A prognostic gene signature in advanced ovarian cancer reveals a microfibril-associated protein (MAGP2) as a promoter of tumor cell survival and angiogenesis

Ovarian cancer is the most lethal gynecologic cancer, and this is largely related to its late diagnosis. High grade serous cancers often initially respond to chemotherapy, resulting in a better survival rate, compared to other ovarian carcinoma subtypes. We review recent work identifying a survival-associated gene expression profile for advanced serous ovarian cancer. Within this signature, the authors identified MAGP2, also known as microfibrillar associated protein 5 (MFAP5), as a highly significant indicator of survival and chemosensitivity. MAGP2 is a multifunctional secreted protein—important for elastic microfibril assembly and modulating endothelial cell behavior—with a newly identified role in cell survival. Through α_Vβ₃ integrin-mediated signaling, MAGP2 promotes tumor and endothelial cell survival and endothelial cell motility, providing a potential mechanistic link between MAGP2 and angiogenesis as well as patient survival.Key words: ovarian cancer, cell survival, MAGP2, α_Vβ₃, endothelial cellAmong reproductive cancers, ovarian cancer has the highest associated mortality rate-the majority of patients present with late—stage disease that has already spread beyond the ovaries.¹ Surgery and chemotherapy are used to eliminate localized and metastatic cancer cells, and the size of the residual legion after surgery is the main prognostic factor for survival. Biomarkers or gene expression profiles associated with prognosis or chemosensitivity in different ovarian cancer subtypes could aid treatment decisions—perhaps improving patient mortality rates by identifying patients that will not respond to traditional surgical or chemotherapeutic approaches. While the underlying mechanisms for ovarian cancer are incompletely understood, cellular alterations important for cancer initiation and progression have been described.² Distinguishing features of cancer cells include the ability to survive and proliferate under adverse conditions, induce and maintain angiogenesis, and invade into the surrounding tissues. The contribution of secreted proteins to these processes is of particular interest, as these proteins can modulate cell signaling in both tumor and normal cells. This review summarizes recent work by Mok et al.³ describing the identification of a gene expression signature correlating with serous ovarian cancer survival and chemosensitivity. They validate the secreted protein, Microfibril-Associated Glycoprotein 2 (MAGP2), gene name, MFAP5 (also known as microfibrillar associated protein 5), as an independent prognostic biomarker of survival and chemosensitivity and report a role for MAGP2 in cell survival.To identify a gene expression signature predicting survival in late stage, high-grade papillary serous ovarian tumors, the authors used laser capture microdissection to isolate epithelial cancer cells from paraffin-embedded tissue and analyzed gene expression changes using microarrays. The authors were able to identify and validate a gene expression signature that correlated with survival in this patient cohort. The median hazard ratio was used as a threshold to group high- and low-risk patients and the representative Kaplan Meier curves were compared using the log-rank test, revealing a significant association between the high-risk group and shorter survival times. Of all the genes examined, microfibril-associated glycoprotein 2 (MAGP2) had the highest correlation with poor patient prognosis. MAGP2 expression was further explored as an independent prognostic marker, at both the mRNA and protein levels. Mok and colleagues confirmed increased MAGP2 expresssion by both real time PCR and immunohistochemical staining. Both high MAGP2 mRNA and protein levels stratified the low and high risk patient groups, with positive MAGP2 predicting a poor prognosis.To explore the signaling pathways affecting ovarian cancer patient survival, the authors compared gene expression profiles from the advanced stage ovarian cancer samples to those from normal ovarian surface epithelial cells and compared them with the patient survival gene signature. The α_Vβ₃ integrin receptor was found to be a central signaling pathway in this network. MAGP2 binds and activates α_Vβ₃ integrin, and the downstream effectors, focal adhesion