Identification of novel candidate biomarkers of epithelial ovarian cancer by profiling the secretomes of three-dimensional genetic models of ovarian carcinogenesis.

Epithelial ovarian cancer (EOC) is still considered the most lethal gynecological malignancy and improved early detection of ovarian cancer is crucial to improving patient prognoses. To address this need, we tested whether candidate EOC biomarkers can be identified using three-dimensional (3D) in vitro models. We quantified changes in the abundance of secreted proteins in a 3D genetic model of early-stage EOC, generated by expressing CMYC and KRAS(G) (12) (V) in TERT-immortalized normal ovarian epithelial cells. Cellular proteins were labeled in live cells using stable isotopic amino acid analogues, and secreted proteins identified and quantified using liquid chromatography-tandem mass spectrometry. Thirty-seven and 55 proteins were differentially expressed by CMYC and CMYC+KRAS(G) (12) (V) expressing cells respectively (p < 0.05; >2-fold). We evaluated expression of the top candidate biomarkers in ∼210 primary EOCs: CHI3L1 and FKBP4 are both expressed by >96% of primary EOCs, and FASN and API5 are expressed by 86 and 75% of cases. High expression of CHI3L1 and FKBP4 was associated with worse patient survival (p = 0.042 and p = 0.002, respectively). Expression of LGALS3BP was positively associated with recurrence (p = 0.0001) and suboptimal debulking (p = 0.018) suggesting that these proteins may be novel prognostic biomarkers. Furthermore, within early stage tumours (I/II), high expression of API5, CHI3L1 and FASN was associated with high tumour grade (p = 3 × 10(-4) , p = 0.016, p = 0.010, respectively). We show in vitro cell biology models of early-stage cancer development can be used to identify novel candidate biomarkers for disease, and report the identification of proteins that represent novel potential candidate diagnostic and prognostic biomarkers for this highly lethal disease.

HIF activation identifies early lesions in VHL kidneys: evidence for site-specific tumor suppressor function in the nephron.

Mutations in the von Hippel-Lindau (VHL) gene are associated with hereditary and sporadic clear cell renal carcinoma. VHL acts in a ubiquitin ligase complex regulating hypoxia-inducible factor-1 (HIF-1), but the link between this function and cancer development is unclear. Here we show that in the kidneys of patients with VHL disease, HIF activation is an early event occurring in morphologically normal single cells within the renal tubules. In comparison, dysplastic lesions, cystic lesions, and tumors showed evidence of additional mechanisms that amplify HIF activation. Detection of cells with constitutive HIF activation identified a large number of previously unrecognized foci of VHL inactivation. In proximal tubules these were almost entirely unicellular, whereas multicellular foci were almost exclusively seen in the distal nephron.

The role of Eag and HERG channels in cell proliferation and apoptotic cell death in SK-OV-3 ovarian cancer cell line.

BACKGROUND: The voltage gated potassium (K+) channels Eag and HERG have been implicated in the pathogenesis of various cancers, through association with cell cycle changes and programmed cell death. The role of these channels in the onset and progression of ovarian cancer is unknown. An understanding of mechanism by which Eag and HERG channels affect cell proliferation in ovarian cancer cells is required and therefore we investigated their role in cell proliferation and their effect on the cell cycle and apoptosis of ovarian cancer cells. METHODS: The presence of Eag and HERG was determined in SK-OV-3 cells using immunofluorescence and western blotting. The effect of the Eag blockers (imipramine and clofilium) and HERG blockers (E-4031 and ergtoxin) on cell proliferation was assessed using the MTS assay with further investigation of their role in the cell cycle and apoptosis determined by flow cytometry. RESULTS: Eag and HERG channels were present in the cytoplasm and nuclei of SK-OV-3 cells. There was significant inhibition of proliferation of SK-OV-3 cells by imipramine (P < 0.001) and ergtoxin (P < 0.05) at 72 hours of culture. Incubation of cells with ergtoxin led to the accumulation of cells in the S and G2/M phase, while cells accumulated in S phase after incubation with E-4031, with no effect on apoptosis. Imipramine did not affect the cell cycle but increased the proportion of SK-OV-3 cells undergoing early apoptosis. CONCLUSION: Both Eag and HERG channels are expressed in SK-OV-3 ovarian cancer cells and have a role in cell proliferation. HERG channels affect the cell cycle while Eag channels are implicated in the inhibition of apoptosis of ovarian cancer cells. The family of Eag channels may represent a new therapeutic target for the treatment of ovarian cancer.

