Gene expression profiling of human ovarian epithelial tumors by digo nucleotide microarray

Gene expression of human ovarian carcinoma cell lines and epithelial ovarian tumors was examined by oligonucleotide microarray for about 6000 human cDNAs. (1) Comparison of gene expression between CDDP-sensitive human ovarian serous adenocarcinoma cell lines and CDDP-resistant cell lines revealed that gamma-glutamylcysteine synthetase, glutathione peroxidase-like protein, dehydrogenase (UGDH), NAD(P)H: quinoneoxireductase, glucose-6-phosphatase, ornithine decarboxylase and dihydrodiol dehydrogenase were associated with a mechanism of CDDP-resistance. Comparison of gene expression between taxol-sensitive human ovarian cell lines and taxol-resistant cell lines showed that up-regulation of 30 kinds of gene expression including MDR and semaphorin E in taxol-resistant cell lines. (2) Comparison of gene expression among serous adenocarcinomas, clear cell adenocarcinomas and non-cancerous ovarian tissues by hierarchical clustering demonstrated that clear difference between carcinomas and non-cancerous ovarian tissues but not obvious difference between serous and clear adenocarcinomas. Genes that were up- and down-regulated specifically in these two types of ovarian carcinomas were further selected by the criteria that difference in the mRNA level by more than 4-fold between tumors and non-cancerous tissues. Tissue type specific alterations of gene expression are likely to play important roles in the carcinogenesis of epithelial ovarian tumors. cDNA microarray is a powerful and high-throughput tool to analyze gene expression of cancer development.     

Transcriptional profiling develops molecular signatures for ovarian tumors.

Of the cancers unique to women, ovarian cancer has the highest mortality rate. Over 26,000 women are diagnosed with this disease in the U.S. annually, and 60% of those diagnosed will die of the disease. One of the greatest problems with this disease is the lack of strong early warning signs or symptoms resulting in advanced stage at presentation in most women, followed by the outgrowth of chemotherapy-resistant disease in the majority of patients. The 5-year survival for patients with early stage disease ranges from 50-90%, but it is less than 25% for advanced-stage disease. In collaboration with researchers at Millennium Predictive Medicine (Cambridge, MA), the Ovarian Cancer Program of the Mayo Clinic Cancer Center analyzed gene expression in over 50 primary ovarian tumors, as compared with normal ovarian epithelial cells. The technologies utilized included microarray analysis with nitrocellulose filters containing 25,000 arrayed human cDNAs, as well as the construction of subtraction suppression hybridization cDNA libraries and their subsequent sequencing. Our specific focus has been on genes that are underexpressed during the development of ovarian cancer, although this analysis has revealed a large number of consistently up- and down-regulated genes. There were more down-regulated genes in ovarian tumors than up-regulated genes. In addition, the number of genes that had altered expression levels was quite large. For example, we found 409 genes down-regulated at least 5-fold, and 72 genes up-regulated at least 5-fold in 33% of the tumors analyzed. We also observed that most of the expression alterations observed in later stage (Stages III/IV) tumors were also observed in early-stage tumors (Stages I/II). This was corroborated using comparative genomic hybridization analysis on the same tumors that were expression profiled. This analysis revealed that the late-stage tumors had more gene amplification than early-stage tumors, but most regions of change (either increases or decreases) were in common between different stage tumors. We also have verified the altered expression levels of several of these genes using several complementary strategies. Finally, we are taking top candidate genes that are consistently under-expressed in ovarian tumors and attempting to determine their functional role in the development of ovarian cancer.     

Monitoring gene expression profile changes in ovarian carcinomas using cDNA microarray

The development of cancer is the result of a series of molecular changes occurring in the cell. These events lead to changes in the expression level of numerous genes that result in different phenotypic characteristics of tumors. In this report we describe the assembly and utilization of a 5766 member cDNA microarray to study the differences in gene expression between normal and neoplastic human ovarian tissues. Several genes that may have biological relevance in the process of ovarian carcinogenesis have been identified through this approach. Analyzing the results of microarray hybridizations may provides new leads for tumor diagnosis and intervention.     

