small cell lung cancer), colon cancer cells (SW480: colorectal adenocarcinoma, SW620: colorectal adenocarcinoma derived from metastatic lymph node) and tumor cells isolated from the ascites fluid of ovarian cancer patients. Tumor cells isolated from ascites fluids were also labeled with a DCLK-1 specific antibody and sorted using Fluorescence-activated cell sorting (FACS). Both DCLK-1 positive and negative cells from the cell sorting were stained by immunofluorescence and images were taken using inverted microscopy. Our data demonstrate that a small population of cells in the tumor cell lines and the tumor cells isolated from the ascites fluid of ovarian cancer patients have cancer stem cell properties. Furthermore, these cells also express stem/progenitor and EMT markers. These findings suggest that using these markers may be more sensitive and informative on patient prognosis than the use of EpCAM and cytokeratins for the detection and enumeration of circulating tumor cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 433. doi:1538-7445.AM2012-433

Abstract 1931: A potential antitumor herb medicine, Corilagin, inhibits ovarian cancer cell growth by blockage of TGF-β/AKT/ERK signaling pathways

Abstract Recently, we have identified that Corilagin is a major anti-tumor active component extracted from a well-known hepatoprotective and antiviral medicinal herb, Phyllanthus niruri L. Corilagin distinctly inhibited the growth of ovarian cancer cell lines SKOv3ip, Hey and HO-8910PM, with IC50s less than 20µg/ml, while displayed the low toxicity against normal ovarian surface epithelium (OSE), with IC50s around 100µg/ml. Corilagin induced cell cycle arrest at G2 stage and enhanced apoptosis in Hey cancer cells. To explore its anti-tumor activity in vivo, Corilagin was delivered intra-peritoneally to the mice bearing SKOv3ip xenograft. Although no statistical difference observed on mice weight between untreated and Corilagin treated groups, xenograft tumor growth was significantly restarted by 15µg/mL of Corilagin treated group comparing to untreated control group (p&amp;lt;0.05). To understand the anti-tumor mechanisms of Corilagin, we performed reverse phase protein array (RPPA) analysis using untreated and Corilagen treated HO-8910PM cells, and the results were confirmed by Western blots. We have found that Corilagin blocked the activation of multiple signaling cascades, such as, pAKT and pERK. Particularly, Corilagin inhibits TGF-β secretion in the culture supernatant of all tested ovarian cancer cell lines and blocks the stabilization of Snail induced by TGF-β in HO-8910PM cells. In contrast, reduction of TGF-β secretion was not observed in cytotoxic drug Taxol-treated cancer cells, suggesting that Corilagin specifically targets TGF-β secretion. TGF-β signaling plays a critical role in epithelial-mesenchymal transition (EMT) and cancer metastasis. High concentration of TGF-β had been detected in ascites, blood and other body fluids of ovarian cancer patients. In addition, our recent studies identified that TGF-β being the most important inflammation factor in ovarian cancer, which stabilizes at the protein level of Snail, an inducer of EMT, and further enhanced Snail expression when combined with other inflammation factors. TGF-β is regulated by Smads, but could also be regulated by AKT/ERK signaling pathways. Our observations indicate that Corilagin extracted from Phyllanthus niruri L. acts as an effective natural targeted therapeutic agent against the growth of ovarian cancer cells via TGF-β/AKT/ERK signaling pathways. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1931. doi:1538-7445.AM2012-1931

Abstract 3573: Urine and serum biomarkers as early detection tools for ovarian cancer

