Cisplatin Resistant Spheroids Model Clinically Relevant Survival Mechanisms in Ovarian Tumors

The majority of ovarian tumors eventually recur in a drug resistant form. Using cisplatin sensitive and resistant cell lines assembled into 3D spheroids we profiled gene expression and identified candidate mechanisms and biological pathways associated with cisplatin resistance. OVCAR-8 human ovarian carcinoma cells were exposed to sub-lethal concentrations of cisplatin to create a matched cisplatin-resistant cell line, OVCAR-8R. Genome-wide gene expression profiling of sensitive and resistant ovarian cancer spheroids identified 3,331 significantly differentially expressed probesets coding for 3,139 distinct protein-coding genes (Fc >2, FDR < 0.05) (S2 Table). Despite significant expression changes in some transporters including MDR1, cisplatin resistance was not associated with differences in intracellular cisplatin concentration. Cisplatin resistant cells were significantly enriched for a mesenchymal gene expression signature. OVCAR-8R resistance derived gene sets were significantly more biased to patients with shorter survival. From the most differentially expressed genes, we derived a 17-gene expression signature that identifies ovarian cancer patients with shorter overall survival in three independent datasets. We propose that the use of cisplatin resistant cell lines in 3D spheroid models is a viable approach to gain insight into resistance mechanisms relevant to ovarian tumors in patients. Our data support the emerging concept that ovarian cancers can acquire drug resistance through an epithelial-to-mesenchymal transition.     

Genomic and proteomic characterization of YDOV-157, a newly established human epithelial ovarian cancer cell line

The existence of several model systems with which to investigate a particular disease is advantageous for researchers. This is especially true for ovarian cancer, which, due to its complex and heterogeneous nature, inherently requires a large number of model systems. Here, we report a new ovarian serous adenocarcinoma cell line, designated YDOV-157, and characterized via post genomics and post proteomics. In this study, primary culture of tumor cells from ascites was performed and the cells were immortalized up to at least 60 passages in vitro. We studied the morphologies, cell proliferation, BRCA1/2 mutations, tumorigenesis capacity, and chemosensitivity of YDOV-157. Using a cDNA microarray, differentially expressed genes were identified and some of them were validated. Using proteomic analysis, we identified proteins that were differentially expressed in YDOV-157. The newly derived cell line, designated YDOV-157, grew as a monolayer and the doubling time was 102 h. When transplanted into nude mice, it initiated the formation of tumor masses with microscopic findings identical to those of the primary tumor. Chemosensitivity test showed that paclitaxel induced the highest chemosensitivity index. In microarray analysis, 2,520 probes were differently expressed, compared to human ovarian surface epithelial cells (HOSEs). In SYBR Green real-time PCR, the expression of E2F2 (P = 0.040) and CRABP2 genes (P = 0.030) was significantly higher in the ovarian cancer cell lines than in HOSEs. Furthermore, proteomic analysis showed that expression of 28 spots was significantly altered between YDOV-157 and HOSE. In conclusion, the newly derived YDOV-157 cell line may be an important research resource for studying cancer cell biology and should also be very useful for developing new strategies that inhibit cancer cell growth and progression.     

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.     

Large scale identification of human hepatocellular carcinoma-associated antigens by autoantibodies

Autoantibodies are often detected in hepatocellular carcinoma (HCC), and these responses may represent recognition of tumor Ags that are associated with transformation events. The identities of these Ags, however, are less well known. Using serological analysis of recombinant cDNA expression libraries (SEREX) from four HCC patients, we identified 55 independent cDNA sequences potentially encoding HCC tumor Ags. Of these genes, 15 are novel. Two such proteins, HCA587 and HCA661, were predominantly detected in testis, but not in other normal tissues, except for a weak expression in normal pancreas. In addition to HCC, these two Ags can be found in cancers of other histological types. Therefore, they can be categorized as cancer-testis (CT) Ags. Two other Ags (HCA519 and HCA90) were highly overexpressed in HCC and also expressed in cancer cell lines of lung, prostate, and pancreas, but not in the respective normal tissues. Four other Ags were identified to be expressed in particular types of cancer cell lines (HCA520 in an ovarian cancer cell line, HCA59 and HCA67 in a colon cancer cell line, HCA58 in colon and ovarian cancer cell lines), but not in the normal tissue counterpart(s). In addition, abundant expression of complement inactivation factors was found in HCC. These results indicate a broad range expression of autoantigens in HCC patients. Our findings open an avenue for the study of autoantigens in the transformation, metastasis, and immune evasion in HCC.     

