Pre-operative levels of angiopoietin protein-like 3 (ANGPTL3) in women diagnosed with high-grade serous carcinoma of the ovary.

Cancer cells need constant supplies of lipids to survive and grow. Lipid dependence has been observed in various types of cancer, including high-grade serous ovarian carcinomas (HGSOC), which is a lethal form of gynecological malignancy. ANGPTL3, PCSK9, and Apo CIII are pivotal lipid-modulating factors, and therapeutic antibodies have been developed against each one (Evinacumab, Evolocumab and Volanesorsen, respectively). The roles -if any- of ANGPTL3, PCSK9, and Apo CIII in HGSOC are unclear. Moreover, levels of these lipid-modulating factors have never been reported before in HGSOC. In this study, circulating levels of ANGPTL3, PCSK9, and Apo CIII, along with lipid profiles, are examined to verify whether one or many of these lipid-regulating factors are associated with HGSOC. Methods ELISA kits were used to measure ANGPTL3, PCSK9 and Apo CIII levels in plasma samples from 31 women with HGSOC and 40 women with benign ovarian lesions (BOL) before treatment and surgery. A Roche Modular analytical platform measured lipid panels, Apo B and Lp(a) levels.Results ANGPTL3 levels were higher in women with HGSOC (84 ng/mL, SD: 29 ng/mL, n = 31) than in women with BOL (67 ng/mL, SD: 31 ng/mL, n = 40; HGSOC vs. BOL P = 0.019). Associations between the lipid panel and ANGPTL3, and the inverse relationship between HDL-cholesterol and triglycerides, were present in women with BOL but not with HGSOC. PCSK9 and Apo CIII were not associated with HGSOC.Conclusions In this cohort of 71 women, ANGPTL3 levels were increased in HGSOC patients. The presence of HGSOC disrupted the classic inverse relationship between HDL and triglycerides, as well as the association between the lipid panel and ANGPTL3. These associations were only maintained in cancer-free women. Given the availability of Evinacumab, a therapeutic antibody against ANGPTL3, the current finding prompts an assessment of whether ANGPTL3 inhibition has therapeutic potential in HGSOC.

Development of a 3D functional assay and identification of biomarkers, predictive for response of high-grade serous ovarian cancer (HGSOC) patients to poly-ADP ribose polymerase inhibitors (PARPis): targeted therapy.

BACKGROUND: Poly(ADP-ribose) polymerase inhibitors (PARPis) specifically target homologous recombination deficiency (HRD) cells and display good therapeutic effect in women with advanced-stage BRCA1/2-mutated breast and epithelial ovarian cancer (EOC). However, about 50% of high grade serous ovarian cancers (HGSOC) present with HRD due to epigenetic BRCA1 inactivation, as well as genetic/epigenetic inactivation(s) of other HR genes, a feature known as "BRCAness". Therefore, there is a potential for extending the use of PARPis to these patients if HR status can be identified. METHODS: We have developed a 3D (spheroid) functional assay to assess the sensitivity of two PARPis (niraparib and olaparib) in ascites-derived primary cell cultures (AsPCs) from HGSOC patients. A method for AsPCs preparation was established based on a matrix (agarose), allowing for easy isolation and successive propagation of monolayer and 3D AsPCs. Based on this method, we performed cytotoxicity assays on 42 AsPCs grown both as monolayers and spheroids. RESULTS: The response to PARPis treatment in monolayer AsPCs, was significantly higher, compared to 3D AsPCs, as 88% and 52% of the monolayer AsPCs displayed sensitivity to niraparib and olaparib respectively, while 66% of the 3D AsPCs were sensitive to niraparib and 38% to olaparib, the latter being more consistent with previous estimates of HRD (40%-60%) in EOC. Moreover, niraparib displayed a significantly stronger cytotoxic effect in both in 3D and monolayer AsPCs, which was confirmed by consecutive analyses of the HR pathway activity (γH2AX foci formation) in PARPis-sensitive and resistant AsPCs. Global gene expression comparison of 6 PARPi-resistant and 6 PARPi-sensitive 3D AsPCs was indicative for the predominant downregulation of numerous genes and networks with previously demonstrated roles in EOC chemoresistance, suggesting that the PARPis-sensitive AsPCs could display enhanced sensitivity to other chemotherapeutic drugs, commonly applied in cancer management. Microarray data validation identified 24 potential gene biomarkers associated with PARPis sensitivity. The differential expression of 7 selected biomarkers was consecutively confirmed by immunohistochemistry in matched EOC tumor samples. CONCLUSION: The application of this assay and the potential biomarkers with possible predictive significance to PARPis therapy of EOC patients now need testing in the setting of a clinical trial.