kinase (FAK), paxillin (PXN), growth factor receptor-bound protein 2 (GRB2) and son of sevenless homolog 1 (SOS 1) were overexpressed. These may contribute toward cell cycle progression and increased cell survival. Changes in a subset of these genes, including FAK and MAGP2, were verified using quantitative Real Time-PCR, indicating that the α_Vβ₃ signaling pathway is over-activated in ovarian cancer. Given that α_Vβ₃ integrin signaling correlated with chemotherapy resistance in ovarian cancer cell lines,⁴ the authors also examined the utility of MAGP2 as an indicator of chemosensitivity. By stratifying patients based on response to chemotherapy, the authors determined that high MAGP2 levels correlate with chemoresistance—suggesting that these patients might require alternative treatment strategies to improve patient outcome.To characterize the role of secreted MAGP2 in modulating tumor and endothelial cells via α_Vβ₃ integrin, an important mediator of cell adhesion and survival, the authors initially employed an in vitro approach. Ovarian cancer cell lines were screened for MAGP2 and α_Vβ₃ integrin expression and were evaluated for their ability to adhere to synthetic MAGP2 protein. Cell lines with (A224, OVCA429 and SKOV3) and without (UCI107) α_Vβ₃ integrin were used. A224 cell adhesion to MAGP2 was diminished in the presence of a α_Vβ₃ integrin blocking antibody, whereas no change in UCI107 cell adhesion was observed, demonstrating a role for MAGP2 in α_Vβ₃ integrin-mediated adhesion. Improved OVCA429 cell survival in serum-free media was also observed in the presence of recombinant MAGP2, suggesting that MAGP2 is important for both α_Vβ₃ integrin-mediated tumor cell survival and adhesion (Fig. 1).Open in a separate windowFigure 1Ovarian serous cancer cells secrete MAGP2, which acts to increase tumor cell survival and endothelial cell motility and survival through the α_Vβ₃ integrin.Previous reports have demonstrated a role for MAGP2 in elastic fiber assembly⁵ and endothelial cell behavior.^6,7 The ability of MAGP2 to modulate endothelial cell behavior via α_Vβ₃ integrin was next examined. The authors observed an increase in human umbilical vein endothelial cell (HUVEC) adhesion to recombinant MAGP2, which was inhibited by pre-incubation with a α_Vβ₃ integrin antibody. HUVEC survival in the absence of serum, and cell motility and invasion were also increased by MAGP2 (Fig. 1). The MAGP2 protein sequence includes the classical integrin-binding RGD domain. To determine whether this domain was important for MAGP2-mediated alterations in endothelial cell behavior, the authors generated recombinant proteins with amino acid substitutions in the RGD motif. Enhanced MAGP2-mediated cell motility was diminished in the presence of α_Vβ₃ integrin blocking antibody or mutated recombinant MAGP2, but not with α₅ or β₁ integrin blocking antibodies, demonstrating the MAGP2 RGD motif was important for α_Vβ₃ integrin signaling in endothelial cells. To further explore MAGP2-mediated changes in endothelial cells, microarray analysis was used to compare gene expression profiles in MAGP2-treated and untreated HUVEC cells. Increased expression of genes involved in cell adhesion such as FAK (focal adhesion kinase) and ITGAV (integrin alpha v) were observed, together with increased cell motility genes, such as CDC42 and APC, while an increase in phosphorylated FAK was observed via western blotting. These data provide mechanistic insight into MAGP2-modulation of cellular activity.To understand MAGP2 function during tumorigenesis, the authors generated SKOV3 ovarian cancer clonal cell lines stably expressing either an empty vector or shRNA against MAGP2. The authors evaluated the ability of subcutaneously injected cells to form tumors in nude mice, showing that cells with reduced MAGP2 expression formed smaller tumors than controls. This effect was confirmed using additional MAGP2-targeting shRNAs in both SKOV3 and OVCAR3 cells. Finally, as MAGP2 affects endothelial cell behavior, the effects on the angiogenic microenvironment was evaluated. Immunohistochemical analysis of MAGP2-knockdown tumor tissue in mice revealed decreased microvessel density (MVD) as visualized by CD34⁺ staining, while MVD and MAGP2 expression significantly correlated in 30 human serous ovarian cancer tissue specimens. Together these data suggest an in vivo role for MAGP2 in promoting angiogenesis.     