A negative genetic interaction map in isogenic cancer cell lines reveals cancer cell vulnerabilities

Improved efforts are necessary to define the functional product of cancer mutations currently being revealed through large‐scale sequencing efforts. Using genome‐scale pooled shRNA screening technology, we mapped negative genetic interactions across a set of isogenic cancer cell lines and confirmed hundreds of these interactions in orthogonal co‐culture competition assays to generate a high‐confidence genetic interaction network of differentially essential or differential essentiality (DiE) genes. The network uncovered examples of conserved genetic interactions, densely connected functional modules derived from comparative genomics with model systems data, functions for uncharacterized genes in the human genome and targetable vulnerabilities. Finally, we demonstrate a general applicability of DiE gene signatures in determining genetic dependencies of other non‐isogenic cancer cell lines. For example, the PTEN^?/? DiE genes reveal a signature that can preferentially classify PTEN‐dependent genotypes across a series of non‐isogenic cell lines derived from the breast, pancreas and ovarian cancers. Our reference network suggests that many cancer vulnerabilities remain to be discovered through systematic derivation of a network of differentially essential genes in an isogenic cancer cell model.     

Identification of differentially expressed genes associated with HER-2/neu overexpression in human breast cancer cells.

Amplification and resulting overexpression of the HER-2/ neu proto-oncogene is found in approximately 30% of human breast and 20% of human ovarian cancers. To better understand the molecular events associated with overexpression of this gene in human breast cancer cells, differential hybridization was used to identify genes whose expression levels are altered in cells overexpressing this receptor. Of 16 000 clones screened from an overexpression cell cDNA library, a total of 19 non-redundant clones were isolated including seven whose expression decreases (C clones) and 12 which increase (H clones) in association with HER-2/ neu overexpression. Of these, five C clones and 11 H clones have been confirmed to be differentially expressed by northern blot analysis. This group includes nine genes of known function, three previously sequenced genes of relatively uncharacterized function and four novel genes without a match in GenBank. Examination of the previously characterized genes indicates that they represent sequences known to be frequently associated with the malignant phenotype, suggesting that the subtraction cloning strategy used identified appropriate target genes. In addition, differential expression of 12 of 16 (75%) cDNAs identified in the breast cancer cell lines are also seen in HER-2/ neu -overexpressing ovarian cancer cells, indicating that they represent generic associations with HER-2/ neu overexpression. Finally, up-regulation of two of the identified cDNAs, one novel and one identified but as yet uncharacterized gene, was confirmed in human breast cancer specimens in association with HER-2/ neu overexpression. Further characterization of these genes may yield insight into the fundamental biology and pathogenetic effects of HER-2/ neu overexpression in human breast and ovarian cancer cells.     

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.     

Proteomic search for potential diagnostic markers and therapeutic targets for ovarian clear cell adenocarcinoma

Clear cell adenocarcinoma (CCA) has a highly malignant potential in human epithelial ovarian cancer. The serum CA-125 is widely used as a marker for ovarian cancer, but the level is relatively low in CCA. Therefore, new sensitive biomarkers are required. In this report, we describe a promising proteomic analysis that is differentially expressed in CCA when compared to mucinous adenocarcinoma, using the ovarian cultured cell lines OVISE, OVTOKO, and MCAS. The disease-associated proteins were identified by 2-D differential gel electrophoresis (2-D DIGE) and MS. In this analysis, 18 up-regulated and 31 down-regulated spots were observed that had at least two-fold differences in the two CCA cell lines than in MCAS as control cells. Some of the proteins differentially expressed in CCA were previously observed as alternative expression levels in ovarian and/or other cancers in clinical samples. In a subsequent preliminary differential study using surgical specimens from patients with CCA, it was demonstrated that the identified proteins were expressed differentially in actual tissues, as well as in the CCA culture cells. The results from this investigation show the potentiality of a proteomic approach for identifying disease-associated proteins, which may eventually serve as diagnostic markers or therapeutic targets in CCA.     

