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Introduction: High-grade serous ovarian cancer (HGSOC) is characterized by high mortality and prevalent recurrences. This study investigates the prognostic value of phosphoglycerate dehydrogenase (PHGDH) in HGSOC which has been linked to metabolic reprogramming and recurrences in other cancers., Methods: Data from 306 patients with advanced-stage HGSOC treated between 2008 and 2015 were analyzed. PHGDH expression levels were determined using immunohistochemistry and categorized as "low" or "high.", Results: PHGDH-high was associated with higher FIGO stage and increased use of neoadjuvant chemotherapy. Patients with PHGDH-high tumors had significantly worse survival than PHDH-low, even after adjusting for confounding factors., (© The Author(s) 2024. Published by Oxford University Press.)

The enzyme iodothyronine deiodinase type 3 (DIO3) contributes to cancer proliferation by inactivating the tumor-suppressive actions of thyroid hormone (T3). We recently established DIO3 involvement in the progression of high-grade serous ovarian cancer (HGSOC). Here we provide a link between high DIO3 expression and lower survival in patients, similar to common disease markers such as Ki67, PAX8, CA-125, and CCNE1. These observations suggest that DIO3 is a logical target for inhibition. Using a DIO3 mimic, we developed original DIO3 inhibitors that contain a core of dibromomaleic anhydride (DBRMD) as scaffold. Two compounds, PBENZ-DBRMD and ITYR-DBRMD, demonstrated attenuated cell counts, induction in apoptosis, and a reduction in cell proliferation in DIO3-positive HGSOC cells (OVCAR3 and KURAMOCHI), but not in DIO3-negative normal ovary cells (CHOK1) and OVCAR3 depleted for DIO3 or its substrate, T3. Potent tumor inhibition with a high safety profile was further established in HGSOC xenograft model, with no effect in DIO3-depleted tumors. The antitumor effects are mediated by downregulation in an array of pro-cancerous proteins, the majority of which known to be repressed by T3. To conclude, using small molecules that specifically target the DIO3 enzyme we present a new treatment paradigm for ovarian cancer and potentially other DIO3-dependent malignancies., (© 2021. The Author(s), under exclusive licence to Springer Nature Limited.)

Background: Aurora-A kinase is important for cellular proliferation and is implicated in the tumorigenesis of several malignancies, including of the ovary. Information regarding the expression patterns of Aurora-A in normal Müllerian epithelium as well as benign, borderline and malignant epithelial ovarian neoplasms is limited., Methods: We investigated Aurora-A expression by immunohistochemistry in 15 benign, 19 borderline and 17 malignant ovarian serous tumors, and 16 benign, 8 borderline, and 2 malignant ovarian mucinous tumors. Twelve fimbriae from seven patients served as normal Müllerian epithelium controls. We also examined Aurora-A protein expression by western blot in normal fimbriae and tumor specimens., Results: All normal fimbriae (n = 12) showed nuclear but not cytoplasmic Aurora-A immunoreactivity by immunohistochemistry. Benign ovarian tumors also showed strong nuclear Aurora-A immunoreactivity. Forty-eight percent (13/27) of borderline tumors demonstrated nuclear Aurora-A immunoreactivity, while the remainder (52%, 14/27) lacked Aurora-A staining. Nuclear Aurora-A immunoreactivity was absent in all malignant serous tumors, however, 47% (8/17) demonstrated perinuclear cytoplasmic staining. These results were statistically significant when tumor class (benign/borderline/malignant) was compared to immunoreactivity localization or intensity (Fisher Exact Test, p < 0.01). Western blot analysis confirmed the greater nuclear Aurora-A expression in control Müllerian epithelium compared to borderline and malignant tumors., Conclusion: Aurora-A kinase is differentially expressed across normal Müllerian epithelium, benign and borderline serous and mucinous ovarian epithelial neoplasms and malignant serous ovarian tumors., with nuclear expression of unphosphorylated Aurora-A being present in normal and benign neoplastic epithelium, and lost in malignant serous neoplasms. Further studies of the possible biological and clinical implications of the loss of nuclear Aurora-A expression in ovarian tumors, and its role in ovarian carcinogenesis are warranted., (© 2021. The Author(s).)