Identification of a functional proprotein convertase cleavage site in microfibril-associated glycoprotein 2

Microfibril-associated glycoprotein 2 (MAGP2) is a secreted protein associated with multiple cellular activities including the organization of elastic fibers in the extracellular matrix (ECM), angiogenesis, as well as regulating Notch and integrin signaling. Importantly, increases in MAGP2 positively correlate with poor prognosis for some ovarian cancers. It has been assumed that full-length MAGP2 is responsible for all reported effects; however, here we show MAGP2 is a substrate for the proprotein convertase (PC) family of endoproteases. Proteolytic processing of MAGP2 by PC cleavage could serve to regulate secretion and thus, activity and function as reported for other extracellular and cell-surface proteins. In support of this idea, MAGP2 contains an evolutionarily conserved PC consensus cleavage site, and amino acid sequencing of a newly identified MAGP2 C-terminal cleavage product confirmed functional PC cleavage. Additionally, mutagenesis of the MAGP2 PC consensus cleavage site or treatment with PC inhibitors prevented MAGP2 proteolytic processing. Finally, both cleaved and uncleaved MAGP2 were detected extracellularly and MAGP2 secretion appeared independent of PC cleavage, suggesting that PC processing occurs mainly outside the cell. Our characterization of alternative forms of MAGP2 present in the extracellular space not only enhances diversity of this ECM protein but also provides a previously unrecognized molecular mechanism for regulation of MAGP2 biological activity.     

Identification of a matrix-binding domain in MAGP1 and MAGP2 and intracellular localization of alternative splice forms.

MAGP1 is a small molecular mass protein associated with microfibrils in the extracellular matrix (ECM). To identify the molecular basis of its interaction with other microfibrillar proteins, deletion constructs of MAGP1 were expressed in a mammalian cell system that served as a model for microfibril assembly. This study identified a 54-amino acid sequence in the carboxyl-terminal region of the protein that defines a matrix-binding domain that is sufficient to target MAGP1 to the ECM. Site-directed mutagenesis demonstrated that binding activity is dependent on the presence of 7 cysteine residues in this sequence. MAGP2 contains a sequence similar to the matrix-binding domain of MAGP1, but could not associate with the ECM because of a single amino acid change. Two naturally occurring MAGP1 splice variants, MAGP1B (human-specific) and MAGP1D (found in mice), localized intracellularly when expressed as chimeric proteins with green fluorescent protein in rat lung fibroblasts. This suggests a second action site for MAGP1.     

Comparison of expression profiles in ovarian epithelium in vivo and ovarian cancer identifies novel candidate genes involved in disease pathogenesis

Molecular events leading to epithelial ovarian cancer are poorly understood but ovulatory hormones and a high number of life-time ovulations with concomitant proliferation, apoptosis, and inflammation, increases risk. We identified genes that are regulated during the estrous cycle in murine ovarian surface epithelium and analysed these profiles to identify genes dysregulated in human ovarian cancer, using publically available datasets. We identified 338 genes that are regulated in murine ovarian surface epithelium during the estrous cycle and dysregulated in ovarian cancer. Six of seven candidates selected for immunohistochemical validation were expressed in serous ovarian cancer, inclusion cysts, ovarian surface epithelium and in fallopian tube epithelium. Most were overexpressed in ovarian cancer compared with ovarian surface epithelium and/or inclusion cysts (EpCAM, EZH2, BIRC5) although BIRC5 and EZH2 were expressed as highly in fallopian tube epithelium as in ovarian cancer. We prioritised the 338 genes for those likely to be important for ovarian cancer development by in silico analyses of copy number aberration and mutation using publically available datasets and identified genes with established roles in ovarian cancer as well as novel genes for which we have evidence for involvement in ovarian cancer. Chromosome segregation emerged as an important process in which genes from our list of 338 were over-represented including two (BUB1, NCAPD2) for which there is evidence of amplification and mutation. NUAK2, upregulated in ovarian surface epithelium in proestrus and predicted to have a driver mutation in ovarian cancer, was examined in a larger cohort of serous ovarian cancer where patients with lower NUAK2 expression had shorter overall survival. In conclusion, defining genes that are activated in normal epithelium in the course of ovulation that are also dysregulated in cancer has identified a number of pathways and novel candidate genes that may contribute to the development of ovarian cancer.     