LY75 Suppression in Mesenchymal Epithelial Ovarian Cancer Cells Generates a Stable Hybrid EOC Cellular Phenotype, Associated with Enhanced Tumor Initiation, Spreading and Resistance to Treatment in Orthotopic Xenograft Mouse Model.

The implications of the epithelial-mesenchymal transition (EMT) mechanisms in the initiation and progression of epithelial ovarian cancer (EOC) remain poorly understood. We have previously shown that suppression of the antigen receptor LY75 directs mesenchymal-epithelial transition (MET) in EOC cell lines with the mesenchymal phenotype, associated with the loss of Wnt/ß-catenin signaling activity. In the present study, we used the LY75-mediated modulation of EMT in EOC cells as a model in order to investigate in vivo the specific role of EOC cells, with an epithelial (E), mesenchymal (M) or mixed epithelial plus mesenchymal (E+M) phenotype, in EOC initiation, dissemination and treatment response, following intra-bursal (IB) injections of SKOV3-M (control), SKOV3-E (Ly75KD) and a mixed population of SKOV3-E+M cells, into severe combined immunodeficiency (SCID) mice. We found that the IB-injected SKOV3-E cells displayed considerably higher metastatic potential and resistance to treatment as compared to the SKOV3-M cells, due to the acquisition of a Ly75KD-mediated hybrid phenotype and stemness characteristics. We also confirmed in vivo that the LY75 depletion directs suppression of the Wnt/ß-catenin pathway in EOC cells, suggestive of a protective role of this pathway in EOC etiology. Moreover, our data raise concerns regarding the use of LY75-targeted vaccines for dendritic-cell EOC immunotherapy, due to the possible occurrence of undesirable side effects.

LY75 Ablation Mediates Mesenchymal-Epithelial Transition (MET) in Epithelial Ovarian Cancer (EOC) Cells Associated with DNA Methylation Alterations and Suppression of the Wnt/ß-Catenin Pathway.

Growing evidence demonstrates that epithelial-mesenchymal transition (EMT) plays an important role in epithelial ovarian cancer (EOC) progression and spreading; however, its molecular mechanisms remain poorly defined. We have previously shown that the antigen receptor LY75 can modulate EOC cell phenotype and metastatic potential, as LY75 depletion directed mesenchymal-epithelial transition (MET) in EOC cell lines with mesenchymal phenotype. We used the LY75-mediated modulation of EMT as a model to investigate for DNA methylation changes during EMT in EOC cells, by applying the reduced representation bisulfite sequencing (RRBS) methodology. Numerous genes have displayed EMT-related DNA methylation patterns alterations in their promoter/exon regions. Ten selected genes, whose DNA methylation alterations were further confirmed by alternative methods, were further identified, some of which could represent new EOC biomarkers/therapeutic targets. Moreover, our methylation data were strongly indicative for the predominant implication of the Wnt/ß-catenin pathway in the EMT-induced DNA methylation variations in EOC cells. Consecutive experiments, including alterations in the Wnt/ß-catenin pathway activity in EOC cells with a specific inhibitor and the identification of LY75-interacting partners by a proteomic approach, were strongly indicative for the direct implication of the LY75 receptor in modulating the Wnt/ß-catenin signaling in EOC cells.

Performance of preoperative plasma tumor markers HE4 and CA125 in predicting ovarian cancer mortality in women with epithelial ovarian cancer.