The value of epithelial membrane antigen in the diagnosis of ovarian tumors.

The value of epithelial membrane antigen (EMA) in the diagnosis of ovarian tumors was investigated using an indirect immunoperoxidase staining technique on 91 histologic sections (88 tumors and 3 normal tissues) and 39 ascitic fluid smears (28 from patients with epithelial ovarian tumors and 11 from cases of myoma uteri). The rate of positive EMA staining was highest in malignant tumors (89.2%), second highest in tumors of low malignant potential (33.3%) and lowest in benign tumors (25.0%); normal ovarian tissues were negative for EMA. Of the malignant tumors, all 48 serous cystadenocarcinomas (100%) and 18 of 26 mucinous cystadenocarcinomas (69.2%) stained positively for EMA. In serous cystadenocarcinomas, the EMA staining was mainly localized on the luminal membrane of cells in well-differentiated tumors, but appeared on the entire cell surface and cytoplasm of cells in poorly differentiated tumors. The results of EMA staining on ascitic fluid smears were almost the same as the results for the histologic sections. The intensity and the localization of EMA staining were related to the grade of malignancy in these ovarian tumors. In comparison with staining for other antigens (carcinoembryonic antigen, CA-125 and human keratin protein), EMA was found to be one of the most sensitive markers for the diagnosis of ovarian cancer.     

Complementary DNA cloning and sequencing of rat ovarian basic fibroblast growth factor and tissue distribution study of its mRNA

Three cDNA clones encoding rat basic fibroblast growth factor (FGF) were isolated from 10(6) independent clones prepared from a pregnant mare serum gonadotropin (PMSG)-stimulated rat ovarian cDNA library. One of the cDNA clones contained the entire coding sequence for basic FGF. The other two possessed the sequence coding the carboxy terminal 61 amino acids of rat basic FGF, the putative upstream intron sequence, and a 3'-noncoding region. The cDNAs encoding rat basic FGF predict a molecule consisting of 154 amino acid residues, which is one amino acid shorter than the human and bovine basic FGF. Otherwise, there are only 5 conservative amino acid substitutions between the rat and the human/bovine sequences. Poly A+ RNA from brain cortex and hypothalamus show a single 6.0 kb band that hybridizes to the cloned cDNA probe by Northern analyses. The observation that basic FGF mRNA is below the limits of detection in adrenal, spleen, heart, lung, kidney, liver, stomach, small intestine, large intestine, testis, and ovary support the notion that the that the high levels of the protein found in these tissues is due to storage of the mitogen in the extracellular matrix and not continuous gene expression. The significance of the abundance of mRNA in tissues which are not undergoing either active angiogenesis or cell proliferation (hypothalamus and brain cortex) is unclear but emphasizes the potential neuronotrophic function of basic FGF.     

miR-145 promotes miR-133b expression through c-myc and DNMT3A-mediated methylation in ovarian cancer cells

Ovarian cancer presents as malignant tumors in the female reproductive system with high mortality. MicroRNAs are involved in the progression of ovarian cancer; however, the regulatory relationship among miRs remains unclear. In our study, we verified that both miR-145 and miR-133b messenger RNA (mRNA) levels in ovarian cancer tissues were lower than in normal ovarian tissues, and their mRNA level in serum of patients with ovarian cancer was reduced. We demonstrated miR-145 targeted c-myc, and c-myc interacted physically with DNMT3A in ovarian cancer cells. We confirmed that c-myc recruited DNMT3A to the miR-133b promoter. miR-133b overexpression also inhibited target gene PKM2 expression along with the Warburg effect. Our results indicate that miR-145 inhibited the Warburg effect through miR-133b/PKM2 pathways, which may improve approaches to ovarian cancer diagnosis and treatment.     