PRP4K is a HER2-regulated modifier of taxane sensitivity

The taxanes are used alone or in combination with anthracyclines or platinum drugs to treat breast and ovarian cancer, respectively. Taxanes target microtubules in cancer cells and modifiers of taxane sensitivity have been identified in vitro, including drug efflux and mitotic checkpoint proteins. Human epidermal growth factor receptor 2 (HER2/ERBB2) gene amplification is associated with benefit from taxane therapy in breast cancer yet high HER2 expression also correlates with poor survival in both breast and ovarian cancer. The pre-mRNA splicing factor 4 kinase PRP4K (PRPF4B), which we identified as a component of the U5 snRNP also plays a role in regulating the spindle assembly checkpoint (SAC) in response to microtubule-targeting drugs. In this study, we found a positive correlation between PRP4K expression and HER2 status in breast and ovarian cancer patient tumors, which we determined was a direct result of PRP4K regulation by HER2 signaling. Knock-down of PRP4K expression reduced the sensitivity of breast and ovarian cancer cell lines to taxanes, and low PRP4K levels correlated with in vitro-derived and patient acquired taxane resistance in breast and ovarian cancer. Patients with high-grade serous ovarian cancer and high HER2 levels had poor overall survival; however, better survival in the low HER2 patient subgroup treated with platinum/taxane-based therapy correlated positively with PRP4K expression (HR = 0.37 [95% CI 0.15-0.88]; p = 0.03). Thus, PRP4K functions as a HER2-regulated modifier of taxane sensitivity that may have prognostic value as a marker of better overall survival in taxane-treated ovarian cancer patients.     

Activation of the PI3K/AKT pathway mediates FSH-stimulated VEGF expression in ovarian serous cystadenocarcinoma

There is evidence to suggest that follicle-stimulating hormone （FSH） can facilitate the neovascularization of ovarian cancers by increasing vascular endothelial growth factor （VEGF） expression in cancer cells, although the underlying molecular mechanism of this process is not well known. Therefore, we investigated the effect of FSH on VEGF expression in the ovarian cancer cell lines SKOV-3 and ES-2. Treatment with FSH significantly increased VEGF expression in a dose- and time-dependent manner. In addition, FSH treatment enhanced the expression of survivin and hypoxlainducible factor-1 （HIF-1α）. Knockdown of survivin or HIF-1α suppressed VEGF expression, but only knockdown of survivin inhibited FSH-stimulated VEGF expression. Pretreatment with LY294002, a phosphoinositide 3-kinase （PI3K）/AKT inhibitor, neutralized the enhanced expression of survivin induced by FSH, but treatment with U0126, a mitogen-activated protein kinase/extracellular signal-regulated kinase inhibitor, had no such effect. We further showed that ovarian serous cystadenocarcinoma samples had much higher incidence of positive AKT and phosphorylated AKT （pAKT） protein staining than did benign ovarian cystadenoma samples （p 〈 0.01）. The 5-year survival rate was only about 15% in patients with ovarian serous cystadenocarcinoma who had AKT and pAKT expression, whereas it was about 80% in those who did not have AKT or pAKT expression. Taken together, these results indicate that FSH increases the expression of VEGF by upregulating the expression of survivin, which is activated by the PI3K/AKT signaling pathway. Understanding the role of the PI3K/AKT pathway in FSH-stimulated expression of survivin and VEGF will be beneficial for evaluating the prognosis for patients with ovarian serous cystadenocarcinoma and for pursulug effective treatment against this disease.     