Eag and HERG potassium channels as novel therapeutic targets in cancer.

Voltage gated potassium channels have been extensively studied in relation to cancer. In this review, we will focus on the role of two potassium channels, Ether à-go-go (Eag), Human ether à-go-go related gene (HERG), in cancer and their potential therapeutic utility in the treatment of cancer. Eag and HERG are expressed in cancers of various organs and have been implicated in cell cycle progression and proliferation of cancer cells. Inhibition of these channels has been shown to reduce proliferation both in vitro and vivo studies identifying potassium channel modulators as putative inhibitors of tumour progression. Eag channels in view of their restricted expression in normal tissue may emerge as novel tumour biomarkers.

Contrasting properties of hypoxia-inducible factor 1 (HIF-1) and HIF-2 in von Hippel-Lindau-associated renal cell carcinoma.

Defective function of the von Hippel-Lindau (VHL) tumor suppressor ablates proteolytic regulation of hypoxia-inducible factor alpha subunits (HIF-1alpha and HIF-2alpha), leading to constitutive activation of hypoxia pathways in renal cell carcinoma (RCC). Here we report a comparative analysis of the functions of HIF-1alpha and HIF-2alpha in RCC and non-RCC cells. We demonstrate common patterns of HIF-alpha isoform transcriptional selectivity in VHL-defective RCC that show consistent and striking differences from patterns in other cell types. We also show that HIF-alpha isoforms display unexpected suppressive interactions in RCC cells, with enhanced expression of HIF-2alpha suppressing HIF-1alpha and vice-versa. In VHL-defective RCC cells, we demonstrate that the protumorigenic genes encoding cyclin D1, transforming growth factor alpha, and vascular endothelial growth factor respond specifically to HIF-2alpha and that the proapoptotic gene encoding BNip3 responds positively to HIF-1alpha and negatively to HIF-2alpha, indicating that HIF-1alpha and HIF-2alpha have contrasting properties in the biology of RCC. In keeping with this, HIF-alpha isoform-specific transcriptional selectivity was matched by differential effects on the growth of RCC as tumor xenografts, with HIF-1alpha retarding and HIF-2alpha enhancing tumor growth. These findings indicate that therapeutic approaches to targeting of the HIF system, at least in this setting, will need to take account of HIF isoform-specific functions.

The PAX8 cistrome in epithelial ovarian cancer.

PAX8 is a lineage-restricted transcription factor that is expressed in epithelial ovarian cancer (EOC) precursor tissues, and in the major EOC histotypes. Frequent overexpression of PAX8 in primary EOCs suggests this factor functions as an oncogene during tumorigenesis, however, the biological role of PAX8 in EOC development is poorly understood. We found that stable knockdown of PAX8 in EOC models significantly reduced cell proliferation and anchorage dependent growth in vitro, and attenuated tumorigenicity in vivo. Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq) and transcriptional profiling were used to create genome-wide maps of PAX8 binding and putative target genes. PAX8 binding sites were significantly enriched in promoter regions (p < 0.05) and superenhancers (p < 0.05). MEME-ChIP analysis revealed that PAX8 binding sites overlapping superenhancers or enhancers, but not promoters, were enriched for JUND/B and ARNT/AHR motifs. Integrating PAX8 ChIP-seq and gene expression data identified PAX8 target genes through their associations within shared topological association domains. Across two EOC models we identified 62 direct regulatory targets based on PAX8 binding in promoters and 1,330 putative enhancer regulatory targets. SEPW1, which is involved in oxidation-reduction, was identified as a PAX8 target gene in both cell line models. While the PAX8 cistrome exhibits a high degree of cell-type specificity, analyses of PAX8 target genes and putative cofactors identified common molecular targets and partners as candidate therapeutic targets for EOC.

A mycobacterial iron chelator, desferri-exochelin, induces hypoxia-inducible factors 1 and 2, NIP3, and vascular endothelial growth factor in cancer cell lines.

