Isabelle Matte

Prognostic significance of IL-6 and IL-8 ascites levels in ovarian cancer patients

BackgroundThe acellular fraction of epithelial ovarian cancer (EOC) ascites promotes de novo resistance of tumor cells and thus supports the idea that tumor cells may survive in the surrounding protective microenvironment contributing to disease recurrence. Levels of the pro-inflammatory cytokines IL-6 and IL-8 are elevated in EOC ascites suggesting that they could play a role in tumor progression.MethodsWe measured IL-6 and IL-8 levels in the ascites of 39 patients with newly diagnosed EOC. Commercially available enzyme-linked immunosorbent assay (ELISA) was used to determine IL-6 and IL-8 ascites levels. Ascites cytokine levels were correlated with clinicopathological parameters and progression-free survival.ResultsMean ascites levels for IL-6 and IL-8 were 6419 pg/ml (SEM: 1409 pg/ml) and 1408 pg/ml (SEM: 437 pg/ml) respectively. The levels of IL-6 and IL-8 in ascites were significantly lower in patients that have received prior chemotherapy before the surgery (Mann-Whitney U test, P = 0.037 for IL-6 and P = 0.008 for IL-8). Univariate analysis revealed that high IL-6 ascites levels (P = 0.021), serum CA125 levels (P = 0.04) and stage IV (P = 0.009) were significantly correlated with shorter progression-free survival. Including these variables in a multivariate analysis revealed that elevated IL-6 levels (P = 0.033) was an independent predictor of shorter progression-free survival.ConclusionElevated IL-6, but not IL-8, ascites level is an independent predictor of shorter progression-free survival.

MUC16 (CA125) regulates epithelial ovarian cancer cell growth, tumorigenesis and metastasis

OBJECTIVESnMUC16 (CA125) protein is a high molecular weight mucin overexpressed in the majority of epithelial ovarian cancers (EOC) but not in the epithelium of normal ovaries suggesting that it might play a role in EOC pathogenesis. Here, we explored the phenotypic consequences of MUC16 knockdown and expression of its C-terminal domain with the aim of establishing a role for MUC16 in tumorigenesis.nnnMETHODSnMUC16 was down-regulated by stably expressing an anti-MUC16 endoplasmic reticulum-targeted single-chain antibody which prevented MUC16 cell surface localization in NIH:OVCAR3 cells. In addition, we generated epitope tagged, N-terminal region-deleted MUC16 constructs with (MUC16TMU) and without (MUC16CTD) cytoplasmic tail deletions and stably expressed them in SKOV3 cells.nnnRESULTSnAlthough MUC16 knockdown did not affect the cell growth rate, knockdown cells reached a stationary growth phase after 4 days whereas control cells continued to grow for up to 7 days. Colony formation assays in soft agar demonstrated that MUC16 knockdown cells had >8-fold reduction in their ability to form colonies. Importantly, MUC16 knockdown completely prevents the formation of subcutaneous tumors in nude mice. Conversely, we show that ectopic expression of the MUC16CTD enhances SKOV3 tumor cell growth, colony formation in soft agar and enhances tumor growth and metastases in SCID mice. In addition, MUC16CTD expression increases cell motility, invasiveness, and metastatic property. Deletion of the cytoplasmic tail from the MUC16CTD completely abolished its ability to enhance tumor cell growth, cell motility and invasiveness. Furthermore, the increased invasive properties of MUC16CTD-expressing cells correlated with decreased expression of E-cadherin and increased expression of N-cadherin and vimentin.nnnCONCLUSIONnThese findings provide the first evidence for a critical role of MUC16 in tumor cell growth, tumorigenesis and metastases.

Downregulation of cell surface CA125/MUC16 induces epithelial-to-mesenchymal transition and restores EGFR signalling in NIH:OVCAR3 ovarian carcinoma cells

Background:Epithelial ovarian cancer (EOC) cells are prone to metastasise throughout the peritoneal cavity. The epithelial-to-mesenchymal transition (EMT) is a necessary step towards metastatic tumour progression. CA125/MUC16 mucin is a high-molecular-weight glycoprotein overexpressed in the majority of serous carcinomas, suggesting a possible role in the pathogenesis of these cancers.Methods:The role of CA125/MUC16 in EMT was investigated using single-chain antibody-mediated knockdown of cell surface CA125/MUC16 in overexpressing EOC NIH:OVCAR3 cells.Results:CA125/MUC16 knockdown was associated with morphological alterations along with decreased surface expression of epithelial markers (E-cadherin, cytokeratin-18) and increased expression of mesenchymal markers (N-cadherin, vimentin). Co-immunoprecipitation experiments revealed that CA125/MUC16 binds to E-cadherin and β-catenin complexes. The in vitro studies showed disruption of cell–cell junctions, enhanced motility, migration and invasiveness in CA125/MUC16 knockdown cells. Enhanced epidermal growth factor receptor (EGFR) activation was observed in CA125/MUC16 knockdown cells along with increased Akt and ERK1/2 phosphorylation, which are downstream effectors of EGFR, and increased MMP-2 and MMP-9 expression and activities. Epidermal growth factor receptor inhibition strongly inhibited the motility of CA125/MUC16 knockdown cells.Conclusions:Our findings suggest that CA125/MUC16 plays a role in EMT, presumably through its interaction with E-cadherin and β-catenin complexes and by modulating EGFR and its downstream signalling pathway in NIH:OVCAR3 cells.