Abstract Background: The measurement of biomarkers present in the bodily fluids of ovarian cancer patients represents an emerging strategy for early detection. To date, a great deal of related work has been devoted to blood, given its systemic exposure and extensive availability through tissue banks. However, blood is a dynamic biofluid with a proteome under continuous metabolic and homeostatic regulation. Alternatively, urine represents a biofluid that is inherently quiescent in that all molecular and proteolytic activity is largely complete upon sampling. Additional characteristics of urine, including its high level of temperature stability and low-complexity matrix resulting from renal filtration, suggest that this biofluid may serve as an effective alternative or companion to blood in biomarker analyses. Methods: A set of urines obtained from over 700 women, including patients diagnosed with ovarian cancer, patients diagnosed with benign pelvic lesions, and healthy donors, was tested for 39 biomarkers which demonstrated previous associations with ovarian cancer. In an effort to identify additional useful biomarkers, 122 candidate biomarkers were evaluated in a separate set of 150 sera obtained from ovarian cancer patients along with benign and healthy controls. Significantly altered biomarkers were identified in each analysis and the full biomarker dataset was examined by multivariate analysis to identify multianalyte panels capable of discriminating ovarian cancer patients from controls with high accuracy. Results: Numerous significantly altered biomarkers were identified in both urine and serum. The classification performance of several biomarkers was found to differ in urine vs. serum with an overall trend in favor of urine biomarkers. Separate urine and serum biomarker panels consisting of 3-4 analytes were identified which could discriminate ovarian cancer from healthy controls at a sensitivity of 96-97% at 95% specificity. Improvement upon this classification efficiency was observed when the best urine and serum biomarkers were combined into a single panel. The combination of urine HE4 and serum CA 125 was most effective in the discrimination of ovarian cancer from benign controls. Conclusions: Urine and serum biomarkers offer complementary information regarding ovarian cancer status. Their combined use may lead to the development of effective early detection tools. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3573. doi:1538-7445.AM2012-3573

Circulating biomarker tissue kallikrein-related peptidase KLK5 impacts ovarian cancer patients’ survival

BackgroundEffective cancer biomarkers for early detection, prognosis, or therapy response prediction are urgently needed in ovarian cancer. Kallikrein-related peptidases, including KLK5, have been reported to play an important role in the course of the disease. Patients and methodsKLK5 antigen content was determined by enzyme-linked immunosorbent assay in ovarian cancer patients’ [FIGO (International Federation of Gynecology and Obstetrics) stages I–IV, n = 52] serum as well as ascitic fluid and compared with KLK5 content in serum of patients with benign ovarian tumors (n = 45). ResultsKLK5 antigen content was significantly elevated in the serum of ovarian cancer patients compared with the serum of patients with benign ovarian tumors. Forty-two of 52 ovarian cancer serum samples, 42 of 43 benign ovarian tumor serum samples, and all 41 ascitic fluid samples were KLK5 positive. Elevated KLK5 antigen in serum and ascitic fluid of ovarian cancer patients was a prognostic factor for progression-free survival. ConclusionsOur data support the finding that ovarian cancer patients release significant amounts of KLK5 into serum and ascitic fluid but KLK5 antigen is low in serum of patients with benign ovarian tumors. Increased serum and ascitic fluid KLK5 levels are associated with poor patient outcome, thus underlining the importance of KLK5 as a biomarker for early detection as well as for disease management in ovarian cancer.

Lysophosphatidic Acid Stimulates the Proliferation of Ovarian Cancer Cells via the gep Proto-Oncogene G 12

Lysophosphatidic acid (LPA), an agonist that activates specific G protein-coupled receptors, is present at an elevated concentration in the serum and ascitic fluid of ovarian cancer patients. Although the increased levels of LPA have been linked to the genesis and progression of different cancers including ovarian carcinomas, the specific signaling conduit utilized by LPA in promoting different aspects of oncogenic growth has not been identified. Here, we show that LPA stimulates both migration and proliferation of ovarian cancer cells. Using multiple approaches, we demonstrate that the stimulation of ovarian cancer cells with LPA results in a robust and statistically significant proliferative response. Our results also indicate that Gα(12), the gep proto-oncogene, which can be stimulated by LPA via specific LPA receptors, is overtly activated in a large array of ovarian cancer cells. We further establish that LPA stimulates the rapid activation of Gα(12) in SKOV-3 cells and the expression of CT12, an inhibitory minigene of Gα(12) that disrupts LPAR-Gα(12) interaction and potently inhibits such activation. Using this inhibitory molecule as well as the shRNA approach, we show that the inhibition of Gα(12) or silencing of its expression drastically and significantly attenuates LPA-mediated proliferation of ovarian cancer cell lines such as SKOV3, Hey, and OVCAR-3. Together with our findings that the silencing of Gα(12) does not have any significant effect on LPA-mediated migratory response of SKOV3 cells, our results point to a critical role for LPA-LPAR-Gα(12) signaling in ovarian cancer cell proliferation and not in migration. Thus, results presented here for the first time demonstrate that the gep proto-oncogene forms a specific node in LPA-LPAR-mediated mitogenic signaling in ovarian cancer cells.