Clinical utility of new biomarkers often requires the identification of their optimal threshold. This external validation study was conducted to assess the performance of the preoperative plasma tumor markers HE4 and CA125 optimal cut-offs to predict cancer mortality in women with epithelial ovarian cancer (EOC). Participating women had upfront debulking surgery in the University Hospital of Quebec City (Canada) between 1998 and 2013. A total of 136 women participated in the training cohort (cohort 1) and 177 in the validation cohort (cohort 2). Preoperative plasma HE4 and CA125 levels were measured by Elecsys. Optimal thresholds were identified in the cohort 1 using time-dependent receiver operating characteristic (ROC) curves. Multivariate Cox models were used to validate the biomarkers using their optimal cut-offs in the cohort 2. The likelihood ratio (LR) test was done to test whether the biomarkers added prognostic information beyond that provided by standard prognostic factors. The Areas Under the Curves (AUC) for HE4 and CA125 were respectively 64.2 (95% CI: 54.7-73.6) and 63.1 (95%CI: 53.6-72.6). The optimal thresholds were 277 pmol/L for HE4 and 282 U/ml for CA125. Preoperative plasma HE4 (≥277 pmol/L) was significantly associated with EOC mortality (adjusted hazard ratio (aHR): 1.90; 95% CI:1.09-3.29). The prognostic effect of HE4 was strongest in the subgroup of women with serous ovarian cancer (aHR: 2.42; 95% CI: 1.25-4.68). Using a multivariate model including all standard prognostic factors, this association was maintained (aHR: 2.21; 95% CI: 1.15-4.23). In addition, preoperative plasma HE4 added prediction for death over the standard prognostic markers in women with serous tumors (p-value for LR-test: 0.01). Preoperative CA125 was not associated with cancer mortality, both in women with EOC and in those with serous tumors. Preoperative HE4 is a promising prognostic biomarker in EOC, especially in serous tumor.

The polypeptide GALNT6 Displays Redundant Functions upon Suppression of its Closest Homolog GALNT3 in Mediating Aberrant O-Glycosylation, Associated with Ovarian Cancer Progression.

Epithelial ovarian cancer (EOC) represents the most lethal gynecologic malignancy; a better understanding of the molecular mechanisms associated with EOC etiology could substantially improve EOC management. Aberrant O-glycosylation in cancer is attributed to alteration of N-acetylgalactosaminyltransferases (GalNAc-Ts). Reports suggest a genetic and functional redundancy between GalNAc-Ts, and our previous data are indicative of an induction of GALNT6 expression upon GALNT3 suppression in EOC cells. We performed single GALNT3 and double GALNT3/T6 suppression in EOC cells, using a combination of the CRISPR-Cas9 system and shRNA-mediated gene silencing. The effect of single GALNT3 and double GALNT3/T6 inhibition was monitored both in vitro (on EOC cells roliferation, migration, and invasion) and in vivo (on tumor formation and survival of experimental animals). We confirmed that GALNT3 gene ablation leads to strong and rather compensatory GALNT6 upregulation in EOC cells. Moreover, double GALNT3/T6 suppression was significantly associated with stronger inhibitory effects on EOC cell proliferation, migration, and invasion, and accordingly displayed a significant increase in animal survival rates compared with GALNT3-ablated and control (Ctrl) EOC cells. Our data suggest a possible functional redundancy of GalNAc-Ts (GALNT3 and T6) in EOC, with the perspective of using both these enzymes as novel EOC biomarkers and/or therapeutic targets.

Proteases and their inhibitors as prognostic factors for high-grade serous ovarian cancer.

Ovarian carcinoma is one of the most lethal malignancies, but only very few prognostic biomarkers are known. The degradome, comprising proteases, protease non-proteolytic homologues and inhibitors, have been involved in the prognosis of many cancer types, including ovarian carcinoma. The prognostic significance of the whole degradome family has not been specifically studied in high-grade serous ovarian cancer. A targeted DNA microarray known as the CLIP-CHIP microarray was used to identify potential prognostic factors in ten high-grade serous ovarian cancer women who had early recurrence (<1.6 years) or late/no recurrence after first line surgery and chemotherapy. In women with early recurrence, we identified seven upregulated genes (TMPRSS4, MASP1/3, SPC18, PSMB1, IGFBP2, CFI - encoding Complement Factor I - and MMP9) and one down-regulated gene (ADAM-10). Using immunohistochemistry, we evaluated the prognostic effect of these 8 candidate genes in an independent cohort of 112 high-grade serous ovarian cancer women. Outcomes were progression, defined according to CA-125 criteria, and death. Multivariate Cox proportional hazard regression models were done to estimate the associations between each protein and each outcome. High ADAM-10 expression (intensity of 2-3) was associated with a lower risk of progression (adjusted hazard ratio (HR): 0.51; 95% confidence interval (CI): 0.29-0.87). High complement factor I expression (intensity 2-3) was associated with a higher risk of progression (adjusted HR: 2.30, 95% CI: 1.17-4.53) and death (adjusted HR: 3.42; 95% CI: 1.72-6.79). Overall, we identified the prognostic value of two proteases, ADAM-10 and complement factor I, for high-grade serous ovarian cancer which could have clinical significance.