Deregulated full-length anaplastic lymphoma kinase (ALK) overexpression has been found in some primary solid tumors, but little is known about its role in ovarian high-grade serous carcinoma (HGSC). The current study focused on the functional roles of ALK in HGSC. Cytoplasmic ALK immunoreactivity without chromosomal rearrangement and gene mutations was significantly higher in HGSC compared with non-HGSC-type ovarian carcinomas, and was significantly associated with several unfavorable clinicopathologic factors and poor prognosis. HGSC cell lines stably overexpressing ALK exhibited increased cell proliferation, enhanced cancer stem cell features, and accelerated cell mobility, whereas these phenotypes were abrogated in ALK-knockdown cells. Expression of the nervous system-associated gene, ELAVL3, and the corresponding protein (commonly known as HuC) was significantly increased in cells overexpressing ALK. Expression of SRY-box transcription factor (Sox)2 and Sox3 (genes associated with the neural progenitor population) increased in ALK-overexpressing but not ALK-knockdown cells. Furthermore, overexpression of Sox2 or Sox3 enhanced both ALK and ELAVL3 promoter activities, suggesting the existence of ALK/Sox/HuC signaling loops. Finally, ALK overexpression was attributed to increased expression of neuroendocrine markers, including synaptophysin, CD56, and B-cell lymphoma 2, in HGSC tissues. These findings suggest that overexpression of full-length ALK may influence the biological behavior of HGSC through cooperation with ELAVL3 and Sox factors, leading to the establishment and maintenance of the aggressive phenotypic characteristics of HGSC., (Copyright © 2021 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Purpose: Low-grade serous ovarian carcinomas (LGSOCs) have historically low chemotherapy responses. Alterations affecting the MAPK pathway, most commonly KRAS/BRAF, are present in 30%-60% of LGSOCs. The purpose of this study was to evaluate binimetinib, a potent MEK1/2 inhibitor with demonstrated activity across multiple cancers, in LGSOC., Methods: This was a 2:1 randomized study of binimetinib (45 mg twice daily) versus physician's choice chemotherapy (PCC). Eligible patients had recurrent measurable LGSOC after ≥ 1 prior platinum-based chemotherapy but ≤ 3 prior chemotherapy lines. The primary end point was progression-free survival (PFS) by blinded independent central review (BICR); additional assessments included overall survival (OS), overall response rate (ORR), duration of response (DOR), clinical-benefit rate, biomarkers, and safety., Results: A total of 303 patients were randomly assigned to an arm of the study at the time of interim analysis (January 20, 2016). Median PFS by BICR was 9.1 months (95% CI, 7.3 to 11.3) for binimetinib and 10.6 months (95% CI, 9.2 to 14.5) for PCC (hazard ratio,1.21; 95%CI, 0.79 to 1.86), resulting in early study closure according to a prespecified futility boundary after 341 patients had enrolled. Secondary efficacy end points were similar in the two groups: ORR 16% (complete response [CR]/partial responses[PRs], 32) versus 13% (CR/PRs, 13); median DOR, 8.1 months (range, 0.03 to ≥ 12.0 months) versus 6.7 months (0.03 to ≥ 9.7 months); and median OS, 25.3 versus 20.8 months for binimetinib and PCC, respectively. Safety results were consistent with the known safety profile of binimetinib; the most common grade ≥ 3 event was increased blood creatine kinase level (26%). Post hoc analysis suggests a possible association between KRAS mutation and response to binimetinib. Results from an updated analysis (n = 341; January 2019) were consistent., Conclusion: Although the MEK Inhibitor in Low-Grade Serous Ovarian Cancer Study did not meet its primary end point, binimetinib showed activity in LGSOC across the efficacy end points evaluated. A higher response to chemotherapy than expected was observed and KRAS mutation might predict response to binimetinib.