Clinicopathological study of metallothionein immunohistochemical expression, in benign, borderline and malignant ovarian epithelial tumors

Metallothioneins (MTs) are a family of cystein-rich metal-binding proteins, which are expressed in normal cells during fetal and postnatal life but also in a variety of human neoplasms. MT expression in human tumors has been linked to resistance to anticancer drugs and differentiation and progression in some types of tumors. This study examined the immunohistochemical expression of MTs in benign, borderline and malignant tumors of ovarian surface epithelium and the possible correlations with clinicopathological parameters and survival. A total of 87 cases with diagnosis of ovarian surface epithelial tumors were included. Specifically, 21 cases of benign cystadenomas (11 serous and 10 mucinous), 14 borderline (low malignant potential tumors, 8 mucinous and 6 serous) and 52 cases of ovarian cancer were analysed. Immunohistochemical expression of MT (cut-off level > 10% of tumor cells) was clearly associated with malignancy. A statistically significant correlation was found between the expression of MT in cancer cases and benign tumors (p < 0.0001) and cancer cases and borderline tumors p = 0.003. In cancer cases a difference was observed between grade I and III (p = 0.002). There was no correlation of MT overexpression with survival in the small number of ovarian carcinoma patients where it was analysed. MT constitutes a marker that characterizes aggressiveness and a high malignant potential in ovarian epithelial tumors. In diagnostic problems MT may help distinguish between benign, borderline and malignant tumors.     

Changes in tumorigenesis- and angiogenesis-related gene transcript abundance profiles in ovarian cancer detected by tailored high density cDNA arrays

BACKGROUND: Complementary DNA array analysis of gene expression has a potential application for clinical diagnosis of disease processes. However, accessibility, affordability, reproducibility of results, and management of the data generated remain issues of concern. Use of cDNA arrays tailored for studies of specific pathways, tissues, or disease states may render a cost- and time-effective method to define potential hallmark genotype alterations. MATERIALS AND METHODS: We produced a 332-membered human cDNA array on nylon membranes tailored for studies of angiogenesis and tumorigenesis in reproductive disease. We tested the system for reproducibility using a novel statistical approach for analysis of array data and employed the arrays to investigate gene expression alterations in ovarian cancer. RESULTS: Intra-assay analysis and removal of agreement outliers was shown to be a critical step prior to interpretation of cDNA array data. The system revealed highly reproducible results, with intermembrane coefficient of reproducibility of +/- 0.98. Comparison of placental and ovarian sample data confirmed expected differences in angiogenic profiles and tissue-specific markers, such as human placental lactogen (hPL). Analysis of expression profiles of five normal ovary and four poorly differentiated serous papillary ovarian adenocarcinoma samples revealed an overall increase in angiogenesis-related markers, including vascular endothelial growth factor (VEGF) and angiopoietin-1 in the diseased tissue. These were accompanied by increases in immune response mediators (e.g. HLA-DR, Ron), apoptotic and neoplastic markers (e.g. BAD protein, b-myb), and novel potential markers of ovarian cancer, such as cofilin, moesin, and neuron-restrictive silencer factor (REST) protein. CONCLUSIONS: In-house production of tailored cDNA arrays, coupled to comprehensive analysis of resulting hybridization profiles, provides an accessible, reliable, and highly effective method of applying array technology to study disease processes. In the ovary, abundance of specific tumor markers, increased macrophage recruitment mediators, a late-stage angiogenesis profile, and the presence of chemoresistance-related markers distinguished normal and advanced ovarian cancer tissue samples. Detection of such parallel changes in pathway- and tissue-specific markers may prove a hallmark ready for application in reproductive disease diagnostic and therapeutic developments.     