Characteristics and outcome of the COEUR Canadian validation cohort for ovarian cancer biomarkers.

BACKGROUND: Ovarian carcinoma is the most lethal gynecological malignancy due to early dissemination and acquired resistance to platinum-based chemotherapy. Reliable markers that are independent and complementary to clinical parameters are needed to improve the management of patients with this disease. The Canadian Ovarian Experimental Unified Resource (COEUR) provides researchers with biological material and associated clinical data to conduct biomarker validation studies. Using standards defined by the Canadian Tissue Repository Network (CTRNet), we have previously demonstrated the quality of the biological material from this resource. Here we describe the clinical characteristics of the COEUR cohort. METHODS: With support from 12 Canadian ovarian cancer biobanks in Canada, we created a central retrospective cohort comprised of more than 2000 patient tissue samples with associated clinical data, including 1246 high-grade serous, 102 low-grade serous, 295 endometrioid, 259 clear cell and 89 mucinous carcinoma histotypes. A two-step reclassification process was applied to assure contemporary histological classification (histotyping). For each histotypes individually, we evaluated the association between the known clinico-pathological parameters (stage, cytoreduction, chemotherapy treatment, BRCA1 and BRCA2 mutation) and patient outcome by using Kaplan-Meier and Cox proportional hazard regression analyses. RESULTS: The median follow-up time of the cohort was 45 months and the 5-year survival rate for patients with high-grade serous carcinomas was 34%, in contrast to endometrioid carcinomas with 80% at 5 years. Survival profiles differed by histotype when stratified by stage or cytoreduction. Women with mucinous or clear cell carcinomas at advanced stage or with non-optimally debulked disease had the worst outcomes. In high-grade serous carcinoma, we observed significant association with longer survival in women harboring BRCA1 or BRCA2 mutation as compared to patients without detectable mutation. CONCLUSIONS: Our results show the expected survival rates, as compared with current literature, in each histotype suggesting that the cohort is an unbiased representation of the five major histotypes. COEUR, a one stop comprehensive biorepository, has collected mature outcome data and relevant clinical data in a comprehensive manner allowing stratified analysis.

Hic-5 regulates epithelial to mesenchymal transition in ovarian cancer cells in a TGFß1-independent manner.

The molecular basis of epithelial ovarian cancer (EOC) dissemination is still poorly understood. We have previously identified the hydrogen peroxide-inducible clone-5 (Hic-5) gene as hypomethylated in high-grade (HG) serous EOC tumors, compared to normal ovarian tissues. Hic-5 is a focal adhesion scaffold protein and has been primarily studied for its role as a key mediator of TGF-ß-induced epithelial-to-mesenchymal transition (EMT) in epithelial cells of both normal and malignant origin; however, its role in EOC has been never investigated. Here we demonstrate that Hic-5 is overexpressed in advanced EOC, and that Hic-5 is upregulated upon TGFß1 treatment in the EOC cell line with epithelial morphology (A2780s), associated with EMT induction. However, ectopic expression of Hic-5 in A2780s cells induces EMT independently of TGFß1, accompanied with enhancement of cellular proliferation rate and migratory/invasive capacity and increased resistance to chemotherapeutic drugs. Moreover, Hic-5 knockdown in the EOC cells with mesenchymal morphology (SKOV3) was accompanied by induction of mesenchymal-to-epithelial transition (MET), followed by a reduction of their proliferative, migratory/invasive capacity, and increased drugs sensitivity in vitro, as well as enhanced tumor cell colonization and metastatic growth in vivo. The modulation of Hic-5 expression in EOC cells resulted in altered regulation of numerous EMT-related canonical pathways and was indicative for a possible role of Hic-5 in controlling EMT through a RhoA/ROCK mediated mechanism. To our knowledge, this is the first report examining the role of Hic-5 in EOC, and its role in maintaining the mesenchymal phenotype of EOC cells independently of exogenous TGFß1 treatment.

Altered expression of different GalNAc­transferases is associated with disease progression and poor prognosis in women with high-grade serous ovarian cancer.

