UGT2B28 accelerates prostate cancer progression through stabilization of the endocytic adaptor protein HIP1 regulating AR and EGFR pathways.

The androgen inactivating UGT2B28 pathway emerges as a predictor of progression in prostate cancer (PCa). However, the clinical significance of UGT2B28 tumoral expression and its contribution to PCa progression remain unclear. Using the Canadian Prostate Cancer Biomarker Network biobank (CPCBN; n = 1512), we analyzed UGT2B28 tumor expression in relation to clinical outcomes in men with localized PCa. UGT2B28 was overexpressed in tumors compared to paired normal adjacent prostatic tissue and was associated with inferior outcomes. Functional analyses indicated that UGT2B28 promoted cell proliferation, and its expression was regulated by the androgen receptor (AR)/ARv7. Mechanistically, UGT2B28 was shown to be a protein partner of the endocytic adaptor protein huntingtin-interacting protein 1 (HIP1), increasing its stability and priming AR/epidermal growth factor receptor (EGFR) pathways, leading to ERK1/2 activation triggering cell proliferation and epithelial-to-mesenchymal transition (EMT). HIP1 knockdown in UGT2B28 positive cells, and dual pharmacological targeting of AR and EGFR pathways, abolished cell proliferative advantages conferred by UGT2B28. In conclusion, UGT2B28 is a prognosticator of progression in localized PCa, regulates both AR and EGFR oncogenic signaling pathways via HIP1, and therefore can be therapeutically targeted by using combination of existing AR/EGFR inhibitors.

CdGAP promotes prostate cancer metastasis by regulating epithelial-to-mesenchymal transition, cell cycle progression, and apoptosis.

High mortality of prostate cancer patients is primarily due to metastasis. Understanding the mechanisms controlling metastatic processes remains essential to develop novel therapies designed to prevent the progression from localized disease to metastasis. CdGAP plays important roles in the control of cell adhesion, migration, and proliferation, which are central to cancer progression. Here we show that elevated CdGAP expression is associated with early biochemical recurrence and bone metastasis in prostate cancer patients. Knockdown of CdGAP in metastatic castration-resistant prostate cancer (CRPC) PC-3 and 22Rv1 cells reduces cell motility, invasion, and proliferation while inducing apoptosis in CdGAP-depleted PC-3 cells. Conversely, overexpression of CdGAP in DU-145, 22Rv1, and LNCaP cells increases cell migration and invasion. Using global gene expression approaches, we found that CdGAP regulates the expression of genes involved in epithelial-to-mesenchymal transition, apoptosis and cell cycle progression. Subcutaneous injection of CdGAP-depleted PC-3 cells into mice shows a delayed tumor initiation and attenuated tumor growth. Orthotopic injection of CdGAP-depleted PC-3 cells reduces distant metastasic burden. Collectively, these findings support a pro-oncogenic role of CdGAP in prostate tumorigenesis and unveil CdGAP as a potential biomarker and target for prostate cancer treatments.

Expression of ERBB Family Members as Predictive Markers of Prostate Cancer Progression and Mortality.

BACKGROUND: EGFR, ERBB2, ERBB3, and ERBB4 are growth receptors of the ERBB family implicated in the development of epithelial cancers. Studies have suggested a role for EGFR and ERBB3 in the development of prostate cancer (PC), while the involvement of ERBB2 and ERBB4 remains unclear. In this study, we evaluated the expression of all members of the ERBB family in PC tissue from a large cohort and determined their contribution, alone or in combination, as prognostic markers. METHODS: Using immunofluorescence coupled with digital image analyses, we quantified the expression of EGFR, ERBB2, ERBB3, and ERBB4 on radical prostatectomy specimens (n = 285) arrayed on six tissue microarrays. By combining EGFR, ERBB2, and ERBB3 protein expression in a decision tree model, we identified an association with biochemical recurrence (log rank = 25.295, p < 0.001), development of bone metastases (log rank = 23.228, p < 0.001), and cancer-specific mortality (log rank = 24.586, p < 0.001). CONCLUSIONS: Our study revealed that specific protein expression patterns of ERBB family members are associated with an increased risk of PC progression and mortality.

PUMA and NOXA Expression in Tumor-Associated Benign Prostatic Epithelial Cells Are Predictive of Prostate Cancer Biochemical Recurrence.