Abstract 2223: ELISA coupled anion exchange methodology for separation of KLK6 glycoprotein subpopulations in biological fluids

Abstract Ovarian cancer is the leading cause of death among all gynecological disorders. Elevated levels of Kallikrein 6 (KLK6), a secreted trypsin-like N-glycosyalted protease, have been shown to have unfavourable prognostic value in ovarian cancer patients. It has been revealed to be aberrantly up regulated at both transcriptional and translational levels in ovarian cancer. Aberrant glycosylation, or more specifically, increased sialylation of proteins has been observed in the cancer of the ovary. KLK6 isolated from ascites fluid of ovarian cancer patients has been shown to have enrichment of terminal alpha 2-6 linked sialic acid at its single N-glycosylation site when compared to protein isolated from cerebrospinal fluid (CSF), which is believed to be the major source of KLK6 present in circulation. However, due to the relatively low levels of KLK6 in serum (ng/ml range) neither mass spectrometry nor lectin based characterization of this protein found in serum has been achieved. Here, we report a novel HPLC anion exchange method, coupled to a KLK6 specific ELISA, capable of differentiating KLK6 glycoform subgroups in biological fluids, including serum, at physiologically relevant levels. In brief, biological fluids were injected onto a MonoQ bead matrix (GE Healthcare), bound proteins were eluted over a salt gradient and the resulting fractions were analyzed for KLK6 content resulting in a chromatographic elution profile containing four distinct peaks. Utilizing this assay, the KLK6 elution profile and distribution across peaks of a small set (n=8) of ovarian cancer patient matched serum and ascites fluid samples was found to be different than the profile of serum and CSF of normal individuals (n=10). Utilizing tandem mass spectrometry (MS/MS), recombinant KLK6 (rKLK6) purified from an immortalized human cell line was characterized and found to have a highly heterogeneous KLK6 population, encompassing the majority of glycoforms previously shown to be present in the protein from CSF and ovarian cancer ascites. This protein was subjected to the developed assay and was found to contain all of the four diagnostic KLK6 peaks present in the previously assayed biological fluids. Due to the available high quantity of protein from this source (mg levels) we were able to analyze the rKLK6 glycoform composition of each peak utilizing lectin affinity and MS/MS based glycopeptide quantification by single reaction monitoring. The combined results showed a significant increase in terminal alpha 2-6 linked sialic acid on the N-glycans found on KLK6 from ovarian cancer serum and ascites, as opposed to CSF and serum of normal individuals. Therefore, further development and application of this methodology might lead to improvement of KLK6 as a serum ovarian cancer biomarker. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2223. doi:10.1158/1538-7445.AM2011-2223

Reactive oxygen species mediate lysophosphatidic acid induced signaling in ovarian cancer cells

Lysophosphatidic acid (LPA) is produced by tumor cells and is present in the ascites fluid of ovarian cancer patients. To determine the role of endogenous LPA in the ovarian cancer cell line SKOV3, we treated cells with the LPA receptor antagonist VPC32183 and found that it inhibited cell growth and induced apoptosis. Exogenous LPA further stimulated ERK and Akt phosphorylation and NF-κB activity. To determine if reactive oxygen species (ROS), which have been implicated as second messengers in cell signaling, were also involved in LPA signaling, we treated cells with the NADPH oxidase inhibitor diphenyleneiodonium (DPI), and antioxidants N-acetyl cysteine, EUK-134 and curcumin, and showed that all blocked LPA-dependent NF-κB activity and cell proliferation. DPI and EUK-134 also inhibited Akt and ERK phosphorylation. LPA was shown to stimulate dichlorofluorescein fluorescence, though not in the presence of DPI, apocynin (an inhibitor of NADPH oxidase), VPC32183, or PEG-catalase. Akt phosphorylation was also inhibited by PEG-catalase and apocynin. These data indicate that NADPH oxidase is a major source of ROS and H 2O 2 is critical for LPA-mediated signaling. Thus, LPA acts as a growth factor and prevents apoptosis in SKOV3 cells by signaling through redox-dependent activation of ERK, Akt, and NF-κB-dependent signaling pathways.