Serum S100A6 Concentration Predicts Peritoneal Tumor Burden in Mice with Epithelial Ovarian Cancer and Is Associated with Advanced Stage in Patients

Background

Ovarian cancer is the 5th leading cause of cancer related deaths in women. Five-year survival rates for early stage disease are greater than 94%, however most women are diagnosed in advanced stage with 5 year survival less than 28%. Improved means for early detection and reliable patient monitoring are needed to increase survival.

Methodology and Principal Findings

Applying mass spectrometry-based proteomics, we sought to elucidate an unanswered biomarker research question regarding ability to determine tumor burden detectable by an ovarian cancer biomarker protein emanating directly from the tumor cells. Since aggressive serous epithelial ovarian cancers account for most mortality, a xenograft model using human SKOV-3 serous ovarian cancer cells was established to model progression to disseminated carcinomatosis. Using a method for low molecular weight protein enrichment, followed by liquid chromatography and mass spectrometry analysis, a human-specific peptide sequence of S100A6 was identified in sera from mice with advanced-stage experimental ovarian carcinoma. S100A6 expression was documented in cancer xenografts as well as from ovarian cancer patient tissues. Longitudinal study revealed that serum S100A6 concentration is directly related to tumor burden predictions from an inverse regression calibration analysis of data obtained from a detergent-supplemented antigen capture immunoassay and whole-animal bioluminescent optical imaging. The result from the animal model was confirmed in human clinical material as S100A6 was found to be significantly elevated in the sera from women with advanced stage ovarian cancer compared to those with early stage disease.

Conclusions

S100A6 is expressed in ovarian and other cancer tissues, but has not been documented previously in ovarian cancer disease sera. S100A6 is found in serum in concentrations that correlate with experimental tumor burden and with clinical disease stage. The data signify that S100A6 may prove useful in detecting and/or monitoring ovarian cancer, when used in concert with other biomarkers.     

Expression and methylation status of 14-3-3 sigma gene can characterize the different histological features of ovarian cancer

We hypothesize that 14-3-3 sigma gene expression and its regulation by methylation can characterize histological types of primary human epithelial ovarian cancer. To test this hypothesis, ovarian cancer cell lines and 54 ovarian cancer tissue samples were analyzed for expression and methylation of 14-3-3 sigma gene using methylation specific PCR. The results of our experiments demonstrate that 14-3-3 sigma gene was methylated and inactivated in ES-2 ovarian cell line, which was derived from clear cell adenocarcinoma. Treatment of this cell line with demethylating agent 5-aza-2'-deoxycytidine restored the expression of 14-3-3 sigma gene. In human ovarian cancer tissues, the expression of 14-3-3 sigma protein was inactivated in most of the ovarian clear cell carcinoma tissues. Interestingly, 14-3-3 sigma protein expression was positive in significantly higher percentages of serous (89.5%), endometrioid (90%), and mucinous (81.8%) ovarian adenocarcinoma tissues. The ovarian clear cell carcinoma samples with inactivated 14-3-3 sigma protein were highly methylated, suggesting that inactivation of 14-3-3 sigma gene is through DNA methylation. Using direct DNA sequencing, 14-3-3 sigma gene methylation on all the 17 CpG sites was significantly higher in ovarian clear cell carcinoma as compared to other histological types of ovarian cancer (serous, endometrioid, and mucinous). This is the first report suggesting that 14-3-3 sigma gene expression and methylation status can characterize histological features of different types of ovarian cancer.     