Hypoxia is a key phenomenon in tumor behavior, selecting for resistance to apoptosis, conferring resistance to radiotherapy and chemotherapy, and also inducing angiogenic factors such as vascular endothelial growth factor (VEGF). Exochelins are naturally evolved iron chelators produced by Mycobacterium tuberculosis. Because iron chelation has been reported to activate the hypoxia-inducible factor (HIF), we investigated the effects of an exochelin [desferri-exochelin (DFE) 772SM] on this hypoxia-inducible pathway and downstream target genes. DFE induced HIF-1alpha and HIF-2alpha transcription factors regulating the hypoxic response in the breast tumor cell line MDA468. DFE was 10 times more potent and more rapid in onset of effect than the clinically used iron chelator deferoxamine. The expression of downstream hypoxia-responsive target genes VEGF and the proapoptotic protein NIP3 was activated by transcription. MDA468 proliferation was inhibited via HIF-independent pathways, related to other effects of iron chelation. DFE inhibited effects of VEGF on endothelial cell proliferation. DFE potentially could be useful in cancer therapy by inducing apoptosis via NIP3 in conjunction with other non-HIF-related growth inhibitory pathways and blocking endothelial proliferation despite the presence of VEGF.

Predominant role of hypoxia-inducible transcription factor (Hif)-1alpha versus Hif-2alpha in regulation of the transcriptional response to hypoxia.

Tumor hypoxia induces the up-regulation of a gene program associated with angiogenesis, glycolysis, adaptation to pH, and apoptosis via the hypoxia-inducible transcription factors (Hifs) 1 and 2. Disruption of this pathway has been proposed as a cancer therapy. Here, we use short interfering RNAs to compare specific inactivation of Hif-1alpha or Hif-2alpha and show markedly different cell type-specific effects on gene expression and cell migration. Remarkably, among a panel of hypoxia-inducible genes, responses were critically dependent on Hif-1 alpha but not Hif-2 alpha in both endothelial and breast cancer cells but critically dependent on Hif-2 alpha in renal carcinoma cells.

Analysis of gene expression in ductal carcinoma in situ of the breast.

PURPOSE: The risk of recurrence and progression of ductal carcinoma in situ (DCIS) of the breast is best designated by morphological indicators, including the presence of necrosis. Our purpose was to identify molecular alterations underlying progression of DCIS. EXPERIMENTAL DESIGN: We have compared gene expression within a cohort of six cases of DCIS with necrosis (DCIS(necrosis+)) and four cases without necrosis (DCIS(necrosis-)) using microdissection and cDNA microarray. RESULTS: A set of 69 cDNAs from a group of 1,181 was identified that were consistently differentially expressed. Among this set, the mRNA for angio-associated migratory cell protein and a serine threonine protein kinase, nuclear Dbf2 related, were consistently higher in DCIS(necrosis+) and were also found to be overexpressed in the T47D breast cancer cell line subjected to hypoxia. Further study of angio-associated migratory cell protein by quantitative reverse transcriptase-PCR and in situ hybridization analysis of 37 cases of DCIS confirmed higher mRNA expression in DCIS(necrosis+) (P = 0.0095). CONCLUSIONS: This study shows that although levels of gene expression are mostly similar between morphologically different DCIS, consistent differences in expression of a subset of genes can be identified between DCIS with and without necrosis.

BNIP3 expression is linked with hypoxia-regulated protein expression and with poor prognosis in non-small cell lung cancer.

BNIP3 is a proapoptotic protein regulated by hypoxia-inducible factor 1. We analyzed BNIP3 expression in 105 tumor samples from early operable, non-small lung cancer and the relationship of expression to hypoxia-inducible factor 1alpha, other hypoxia-regulated pathways, and prognosis. There was strong cytoplasmic expression in >10% of cells in 40 of 105 cases. BNIP3 expression was associated significantly with high hypoxia-inducible factor 1alpha (P = 0.003), carbonic anhydrase 9 (P = 0.04), and was inversely associated with bcl-2 expression (P = 0.009). High BNIP3 expression was a major independent factor for overall survival. Thus, high expression of a hypoxia regulated proapoptotic pathway was associated with a selection of an aggressive phenotype in vivo.

Expression and prognostic significance of the oncogenic K2P potassium channel KCNK9 (TASK-3) in ovarian carcinoma.