High grade serous ovarian cancer (HGSOC) is a fatal gynecologic malignancy in the U.S. with limited treatment options. New therapeutic strategies include targeting of the cell cycle checkpoints, e.g., ATR and CHK1. We recently reported a promising clinical activity of the CHK1 inhibitor (CHK1i) prexasertib monotherapy in BRCA wild-type (BRCAwt) HGSOC patients. In this study, biopsies of treated patients and cell line models were used to investigate possible mechanisms of resistance to CHK1i. We report that BRCAwt HGSOC develops resistance to prexasertib monotherapy via a prolonged G2 delay induced by lower CDK1/CyclinB1 activity, thus preventing cells from mitotic catastrophe and cell death. On the other hand, we noted CHK1's regulation on RAD51-mediated homologous recombination (HR) repair was not altered in CHK1i-resistant cells. Therefore, CHK1i sensitizes CHK1i-resistant cells to DNA damaging agents such as gemcitabine or hydroxyurea by inhibition of HR. In summary, our results demonstrate new mechanistic insights of functionally distinct CHK1 activities and highlight a potential combination treatment approach to overcome CHK1i resistance in BRCAwt HGSOC.

Identifying cancer drivers and actionable mutations is critical for precision oncology. In epithelial ovarian cancer (EOC) the majority of mutations lack biological or clinical validation. We fully characterized 43 lines of Patient-Derived Xenografts (PDXs) and performed copy number analysis and whole exome sequencing of 12 lines derived from naïve, high grade EOCs. Pyrosequencing allowed quantifying mutations in the source tumours. Drug response was assayed on PDX Derived Tumour Cells (PDTCs) and in vivo on PDXs. We identified a PIK3R1 W624R variant in PDXs from a high grade serous EOC. Allele frequencies of PIK3R1 W624R in all the passaged PDXs and in samples of the source tumour suggested that it was truncal and thus possibly a driver mutation. After inconclusive results in silico analyses, PDTCs and PDXs allowed the showing actionability of PIK3R1 W624R and addiction of PIK3R1 W624R carrying cells to inhibitors of the PI3K/AKT/mTOR pathway. It is noteworthy that PIK3R1 encodes the p85α regulatory subunit of PI3K, that is very rarely mutated in EOC. The PIK3R1 W624R mutation is located in the cSH2 domain of the p85α that has never been involved in oncogenesis. These data show that patient-derived models are irreplaceable in their role of unveiling unpredicted driver and actionable variants in advanced ovarian cancer., Competing Interests: The authors declare no competing interests.

Ovarian cancer has a high death rate and is often not detected until late stages. While some studies found high expressions of MMPs correlated with cancer invasion, metastasis, and poor prognosis, however, several other studies indicated MMPs might inhibit cancer rather than promote cancer in certain situations. Thus, the role of different MMPs in different cancer types needs a systematic re-evaluation. In this study, we used RNA-Seq and corresponding clinical data downloaded from TCGA and analyzed the correlations between MMP expressions and the clinicopathologic characteristics and outcome in ovarian serous cystadenocarcinoma (OSC) patients. Among the MMPs investigated, MMP-3 was significantly increased in high-grade and high-stage tumors compared with low-grade and low-stage ones. Using univariate analysis and multivariate Cox model, high expressions of MMP-19 and -20 were found to associate with poor overall survival independent of clinicopathologic characteristics. Moreover, using in vitro studies, we found ovarian cancer cell lines with higher MMP-19 and -20 protein expressing levels were associated with anti-cancer drugs resistance, while knockdown of MMP-19 or -20 increased ovarian cancer cell sensitivities to several clinical using chemotherapy agents. Finally, knockdown of MMP-19 or -20 also decreased the invasion abilities of several ovarian cancer cell lines. These in vitro studies provided potential mechanisms of high MMP-19 and -20 expressions in the poor prognosis of ovarian cancer.

