ABSTRACT
AIM: In recent years, the Wnt signalling pathway has been implicated in epithelial ovarian cancer and its members have potential as diagnostic, prognostic and therapeutic targets. Here we investigated the role of two Wnt receptor tyrosine kinases (RTKs), ROR1 and ROR2, and their putative ligand, Wnt5a, in ovarian cancer. METHODS: Immunohistochemistry for ROR2 was performed in a large patient cohort, including benign controls, borderline tumours and epithelial ovarian cancer. In addition, siRNA was used to silence ROR1, ROR2 and Wnt5a individually, and together, in two ovarian cancer cell lines, and the effects on cell proliferation, adhesion, migration and invasion were measured. RESULTS: ROR2 expression is significantly increased in ovarian cancer patients compared to patients with benign disease. In vitro assays showed that silencing either receptor inhibits ovarian cancer cell migration and invasion, and concurrently silencing both receptors has an even stronger inhibitory effect on proliferation, migration and invasion. CONCLUSIONS: ROR2 expression is increased in epithelial ovarian cancer, and silencing ROR2 and its sister receptor ROR1 has a strong inhibitory effect on the ability of ovarian cancer cells to proliferate, migrate and invade through an extracellular matrix.
Subject(s)
Cell Movement/genetics , Neoplasms, Glandular and Epithelial/genetics , Ovarian Neoplasms/genetics , RNA Interference , Receptor Tyrosine Kinase-like Orphan Receptors/genetics , Carcinoma, Ovarian Epithelial , Cell Adhesion/genetics , Cell Line, Tumor , Cell Proliferation/genetics , Cohort Studies , Disease-Free Survival , Female , Gene Expression Regulation, Neoplastic , Humans , Immunoblotting , Immunohistochemistry , Neoplasm Invasiveness , Neoplasm Recurrence, Local , Neoplasms, Glandular and Epithelial/metabolism , Neoplasms, Glandular and Epithelial/pathology , Ovarian Neoplasms/metabolism , Ovarian Neoplasms/pathology , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins/metabolism , Receptor Tyrosine Kinase-like Orphan Receptors/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Wnt Proteins/genetics , Wnt Proteins/metabolism , Wnt-5a ProteinABSTRACT
Epithelial ovarian cancer has the highest mortality rate among gynecological cancers. Altered glycosylation is associated with oncogenic transformation producing tumor-associated carbohydrate antigens. We investigated the potential of natural occurring antiglycan antibodies in the diagnosis of ovarian cancer by using printed glycan array. Antiglycan antibodies bound to 203 chemically synthesized printed glycans were detected via biotin-streptavidin fluorescence system in serum of women with normal operative findings (healthy controls; n = 24) and nonmucinous borderline or ovarian cancer of various FIGO stages (n = 33). Data were validated measuring blood group associated di-, tri and tetrasaccharide antigens on known ABO blood groups. Antiglycan antibodies demonstrated high reproducibility (r(c) > 0.9). Cluster analysis identified repetitive patterns of specific core carbohydrate structures: 11 N-linked glycans, 3 O-linked glycans and 2 glycosphingolipids. Biomarker detection revealed 24 glycans including P(1) (Galα1-4Galß1-4GlcNAcß; p < 0.001) significantly discriminating between (low-) malignant tumors and healthy controls. Comparable sensitivity and specificity with tumor marker CA125 was achieved by a panel of multivariate selected and linear combined antiglycan antibody signals (79.2 and 84.8%, respectively). Our findings demonstrate the potential of glycan arrays in the development of a new generation of biomarkers for ovarian cancer.
Subject(s)
Biomarkers, Tumor/genetics , Immunoglobulin G/blood , Immunoglobulin G/immunology , Ovarian Neoplasms/metabolism , Polysaccharides/analysis , Polysaccharides/immunology , Adenocarcinoma, Clear Cell/immunology , Adenocarcinoma, Clear Cell/metabolism , Biomarkers, Tumor/blood , Carcinoma, Transitional Cell/immunology , Carcinoma, Transitional Cell/metabolism , Case-Control Studies , Cystadenocarcinoma, Serous/immunology , Cystadenocarcinoma, Serous/metabolism , Endometrial Neoplasms/immunology , Endometrial Neoplasms/metabolism , Female , Follow-Up Studies , Glycosylation , Humans , Microarray Analysis , Middle Aged , Neoplasm Grading , Neoplasm Staging , Ovarian Neoplasms/immunology , Polysaccharides/blood , Prognosis , Prospective Studies , ProteomicsABSTRACT
Anti-glycan antibodies represent a vast and yet insufficiently investigated subpopulation of naturally occurring and adaptive antibodies in humans. Recently, a variety of glycan-based microarrays emerged, allowing high-throughput profiling of a large repertoire of antibodies. As there are no direct approaches for comparison and evaluation of multi-glycan assays we compared three glycan-based immunoassays, namely printed glycan array (PGA), fluorescent microsphere-based suspension array (SA) and ELISA for their efficacy and selectivity in profiling anti-glycan antibodies in a cohort of 48 patients with and without ovarian cancer. The ABO blood group glycan antigens were selected as well recognized ligands for sensitivity and specificity assessments. As another ligand we selected P(1), a member of the P blood group system recently identified by PGA as a potential ovarian cancer biomarker. All three glyco-immunoassays reflected the known ABO blood groups with high performance. In contrast, anti-P(1) antibody binding profiles displayed much lower concordance. Whilst anti-P(1) antibody levels between benign controls and ovarian cancer patients were significantly discriminated using PGA (p=0.004), we got only similar results using SA (p=0.03) but not for ELISA. Our findings demonstrate that whilst assays were largely positively correlated, each presents unique characteristic features and should be validated by an independent patient cohort rather than another array technique. The variety between methods presumably reflects the differences in glycan presentation and the antigen/antibody ratio, assay conditions and detection technique. This indicates that the glycan-antibody interaction of interest has to guide the assay selection.