BACKGROUND: Given that treatment decisions in prostate cancer (PC) are often based on risk, there remains a need to find clinically relevant prognostic biomarkers to stratify PC patients. We evaluated PUMA and NOXA expression in benign and tumor regions of the prostate using immunofluorescence techniques and determined their prognostic significance in PC. METHODS: PUMA and NOXA expression levels were quantified on six tissue microarrays (TMAs) generated from radical prostatectomy samples (n = 285). TMAs were constructed using two cores of benign tissue and two cores of tumor tissue from each patient. Association between biomarker expression and biochemical recurrence (BCR) at 3 years was established using log-rank (LR) and multivariate Cox regression analyses. RESULTS: Kaplan-Meier analysis showed a significant association between BCR and extreme levels (low or high) of PUMA expression in benign epithelial cells (LR = 8.831, p = 0.003). Further analysis revealed a significant association between high NOXA expression in benign epithelial cells and BCR (LR = 14.854, p < 0.001). The combination of extreme PUMA and high NOXA expression identified patients with the highest risk of BCR (LR = 16.778, p < 0.001) in Kaplan-Meier and in a multivariate Cox regression analyses (HR: 2.935 (1.645-5.236), p < 0.001). CONCLUSIONS: The combination of PUMA and NOXA protein expression in benign epithelial cells was predictive of recurrence following radical prostatectomy and was independent of PSA at diagnosis, Gleason score and pathologic stage.

V-ATPase-associated prorenin receptor is upregulated in prostate cancer after PTEN loss.

Phosphatase and tensin homolog (PTEN) tumor suppressor protein loss is common in prostate cancer (PCa). PTEN loss increases PI3K/Akt signaling, which promotes cell growth and survival. To find secreted biomarkers of PTEN loss, a proteomic screen was used to compare secretomes of cells with and without PTEN expression. We showed that PTEN downregulates Prorenin Receptor (PRR) expression and secretion of soluble Prorenin Receptor (sPRR) in PCa cells and in mouse. PRR is an accessory protein required for assembly of the vacuolar ATPase (V-ATPase) complex. V-ATPase is required for lysosomal acidification, amino acid sensing, efficient mechanistic target of Rapamycin complex 1 (mTORC1) activation, and ß-Catenin signaling. On PCa tissue microarrays, PRR expression displayed a positive correlation with Akt phosphorylation. Moreover, PRR expression was required for proliferation of PCa cells by maintaining V-ATPase function. Further, we provided evidence for a potential clinical role for PRR expression and sPRR concentration in differentiating low from high Gleason grade PCa. Overall, the current study unveils a mechanism by which PTEN can inhibit tumor growth. Lower levels of PRR result in attenuated V-ATPase activity and reduced PCa cell proliferation.

Validation of the prognostic value of NF-κB p65 in prostate cancer: A retrospective study using a large multi-institutional cohort of the Canadian Prostate Cancer Biomarker Network.

BACKGROUND: The identification of patients with high-risk prostate cancer (PC) is a major challenge for clinicians, and the improvement of current prognostic parameters is an unmet clinical need. We and others have identified an association between the nuclear localization of NF-κB p65 and biochemical recurrence (BCR) in PC in small and/or single-centre cohorts of patients. METHODS AND FINDINGS: In this study, we accessed 2 different multi-centre tissue microarrays (TMAs) representing cohorts of patients (Test-TMA and Validation-TMA series) of the Canadian Prostate Cancer Biomarker Network (CPCBN) to validate the association between p65 nuclear frequency and PC outcomes. Immunohistochemical staining of p65 was performed on the Test-TMA and Validation-TMA series, which include PC tissues from patients treated by first-line radical prostatectomy (n = 250 and n = 1,262, respectively). Two independent observers evaluated the p65 nuclear frequency in digital images of cancer tissue and benign adjacent gland tissue. Kaplan-Meier curves coupled with a log-rank test and univariate and multivariate Cox regression models were used for statistical analyses of continuous values and dichotomized data (cutoff of 3%). Multivariate analysis of the Validation-TMA cohort showed that p65 nuclear frequency in cancer cells was an independent predictor of BCR using continuous (hazard ratio [HR] 1.02 [95% CI 1.00-1.03], p = 0.004) and dichotomized data (HR 1.33 [95% CI 1.09-1.62], p = 0.005). Using a cutoff of 3%, we found that this biomarker was also associated with the development of bone metastases (HR 1.82 [95% CI 1.05-3.16], p = 0.033) and PC-specific mortality (HR 2.63 [95% CI 1.30-5.31], p = 0.004), independent of clinical parameters. BCR-free survival, bone-metastasis-free survival, and PC-specific survival were shorter for patients with higher p65 nuclear frequency (p < 0.005). As the small cores on TMAs are a limitation of the study, a backward validation of whole PC tissue section will be necessary for the implementation of p65 nuclear frequency as a PC biomarker in the clinical workflow. CONCLUSIONS: We report the first study using the pan-Canadian multi-centre cohorts of CPCBN and validate the association between increased frequency of nuclear p65 frequency and a risk of disease progression.