Protein glycosylation perturbations are implicated in a variety of diseases, including cancer. Aberrant glycosylation in cancer is frequently attributed to altered expression of polypeptide GalNAc transferases (GalNAc­Ts) - enzymes initiating mucin-type O-glycosylation. A previous study from our group demonstrated that one member of this family (GALNT3) is overexpressed in epithelial ovarian cancer (EOC), and GALNT3 expression correlated with shorter progression-free survival (PFS) in EOC patients with advanced disease. As considerable degree of redundancy between members of the GalNAc­Ts gene family has been frequently observed, we decided to investigate whether other members of this family are essential in EOC progression. In silico analysis based on publically available data was indicative for altered expression of five GalNAc­Ts (GALNT2, T4, T6, T9 and T14) in ovarian high-grade serous carcinoma (HGSC) samples compared to non-tumoral (control) ovarian tissue. We analyzed protein expression of these GalNAc­Ts in EOC cells and tumors by western blotting, followed by immunohistochemical (IHC) evaluation of their expression in EOC tumor and control samples using tissue microarrays (TMAs). Western blot analyses were indicative for low expression of GALNT2 and strong expression of GALNT6, T9 and T14 in both EOC cells and tumors. These observations were confirmed by IHC. GALNT2 displayed significantly lower expression, while GALNT6, GALNT9 and GALNT14 showed significantly higher expression in HGSC tumors compared to control tissue. Importantly, GALNT6 and GALNT14 expression correlated with poor prognosis of serous EOC patients. Moreover, our results suggest for overlapping functions of some GalNAc­Ts, more specifically GALNT3 and GALNT6, in directing EOC progression. Our results are indicative for a possible implication of different members of the GalNAc­T gene family in modulating EOC progression, and the potential use of GALNT6 and GALNT14 as novel prognostic EOC biomarkers. These data warrant future studies on the role of members of the GalNAc­Ts gene family in ovarian tumorigenesis.

Systems biology combining human- and animal-data miRNA and mRNA data identifies new targets in ureteropelvic junction obstruction.

BACKGROUND: Although renal fibrosis and inflammation have shown to be involved in the pathophysiology of obstructive nephropathies, molecular mechanisms underlying evolution of these processes remain undetermined. In an attempt towards improved understanding of obstructive nephropathy and improved translatability of the results to clinical practice we have developed a systems biology approach combining omics data of both human and mouse obstructive nephropathy. RESULTS: We have studied in parallel the urinary miRNome of infants with ureteropelvic junction obstruction and the kidney tissue miRNome and transcriptome of the corresponding neonatal partial unilateral ureteral obstruction (UUO) mouse model. Several hundreds of miRNAs and mRNAs displayed changed abundance during disease. Combination of miRNAs in both species and associated mRNAs let to the prioritization of five miRNAs and 35 mRNAs associated to disease. In vitro and in vivo validation identified consistent dysregulation of let-7a-5p and miR-29-3p and new potential targets, E3 ubiquitin-protein ligase (DTX4) and neuron navigator 1 (NAV1), potentially involved in fibrotic processes, in obstructive nephropathy in both human and mice that would not be identified otherwise. CONCLUSIONS: Our study is the first to correlate a mouse model of neonatal partial UUO with human UPJ obstruction in a comprehensive systems biology analysis. Our data revealed let-7a and miR-29b as molecules potentially involved in the development of fibrosis in UPJ obstruction via the control of DTX4 in both man and mice that would not be identified otherwise.

Suppression of the grainyhead transcription factor 2 gene (GRHL2) inhibits the proliferation, migration, invasion and mediates cell cycle arrest of ovarian cancer cells.

Previously, we have identified the Grainyhead transcription factor 2 gene (GRHL2) as notably hypomethylated in high-grade (HG) serous epithelial ovarian tumors, compared with normal ovarian tissues. GRHL2 is known for its functions in normal tissue development and wound healing. In the context of cancer, the role of GRHL2 is still ambiguous as both tumorigenic and tumor suppressive functions have been reported for this gene, although a role of GRHL2 in maintaining the epithelial status of cancer cells has been suggested. In this study, we report that GRHL2 is strongly overexpressed in both low malignant potential (LMP) and HG serous epithelial ovarian tumors, which probably correlates with its hypomethylated status. Suppression of the GRHL2 expression led to a sharp decrease in cell proliferation, migration and invasion and induced G1 cell cycle arrest in epithelial ovarian cancer (EOC) cells displaying either epithelial (A2780s) or mesenchymal (SKOV3) phenotypes. However, no phenotypic alterations were observed in these EOC cell lines following GRHL2 silencing. Gene expression profiling and consecutive canonical pathway and network analyses confirmed these data, as in both these EOC cell lines, GRHL2 ablation was associated with the downregulation of various genes and pathways implicated in cell growth and proliferation, cell cycle control and cellular metabolism. Taken together, our data are indicative for a strong oncogenic potential of the GRHL2 gene in EOC progression and support recent findings on the role of GRHL2 as one of the major phenotypic stability factors (PSFs) that stabilize the highly aggressive/metastatic hybrid epithelial/mesenchymal (E/M) phenotype of cancer cells.