Glycoprotein profiles of human breast cells demonstrate a clear clustering of normal/benign versus malignant cell lines and basal versus luminal cell lines

Gene expression profiling has defined molecular subtypes of breast cancer including those identified as luminal and basal. To determine if glycoproteins distinguish various subtypes of breast cancer, we obtained glycoprotein profiles from 14 breast cell lines. Unsupervised hierarchical cluster analysis demonstrated that the glycoprotein profiles obtained can serve as molecular signatures to classify subtypes of breast cancer, as well as to distinguish normal and benign breast cells from breast cancer cells. Statistical analyses were used to identify glycoproteins that are overexpressed in normal versus cancer breast cells, and those that are overexpressed in luminal versus basal breast cancer. Among the glycoproteins distinguishing normal breast cells from cancer cells are several proteins known to be involved in cell adhesion, including proteins previously identified as being altered in breast cancer. Basal breast cancer cell lines overexpressed a number of CD antigens, including several integrin subunits, relative to luminal breast cancer cell lines, whereas luminal breast cancer cells overexpressed carbonic anhydrase 12, clusterin, and cell adhesion molecule 1. The differential expression of glycoproteins in these breast cancer cell lines readily allows the classification of the lines into normal, benign, malignant, basal, and luminal groups.     

Mitochondrial comparative proteomics of human ovarian cancer cells and their platinum-resistant sublines

Resistance to platinum-based chemotherapy is the major obstacle to successful treatment of ovarian cancer. It is evident that mitochondrial defects and the dysfunctions of oxidative phosphorylation and energy production in ovarian cancer cells were directly related to their resistance to platinum drugs. Using 2-D DIGE, we compared mitochondrial proteins from two platinum-sensitive human ovarian cancer cell lines (SKOV3 and A2780) with that of four platinum-resistant sublines (SKOV3/CDDP, SKOV3/CBP, A2780/CDDP, and A2780/CBP). Among the 236 differentially expressed spots, five mitochondrial proteins (ATP-α, PRDX3, PHB, ETF, and ALDH) that participate in the electron transport respiratory chain were identified through mass spectrometry. All of them are downregulated in one or two of the platinum-resistant cell lines. Three proteins (ATP-α, PRDX3, and PHB) were validated by using western blot and immunohistochemistry. There is a significant decrease of PHB in tumor tissues from ovarian cancer patients who were resistant to platinum-based chemotherapies. This is the first direct mitochondrial proteomic comparison between platinum-sensitive and resistant ovarian cancer cells. These studies demonstrated that 2-D DIGE-based proteomic analysis could be a powerful tool to investigate limited mitochondrial proteins, and the association of PHB expression with platinum resistance indicates that mitochondria defects may contribute to platinum resistance in ovarian cancer cells.     

Mutually exclusive antiproliferative effect of cell line-specific HOX inhibition in epithelial ovarian cancer cell lines: SKOV-3 vs RMUG-S

We aimed to discover cell line-specific overexpressed HOX genes responsible for chemoresistance and to identify the mechanisms behind HOX-induced cell line-specific chemoresistance in EOC. Ten HOX genes and eight EOC cell lines were tested for any cell line-specific overexpression that presents a mutually exclusive pattern. Cell viability was evaluated after treatment with cisplatin and/or siRNA for cell line-specific overexpressed HOX genes. Immunohistochemical (IHC) staining for HOXB9 was performed in 84 human EOC tissues. HOXA10 and HOXB9 were identified as cell line-specific overexpressed HOX genes for SKOV-3 and RMUG-S, respectively. Inhibiting the expression of cell line-specific HOX genes, but not of other HOX genes, significantly decreased cell viability. In SKOV-3 cells, cell viability decreased to 46.5% after initial 10 µM cisplatin treatment; however, there was no further decrease upon additional treatment with HOXA10 siRNA. In contrast, cell viability did not significantly decrease upon cisplatin treatment in RMUG-S cells, but decreased to 65.5% after additional treatment with HOXB9 siRNA. In both cell lines, inhibiting cell line-specific HOX expression enhanced apoptosis but suppressed the expression of epithelial-mesenchymal transition (EMT) markers such as vimentin, MMP9, and Oct4. IHC analysis showed that platinum-resistant cancer tissues more frequently had high HOXB9 expression than platinum-sensitive cancer tissues. HOXB9, which is overexpressed in RMUG-S but not in SKOV-3 cells, appeared to be associated with cell line-specific platinum resistance in RMUG-S. Inhibiting HOXB9 overexpression in RMUG-S cells may effectively eliminate platinum-resistant ovarian cancer cells by facilitating apoptosis and inhibiting EMT.