CCN3/Nephroblastoma Overexpressed Is a Functional Mediator of Prostate Cancer Bone Metastasis That Is Associated with Poor Patient Prognosis.

Prostate cancer (PC) commonly metastasizes to the bone, resulting in pathologic fractures and poor prognosis. CCN3/nephroblastoma overexpressed is a secreted protein with a known role in promoting breast cancer metastasis to bone. However, in PC, CCN3 has been ascribed conflicting roles; some studies suggest that CCN3 promotes PC metastasis, whereas others argue a tumor suppressor role for CCN3 in this disease. Indeed, in the latter context, CCN3 has been shown to sequester the androgen receptor (AR) and suppress AR signaling. In the present study, we demonstrate that CCN3 functions as a bone-metastatic mediator, which is dependent on its C-terminal domain for this function. Analysis of tissue microarrays comprising >1500 primary PC patient radical prostatectomy specimens reveals that CCN3 expression correlates with aggressive disease and is negatively correlated with the expression of prostate-specific antigen, a marker of AR signaling. Together, these findings point to CCN3 as a biomarker to predict PC aggressiveness while providing clarity on its role as a functional mediator of PC bone metastasis.

Potential Cross-Talk between Alternative and Classical NF-κB Pathways in Prostate Cancer Tissues as Measured by a Multi-Staining Immunofluorescence Co-Localization Assay.

BACKGROUND: While the classical NF-κB/p65 pathway is known to be involved in prostate cancer progression and is associated with poor patient outcome, the role of the NF-κB /RelB alternative protein is not well defined. Here we analyzed the activation of both NF-κB pathways in prostate cancer tissues and correlate this activation with clinical features of the disease. METHODS: A multiple immunofluorescence technique was employed to concomitantly and quantitatively visualize the nuclear localization of p65 and RelB in 200 paraffin embedded samples. Epithelia were defined using appropriate fluorochrome markers and the resulting immunofluorescent signals were quantified with an automated scoring system. RESULTS: The nuclear frequency of p65 was found to be significantly increased in tumor tissues as compared with normal adjacent tissue, whereas the frequency for RelB was decreased (p < 0.001, Wilcoxon test). As previously reported, p65 nuclear frequency was associated with a risk of biochemical recurrence. Although, RelB nuclear frequency alone did not predict recurrence, the presence of activated RelB reduced the risk of recurrence associated with the activation of p65. CONCLUSION: For the first time p65/RelB co-distribution was assessed in prostate cancer tissues and suggested a negative crosstalk between the two NF-κB pathways in prostate cancer progression.

The ARID1A pathway in ovarian clear cell and endometrioid carcinoma, contiguous endometriosis, and benign endometriosis.

OBJECTIVE: To assess ARID1A-encoded protein (BAF250a) and phosphorylated AKT (pAKT) expression, apoptosis, and the DNA damage response pathway in endometrioid and clear cell ovarian cancers (endometriosis-associated ovarian cancers [EAOCs]), and benign endometriotic ovarian cysts. METHODS: In a retrospective study, tissue samples were reviewed from patients who had undergone surgery for EAOC or endometriotic ovarian cysts at a center in Montreal, QC, Canada, between 2000 and 2012. A tissue microarray including cases of endometrioid carcinoma, clear cell carcinoma, contiguous endometriosis (i.e. apparently benign endometriosis near the EAOC), and benign endometriotic ovarian cysts, was analyzed for the expression of various proteins. RESULTS: Loss of BAF250a expression was seen in 13 (22%) of 59 endometrioid cancers, 17 (47%) of 36 clear cell cases, 8 (44%) of 18 contiguous endometriosis cases, and 3 (8%) of 66 benign endometriotic ovarian cysts. In tissues showing loss of BAF250a, expression of pAKT, γH2AX, BIM, and BAX was higher in EAOC and contiguous endometriosis than in benign endometriosis (P<0.05), whereas expression of pATM, pCHK2, and Bcl2 was low. All proteins except for Bcl2 showed low expression in benign endometriosis. CONCLUSION: Loss of ARID1A-encoded protein seems to be an early event in EOAC, along with pAKT activation, alteration of γH2AX, and concomitant activation of the apoptosis pathway.