Genomic analysis of epithelial ovarian cancer

Ovarian cancer is a major health problem for women in the United States. Despite evidence of considerable heterogeneity, most cases of ovarian cancer are treated in a similar fashion. The molecular basis for the clinicopathologic characteristics of these tumors remains poorly defined. Whole genome expression profiling is a genomic tool, which can identify dysregulated genes and uncover unique sub-classes of tumors. The application of this technology to ovarian cancer has provided a solid molecular basis for differences in histology and grade of ovarian tumors. Differentially expressed genes identified pathways implicated in cell proliferation, invasion, motility, chromosomal instability, and gene silencing and provided new insights into the origin and potential treatment of these cancers. The added knowledge provided by global gene expression profiling should allow for a more rational treatment of ovarian cancers. These techniques are leading to a paradigm shift from empirical treatment to an individually tailored approach. This review summarizes the new genomic data on epithelial ovarian cancers of different histology and grade and the impact it will have on our understanding and treatment of this disease.     

Trophinin is a potent prognostic marker of ovarian cancer involved in platinum sensitivity

Ovarian cancer is the leading cause of death in women with gynecological malignancies, with prognosis of advanced stage tumors determined by chemotherapeutic response and the success of tumor resection. Since aberrant RAS pathway activation is frequent in ovarian cancer, study of in vitro RAS-induced transformation and accompanying genomic expression changes in ovarian surface epithelial cells is imperative for development of new therapeutic modalities and for understanding tumorigenesis. cDNA microarray analysis revealed TROPHONIN (TRO), a homophilic adhesion molecule involved in blastocyst implantation, was among the genes most downregulated by RAS induction. TRO expression is higher in cisplatin-sensitive cancer cell lines and positively correlates with prognoses in ovarian cancers. TRO knockdown by RNA interference conferred cisplatin resistance and led to increased invasiveness of cultured ovarian cancer cells. These findings underscore the importance of TRO in tumorigenesis, and suggest that TRO may be a useful biomarker for cisplatin sensitivity and invasive potential.     

Expression of c-Src and phospho-Src in epithelial ovarian carcinoma

Abnormal c-Src expression and activation has been observed in a number of tumors. To determine the therapeutic potential of Src inhibitors for ovarian cancer patients, this study aimed to explore the expression patterns of c-Src and phospho-Src in epithelial ovarian cancer. A total of 82 patients with epithelial ovarian cancer treated at Sun Yat-sen University Cancer Center from January 1999 to December 2005 were enrolled along with 25 patients with benign ovarian lesions; 20 normal ovarian tissues served as controls. Expression of c-Src and phospho-Src (Tyr416) was examined using immunohistochemistry. Survival analyses were performed using Kaplan–Meier curves. As compared to the control group, a significantly greater proportion of ovarian cancer tissues were positive for c-Src and phospho-Src expression (P < 0.001). c-Src expression was associated with age, while phospho-Src expression was significantly associated with age, FIGO stage, histology grade, and residual tumor size after surgery (all P < 0.05). The mean survival time was associated with phospho-Src expression, but not with c-Src expression. The mean survival times of patients with phospho-Src-positive tumors were significantly greater than those with phospho-Src-negative tumors (87.4 months, 95 % CI = 74.3–100.5 months and 91.5 months, 95 % CI = 84.7–98.2 months, respectively; P = 0.013). The increased c-Src expression and activation in epithelial ovarian cancer suggests that ovarian cancer patients may benefit from tyrosine kinase inhibitors such as Dasatinib. Activation of c-Src through phosphorylation at Tyr416 may play a role in the early stages of ovarian cancer development, and evaluation of its expression may be a useful prognostic marker of epithelial ovarian cancer.     

A critical assessment of SELDI-TOF-MS for biomarker discovery in serum and tissue of patients with an ovarian mass

