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.     

Characterization of DOK1, a candidate tumor suppressor gene, in epithelial ovarian cancer

In attempt to discover novel aberrantly hypermethylated genes with putative tumor suppressor function in epithelial ovarian cancer (EOC), we applied expression profiling following pharmacologic inhibition of DNA methylation in EOC cell lines. Among the genes identified, one of particular interest wаs DOK1, or downstream of tyrosine kinase 1, previously recognized as a candidate tumor suppressor gene (TSG) for leukemia and other human malignancies. Using bisulfite sequencing, we determined that a 5′-non-coding DNA region (located at nt −1158 to −850, upstream of the DOK1 translation start codon) was extensively hypermethylated in primary serous EOC tumors compared with normal ovarian specimens; however, this hypermethylation was not associated with DOK1 suppression. On the contrary, DOK1 was found to be strongly overexpressed in serous EOC tumors as compared to normal tissue and importantly, DOK1 overexpression significantly correlated with improved progression-free survival (PFS) values of serous EOC patients. Ectopic modulation of DOK1 expression in EOC cells and consecutive functional analyses pointed toward association of DOK1 expression with increased EOC cell migration and proliferation, and better sensitivity to cisplatin treatment. Gene expression profiling and consecutive network and pathway analyses were also confirmative for DOK1 association with EOC cell migration and proliferation. These analyses were also indicative for DOK1 protective role in EOC tumorigenesis, linked to DOK1-mediated induction of some tumor suppressor factors and its suppression of pro-metastasis genes. Taken together, our findings are suggestive for a possible tumor suppressor role of DOK1 in EOC; however its implication in enhanced EOC cell migration and proliferation restrain us to conclude that DOK1 represents a true TSG in EOC. Further studies are needed to more completely elucidate the functional implications of DOK1 and other members of the DOK gene family in ovarian tumorigenesis.     

Prognostic Outcome of Mesenchymal Transition Biomarkers in Correlation with EGFR Expression in Epithelial Ovarian Carcinoma Patients

Background: CD44 is an epithelial-mesenchymal transition (EMT) surface receptor that regulates the interactivity between the cells and the extracellular matrix, thereby promoting cell migration. The epidermal growth factor receptor (EGFR) family is a trans-membrane kinase-related protein. It regulates cell adhesion proteins, which may promote cell proliferation and invasiveness. Mesenchymal epithelial transition (MET) is another EMT receptor that stimulates cell proliferation, invasion, survival, and angiogenesis. This study aimed to evaluate the prognostic impact of CD44, EGFR expressions, and MET gene amplification in epithelial ovarian cancer (EOC). Methods: This is a retrospective cohort study, including 85 cases of EOC. CD44 and EGFR expressions were evaluated in both epithelial and stromal cells by immunohistochemistry. Tumor cells also underwent a cytogenetic analysis using fluorescent in situ hybridization (FISH) to detect MET gene amplification. Results: High CD44 expression in tumors was significantly associated with serous subtypes (P=0.001), peritoneal deposits (P=0.002), and advanced stage (P=0.002). EGFR high tumor expression demonstrated a significant association with lymph node metastasis (P=0.038) and the advanced stage of EOC (P=0.016). Increased copy number of the MET gene was significantly associated with partial therapy response (P=0.030).  CD44 and EGFR tumor high expression was associated with poor overall survival (OS). In addition, MET gene gain in tumors was associated with a shorter OS (P=0.000). Conclusion: EMT biomarkers (CD44 and MET) and EGFR expression in EOC are independent prognostic factors for OS. MET gene increase copy number was detected in cases of serous neoplasm and associated with poor survival and minimal therapy response.     

Gene expression patterns of chemoresistant and chemosensitive serous epithelial ovarian tumors with possible predictive value in response to initial chemotherapy