DNA damage signaling and apoptosis in preinvasive tubal lesions of ovarian carcinoma.

OBJECTIVE: High-grade serous ovarian cancer (HGSC) is the most life-threatening gynecological malignancy despite surgery and chemotherapy. A better understanding of the molecular basis of the preinvasive stages might be helpful in early detection and diagnosis. Genetic instability is 1 of the characteristics shared by most human cancers, and its level is variable through precancerous lesions to advanced cancer. Because DNA damage response (DDR) has been described as 1 of the first phases in genomic instability, we investigated the level of DDR activation and the apoptosis pathway in serous tubal intraepithelial carcinoma (STIC), the potential precursor of HGSC. METHODS/MATERIALS: A tissue microarray including 21 benign fallopian tubes, 21 STICs, 17 HGSCs from patients with STICs (associated ovarian cancer [AOC]) from the same individuals, and 30 HGSCs without STICs (non-AOC) was used in this study.Immunohistochemistry was performed to evaluate the level of DDR proteins (pATM, pChk2, γH2AX, 53BP1, and TRF2), apoptosis proteins (Bcl2, BAX, and BIM), and cyclin E. RESULTS: The expression of all DDR proteins increased from benign fallopian tubes to STICs. The level of expression of pATM, pChk2, γH2AX, and TRF2 was also increased in STICs in comparison with AOC. BAX, BIM, and cyclin E expressions were high in STICs, whereas Bcl2 expression was low. Immunohistochemical profiles of AOC and non-AOC were also different. CONCLUSIONS: These results suggest an activation of the DDR and apoptosis pathways in STICs, indicating that genomic instability may occur early in the precancerous lesions of HGSC.

RAN nucleo-cytoplasmic transport and mitotic spindle assembly partners XPO7 and TPX2 are new prognostic biomarkers in serous epithelial ovarian cancer.

PURPOSE: Epithelial ovarian cancer has the highest mortality rate of all gynecological malignancies. We have shown that high RAN expression strongly correlates with high-grade and poor patient survival in epithelial ovarian cancer. However, as RAN is a small GTPase involved in two main biological functions, nucleo-cytoplasmic transport and mitosis, it is still unknown which of these functions associate with poor prognosis. METHODS: To examine the biomarker value of RAN network components in serous epithelial ovarian cancer, protein expression of six specific RAN partners was analyzed by immunohistochemistry using a tissue microarray representing 143 patients associated with clinical parameters. The RAN GDP/GTP cycle was evaluated by the expression of RANBP1 and RCC1, the mitotic function by TPX2 and IMPß, and the nucleo-cytoplasmic trafficking function by XPO7, XPOT and IMPß. RESULTS: Based on Kaplan-Meier analyses, RAN, cytoplasmic XPO7 and TPX2 were significantly associated with poor overall patient survival, and RAN and TPX2 were associated with lower disease free survival in patients with high-grade serous carcinoma. Cox regression analysis revealed that RAN and TPX2 expression were independent prognostic factors for both overall and disease free survival, and that cytoplasmic XPO7 expression was a prognostic factor for overall patient survival. CONCLUSIONS: In this systematic study, we show that RAN and two protein partners involved in its nucleo-cytoplasmic and mitotic functions (XPO7 and TPX2, respectively) can be used as biomarkers to stratify patients based on prognosis. In particular, we reported for the first time the clinical relevance of the exportin XPO7 and showed that TPX2 expression had the strongest prognostic value. These findings suggest that protein partners in each of RAN's functions can discriminate between different outcomes in high-grade serous epithelial ovarian cancer patients. Furthermore, these proteins point to cellular processes that may ultimately be targeted to improve the survival in serous epithelial ovarian cancer.

Expression and role of the angiotensin II AT2 receptor in human prostate tissue: in search of a new therapeutic option for prostate cancer.