Identification of Siglec-9 as the receptor for MUC16 on human NK cells, B cells, and monocytes

BackgroundMUC16 is a cell surface mucin expressed at high levels by epithelial ovarian tumors. Following proteolytic cleavage, cell surface MUC16 (csMUC16) is shed in the extracellular milieu and is detected in the serum of cancer patients as the tumor marker CA125. csMUC16 acts as an adhesion molecule and facilitates peritoneal metastasis of ovarian tumors. Both sMUC16 and csMUC16 also protect cancer cells from cytotoxic responses of natural killer (NK) cells. In a previous study we demonstrated that sMUC16 binds to specific subset of NK cells. Here, we identify the csMUC16/sMUC16 binding partner expressed on immune cells.ResultsAnalysis of immune cells from the peripheral blood and peritoneal fluid of ovarian cancer patients indicates that in addition to NK cells, sMUC16 also binds to B cells and monocytes isolated from the peripheral blood and peritoneal fluid. I-type lectin, Siglec-9, is identified as the sMUC16 receptor on these immune cells. Siglec-9 is expressed on approximately 30-40% of CD16pos/CD56dim NK cells, 20-30% of B cells and >95% of monocytes. sMUC16 binds to the majority of the Siglec-9pos NK cells, B cells and monocytes. sMUC16 is released from the immune cells following neuraminidase treatment. Siglec-9 transfected Jurkat cells and monocytes isolated from healthy donors bind to ovarian tumor cells via Siglec-9-csMUC16 interaction.ConclusionsRecent studies indicate that csMUC16 can act as an anti-adhesive agent that blocks tumor-immune cell interactions. Our results demonstrate that similar to other mucins, csMUC16 can also facilitate cell adhesion by interacting with a suitable binding partner such as mesothelin or Siglec-9. Siglec-9 is an inhibitory receptor that attenuates T cell and NK cell function. sMUC16/csMUC16-Siglec-9 binding likely mediates inhibition of anti-tumor immune responses.

Selective removal of ovarian cancer cells from human ascites fluid using magnetic nanoparticles

A majority of ovarian cancer metastases result from the shedding of malignant cells from the primary tumor into the abdominal cavity. Free-floating cancer cells in serous effusions of late-stage ovarian cancer patients may spread to internal organs, making effective treatment extremely difficult. Selective removal of ovarian cancer cells from serous fluids may abate metastasis and improve long-term prognoses. We have already shown that superparamagnetic nanoparticles conjugated to an ephrin-A1 mimetic peptide with a high affinity for the EphA2 receptor can be used to capture and remove cultured human ovarian cancer cells from the peritonea of experimental mice. Here we demonstrate the potential clinical utility of the methodology by in vitro capture and isolation of cancer cells from the ascites fluid of ovarian cancer patients. From the Clinical Editor Ovarian cancer metastases usually are the result of shedding of malignant cells from the primary tumor into the abdominal cavity. In this paper, a novel nanotechnology-based method is demonstrated for the in vitro capture and isolation of cancer cells from the ascites fluid of ovarian cancer patients.

Differential N-glycosylation of Kallikrein 6 Derived from Ovarian Cancer Cells or the Central Nervous System

Ovarian cancer causes more deaths than any other gynecological disorder. Perturbed glycosylation is one of the hallmarks of this malignancy. Kallikrein 6 (KLK6) elevation in serum is a diagnostic and prognostic indicator in ovarian cancer. The majority of ovarian carcinomas express high levels of KLK6, which diffuses into the circulation. Under physiological conditions, KLK6 is expressed highly in the central nervous system and found at high levels in cerebrospinal fluid from where it enters the circulation. Our aim was to characterize and compare the N-glycosylation status of this protein in ovarian cancer ascites fluid and cerebrospinal fluid. Anion-exchange chromatography was used to reveal different post-translational modifications on the two isoforms. Mobility gel shift Western blot analysis coupled with glycosidase digestion showed that the molecular weight difference between the two isoforms was because of differential glycosylation patterns. The presence of a single N-glycosylation site on KLK6 was confirmed by site-directed mutagenesis. Using a Sambucus nigra agglutinin-monoclonal antibody sandwich enzyme-linked immunosorbent assay approach, it was shown that ovarian cancer-derived KLK6 was modified with alpha2-6-linked sialic acid. The structure and composition of glycans of both KLK6 isoforms was elucidated by glycopeptide monitoring with electrospray ionization-Orbitrap tandem mass spectrometry. Therefore, the extensive and almost exclusive sialylation of KLK6 from ovarian cancer cells could lead to the development of an improved biomarker for the early diagnosis of ovarian carcinoma.