ABSTRACT: BACKGROUND: Less than 25% of patients with a pelvic mass who are presented to a gynecologist will eventually be diagnosed with epithelial ovarian cancer. Since there is no reliable test to differentiate between different ovarian tumors, accurate classification could facilitate adequate referral to a gynecological oncologist, improving survival. The goal of our study was to assess the potential value of a SELDI-TOF-MS based classifier for discriminating between patients with a pelvic mass. METHODS: Our study design included a well-defined patient population, stringent protocols and an independent validation cohort. We compared serum samples of 53 ovarian cancer patients, 18 patients with tumors of low malignant potential, and 57 patients with a benign ovarian tumor on different ProteinChip arrays. In addition, from a subset of 84 patients, tumor tissues were collected and microdissection was used to isolate a pure and homogenous cell population. RESULTS: Diagonal Linear Discriminant Analysis (DLDA) and Support Vector Machine (SVM) classification on serum samples comparing cancer versus benign tumors, yielded models with a classification accuracy of 71-81% (cross-validation), and 73-81% on the independent validation set. Cancer and benign tissues could be classified with 95-99% accuracy using cross-validation. Tumors of low malignant potential showed protein expression patterns different from both benign and cancer tissues. Remarkably, none of the peaks differentially expressed in serum samples were found to be differentially expressed in the tissue lysates of those same groups. CONCLUSION: Although SELDI-TOF-MS can produce reliable classification results in serum samples of ovarian cancer patients, it will not be applicable in routine patient care. On the other hand, protein profiling of microdissected tumor tissue may lead to a better understanding of oncogenesis and could still be a source of new serum biomarkers leading to novel methods for differentiating between different histological subtypes.     

Epigenetic analysis of sporadic and Lynch-associated ovarian cancers reveals histology-specific patterns of DNA methylation

Diagnosis and treatment of epithelial ovarian cancer is challenging due to the poor understanding of the pathogenesis of the disease. Our aim was to investigate epigenetic mechanisms in ovarian tumorigenesis and, especially, whether tumors with different histological subtypes or hereditary background (Lynch syndrome) exhibit differential susceptibility to epigenetic inactivation of growth regulatory genes. Gene candidates for epigenetic regulation were identified from the literature and by expression profiling of ovarian and endometrial cancer cell lines treated with demethylating agents. Thirteen genes were chosen for methylation-specific multiplex ligation-dependent probe amplification assays on 104 (85 sporadic and 19 Lynch syndrome-associated) ovarian carcinomas. Increased methylation (i.e., hypermethylation) of variable degree was characteristic of ovarian carcinomas relative to the corresponding normal tissues, and hypermethylation was consistently more prominent in non-serous than serous tumors for individual genes and gene sets investigated. Lynch syndrome-associated clear cell carcinomas showed the highest frequencies of hypermethylation. Among endometrioid ovarian carcinomas, lower levels of promoter methylation of RSK4, SPARC, and HOXA9 were significantly associated with higher tumor grade; thus, the methylation patterns showed a shift to the direction of high-grade serous tumors. In conclusion, we provide evidence of a frequent epigenetic inactivation of RSK4, SPARC, PROM1, HOXA10, HOXA9, WT1-AS, SFRP2, SFRP5, OPCML, and MIR34B in the development of non-serous ovarian carcinomas of Lynch and sporadic origin, as compared to serous tumors. Our findings shed light on the role of epigenetic mechanisms in ovarian tumorigenesis and identify potential targets for translational applications.     

Epigenetic analysis of sporadic and Lynch-associated ovarian cancers reveals histology-specific patterns of DNA methylation

Diagnosis and treatment of epithelial ovarian cancer is challenging due to the poor understanding of the pathogenesis of the disease. Our aim was to investigate epigenetic mechanisms in ovarian tumorigenesis and, especially, whether tumors with different histological subtypes or hereditary background (Lynch syndrome) exhibit differential susceptibility to epigenetic inactivation of growth regulatory genes. Gene candidates for epigenetic regulation were identified from the literature and by expression profiling of ovarian and endometrial cancer cell lines treated with demethylating agents. Thirteen genes were chosen for methylation-specific multiplex ligation-dependent probe amplification assays on 104 (85 sporadic and 19 Lynch syndrome-associated) ovarian carcinomas. Increased methylation (i.e., hypermethylation) of variable degree was characteristic of ovarian carcinomas relative to the corresponding normal tissues, and hypermethylation was consistently more prominent in non-serous than serous tumors for individual genes and gene sets investigated. Lynch syndrome-associated clear cell carcinomas showed the highest frequencies of hypermethylation. Among endometrioid ovarian carcinomas, lower levels of promoter methylation of RSK4, SPARC, and HOXA9 were significantly associated with higher tumor grade; thus, the methylation patterns showed a shift to the direction of high-grade serous tumors. In conclusion, we provide evidence of a frequent epigenetic inactivation of RSK4, SPARC, PROM1, HOXA10, HOXA9, WT1-AS, SFRP2, SFRP5, OPCML, and MIR34B in the development of non-serous ovarian carcinomas of Lynch and sporadic origin, as compared to serous tumors. Our findings shed light on the role of epigenetic mechanisms in ovarian tumorigenesis and identify potential targets for translational applications.     