The prosurvival activity of ascites against TRAIL is associated with a shorter disease-free interval in patients with ovarian cancer

BackgroundThe production of ascites is a common complication of ovarian cancer. Ascites constitute a unique tumor microenvironment that may affect disease progression. In this context, we recently showed that ovarian cancer ascites may protect tumor cells from TRAIL-induced apoptosis. In this study, we sought to determine whether the prosurvival effect of ascites affects disease-free intervals.MethodsPeritoneal fluids were obtained from 54 women undergoing intra-abdominal surgery for suspected ovarian cancer (44 cancers and 10 benign diseases). The ability of peritoneal fluids to protect from TRAIL was assessed in the ovarian cancer cell line CaOV3, and IC50 were determined. The anti-apoptotic activity of 6 ascites against cisplatin, paclitaxel, doxorubicin, etoposide and vinorelbine was also assessed in CaOV3 cells, and the prosurvival activity of two ascites was assessed in 9 primary ovarian cancer cultures.ResultsAmong the 54 peritoneal fluids tested, inhibition of TRAIL cytotoxicity was variable. Fluids originating from ovarian cancer were generally more protective than fluids from non-malignant diseases. Most of the 44 ovarian cancer ascites increased TRAIL IC50 and this inhibitory effect did not correlate strongly with the protein concentration in these ascites or the levels of serum CA125, a tumor antigen which is used in the clinic as a marker of tumor burden. The effect of ascites on cisplatin- and paclitaxel-induced cell death was assessed with 4 ascites having inhibitory effect on TRAIL-induced cell death and 2 that do not. The four ascites with prosurvival activity against TRAIL had some inhibitory on cisplatin and/or paclitaxel. Two ovarian cancer ascites, OVC346 and OVC509, also inhibited TRAIL cytotoxicity in 9 primary cultures of ovarian tumor and induced Akt activation in three of these primary cultures. Among a cohort of 35 patients with ascites, a threshold of TRAIL IC50 with ascites/IC50 without ascites > 2 was associated with shorter disease-free interval.ConclusionsThe prosurvival activity of ascites against TRAIL is associated with shorter disease-free interval, which may be explained, at least in part, by ascites-induced cisplatin/paclitaxel resistance. Our findings suggest that ascites may contain prosurvival factors that protect against TRAIL and chemotherapy and consequently affect disease progression.

Ovarian cancer ascites increase Mcl-1 expression in tumor cells through ERK1/2-Elk-1 signaling to attenuate TRAIL-induced apoptosis

BackgroundAscites may affect the progression of ovarian cancer (OC). In particular, soluble factors present in OC ascites can create a protective environment for tumor cells that promote de novo resistance to drug- and death receptor-induced apoptosis. However, the underlying molecular mechanisms responsible for ascites-induced drug resistance are not well characterized.MethodsUsing human OC cell lines and tissues microarrays of human OC biopsies, we assessed the mechanism by which OC ascites increase Mcl-1 expression using Western blots, chemical inhibitors of ERK and small-inhibitory RNA treatments.ResultsIn the present study, we found that both Mcl-1 mRNA and protein levels were upregulated within 2 h upon treatment of OC cells with ascites obtained from women with advanced OC. In contrast, the expression of other Bcl-2 family antiapoptotic members such as Bcl-2 and Bcl-XL was not affected by ascites. An increase of Mcl-1 expression was consistently observed across different ascites from women with advanced serous OC. The knockdown of Mcl-1 significantly blocked ascites-induced Mcl-1 upregulation and ascites-mediated inhibition of TRAIL-induced apoptosis. Ascites induced a rapid phosphorylation of ERK1/2 and Elk-1 transcription factor. Furthermore, we found that ERK1/2 inhibition or Elk-1 knockdown was sufficient to block ascites-induced Mcl-1 expression. In high grade serous OC, we found a positive correlation between phosphorylated ERK1/2 and Mcl-1 expression.ConclusionsThese results indicate that ascites-induced ERK1/2/Elk-1 signaling is critical for Mcl-1 expression and for the ascites-mediated attenuation of TRAIL-induced apoptosis. The ERK1/2/Elk-1/Mcl-1 pathway represents a novel mechanism by which ascites induce de novo TRAIL resistance in OC cells.

