ABSTRACT
BACKGROUND: Current prognostic information in ovarian cancer is based on tumor stage, tumor grade, and postoperative tumor size. Reliable molecular prognostic markers are scarce. In this article, the authors describe epigenetic events in a frequently deleted region on chromosome 8p22 that influence the expression of tumor suppressor candidate 3 (TUSC3), a putative tumor suppressor gene in ovarian cancer. METHODS: Messenger RNA expression and promoter hypermethylation of TUSC3 were studied in ovarian cancer cell lines and in tumor samples from 2 large, independent ovarian cancer cohorts using polymerase chain reaction-based methods. RESULTS: The results indicated that TUSC3 expression is decreased significantly because of promoter methylation in malignant ovarian tumors compared with benign controls. Almost 33% of ovarian cancer samples had detectable TUSC3 promoter methylation. Furthermore, methylation status of the TUSC3 promoter had a significant and independent influence on progression-free and overall survival. CONCLUSIONS: TUSC3 hypermethylation predicted progression-free and overall survival in ovarian cancer. The current observations suggested a role for N-glycosylating events in ovarian cancer pathogenesis in general and identified the epigenetic silencing of TUSC3 as a prognostic factor in this disease.
Subject(s)
DNA Methylation , Membrane Proteins/genetics , Ovarian Neoplasms/genetics , Tumor Suppressor Proteins/genetics , Adult , Cell Line, Tumor , Disease-Free Survival , Female , Genes, Tumor Suppressor , Humans , Middle Aged , Ovarian Neoplasms/mortality , Prognosis , Promoter Regions, GeneticABSTRACT
PURPOSE: Although prognostic and predictive factors in ovarian cancer have been extensively studied for decades, only few have been identified and introduced to clinical practice. Here, we evaluate hVps37A (HCRP1) as a possible novel predictive marker for ovarian cancer. hVps37A was originally described as a member of the membrane-trafficking ESCRT-I complex mediating the internalization and degradation of ubiquitinated membrane receptors. EXPERIMENTAL DESIGN: We analyzed an ovarian cancer tissue microarray for HCRP1, EGFR, and HER2 expression. We used a tetracycline inducible ovarian cancer cell culture model to show the effects of hVps37A knockdown in vitro and in vivo. In addition, we studied the effects of epidermal growth factor receptor (EGFR) inhibitors cetuximab and lapatinib on ovarian cancer cells under conditions of hVps37A knockdown. RESULTS: We find that hVps37A is significantly downregulated in ovarian cancer and modifies the prognostic value of EGFR and HER2 expression. In addition, hVps37A downregulation in ovarian cancer cells leads to cytoplasmic pEGFR retention and hyperactivation of downstream pathways and is associated with enhanced xenograft growth in nude mice and invasion of the collagen matrix. Furthermore, due to subsequent sustained Akt- and MAPK-pathway activation, hVps37A-deficient cells become irresponsive to inhibition by the therapeutic antibody cetuximab. CONCLUSION: We propose that hVps37A status could become a novel prognostic and therapeutic marker for EGFR or HER2 driven tumors.