Biocompatible Lipid Nanoparticles as Carriers To Improve Curcumin Efficacy in Ovarian Cancer Treatment.

Curcumin is a natural molecule with proved anticancer efficacy on several human cancer cell lines. However, its clinical application has been limited due to its poor bioavailability. Nanocarrier-based drug delivery approaches could make curcumin dispersible in aqueous media, thus overtaking the limits of its low solubility. The aim of this study was to increase the bioavailability and the antitumoral activity of curcumin, by entrapping it into nanostructured lipid carriers (NLCs). For this purpose here we describe the preparation and characterization of three kinds of curcumin-loaded NLCs. The nanosystems allowed the achievement of a controlled release of curcumin, the amounts of curcumin released after 24 h from Compritol-Captex, Compritol-Miglyol, and Compritol NLCs being, respectively, equal to 33, 28, and 18% w/w on the total entrapped curcumin. Considering the slower curcumin release profile, Compritol NLCs were chosen to perform successive in vitro studies on ovarian cancer cell lines. The results show that curcumin-loaded NLCs maintain anticancer activity, and reduce cell colony survival more effectively than free curcumin. As an example, the ability of A2780S cells to form colonies was decreased after treatment with 5 µM free curcumin by 50% ± 6, whereas, at the same concentration, the delivery of curcumin with NLC significantly (p < 0.05) inhibited colony formation to approximately 88% ± 1, therefore potentiating the activity of curcumin to inhibit A2780S cell growth. The obtained results clearly suggest that the entrapment of curcumin into NLCs increases curcumin efficacy in vitro, indicating the potential use of NLCs as curcumin delivery systems.

Proteomic dataset for altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells.

This article contains raw and processed data related to research published in "Role of the polypeptide N-acetylgalactosaminyltransferase 3 in ovarian cancer progression: possible implications in abnormal mucin O-glycosylation" [1]. The data presented here was obtained with the application of a bioorthogonal chemical reporter strategy analyzing differential glycoprotein expression following the knock-down (KD) of the GALNT3 gene in the epithelial ovarian cancer (EOC) cell line A2780s. LC-MS/MS mass spectrometry analysis was then performed and the processed data related to the identified glycoproteins show that several hundred proteins are differentially expressed between control and GALNT3 KD A2780s cells. The obtained data also uncover numerous novel glycoproteins; some of which could represent new potential EOC biomarkers and/or therapeutic targets.

A metabolic labeling approach for glycoproteomic analysis reveals altered glycoprotein expression upon GALNT3 knockdown in ovarian cancer cells.

UNLABELLED: Epithelial ovarian cancer (EOC) is a disease responsible for more deaths among women in the Western world than all other gynecologic malignancies. There is urgent need for new therapeutic targets and a better understanding of EOC initiation and progression. We have previously identified the polypeptide N-acetylgalactosaminyltransferase 3 (GALNT3) gene, a member of the GalNAc-transferases (GalNAc-Ts) gene family, as hypomethylated and overexpressed in high-grade serous EOC tumors, compared to low malignant potential EOC tumors and normal ovarian tissues. This data also suggested for a role of GALNT3 in aberrant EOC glycosylation, possibly implicated in disease progression. To evaluate differential glycosylation in EOC caused by modulations in GALNT3 expression, we used a metabolic labeling strategy for enrichment and mass spectrometry-based characterization of glycoproteins following GALNT3 gene knockdown (KD) in A2780s EOC cells. A total of 589 differentially expressed glycoproteins were identified upon GALNT3 KD. Most identified proteins were involved in mechanisms of cellular metabolic functions, post-translational modifications, and some have been reported to be implicated in EOC etiology. The GALNT3-dependent glycoproteins identified by this metabolic labeling approach support the oncogenic role of GALNT3 in EOC dissemination and may be pursued as novel EOC biomarkers and/or therapeutic targets. BIOLOGICAL SIGNIFICANCE: Knowledge of the O-glycoproteome has been relatively elusive, and the functions of the individual polypeptide GalNAc-Ts have been poorly characterized. Alterations in GalNAc-Ts expression were shown to provide huge variability in the O-glycoproteome in various pathologies, including cancer. The application of a chemical biology approach for the metabolic labeling and subsequent characterization of O-glycoproteins in EOC using the Ac4GalNAz metabolite has provided a strategy allowing for proteomic discovery of GalNAc-Ts specific functions. Our study supports an essential role of one of the GalNAc-Ts - GALNT3, in EOC dissemination, including its implication in modulating PTMs and EOC metabolism. Our approach validates the use of the applied metabolic strategy to identify important functions of GalNAc-Ts in normal and pathological conditions.