Chemotherapy (CT) resistance in ovarian cancer is broad and encompasses diverse, unrelated drugs, suggesting more than one mechanism of resistance. We aimed to analyze the gene expression patterns in primary serous epithelial ovarian cancer (EOC) samples displaying different responses to first-line CT in an attempt to identify specific molecular signatures associated with response to CT. Initially, the expression profiles of 15 chemoresistant serous EOC tumors [time to recurrence (TTR) or =30 months) were independently analyzed which allowed the identification of specific sets of differentially expressed genes that might be functionally implicated in the evolution of the chemoresistant or the chemosensitive phenotype. Our data suggest that the intrinsic chemoresistance in serous EOC cells may be attributed to the combined action of different molecular mechanisms and factors linked with drug influx and efflux and cell proliferation, as possible implications of other molecular events including altered metabolism, apoptosis and inflammation cannot be excluded. Next, gene expression comparison using hierarchical clustering clearly distinguished chemosensitive and chemoresistant tumors from the 25 serous EOC samples (training set), and consecutive class prediction analysis was used to develop a 43-gene classifier that was further validated in an independent cohort of 15 serous EOC patients and 2 patients with other ovarian cancer histotypes (test set). The 43-gene predictor set properly classified serous EOC patients at high risk for early (< or =22 months) versus late (>22 months) relapse after initial CT. Thus, gene expression array technology can effectively classify serous EOC tumors according to CT response. The proposed 43-gene model needs further validation.     

Expression of MUC1 and MUC2 mucin gene products in human ovarian carcinomas

BACKGROUND: Aberrations in expression of mucin glycoproteins have been observed during malignant transformation of human ovarian epithelium. To date, several secretory mucin genes designated the MUC gene family have been identified, of which MUC1 encodes a mammary-type and MUC2 an intestinal-type epithelial mucin. However, information on the expression and potential value of MUC1 and MUC2 mucins in ovarian cancer is limited. METHODS: This study investigated immunohistochemical expressions of MUC1 and MUC2 mucins in 23 benign and 45 malignant human ovarian tumors to assess their clinicopathological relevance. RESULTS: All benign serous tumors and also associated normal-appearing epithelia expressed MUC1 mucin on the cell surfaces. Benign mucinous tumors occasionally expressed MUC1 and MUC2 mucins. Most serous carcinomas (19/21; 90%) expressed MUC1 but not MUC2 mucin. Of the 16 mucinous carcinomas, 10 (62%) and five (31%) expressed MUC1 and MUC2 mucins, respectively. Four of the five clear cell and the three endometroid type carcinomas expressed MUC-1 but not MUC-2 mucin. A significant association was found between a high expression of MUC1 and histological grade (P = 0.005) and also disease stage (P = 0.001). CONCLUSION: These results suggest that a high expression of MUC1 may contribute to a poor prognosis in ovarian carcinoma.     

Identification of novel variant, 1484delG in the 3'UTR of H3F3B, a member of the histone 3B replacement family, in ovarian tumors

Previous studies have implicated the chromosomal region at 17q25 as harboring tumor suppressor genes based on the frequent loss of heterozygosity (LOH) observed in epithelial ovarian cancers (EOC). RT-PCR validation of Affymetrix GeneChip expression of H3F3B, a member of the 3B histone family that maps to 17q25.1, revealed a doublet band in cDNA from one of four EOC cell lines, OV90. In contrast to three other EOC cell lines (TOV81D, TOV112D and TOV21G) and primary cultures derived from normal ovarian surface epithelial cells (NOSE), sequence analysis of the cDNA revealed a deletion of G at position 1484 of the transcribed sequence which is located within the 3'UTR of H3F3B. OV90 was derived from ascites fluid of an undifferentiated adenocarcinoma of ovarian origin. The variant allele was identified in 1 of 65 (2%) healthy women with no prior history of cancer and in 5 participants with ovarian tumors comprising of 4 of 79 (5%) malignant EOC, none of 10 low malignancy potential tumors, and 1 of 8 (13%) benign tumors. All carriers of the variant alleles were heterozygous and tumor samples did not exhibit preferential LOH of the normal allele. The variant allele was identified in EOC samples of clear cell (1 of 20), mucinous (1 of 8), mixed cell (1 of 3) and undifferentiated (1 of 2) histopathological subtypes but none of 34 serous or 12 endometrioid subtype tumors. One of 3 mucinous benign tumors also harbored the variant allele. The functional significance of the variant is unknown, however its presence in rare subtypes of ovarian epithelial tumors warrants further investigation.     