Osteoprotegerin (OPG) activates integrin, focal adhesion kinase (FAK), and Akt signaling in ovarian cancer cells to attenuate TRAIL-induced apoptosis

BackgroundResistance to apoptosis is a major problem in ovarian cancer (OC) and correlates with poor prognosis. Osteoprotegerin (OPG) is a soluble secreted factor that acts as a decoy receptor for receptor activator of NF-κB ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). OPG has been reported to attenuate TRAIL-induced apoptosis in a variety of cancer cells, including OC cells. OPG-mediated protection against TRAIL has been attributed to its decoy receptor function. However, OPG activates integrin/focal adhesion kinase (FAK) signaling in endothelial cells. In OC cells, activation of integrin/FAK signaling inhibits TRAIL-induced apoptosis. Based on these observations, we hypothesized that OPG could attenuate TRAIL-induced apoptosis in OC cells through integrin/FAK signaling.MethodsIn vitro experiments including immunoblots, colony formation assays, and apoptosis measurements were used to assess the effect of OPG on TRAIL-induced apoptosis.ResultsExogenous OPG protected from TRAIL-induced apoptosis in a TRAIL binding-independent manner and OPG protection was αvβ3 and αvβ5 integrin/FAK signaling-dependent. Moreover, OPG-mediated activation of integrin/FAK signaling resulted in the activation of Akt. Inhibition of both integrin/FAK and Akt signaling significantly inhibited OPG-mediated attenuation of TRAIL-induced apoptosis. Although OPG also stimulated ERK1/2 phosphorylation, inhibition of ERK1/2 signaling did not significantly altered OPG protection.ConclusionsOur studies provide evidence, for the first time, that OPG can attenuate TRAIL-induced apoptosis in a TRAIL binding-independent manner through the activation of integrin/FAK/Akt signaling in OC cells.

Osteoprotegerin (OPG) protects ovarian cancer cells from TRAIL-induced apoptosis but does not contribute to malignant ascites-mediated attenuation of TRAIL-induced apoptosis

BackgroundResistance to apoptosis is a major problem in ovarian cancer and correlates with poor prognosis. Osteoprotegerin (OPG) is a secreted factor in malignant ascites and acts as a decoy receptor for receptor activator of NF-κB ligand (RANKL) and tumor necrosis factor-related apoptosis-inducing ligand (TRAIL). TRAIL promotes apoptosis in ovarian cancer cells. Ovarian cancer ascites attenuate TRAIL-induced apoptosis raising the possibility that OPG contained in ascites may abrogate the anti-tumor activity of TRAIL.MethodsDetermination of OPG levels in ascites was measured by ELISA. Effect of OPG on TRAIL-induced cell death was determined by XTT and colony forming assays in ovarian cancer cell lines and primary tumor cells. Apoptosis was assessed by ELISA.ResultsWe found that recombinant OPG and malignant ascites attenuates TRAIL-induced cell death and apoptosis in a dose-dependent manner in ovarian cancer cell lines and primary ovarian tumor cells. OPG is present at high levels in the ascites of patients with ovarian cancer. We found a positive correlation between the levels of OPG in ascites and the ability of the ascites to attenuate TRAIL-induced cell death. The anti-apoptotic effect of ascites was not reversed by co-incubation with an OPG blocking antibody.ConclusionsOPG and malignant ascites protect ovarian cancer cells from TRAIL-induced apoptosis. Although malignant ascites contain high levels of OPG, OPG is not a critical component that contributes to ascites-mediated attenuation of TRAIL-induced apoptosis.

Antiapoptotic proteins Bcl-2 and Bcl-XL inhibit Clostridium difficile toxin A-induced cell death in human epithelial cells.

ABSTRACT It has been well established that Clostridium difficile toxin A (TcdA) induces cell death in human epithelial cells. However, the mechanism of TcdA-induced cell death remains to be fully characterized. Here, we show that TcdA induces dose-dependent cell death in ovarian carcinoma and colonic carcinoma cell lines. TcdA-mediated cell death, as well as caspase 8 and caspase 3 activation, were specifically abrogated by anti-toxin antibodies. Although caspase 8 and caspase 3 were activated by TcdA in OVCAR3 ovarian carcinoma and T84 colonic cancer cells, pancaspase and caspase 8, 3, and 9 inhibitors did not block TcdA-induced cell death. In contrast, tumor necrosis factor-related apoptosis-inducing ligand-induced cell death was nearly completely blocked by caspase inhibitors in OVCAR3 cells. In these cells, TcdA induces the mitochondrial pathway of apoptosis, as demonstrated by changes in mitochondrial outer membrane permeabilization (MOMP). Furthermore, overexpression of the antiapoptotic proteins Bcl-2 and Bcl-XL significantly inhibited TcdA-induced cell death, as well as TcdA-induced MOMP. Conversely, small interfering RNA-mediated inhibition of Bcl-XL in TcdA-resistant SKOV3ip1 cells enhanced TcdA-induced cell death. Overexpression of the antiapoptotic proteins Bcl-2 and Bcl-XL in T84 cells also inhibited TcdA-induced cell death. Altogether, our data demonstrate that TcdA induces cell death in both ovarian and colonic cancer cells preferentially via the mitochondrial pathway of apoptosis by a death receptor-independent and a caspase-independent mechanism. This process is regulated by antiapoptotic members of the Bcl-2 family.