BACKGROUND/AIMS: The TWIK-related acid sensitive K(+) channel-3 (TASK-3) is an oncogenic potassium channel. We investigated the expression of TASK-3 in human ovaries, examined its prognostic significance, and determined effects of TASK-3 blockers on cell proliferation and apoptosis. MATERIALS AND METHODS: Immunofluorescence and western blotting were used to investigate TASK-3 expression in two ovarian cancer cell lines, normal ovarian surface epithelium and cancer. Immunohistochemistry quantified expression in an ovarian cancer tissue microarray. The effect of TASK-3 blocking agents on cell proliferation was investigated with the CellTiter 96® Aqueous Non-Radioactive Cell Proliferation assay and on apoptosis with flow cytometry. RESULTS: TASK-3 expression was confirmed by immunofluorescence in the SKOV-3 and OVCAR-3 cell lines, normal ovaries (n=4) and ovarian tumours (n=4) and by western blotting in normal ovaries (n=6) and ovarian tumours (n=22). Immunohistochemistry demonstrated immunostaining in 99% of tumours (n=230). Increased immunostaining conferred a survival advantage (p=0.002; median survival of >24 months). TASK-3 blockers caused a significant reduction in cell proliferation and an increase in apoptosis in the SKOV-3 and OVCAR-3 cell lines. CONCLUSION: TASK-3 is expressed in epithelial ovarian cancer, conferring a significant survival advantage on patients with increased expression. TASK-3-modulating agents have a significant effect on cell proliferation and apoptosis. Based on these results, we propose that TASK-3 could prove to be both a novel tumour marker and a new therapeutic target in ovarian cancer, but further investigation is required.

The Eag potassium channel as a new prognostic marker in ovarian cancer.

BACKGROUND: Ovarian cancer is the second most common cancer of the female genital tract in the United Kingdom (UK), accounting for 6% of female deaths due to cancer. This cancer is associated with poor survival and there is a need for new treatments in addition to existing chemotherapy to improve survival. Potassium (K+) channels have been shown to be overexpressed in various cancers where they appear to play a role in cell proliferation and progression. OBJECTIVES: To determine the expression of the potassium channels Eag and HERG in ovarian cancer tissue and to assess their role in cell proliferation. METHODS: The expression of Eag and HERG potassium channels was examined in an ovarian cancer tissue microarray. Their role in cell proliferation was investigated by blocking voltage-gated potassium channels in an ovarian cancer cell line (SK-OV-3). RESULTS: We show for the first time that high expression of Eag channels in ovarian cancer patients is significantly associated with poor survival (P = 0.016) unlike HERG channel expression where there was no correlation with survival. There was also a significant association of Eag staining with high tumour grade (P = 0.014) and presence of residual disease (P = 0.011). Proliferation of SK-OV-3 cells was significantly (P < 0.001) inhibited after treatment with voltage gated K+ channel blockers. CONCLUSION: This novel finding demonstrates a role for Eag as a prognostic marker for survival in patients with ovarian cancer.

Microcell-mediated chromosome transfer identifies EPB41L3 as a functional suppressor of epithelial ovarian cancers.

We used a functional complementation approach to identify tumor-suppressor genes and putative therapeutic targets for ovarian cancer. Microcell-mediated transfer of chromosome 18 in the ovarian cancer cell line TOV21G induced in vitro and in vivo neoplastic suppression. Gene expression microarray profiling in TOV21G(+18) hybrids identified 14 candidate genes on chromosome 18 that were significantly overexpressed and therefore associated with neoplastic suppression. Further analysis of messenger RNA and protein expression for these genes in additional ovarian cancer cell lines indicated that EPB41L3 (erythrocyte membrane protein band 4.1-like 3, alternative names DAL-1 and 4.1B) was a candidate ovarian cancer-suppressor gene. Immunoblot analysis showed that EPB41L3 was activated in TOV21G(+18) hybrids, expressed in normal ovarian epithelial cell lines, but was absent in 15 (78%) of 19 ovarian cancer cell lines. Using immunohistochemistry, 66% of 794 invasive ovarian tumors showed no EPB41L3 expression compared with only 24% of benign ovarian tumors and 0% of normal ovarian epithelial tissues. EPB41L3 was extensively methylated in ovarian cancer cell lines and primary ovarian tumors compared with normal tissues (P = .00004), suggesting this may be the mechanism of gene inactivation in ovarian cancers. Constitutive reexpression of EPB41L3 in a three-dimensional multicellular spheroid model of ovarian cancer caused significant growth suppression and induced apoptosis. Transmission and scanning electron microscopy demonstrated many similarities between EPB41L3-expressing cells and chromosome 18 donor-recipient hybrids, suggesting that EPB41L3 is the gene responsible for neoplastic suppression after chromosome 18 transfer. Finally, an inducible model of EPB41L3 expression in three-dimensional spheroids confirmed that reexpression of EPB41L3 induces extensive apoptotic cell death in ovarian cancers.