High grade serous ovarian cancer (HGSOC) is the most common and aggressive ovarian cancer subtype with the worst clinical outcome due to intrinsic or acquired drug resistance. Standard treatment involves platinum compounds. Cancer development and chemoresistance is often associated with an increase in histone deacetylase (HDAC) activity. The purpose of this study was to examine the potential of HDAC inhibitors (HDACi) to increase platinum potency in HGSOC. Four HGSOC cell lines with different cisplatin sensitivity were treated with combinations of cisplatin and entinostat (class I HDACi), panobinostat (pan-HDACi), or nexturastat A (class IIb HDACi), respectively. Inhibition of class I HDACs by entinostat turned out superior in increasing cisplatin potency than pan-HDAC inhibition in cell viability assays (MTT), apoptosis induction (subG1), and caspase 3/7 activation. Entinostat was synergistic with cisplatin in all cell lines in MTT and caspase activation assays. MTT assays gave combination indices (CI values) < 0.9 indicating synergism. The effect of HDAC inhibitors could be attributed to the upregulation of pro-apoptotic genes ( CDNK1A , APAF1 , PUMA , BAK1 ) and downregulation of survivin . In conclusion, the combination of entinostat and cisplatin is synergistic in HGSOC and could be an effective strategy for the treatment of aggressive ovarian cancer.

Objective: The objective of this study was to analyze the expression level and clinical role of soluble AXL (sAXL) in cancers affecting the serosal surfaces, with focus on ovarian carcinoma., Methods: sAXL protein expression by ELISA was analyzed in 572 effusion supernatants, including 424 peritoneal, 147 pleural and 1 pericardial specimens., Results: sAXL was overexpressed in peritoneal effusions compared to pleural and pericardial specimens (p < 0.001). sAXL levels were additionally significantly higher in effusions from patients with ovarian carcinoma, malignant mesothelioma and breast carcinoma compared to specimens from patients with other cancers (predominantly carcinomas of lung, gastrointestinal or uterine corpus/cervix origin) or benign reactive effusions (p < 0.001). sAXL was further overexpressed in high-grade serous carcinoma (HGSC; n = 373) compared to low-grade serous carcinoma (LGSC; n = 32; p = 0.036). In HGSC, sAXL levels were significantly lower in post-chemotherapy effusions compared to primary diagnosis pre-chemotherapy specimens (p = 0.002). sAXL levels in HGSC were unrelated to chemoresponse at diagnosis, progression-free survival or overall survival. Levels were similarly unrelated to survival in LGSC and breast carcinoma., Conclusions: sAXL is widely expressed in malignant effusions, particularly in ovarian and breast carcinoma and in malignant mesothelioma. sAXL is overexpressed in HGSC compared to LGSC and its levels are lower following exposure to chemotherapy. However, sAXL levels are not informative of chemoresponse or survival., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Aim: To explore the expression profile of some DHRS genes in high-grade serous ovarian cancer (SOVC) and to study their prognostic values., Patients & Methods: A retrospective bioinformatic analysis was performed using data in the Gene Expression Omnibus, the Human Protein Atlas and the Cancer Genome Atlas-Ovarian Cancer., Results: Increased DHRS12 expression was an independent indicator of poor overall survival (hazard ratio [HR]: 1.265, 95% CI: 1.075-1.488; p = 0.005) and recurrence-free survival (RFS; HR: 2.242, 95%CI: 1.464-3.432; p < 0.001) in patients with high-grade SOVC. DNA deletion was associated with decreased DHRS12 expression, as well as the best overall survival and RFS among the three copy number alteration groups., Conclusion: DHRS12 might serve as a valuable prognostic biomarker in high-grade SOVC.