Identification of novel therapeutic targets in microdissected clear cell ovarian cancers

Clear cell ovarian cancer is an epithelial ovarian cancer histotype that is less responsive to chemotherapy and carries poorer prognosis than serous and endometrioid histotypes. Despite this, patients with these tumors are treated in a similar fashion as all other ovarian cancers. Previous genomic analysis has suggested that clear cell cancers represent a unique tumor subtype. Here we generated the first whole genomic expression profiling using epithelial component of clear cell ovarian cancers and normal ovarian surface specimens isolated by laser capture microdissection. All the arrays were analyzed using BRB ArrayTools and PathwayStudio software to identify the signaling pathways. Identified pathways validated using serous, clear cell cancer cell lines and RNAi technology. In vivo validations carried out using an orthotopic mouse model and liposomal encapsulated siRNA. Patient-derived clear cell and serous ovarian tumors were grafted under the renal capsule of NOD-SCID mice to evaluate the therapeutic potential of the identified pathway. We identified major activated pathways in clear cells involving in hypoxic cell growth, angiogenesis, and glucose metabolism not seen in other histotypes. Knockdown of key genes in these pathways sensitized clear cell ovarian cancer cell lines to hypoxia/glucose deprivation. In vivo experiments using patient derived tumors demonstrate that clear cell tumors are exquisitely sensitive to antiangiogenesis therapy (i.e. sunitinib) compared with serous tumors. We generated a histotype specific, gene signature associated with clear cell ovarian cancer which identifies important activated pathways critical for their clinicopathologic characteristics. These results provide a rational basis for a radically different treatment for ovarian clear cell patients.     

Classifications of ovarian cancer tissues by proteomic patterns

Ovarian cancer is a morphologically and biologically heterogeneous disease. The identification of type-specific protein markers for ovarian cancer would provide the basis for more tailored treatments, as well as clues for understanding the molecular mechanisms governing cancer progression. In the present study, we used a novel approach to classify 24 ovarian cancer tissue samples based on the proteomic pattern of each sample. The method involved fractionation according to pI using chromatofocusing with analytical columns in the first dimension followed by separation of the proteins in each pI fraction using nonporous RP HPLC, which was coupled to an ESI-TOF mass analyzer for molecular weight (MW) analysis. A 2-D mass map of the protein content of each type of ovarian cancer tissue samples based upon pI versus intact protein MW was generated. Using this method, the clear cell and serous ovarian carcinoma samples were histologically distinguished by principal component analysis and clustering analysis based on their protein expression profiles and subtype-specific biomarker candidates of ovarian cancers were identified, which could be further investigated for future clinical study.     

Vasohibin-2 expressed in human serous ovarian adenocarcinoma accelerates tumor growth by promoting angiogenesis

Vasohibin-1 (VASH1) is a VEGF-inducible endothelium-derived angiogenesis inhibitor and VASH2 is its homolog. Our previous analysis revealed that VASH1 is expressed in endothelial cells to terminate angiogenesis, whereas VASH2 is expressed in infiltrating mononuclear cells mobilized from bone marrow to promote angiogenesis in a mouse model of hypoxia-induced subcutaneous angiogenesis. To test the possible involvement of VASH2 in the tumor, we examined human ovarian cancer cells for the presence of VASH2. Immunohistochemical analysis revealed that VASH2 protein was preferentially detected in cancer cells of serous ovarian adenocarcinoma. We then used SKOV-3 and DISS, two representative human serous adenocarcinoma cell lines, and examined the role of VASH2 in the tumor. The knockdown of VASH2 showed little effect on the proliferation of cancer cells in vitro but notably inhibited tumor growth, peritoneal dissemination, and tumor angiogenesis in a murine xenograft model. Next, we stably transfected the human VASH2 gene into two types of murine tumor cells, EL-4 and MLTC-1, in which endogenous VASH2 was absent. When either EL-4 or MLTC-1 cells were inoculated into VASH2 (-/-) mice, the VASH2 transfectants formed bigger tumors when compared with the controls, and the tumor microvessel density was significantly increased. VASH2 stimulated the migration of endothelial cells, and its increased expression in cancer cells is related to the decrease of mir-200b. These results indicate that VASH2 expressed in serous ovarian carcinoma cells promoted tumor growth and peritoneal dissemination by promoting angiogenesis. Mol Cancer Res; 10(9); 1135-46. ?2012 AACR.     