The differential role of L1 in ovarian carcinoma and normal ovarian surface epithelium

Epithelial ovarian carcinoma (EOC) arises from the ovarian surface epithelium (OSE), a monolayer of poorly differentiated epithelial cells that lines the ovary. The molecular mechanisms underlying EOC invasion into the surrounding stroma and dissemination to the peritoneum and to retroperitoneal lymph nodes are still unclear. Here, we analyzed the expression and the functional role of the cell adhesion molecule L1 during EOC development. In patient-derived samples, L1 was expressed both in OSE and in a subset of EOC, in the latter being mostly restricted to the invasive areas of the tumors. The expression of L1 correlated significantly with poor outcome and with unfavorable clinicopathologic features of the disease. The peculiar expression pattern of L1 in normal OSE and invasive EOC raised the possibility that this adhesion molecule serves a different function in nontransformed versus neoplastic ovarian epithelial cells. Indeed, we showed that in OSE cells L1 supports cell-cell adhesion and enhances apoptosis, whereas it has no effect on cell proliferation and invasion. In contrast, L1 inhibits cell-cell adhesion and apoptosis in ovarian carcinoma cells, where it promotes malignancy-related properties, such as cell proliferation, Erk1/2-dependent and phosphoinositide 3-kinase-dependent invasion, and transendothelial migration. Interestingly, a crosstalk with the fibroblast growth factor receptor signaling is implicated in the promalignant function of L1 in tumor cells. Our findings point to L1 as an EOC biomarker correlating with poor prognosis, and highlight a switch in L1 function associated to the neoplastic transformation of ovarian epithelial cells, thus implicating L1 as a potential therapeutic target.     

Expression and functions of galectin-7 in ovarian cancer

There is a critical need to develop effective new strategies for diagnosis and treatment of ovarian cancer. In the present work, we investigated the expression of galectin-7 (gal-7) in epithelial ovarian cancer (EOC) cells and studied its functional relevance. Immunohistochemical analysis of gal-7 expression in tissue microarrays showed that while gal-7 was not detected in normal ovarian tissues, positive cytoplasmic staining of gal-7 was detected in epithelial cells in all EOC histological subtypes but was more frequent in high grade tumors and metastatic samples. Gal-7 expression correlated with a significant difference in the overall survival of patients with ovarian serous cystadenocarcinoma. Furthermore, using human EOC cell lines, we found that gal-7 expression was induced by mutant p53. Mechanistically, Matrigel invasion assays and live cell imaging showed that gal-7 increased the invasive behavior of ovarian cancer cells by inducing MMP-9 and increasing cell motility. EOC cells can also secrete gal-7. Recombinant human gal-7 kills Jurkat T cells and human peripheral T cells, suggesting that gal-7 also has immunosuppressive properties. Taken together, our study validates the clinical significance of gal-7 overexpression in ovarian cancer and provides a rationale for targeting gal-7 to improve the outcome of patients with this disease.     

Roles of KLF6 and KLF6-SV1 in ovarian cancer progression and intraperitoneal dissemination.

PURPOSE: We investigated the role of the KLF6 tumor suppressor gene and its alternatively spliced isoform KLF6-SV1 in epithelial ovarian cancer (EOC). EXPERIMENTAL DESIGN: We first analyzed tumors from 68 females with EOC for KLF6 gene inactivation using fluorescent loss of heterozygosity (LOH) analysis and direct DNA sequencing and then defined changes in KLF6 and KLF6-SV1 expression levels by quantitative real-time PCR. We then directly tested the effect of KLF6 and KLF6-SV1 inhibition in SKOV-3 stable cell lines on cellular invasion and proliferation in culture and tumor growth, i.p. dissemination, ascites production, and angiogenesis in vivo using BALB/c nu/nu mice. All statistical tests were two sided. RESULTS: LOH was present in 59% of samples in a cell type-specific manner, highest in serous (72%) and endometrioid (75%) subtypes, but absent in clear cell tumors. LOH was significantly correlated with tumor stage and grade. In addition, KLF6 expression was decreased in tumors when compared with ovarian surface epithelial cells. In contrast, KLF6-SV1 expression was increased approximately 5-fold and was associated with increased tumor grade regardless of LOH status. Consistent with these findings, KLF6 silencing increased cellular and tumor growth, angiogenesis, and vascular endothelial growth factor expression, i.p. dissemination, and ascites production. Conversely, KLF6-SV1 down-regulation decreased cell proliferation and invasion and completely suppressed in vivo tumor formation. CONCLUSION: Our results show that KLF6 and KLF6-SV1 are associated with key clinical features of EOC and suggest that their therapeutic targeting may alter ovarian cancer growth, progression, and dissemination.     