Purpose Uterine serous carcinoma is a rare, aggressive variant of endometrial cancer. Trastuzumab is a humanized monoclonal antibody that targets human epidermal growth factor receptor 2 (HER2)/neu, a receptor overexpressed in 30% of uterine serous carcinoma. This multicenter, randomized phase II trial compared carboplatin-paclitaxel with and without trastuzumab in patients with advanced or recurrent uterine serous carcinoma who overexpress HER2/neu. Methods Eligible patients had primary stage III or IV or recurrent HER2/neu-positive disease. Participants were randomly assigned to receive carboplatin-paclitaxel (control arm) for six cycles with or without intravenous trastuzumab (experimental arm) until progression or unacceptable toxicity. The primary end point was progression-free survival, which was assessed for differences between treatment arms via one-sided log-rank tests. Results From August 2011 to March 2017, 61 patients were randomly assigned. Forty progression-free survival-related events occurred among 58 evaluable participants. Among all patients, median progression-free survival was 8.0 months (control) versus 12.6 months (experimental; P = .005; hazard ratio [HR], 0.44; 90% CI, 0.26 to 0.76). Similarly, median progression-free survival was 9.3 (control) versus 17.9 (experimental) months among 41 patients with stage III or IV disease undergoing primary treatment ( P = .013; HR, 0.40; 90% CI, 0.20 to 0.80) and 6.0 (control) versus 9.2 months (experimental), respectively, among 17 patients with recurrent disease ( P = .003; HR, 0.14; 90% CI, 0.04 to 0.53). Toxicity was not different between treatment arms, and no unexpected safety signals emerged. Conclusion Addition of trastuzumab to carboplatin-paclitaxel was well tolerated and increased progression-free survival. These encouraging results deserve further investigation to determine their impact on overall survival in patients with advanced or recurrent uterine serous carcinoma who overexpress HER2/neu.

Glutamate-ammonia ligase (GLUL), which is also called GS (glutamine synthetase), is the enzyme that catalyzes the synthesis of glutamine from glutamate and ammonia in an ATP-dependent reaction. Here, we found higher expression of GLUL in the ovarian cancer patients was associated with worse disease-free survival (DFS) and overall survival (OS). In addition, GLUL was heterogeneously expressed in various ovarian cancer cells. The mRNA and protein expression levels of GLUL in NIH:OVCAR-3 and ES-2 cells were obviously higher than that in the other types of ovarian cancer cells. Knockdown of GLUL in NIH:OVCAR-3 or ES-2 cells could significantly decrease the proliferation ability. Furthermore, GLUL knockdown markedly inhibited the p38 MAPK signaling pathway in NIH:OVCAR-3 or ES-2 cells. Our findings suggest that decreasing expression of GLUL may be a new approach that can be used for ovarian cancer treatment., (© 2018 Wiley Periodicals, Inc.)