Comparative proteomics of ovarian epithelial tumors

We analyzed 12 ovarian epithelial tumors using 2D PAGE-based comparative proteomics to construct intra- and inter-tumoral distance map trees and to discover surrogate biomarkers indicative of an ovarian tumor. The analysis was performed after laser microdissection of 12 fresh-frozen tissue samples, including 4 serous, 5 mucinous, and 3 endometrioid tumors, with correlation with their histopathological characteristics. Ovarian epithelial tumors and normal tissues showed an apparent separation on the distance map tree. Mucinous carcinomas were closest to the normal group, whereas serous carcinomas were located furthest from the normal group. All mucinous tumors with aggressive histology were separated from the low malignant potential (LMP) group. The benign-looking cysts adjacent to the intraepithelial carcinoma (IEC) showed an expression pattern identical to that of the IEC area. The extent of change on the lineages leading to the mucinous and serous carcinoma was 1.98-fold different. The overall gene expression profiles of serous or endometrioid carcinomas appeared to be less affected by grade or stage than by histologic type. The potential candidate biomarkers screened in ovarian tumors and found to be significantly up-regulated in comparison to normal tissues were as follows: NM23, annexin-1, protein phosphatase-1, ferritin light chain, proteasome alpha-6, and NAGK (N-acetyl glucosamine kinase). In conclusion, ovarian mucinous tumors are distinct from other ovarian epithelial tumors. LMP mucinous tumors showing histologically aggressive features belong to mucinous carcinoma on the proteomic basis.     

Progesterone signaling mediated through progesterone receptor membrane component-1 in ovarian cells with special emphasis on ovarian cancer

Various ovarian cell types including granulosa cells and ovarian surface epithelial cells express the progesterone (P4) binding protein, progesterone receptor membrane component-1 (PGRMC1). PGRMC1 is also expressed in ovarian tumors. PGRMC1 plays an essential role in promoting the survival of both normal and cancerous ovarian cell in vitro. Given the clinical significance of factors that regulate the viability of ovarian cancer, this review will focus on the role of PGRMC1 in ovarian cancer, while drawing insights into the mechanism of PGRMC1's action from cell lines derived from healthy ovaries as well as ovarian tumors.Studies using PGRMC1siRNA demonstrated that P4's ability to inhibit ovarian cells from undergoing apoptosis in vitro is dependent on PGRMC1. To confirm the importance of PGRMC1, the ability of PGRMC1-deplete ovarian cancer cell lines to form tumors in intact nude mice was assessed. Compared to PGRMC1-expressing ovarian cancer cells, PGRMC1-deplete ovarian cancer cells formed tumors in fewer mice (80% compared to 100% for controls). Moreover, the number of tumors derived from PGRMC1-deplete ovarian cancer cells was 50% of that observed in controls. Finally, the tumors that formed from PGRMC1-deplete ovarian cancer cells were about a fourth the size of tumors derived from ovarian cancer cells with normal levels of PGRMC1. One reason for PGRMC1-deplete tumors being smaller is that they had a poorly developed microvasculature system. How PGRMC1 regulates cell viability and in turn tumor growth is not known but part of the mechanism likely involves the regulation of genes that promote cell survival and inhibit apoptosis.