BACKGROUND: Evidence shows that angiotensin II type 1 receptor (AT1R) blockers may be associated with improved outcome in prostate cancer patients. It has been proposed that part of this effect could be due to angiotensin II type 2 receptor (AT2R) activation, the only active angiotensin II receptor in this situation. This study aimed to characterize the localization and expression of AT2R in prostate tissues and to assess its role on cell morphology and number in prostatic epithelial cells in primary culture. METHODS: AT2R and its AT2R-interacting protein (ATIP) expression were assessed on non-tumoral and tumoral human prostate using tissue microarray immunohistochemistry, binding assay, and Western blotting. AT2R effect on cell number was measured in primary cultures of epithelial cells from non-tumoral human prostate. RESULTS: AT2R was localized at the level of the acinar epithelial layer and its expression decreased in cancers with a Gleason score 6 or higher. In contrast, ATIP expression increased with cancer progression. Treatment of primary cell cultures from non-tumoral prostate tissues with C21/M024, a selective AT2R agonist, alone or in co-incubation with losartan, an AT1R antagonist, significantly decreased cell number compared to untreated cells. CONCLUSIONS: AT2R and ATIP are present in non-tumoral human prostate tissues and differentially regulated according to Gleason score. The decrease in non-tumoral prostate cell number upon selective AT2R stimulation suggests that AT2R may have a protective role against prostate cancer development. Treatment with a selective AT2R agonist could represent a new approach for prostate cancer prevention or for patients on active surveillance.

VGLL3 expression is associated with a tumor suppressor phenotype in epithelial ovarian cancer.

Previous studies have implicated vestigial like 3 (VGLL3), a chromosome 3p12.3 gene that encodes a putative transcription co-factor, as a candidate tumor suppressor gene (TSG) in high-grade serous ovarian carcinomas (HGSC), the most common type of epithelial ovarian cancer. A complementation analysis based on microcell-mediated chromosome transfer (MMCT) using a centric fragment of chromosome 3 (der3p12-q12.1) into the OV-90 ovarian cancer cell line haploinsufficient for 3p and lacking VGLL3 expression was performed to assess the effect on tumorigenic potential and growth characteristics. Genetic characterization of the derived MMCT hybrids revealed that only the hybrid that contained an intact VGLL3 locus exhibited alterations of tumorigenic potential in a nude mouse xenograft model and various in vitro growth characteristics. Only stable OV-90 transfectant clones expressing low levels of VGLL3 were derived. These clones exhibited an altered cytoplasmic morphology characterized by numerous single membrane bound multivesicular-bodies (MVB) that were not attributed to autophagy. Overexpression of VGLL3 in OV-90 was achieved using a lentivirus-based tetracycline inducible gene expression system, which also resulted in MVB formation in the infected cell population. Though there was no significant differences in various in vitro and in vivo growth characteristics in a comparison of VGLL3-expressing clones with empty vector transfectant controls, loss of VGLL3 expression was observed in tumors derived from mouse xenograft models. VGLL3 gene and protein expression was significantly reduced in HGSC samples (>98%, p < 0.05) relative to either normal ovarian surface epithelial cells or epithelial cells of the fallopian tube, possible tissues of origin of HGSC. Also, there appeared to be to be more cases with higher staining levels in stromal tissue component from HGSC cases that had a prolonged disease-free survival. The results taken together suggest that VGLL3 is involved in tumor suppressor pathways, a feature that is characterized by the absence of VGLL3 expression in HGSC samples.

p53 inhibits angiogenesis by inducing the production of Arresten.

Several types of collagen contain cryptic antiangiogenic noncollagenous domains that are released upon proteolysis of extracellular matrix (ECM). Among those is Arresten, a collagen-derived antiangiogenic factor (CDAF) that is processed from α1 collagen IV. However, the conditions under which Arresten is released from collagen IV in vivo or whether the protein functions in tumor suppressor pathways remain unknown. Here, we show that p53 induces the expression of α1 collagen IV and release of Arresten-containing fragments from the ECM. Comparison of the transcriptional activation of COL4A1 with other CDAF-containing genes revealed that COL4A1 is a major antiangiogenic gene induced by p53 in human adenocarinoma cells. p53 directly activated transcription of the COL4A1 gene by binding to an enhancer region 26 kbp downstream of its 3' end. p53 also stabilized the expression of full-length α1 collagen IV by upregulation of α(II) prolyl-hydroxylase and increased the release of Arresten in the ECM through a matrix metalloproteinase (MMP)-dependent mechanism. The resulting upregulation of α1 collagen IV and production of Arresten by the tumor cells significantly inhibited angiogenesis and limited tumor growth in vivo. Furthermore, we show that immunostaining of Arresten correlated with p53 status in human prostate cancer specimens. Our findings, therefore, link the production of Arresten to the p53 tumor suppressor pathway and show a novel mechanism through which p53 can inhibit angiogenesis.