BRCA1 and BRCA2 as ovarian cancer susceptibility genes.

Individuals carrying germline mutations in one allele of the BRCA1 or BRCA2 genes are at significantly increased risk of developing cancer. Although the increased risk of breast cancer is often highlighted, cancer at several other sites is also considerably more common in these individuals. Here, we discuss existing knowledge of the role of BRCA1 and BRCA2 mutation in pre-disposition to ovarian cancer. The risk of an individual with a mutation developing cancer of the ovary appears to be influenced by the position of the mutation within the BRCA gene, the presence of allelic variants of modifying genes and the hormonal exposure of the carrier. Once cancer has developed, the pathology and clinical behaviour of BRCA-associated tumours is distinct from sporadic cases. Comparison of the pathogenesis of breast and ovarian cancers caused by BRCA mutation provides insight into the function of BRCA proteins as tumour suppressors in different cellular environments.

Anoxic induction of ATF-4 through HIF-1-independent pathways of protein stabilization in human cancer cells.

Hypoxia is a key factor in tumor development, contributing to angiogenesis and radiotherapy resistance. Hypoxia-inducible factor-1 (HIF-1) is a major transcription factor regulating the response of cancer cells to hypoxia. However, tumors also contain areas of more severe oxygen depletion, or anoxia. Mechanisms for survival under anoxia are HIF-1alpha independent in Caenorhabditis elegans and, thus, differ from the hypoxic response. Here we report a differential response of cancer cells to hypoxia and anoxia by demonstrating the induction of activating transcription factor-4 (ATF-4) and growth arrest DNA damage 153 (GADD153) protein specifically in anoxia and the lack of induction in hypoxia. By applying RNAi, ATF-4 induction in anoxia was shown to be independent of HIF-1alpha, and desferrioxamine mesylate (DFO) and cobalt chloride induced HIF-1alpha but not ATF-4 or GADD153. Furthermore, the inductive response of ATF-4 and GADD153 was not related to alterations in or arrest of mitochondrial respiration and was independent of von Hippel-Lindau (VHL) disease mutations. In reoxygenated anoxic cells, ATF-4 had a half-life of less than 5 minutes; adding the proteasome inhibitor to normoxic cells up-regulated ATF-4 protein. Extracts from primary human tumors demonstrated more ATF-4 expression in tumors near necrotic areas. Thus, this study demonstrates a novel HIF-1alpha-independent anoxic mechanism that regulates ATF-4 induction at the protein stability level in tumor cells.

Expression of the cell death genes BNip3 and NIX in ductal carcinoma in situ of the breast; correlation of BNip3 levels with necrosis and grade.

Ductal carcinoma in situ (DCIS) of the breast is an early, non-invasive lesion and the prognosis is associated with the extent of necrosis and cell death within the tumour. Two cell death genes, BNip3 and NIX, are up-regulated in response to hypoxia in breast carcinoma cells, although any involvement of either gene in disease progression is currently unknown. This study has analysed the expression of BNip3 and NIX in 56 samples of breast DCIS, as well as in adjacent benign and invasive breast tissue. Both genes are strongly expressed in the epithelial component of a subset of DCIS and invasive disease. The data show a correlation between high expression of BNip3 in the DCIS cells and a high-grade, necrotic lesion that is likely to be associated with invasive tumour. BNip3 was present in tumour-associated macrophages and in apocrine metaplastic lesions. Expression of NIX did not correlate with any of the parameters investigated.