ABCC4/MRP4 contributes to the aggressiveness of Myc-associated epithelial ovarian cancer

Epithelial ovarian cancer (EOC) is a complex disease comprising discrete histological and molecular subtypes, for which survival rates remain unacceptably low. Tailored approaches for this deadly heterogeneous disease are urgently needed. Efflux pumps belonging to the ATP-binding cassette (ABC) family of transporters are known for roles in both drug resistance and cancer biology and are also highly targetable. Here we have investigated the association of ABCC4/MRP4 expression to clinical outcome and its biological function in endometrioid and serous tumors, common histological subtypes of EOC. We found high expression of ABCC4/MRP4, previously shown to be directly regulated by c-Myc/N-Myc, was associated with poor prognosis in endometrioid EOC (P = .001) as well as in a subset of serous EOC with a “high-MYCN” profile (C5/proliferative; P = .019). Transient siRNA-mediated suppression of MRP4 in EOC cells led to reduced growth, migration and invasion, with the effects being most pronounced in endometrioid and C5-like serous cells compared to non-C5 serous EOC cells. Sustained knockdown of MRP4 also sensitized endometrioid cells to MRP4 substrate drugs. Furthermore, suppression of MRP4 decreased the growth of patient-derived EOC cells in vivo. Together, our findings provide the first evidence that MRP4 plays an important role in the biology of Myc-associated ovarian tumors and highlight this transporter as a potential therapeutic target for EOC.     

Treatment issues in clear cell carcinoma of the ovary: a different entity?

BACKGROUND: Ovarian clear cell carcinoma (OCCC) is a distinct histopathologic subtype of epithelial ovarian cancer (EOC) with an incidence of <5% of all ovarian malignancies. Our goal was to review the clinical features and management of patients with OCCC. METHODS AND RESULTS: We performed a PubMed search using the phrase "clear cell ovarian cancer." We reviewed 54 articles referring to OCCC. OCCC patients have a high incidence of stage I disease and frequently present with a large pelvic mass. Recurrences are more frequent with this entity than with other types of EOC. The clinical management of advanced EOC includes maximal cytoreduction and platinum plus paclitaxel-based chemotherapy. The survival rates of patients with advanced OCCC are lower than those of patients with advanced serous EOC (serous subtype). The poor response rate to platinum-based regimens may be related to the intrinsic chemoresistance of these tumors. Despite their aggressive clinical course, OCCCs are still treated similarly to the other EOCs at the present time, because the rarity of these tumors prevents the conduction of randomized studies. CONCLUSION: Novel treatment approaches should be adopted in OCCC. Molecular-targeted therapies and effective new agents without cross-resistance to platinum compounds should be evaluated in a prospective clinical trial in OCCC.     

Validating the impact of a molecular subtype in ovarian cancer on outcomes: A study of the OVCAD Consortium

Most patients with epithelial ovarian cancer (EOC) are diagnosed at advanced stage and have a poor prognosis. However, a small proportion of these patients will survive, whereas others will die very quickly. Clinicopathological factors do not allow precise identification of these subgroups. Thus, we have validated a molecular subclassification as new prognostic factor in EOC. One hundred and ninety‐four patients with Stage II–IV EOC were characterized by whole‐genome expression profiling of tumor tissues and were classified using a published 112 gene set, derived from an International Federation of Gynecology and Obstetrics (FIGO) stage‐directed supervised classification approach. The 194 tumor samples were classified into two subclasses comprising 95 (Subclass 1) and 99 (Subclass 2) tumors. All nine FIGO II tumors were grouped in Subclass 1 (P = 0.001). Subclass 2 (54% of advanced‐stage tumors) was significantly correlated with peritoneal carcinomatosis and non‐optimal debulking. Patients with Subclass 2 tumors had a worse overall survival for both serous and non‐serous histological subtypes, as revealed by univariate analysis (hazard ratios [HR] of 3.17 and 17.11, respectively; P ≤ 0.001) and in models corrected for relevant clinicopathologic parameters (HR 2.87 and 12.42, respectively; P ≤ 0.023). Significance analysis of microarrays revealed 2082 genes that were differentially expressed in advanced‐grade serous tumors of both subclasses and the focal adhesion pathway as the most deregulated pathway. In the present validation study, we have shown that, in advanced‐stage serous ovarian cancer, two approximately equally large molecular subtypes exist, independent of classical clinocopathological parameters and presenting with highly different whole‐genome expression profiles and a markedly different overall survival. Similar results were obtained in a small cohort of patients with non‐serous tumors. (Cancer Sci 2012; 103: 1334–1341)     

Monoclonal antibody targeting MUC1 and increasing sensitivity to docetaxel as a novel strategy in treating human epithelial ovarian cancer