An essential role for Ran GTPase in epithelial ovarian cancer cell survival.

BACKGROUND: We previously identified that Ran protein, a member of the Ras GTPase family, is highly expressed in high grade and high stage serous epithelial ovarian cancers, and that its overexpression is associated with a poor prognosis. Ran is known to contribute to both nucleocytoplasmic transport and cell cycle progression, but its role in ovarian cancer is not well defined. RESULTS: Using a lentivirus-based tetracycline-inducible shRNA approach, we show that downregulation of Ran expression in aggressive ovarian cancer cell lines affects cellular proliferation by inducing a caspase-3 associated apoptosis. Using a xenograft tumor assay, we demonstrate that depletion of Ran results in decreased tumorigenesis, and eventual tumor formation is associated with tumor cells that express Ran protein. CONCLUSION: Our results suggest a role for Ran in ovarian cancer cell survival and tumorigenicity and suggest that this critical GTPase may be suitable as a therapeutic target.

Immunohistochemical profiling of benign, low malignant potential and low grade serous epithelial ovarian tumors.

BACKGROUND: Serous epithelial ovarian tumors can be subdivided into benign (BOV), low malignant potential (LMP) or borderline and invasive (TOV) tumors. Although the molecular characteristics of serous BOV, LMP and low grade (LG) TOV tumors has been initiated, definitive immunohistochemical markers to distinguish between these tumor types have not been defined. METHODS: In the present study, we used a tissue array composed of 27 BOVs, 78 LMPs and 23 LG TOVs to evaluate the protein expression of a subset of selected candidates identified in our previous studies (Ape1, Set, Ran, Ccne1 and Trail) or known to be implicated in epithelial ovarian cancer disease (p21, Ccnb1, Ckd1). RESULTS: Statistically significant difference in protein expression was observed for Ccnb1 when BOV tumors were compared to LMP tumors (p = 0.003). When BOV were compared to LG TOV tumors, Trail was significantly expressed at a higher level in malignant tumors (p = 0.01). Expression of p21 was significantly lower in LG tumors when compared with either BOVs (p = 0.03) or LMPs (p = 0.001). We also observed that expression of p21 was higher in LMP tumors with no (p = 0.02) or non-invasive (p = 0.01) implants compared to the LMP associated with invasive implants. CONCLUSION: This study represents an extensive analyse of the benign and highly differentiated ovarian disease from an immunohistochemical perspective.

An apoptotic molecular network identified by microarray: on the TRAIL to new insights in epithelial ovarian cancer.

BACKGROUND: In a previous microarray expression analysis, the authors identified candidate genes that were expressed differentially between ovarian tumors with low malignant potential and invasive serous epithelial ovarian tumors. Among them, the apoptosis-related candidate genes tumor necrosis factor-related apoptosis-inducing ligand (TRAIL), caspase 8 (CASP8), FLICE-inhibitory protein (FLIP), and cytochrome C (CYC) were identified. METHODS: For the current study, the authors conducted immunohistochemical analyses of a tissue array comprised of 235 serous tumors of different grades and stages to evaluate whether there was differential protein expression for these candidates and for the 4 death cell receptors of Trail: Dr4, Dr5, DcR1, and DcR2. RESULTS: All proteins except DcR1 and DcR2 had significantly differential expression levels between grade 0 tumors (low malignant potential) and grade 2 and 3 tumors. Trail also showed differential expression between grade 0 tumors and grade 1 tumors. When all tumors were compared, the expression levels of Trail, Dr4, Dr5, DcR1, and Flip differed significantly between early-stage and advanced-stage disease. High Dr5 expression was associated with a poor prognosis in patients who had invasive tumors and in the subgroup of patients who had grade 3 tumors. Furthermore, the combinations of 2 proteins (Trail and Dr5, DcR2 and Cyc, Flip and Dr5, Flip and DcR2, DcR1 and Dr5 or Dr4 and Flip) revealed an association with patient prognosis. CONCLUSIONS: The identification of new proteins in the initial diagnosis and prognosis of patients with epithelial ovarian cancer may lead to a better understanding of the disease, highlighting new potential therapeutic targets, and may be useful in patient management.