Subject(s)
Antibodies, Monoclonal/therapeutic use , Endosomal Sorting Complexes Required for Transport/genetics , Ovarian Neoplasms/drug therapy , Animals , Antibodies, Monoclonal/pharmacology , Antibodies, Monoclonal, Humanized , Antineoplastic Agents/pharmacology , Antineoplastic Agents/therapeutic use , Biomarkers, Tumor/genetics , Biomarkers, Tumor/metabolism , Blotting, Western , Cell Culture Techniques , Cell Line, Tumor , Cell Proliferation/drug effects , Cetuximab , Endosomal Sorting Complexes Required for Transport/metabolism , ErbB Receptors/metabolism , Female , Gene Expression Regulation, Neoplastic , Humans , Immunohistochemistry , Kaplan-Meier Estimate , Lapatinib , Mice , Mice, Nude , Ovarian Neoplasms/genetics , Ovarian Neoplasms/pathology , Prognosis , Quinazolines/pharmacology , RNA Interference , Receptor, ErbB-2/metabolism , Reverse Transcriptase Polymerase Chain Reaction , Tissue Array Analysis , Xenograft Model Antitumor Assays/methodsABSTRACT
OBJECTIVE: In the current study, we aimed to investigate the role of the long isoform of cellular Fas-associated death domain-like interleukin-1beta-converting enzyme (FLICE)-like inhibitory protein (c-FLIP(L)) in ovarian cancer (OC) development by using RNA interference (RNAi) in vitro and in vivo. METHODS: TRAIL-resistant human OC cell lines were genetically manipulated by RNAi-mediated suppression of c-FLIP(L). Subsequently, the genetic alteration that was introduced into the various OC cell lines was characterized in vitro and in vivo. RESULTS: We previously showed that about 40% of OC patients express high levels of c-FLIP(L), and that natural killer (NK) cells mediated immunosurveillance in OC. In the present study, we observed that the knockdown of c-FLIP(L) in human OC cell lines not only enhanced their sensitivity to TRAIL-mediated apoptosis, but also inhibited their migratory phenotype in a TRAIL-dependent manner in vitro. Shutdown of c-FLIP(L) in OC cells significantly decreased tumor development by induction of apoptosis and reduction of proliferation in vivo. Importantly, the knockdown of c-FLIP(L) particularly inhibited the invasion of OC cells into the peritoneal cavity, which might be due to high expression of TRAIL by NK cells and NK-cell mediated immunosurveillance. CONCLUSION: These data demonstrate that c-FLIP(L) exhibits multiple functions in OC cells: first by concomitantly evading the natural immunity mediated by TRAIL-induced cell death, and second by augmenting cell motility and invasion in vivo. Our findings indicate that c-FLIP(L) regulates sensitivity of OC to TRAIL-mediated apoptosis and offers possible therapeutical implications for OC in the future.
Subject(s)
CASP8 and FADD-Like Apoptosis Regulating Protein/physiology , Ovarian Neoplasms/immunology , Ovarian Neoplasms/pathology , Animals , Apoptosis/drug effects , CASP8 and FADD-Like Apoptosis Regulating Protein/deficiency , CASP8 and FADD-Like Apoptosis Regulating Protein/genetics , CASP8 and FADD-Like Apoptosis Regulating Protein/immunology , Cell Growth Processes/physiology , Cell Line, Tumor , Cell Movement/physiology , Down-Regulation , Drug Resistance, Neoplasm , Female , Gene Knockdown Techniques , Humans , Immunologic Surveillance , Mice , Mice, Nude , Neoplasm Invasiveness , Ovarian Neoplasms/drug therapy , RNA, Small Interfering/genetics , TNF-Related Apoptosis-Inducing Ligand/pharmacology , Xenograft Model Antitumor AssaysABSTRACT
OBJECTIVE: Transforming growth factor beta (TGF-beta) signaling via Smads plays a central role in carcinogenesis. Bmp and activin membrane-bound inhibitor (BAMBI) was initially described as a pseudoreceptor antagonizing TGF-beta receptor activation, thus impairing signaling. Here we wanted to estimate the role of BAMBI in ovarian cancer. METHODS: The function of BAMBI was studied using a cell line model and intracellular localization experiments. The impact of BAMBI expression on patient outcome was estimated by real-time PCR and immunohistochemistry. RESULTS: We demonstrate for the first time a nuclear co-translocation of BAMBI with Smad2/3 upon TGF-beta treatment. Moreover, overexpression of BAMBI in an in vitro model led to significantly increased proliferation (doubling time -37.0%, P=0.010), migration (+581.2%, P=0.004) and resistance to TGF-beta-mediated apoptosis (decrease of apoptosis from 30% in the control cells to 7% in the BAMBI-overexpressing cells). Although-prima facie-this fits to the thesis of BAMBI as a pseudoreceptor, it may also be explained by modulation of TGF-beta signaling in the nucleus, leading to the observed pro-oncogenic properties. The tumor promoting impact of BAMBI mRNA overexpression in vitro could not be confirmed in primary tumor samples, and while nearly all tumor samples showed up-regulation of BAMBI (37.3% 1+, 39.2% 2+, and 16.7% 3+, respectively) compared to undetectable BAMBI in healthy pre- and post-menopausal ovarian epithelia, no impact of BAMBI expression on recurrence free and overall survival could be observed. CONCLUSION: These findings provide new insights into the Smad-mediated pathway by inferring that BAMBI is a novel modulator of TGF-beta signaling.