The mannose receptor LY75 (DEC205/CD205) modulates cellular phenotype and metastatic potential of ovarian cancer cells.

The molecular basis of epithelial ovarian cancer (EOC) dissemination is still poorly understood. Previously, we identified the mannose receptor LY75 gene as hypomethylated in high-grade (HG) serous EOC tumors, compared to normal ovarian tissues. LY75 represents endocytic receptor expressed on dendritic cells and so far, has been primarily studied for its role in antigen processing and presentation. Here we demonstrate that LY75 is overexpressed in advanced EOC and that LY75 suppression induces mesenchymal-to-epithelial transition (MET) in EOC cell lines with mesenchymal morphology (SKOV3 and TOV112), accompanied by reduction of their migratory and invasive capacity in vitro and enhanced tumor cell colonization and metastatic growth in vivo. LY75 knockdown in SKOV3 cells also resulted in predominant upregulation of functional pathways implicated in cell proliferation and metabolism, while pathways associated with cell signaling and adhesion, complement activation and immune response were mostly suppressed. Moreover, LY75 suppression had an opposite effect on EOC cell lines with epithelial phenotype (A2780s and OV2008), by directing epithelial-to-mesenchymal transition (EMT) associated with reduced capacity for in vivo EOC cell colonization, as similar/identical signaling pathways were reversely regulated, when compared to mesenchymal LY75 knockdown EOC cells.To our knowledge, this is the first report of a gene displaying such pleiotropic effects in sustaining the cellular phenotype of EOC cells and points to novel functions of this receptor in modulating EOC dissemination. Our data also support previous findings regarding the superior capacity of epithelial cancer cells in metastatic colonization of distant sites, compared to cancer cells with mesenchymal-like morphology.

BCAT1 expression associates with ovarian cancer progression: possible implications in altered disease metabolism.

Previously, we have identified the branched chain amino-acid transaminase 1 (BCAT1) gene as notably hypomethylated in low-malignant potential (LMP) and high-grade (HG) serous epithelial ovarian tumors, compared to normal ovarian tissues. Here we show that BCAT1 is strongly overexpressed in both LMP and HG serous epithelial ovarian tumors, which probably correlates with its hypomethylated status. Knockdown of the BCAT1 expression in epithelial ovarian cancer (EOC) cells led to sharp decrease of cell proliferation, migration and invasion and inhibited cell cycle progression. BCAT1 silencing was associated with the suppression of numerous genes and pathways known previously to be implicated in ovarian tumorigenesis, and the induction of some tumor suppressor genes (TSGs). Moreover, BCAT1 suppression resulted in downregulation of numerous genes implicated in lipid production and protein synthesis, suggesting its important role in controlling EOC metabolism. Further metabolomic analyses were indicative for significant depletion of most amino acids and different phospho- and sphingolipids following BCAT1 knockdown. Finally, BCAT1 suppression led to significantly prolonged survival time in xenograft model of advanced peritoneal EOC. Taken together, our findings provide new insights about the functional role of BCAT1 in ovarian carcinogenesis and identify this transaminase as a novel EOC biomarker and putative EOC therapeutic target.

Histone deacetylase 1 and 2 regulate Wnt and p53 pathways in the ureteric bud epithelium.