Differential analysis of ovarian and endometrial cancers identifies a methylator phenotype

Despite improved outcomes in the past 30 years, less than half of all women diagnosed with epithelial ovarian cancer live five years beyond their diagnosis. Although typically treated as a single disease, epithelial ovarian cancer includes several distinct histological subtypes, such as papillary serous and endometrioid carcinomas. To address whether the morphological differences seen in these carcinomas represent distinct characteristics at the molecular level we analyzed DNA methylation patterns in 11 papillary serous tumors, 9 endometrioid ovarian tumors, 4 normal fallopian tube samples and 6 normal endometrial tissues, plus 8 normal fallopian tube and 4 serous samples from TCGA. For comparison within the endometrioid subtype we added 6 primary uterine endometrioid tumors and 5 endometrioid metastases from uterus to ovary. Data was obtained from 27,578 CpG dinucleotides occurring in or near promoter regions of 14,495 genes. We identified 36 locations with significant increases or decreases in methylation in comparisons of serous tumors and normal fallopian tube samples. Moreover, unsupervised clustering techniques applied to all samples showed three major profiles comprising mostly normal samples, serous tumors, and endometrioid tumors including ovarian, uterine and metastatic origins. The clustering analysis identified 60 differentially methylated sites between the serous group and the normal group. An unrelated set of 25 serous tumors validated the reproducibility of the methylation patterns. In contrast, >1,000 genes were differentially methylated between endometrioid tumors and normal samples. This finding is consistent with a generalized regulatory disruption caused by a methylator phenotype. Through DNA methylation analyses we have identified genes with known roles in ovarian carcinoma etiology, whereas pathway analyses provided biological insight to the role of novel genes. Our finding of differences between serous and endometrioid ovarian tumors indicates that intervention strategies could be developed to specifically address subtypes of epithelial ovarian cancer.     

Human SBK1 is dysregulated in multiple cancers and promotes survival of ovary cancer SK-OV-3 cells

Protein kinases are involved in comprehensive cellular processes and also implicated in many human diseases. SH3-binding domain kinase 1 (SBK1) was first cloned and characterized in rat and the human cDNA was cloned in our lab in 2006, but the expression and function of endogenous protein have not been well studied in human. In this follow up study, we screened a panel of cell lines and tissues, as well as a tumor tissue array for SBK1 expression at both RNA and protein levels. To detect the protein, we generated the first rabbit polyclonal antibody against human SBK1. We show that the SBK1 is expressed in most of the cells and tissues examined, and the protein is highly up-regulated in ovarian serous adenocarcinoma while down-regulated in esophagus squamous cell carcinoma and stomach adenocarcinoma. When over-expressed in an ovarian cancer cells SK-OV-3 by adenovirus infection, SBK1 protected the cells from apoptosis induced by the viral infection, therefore promoting cancer cell survival. Given that a missense mutation K92E in human SBK1 was identified recently from ovarian mucinous carcinoma, together, these results suggest that the wide-spread expression pattern of human SBK1 may predict a broad cellular function, and its dysregulated in certain cancers suggests an involvement of the protein in the pathogenesis of human cancers.     