The purpose of this study was to investigate the in vitro effect of anti-MUC1 monoclonal antibody (MAb) C595 alone and in combination with docetaxel, on the growth and survival of different epithelial ovarian cancer (EOC) cell lines. MUC1 expression was assessed on EOC cell lines (OVCAR-3, IGROV-1, A2780, CAOV-3, TOV-21G, TOV-112D, SKOV-3 and OV-90) using immunofluorescence labeling and flow cytometry. The effect of MAb C595 alone or in combination with docetaxel on the cell lines was studied by proliferation, colony and TUNEL assays. Our results indicate that all primary and metastatic EOC cell lines tested were positive to MAb C595 (MUC1); MAb C595 inhibited EOC cell proliferation in a MUC1- and dose-dependent manner; low-dose MAb C595 (1/2 of IC₅₀) combined with docetaxel greatly improved efficiency of cell killing in EOC cells and induced apoptosis; the additive effect of MAb C595 was further confirmed in colony forming assays; and cell death following single or combined treatments was associated with the release of cytochrome c and increased caspase-3 activity. These results suggest that MAb C595 used either alone, or combined with docetaxel, is an attractive strategy for targeting human EOC.     

桥接整合因子1在上皮性卵巢癌组织和细胞中的表达及其临床意义

目的：探讨桥接整合因子1(BIN1)在上皮性卵巢癌(EOC)组织中的表达及其临床意义,以及BIN1对EOC细胞A2780增殖、迁移和侵袭的影响。方法：收集2017年7月至2018年1月河北医科大学第四医院手术切除的67例EOC患者的肿瘤组织及同期因其他妇科疾病手术切除的30例非肿瘤患者的卵巢组织(正常对照组)标本。用免疫组织化学染色法检测EOC组织和非肿瘤卵巢组织中BIN1蛋白的表达水平,χ²检验分析BIN1表达与患者临床病理特征之间的关联,Kaplan-Meier法分析BIN1表达与患者的无病生存期(DFS)和总生存期(OS)之间的关系。用qPCR和WB法检测EOC细胞SKOV3、A2780和人卵巢上皮细胞IOSE80中BIN1 m RNA和蛋白的表达水平。利用基因转染技术将BIN1质粒CMV-MCS-GFP-SV40-NeomycinBIN1和空载体质粒CMV-MCS-GFP-SV40-Neomycin分别转染到A2780细胞以构建过表达BIN1细胞及其对照,用qPCR和WB法分别检测转染细胞中BIN1 mRNA和蛋白的表达水平,CCK-8、划痕愈合和Tran...     

粘蛋白MUC1在卵巢浆液性肿瘤中的表达及其临床意义

目的:研究粘蛋白MUC1在卵巢浆液性肿瘤中的表达及其临床意义。方法:应用免疫组织化学SP法检测53例卵巢浆液性囊腺癌、20例交界性卵巢浆液性囊腺瘤、20例卵巢浆液性囊腺瘤、20例正常卵巢组织中MUC1的表达情况。结果:MUC1在卵巢浆液性囊腺癌中的阳性表达率为88.7%,略高于交界性浆液性囊腺瘤中的表达(70%)(P=0.1172),明显高于良性卵巢浆液性囊腺瘤(35%)及正常卵巢组织(30%)中的表达(P<0.05)。MUC1的阳性表达与卵巢浆液性囊腺癌的临床分期有关(P<0.05),与年龄、组织分级和淋巴结转移无关(P>0.05)。结论:粘蛋白MUC1在卵巢浆液性囊腺癌的发生、发展中起重要作用,MUC1可作为判断卵巢浆液性囊腺癌生物学行为和恶性潜能的参考指标。     

Hypoxia inducible factor 1-alpha expression as a factor predictive of efficacy of taxane/platinum chemotherapy in advanced primary epithelial ovarian cancer

To investigate the impact on survival of HIF 1-alpha expression on primary advanced epithelial ovarian cancer (EOC), we examined the correlations between prognosis and HIF 1-alpha expression by Western blot analysis in 52 cases of stage III/IV EOC. HIF 1-alpha expression was confirmed in 36 cases (69.2%) of EOC, and HIF 1-alpha-expressing tumors had a significantly higher rate of response (p<0.01) to postoperative paclitaxel/carboplatin combination chemotherapy (TC) than tumors without HIF1-alpha expression. Moreover, patients with HIF 1-alpha-expressing tumors with suboptimal resection of stage III/IV tumors indicated for postoperative TC exhibited significantly better survival (p<0.01).