Histone deacetylases (HDACs) regulate a broad range of biological processes through removal of acetyl groups from histones as well as non-histone proteins. Our previous studies showed that Hdac1 and Hdac2 are bound to promoters of key renal developmental regulators and that HDAC activity is required for embryonic kidney gene expression. However, the existence of many HDAC isoforms in embryonic kidneys raises questions concerning the possible specificity or redundancy of their functions. We report here that targeted deletion of both the Hdac1 and Hdac2 genes from the ureteric bud (UB) cell lineage of mice causes bilateral renal hypodysplasia. One copy of either Hdac1 or Hdac2 is sufficient to sustain normal renal development. In addition to defective cell proliferation and survival, genome-wide transcriptional profiling revealed that the canonical Wnt signaling pathway is specifically impaired in UB(Hdac1,2-/-) kidneys. Our results also demonstrate that loss of Hdac1 and Hdac2 in the UB epithelium leads to marked hyperacetylation of the tumor suppressor protein p53 on lysine 370, 379 and 383; these post-translational modifications are known to boost p53 stability and transcriptional activity. Genetic deletion of p53 partially rescues the development of UB(Hdac1,2-/-) kidneys. Together, these data indicate that Hdac1 and Hdac2 are crucial for kidney development. They perform redundant, yet essential, cell lineage-autonomous functions via p53-dependent and -independent pathways.

Akt confers cisplatin chemoresistance in human gynecological carcinoma cells by modulating PPM1D stability.

Ovarian cancer (OVCA) and cervical cancer (CECA) are lethal gynecological malignancies. Cisplatin (CDDP) and platinum derivatives are first line chemotherapeutics and their resistance impedes successful treatment. Understanding the molecular dysregulation underlying chemoresistance is important in developing rational therapeutic strategies. We have established that Protein Phosphatase Magnesium-dependent 1 D (PPM1D) confers CDDP resistance in gynecological cancer cells by deactivating p53. However, whether CDDP regulates intra-cellular PPM1D localization and whether this regulation is different between chemosensitive and chemoresistant cancer cells is unknown. Moreover, whether Akt regulates PPM1D in the context of CDDP resistance has not been studied. To illustrate the role of PPM1D in gynecological cancer cell chemoresistance and its regulation by Akt we have demonstrated that: (a) CDDP induced PPM1D down-regulation through proteasomal degradation in sensitive CECA cells; (b) CDDP induced PPM1D nuclear localization in resistant CECA cells, and nuclear exclusion in sensitive CECA cells and OVCA xenografts; (c) Over-expression of active Akt in sensitive CECA cells stabilized PPM1D content through inhibition of CDDP-induced PPM1D down-regulation; (d) Inhibition of Akt activity in resistant OVCA cells leads to decreased PPM1D stability and CDDP-induced down-regulation in resistant CECA cells; and (e) PPM1D is highly expressed in human ovarian tumor subtypes and in a tissue microarray panel of human ovarian tumors. In conclusion, we have established that PPM1D plays an important role in promoting CDDP resistance and as a novel downstream target of Akt, PPM1D mediates its action in conferring CDDP resistance in gynecological cancer cells.

Genome-wide analysis of gestational gene-environment interactions in the developing kidney.

The G protein-coupled bradykinin B2 receptor (Bdkrb2) plays an important role in regulation of blood pressure under conditions of excess salt intake. Our previous work has shown that Bdkrb2 also plays a developmental role since Bdkrb2(-/-) embryos, but not their wild-type or heterozygous littermates, are prone to renal dysgenesis in response to gestational high salt intake. Although impaired terminal differentiation and apoptosis are consistent findings in the Bdkrb2(-/-) mutant kidneys, the developmental pathways downstream of gene-environment interactions leading to the renal phenotype remain unknown. Here, we performed genome-wide transcriptional profiling on embryonic kidneys from salt-stressed Bdkrb2(+/+) and Bdkrb2(-/-) embryos. The results reveal significant alterations in key pathways regulating Wnt signaling, apoptosis, embryonic development, and cell-matrix interactions. In silico analysis reveal that nearly 12% of differentially regulated genes harbor one or more Pax2 DNA-binding sites in their promoter region. Further analysis shows that metanephric kidneys of salt-stressed Bdkrb2(-/-) have a significant downregulation of Pax2 gene expression. This was corroborated in Bdkrb2(-/-);Pax2(GFP+/tg) mice, demonstrating that Pax2 transcriptional activity is significantly repressed by gestational salt-Bdkrb2 interactions. We conclude that gestational gene (Bdkrb2) and environment (salt) interactions cooperate to impact gene expression programs in the developing kidney. Suppression of Pax2 likely contributes to the defects in epithelial survival, growth, and differentiation in salt-stressed BdkrB2(-/-) mice.