Identification of a potential ovarian cancer stem cell gene expression profile from advanced stage papillary serous ovarian cancer

Identification of gene expression profiles of cancer stem cells may have significant implications in the understanding of tumor biology and for the design of novel treatments targeted toward these cells. Here we report a potential ovarian cancer stem cell gene expression profile from isolated side population of fresh ascites obtained from women with high-grade advanced stage papillary serous ovarian adenocarcinoma. Affymetrix U133 Plus 2.0 microarrays were used to interrogate the differentially expressed genes between side population (SP) and main population (MP), and the results were analyzed by paired T-test using BRB-ArrayTools. We identified 138 up-regulated and 302 down-regulated genes that were differentially expressed between all 10 SP/MP pairs. Microarray data was validated using qRT-PCR and17/19 (89.5%) genes showed robust correlations between microarray and qRT-PCR expression data. The Pathway Studio analysis identified several genes involved in cell survival, differentiation, proliferation, and apoptosis which are unique to SP cells and a mechanism for the activation of Notch signaling is identified. To validate these findings, we have identified and isolated SP cells enriched for cancer stem cells from human ovarian cancer cell lines. The SP populations were having a higher colony forming efficiency in comparison to its MP counterpart and also capable of sustained expansion and differentiation in to SP and MP phenotypes. 50,000 SP cells produced tumor in nude mice whereas the same number of MP cells failed to give any tumor at 8 weeks after injection. The SP cells demonstrated a dose dependent sensitivity to specific γ-secretase inhibitors implicating the role of Notch signaling pathway in SP cell survival. Further the generated SP gene list was found to be enriched in recurrent ovarian cancer tumors.     

Knockdown of TMEM14A expression by RNAi inhibits the proliferation and invasion of human ovarian cancer cells

Transmembrane protein 14A (TMEM14A) is a member of TMEMs. Alterations in TMEMs expression have been identified in several types of cancer, but the expression and function of TMEM14A in ovarian cancer is still unclear. Here, analysis on the expression data of the Cancer Genome Atlas (TCGA) ovarian serous cystadenocarcinoma (OV) dataset demonstrated the overexpression of TMEM14A in ovarian cancer tissues compared with normal tissues, which was consistent with our real-time PCR analysis on ovarian cancer and normal tissues collected from 30 patients. In addition, TMEM14A knockdown in two ovarian cancer cell lines, A2780 and HO-8910, reduced cell proliferation, causes cell cycle arrest and suppressed cell invasion. Moreover, silencing of TMEM14A notably repressed G1/S cell cycle transition and cell invasion via down-regulating the expression of cell cycle related proteins (Cyclin D1, Cyclin E and PCNA) and metastasis-related proteins (MMP-2 and MMP-9), respectively. TMEM14A knockdown significantly reduced the phosphorylation status of Smad2 and Smad3, downstream effectors of TGF-β signalling. In summary, these results indicate that TMEM14A has a pro-tumorigenic effect in ovarian cancer cells, suggesting an important role of this protein in ovarian cancer oncogenesis and metastasis.     

Immunohistochemistry,glycosylation and immunosuppression of glycodelin in human ovarian cancer

Glycodelins (Gds) are glycoproteins with a gender specific glycosylation. Glycodelin A (GdA) is primarily produced in endometrial and decidual tissue and secreted to amniotic fluid. Glycodelins were also identified in several cancer types, including serous ovarian cancer. Gds act as a T-cell inhibitor and are involved in inactivation of human monocytes. With a Gd peptide antibody, derived from a 15 amino acid sequence of human Gd and in situ hybridization experiments, the expression of Gd in serous, mucinous, endometrioid and clear cell ovarian tumors was identified. In contrast to former investigations with antibodies against GdA, a positive immunohistochemical reaction for Gd was observed in all forms of epithelium ovarian cancer. These results were confirmed with in situ hybridization. In addition, Gd is expressed in granulose cell tumors, a non-epithelial form of ovarian cancer. Furthermore, Gd was purified from ascites fluid of ovarian cancer patients. Ascites Gd showed significant differences in its structure of sialyl Lewis-type oligosaccharides compared to GdA. Additionally, ascites Gd inhibits IL-2 stimulated proliferation of peripheral blood leucocytes and inhibits adhesion of SLe^X-positive cells to E-selectin. Therefore, Gd could act as an inhibitor of lymphocyte activation and/or adhesion in ovarian cancer. U. Jeschke, I. Mylonas and C. Kunert-Keil contributed equally to this work.