Inflammation-regulating factors in ascites as predictive biomarkers of drug resistance and progression-free survival in serous epithelial ovarian cancers

BackgroundPlatinum-based combination therapy is the standard first-line treatment for women with advanced serous epithelial ovarian carcinoma (EOC). However, about 20xa0% will not respond and are considered clinically resistant. The availability of biomarkers to predict responses to the initial therapy would provide a practical approach to identify women who would benefit from a more appropriate first-line treatment. Ascites is an attractive inflammatory fluid for biomarker discovery as it is easy and minimally invasive to obtain. The aim of this study was to evaluate whether six selected inflammation-regulating factors in ascites could serve as diagnostic or drug resistance biomarkers in patients with advanced serous EOC.MethodsA total of 53 women with stage III/IV serous EOC and 10 women with benign conditions were enrolled in this study. Eleven of the 53 women with serous EOC were considered clinically resistant to treatment with progression-free survivalu2009<u20096xa0months. Ascites were collected at the time of the debulking surgery and the levels of cytokines were measured by ELISA. The six selected cytokines were evaluated for their ability to discriminate serous EOC from benign controls, and to discriminate platinum resistant from platinum sensitive patients.ResultsMedian ascites levels of IL-6, IL-10 and osteoprotegerin (OPG) were significantly higher in women with advanced serous EOC than in controls (Pu2009≤u20090.012). There were no significant difference in the median ascites levels of leptin, soluble urokinase plasminogen activator receptor (suPAR) and CCL18 among serous EOC women and controls. In Receiver Operator curve (ROC) analysis, IL-6, IL-10 and OPG had a high area under the curve value of 0.905, 0.832 and 0.825 respectively for distinguishing EOC from benign controls. ROC analysis of individual cytokines revealed low discriminating potential to stratify patients according to their sensitivity to first-line treatment. The combination of biomarkers with the highest discriminating potential was with CA125 and leptin (AUCu2009=u20090.936, 95xa0% CI: 0.894–0.978).ConclusionIL-6 was found to be strongly associated with advanced serous EOC and could be used in combination with serum CA125 to discriminate benign and EOC. Furthermore, the combination of serum CA125 and ascites leptin was a strong predictor of clinical resistance to first-line therapy.

Ovarian cancer ascites enhance the migration of patient-derived peritoneal mesothelial cells via cMet pathway through HGF-dependent and -independent mechanisms

Ovarian cancer ascites consist of a proinflammatory environment that is characterized by the presence of abundant human peritoneal mesothelial cells (HPMCs). Cytokines and growth factors in ascites modulate cell activities of tumor cells. The expression of proinflammatory cytokines in ascites is associated with a more aggressive tumor phenotype. The effect of ascites on HPMCs is for the most part unknown but this interplay is thought to be important for epithelial ovarian cancer (EOC) progression. Here, we examine the components of ascites, which stimulate patient‐derived HPMC migration, from women with advanced EOC. We show that ovarian cancer ascites enhanced the migration of HPMCs. This effect was inhibited by heat treatment, hepatocyte growth factor (HGF) blocking antibodies and a HGF receptor (cMet) inhibitor. In ovarian cancer ascites, HGF is present at high concentration compared to benign fluids. Ascites‐mediated activation of cMet was associated with Akt and EKR1/2 phosphorylation. This response was partly inhibited by heat treatment and cMet inhibitor. Ascites‐induced migration and a cMet phosphorylation were strongly inhibited by epidermal growth factor receptor (EGFR) inhibitor PD153035, suggesting the transactivation of cMet by EGFR. Our study suggests that HGF and ligands of EGFR are factors that mediate ovarian cancer ascites‐mediated migration of HPMCs by activating cMet and possibly downstream ERK1/2 and Akt pathways. The study provides evidence for the first time that ascites not only support tumor growth but also enhance the migratory potential of cancer‐associated mesothelial cells, which in turn may support cancer progression.