Objective: To analyze the expression and clinical role of CHK1 and CHK2 in metastatic high-grade serous carcinoma (HGSC)., Methods: HGSC effusions (n = 335; 280 peritoneal, 55 pleural) were analyzed for protein expression of total CHK1 and its phosphorylated forms p-ser317 and p-ser296, as well as total CHK2 and its phosphorylated form p-thr68 using immunohistochemistry. Expression was analyzed for association with clinicopathologic parameters, including chemotherapy response, and survival., Results: Carcinoma cells stained positive, predominantly at the nuclei, in the majority of cases (range 83-100% for the five antibodies), while expression in reactive mesothelial cells and tumor-associated macrophages was more variable. Total CHK1 (p = 0.037), p-CHK1ser317 (p = 0.001), p-CHK1ser296 (p = 0.002) and p-CHK2thr68 (p < 0.001) expression was significantly higher in post-chemotherapy disease recurrence compared to pre-chemotherapy effusions obtained at diagnosis. CHK1, p-CHK1ser296, p-CHK2thr68 and p-CHK1ser317 nuclear expression was positively related to expression of the checkpoint regulator WEE1, previously studied in this cohort (p = 0.003, p = 0.013, p = 0.001 and p = 0.01, respectively). Higher total CHK1 (p = 0.007), p-CHK1ser317 (p = 0.004), CHK2 (p = 0.01) and p-CHK2thr68 (p = 0.048) expression was significantly related to shorter overall survival in univariate analysis, and CHK1ser317 was an independent prognostic marker in multivariate analysis (p = 0.025). Higher p-CHK1ser317 (p = 0.03) and CHK2 (p = 0.034) expression was additionally associated with poor progression-free survival., Conclusions: CHK1 and CHK2 and their activated forms are frequently expressed in HGSC effusions, with higher expression following exposure to chemotherapy, and their expression is related to survival., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Purpose: Gene expression of a variety of the 15 members of the KLK serine protease family is dysregulated in ovarian cancer. We aimed at determining the clinical relevance of KLK13 and KLK14 mRNA expression in tumor tissues of a homogeneous patient cohort afflicted with advanced high-grade serous ovarian cancer (FIGO stage III/IV)., Methods: mRNA expression levels of KLK13 and KLK14 were assessed by quantitative PCR in tumor tissue of 91 patients and related with clinical factors and patients' outcome., Results: There was no significant association of KLK13 and KLK14 mRNA expression with the clinical factors ascitic fluid volume or residual tumor mass. In univariate Cox regression analysis, elevated KLK13 mRNA levels were significantly linked with shorter progression-free (PFS; hazard ratio [HR] = 1.97, P = 0.020) and overall survival (OS; HR = 1.81, P = 0.041). High KLK14 mRNA levels were significantly associated with prolonged PFS (HR = 0.44, P = 0.017) and showed a trend towards significance for OS (HR = 0.55, P = 0.070). In multivariable analysis, including the factors age, residual tumor mass, ascitic fluid volume, KLK13, and KLK14, both KLKs, apart from residual tumor mass, remained statistically independent predictive markers: patients with high KLK13 mRNA expression levels displayed a more than twofold increase risk for shorter PFS (HR = 2.14, P = 0.020) as well as OS (HR = 2.05, P = 0.028), whereas elevated KLK14 mRNA values were found to be significant for both, prolonged PFS (HR = 0.36, P = 0.007) and OS (HR = 0.46, P = 0.037)., Conclusion: These results indicate that in advanced high-grade serous ovarian cancer KLK13 may become proficient for tumor-supporting functions, whereas KLK14 may have adopted tumor-suppressing activity.

Objective: High grade serous ovarian cancer (HGSC) remains one of the most lethal malignancies in females. We previously reported that γ-glutamyl cyclotransferase (GGCT) was significantly upregulated in serous ovarian cancer. The current study was aimed to explore the function and underlying mechanism of GGCT in HGSC., Methods: GGCT expression was assessed by immunohistochemistry in 128 HGSC patients. Stable cell lines with GGCT gene overexpression or knockdown were established to investigate the function of GGCT in HGSC in vitro and in vivo., Results: GGCT is highly upregulated in HGSC tissues and associated with FIGO stage, lymph node metastasis and ascitic fluid volume. High expression of GGCT is associated with poor survival in HGSC patients. The Harrell's c-indexes of the prognostic models for overall survival and progression-free survival prediction were 0.758 and 0.726, respectively. GGCT knockdown suppresses proliferation, clone formation, migration, and invasion of tumor cells in vitro while forced GGCT overexpression presents opposite results. Furthermore, GGCT silencing inhibits tumor growth and spread in vivo. Epithelial-mesenchymal transition (EMT) and PI3K/AKT/mTOR signaling pathway are suppressed in GGCT silenced cells and enhanced in GGCT overexpressed cells. Inactivation of PI3K/AKT/mTOR signaling pathway in GGCT overexpressed cells induces EMT inhibition., Conclusions: Our data reveals an important role of GGCT in regulating EMT and progression of HGSC, providing a valuable prognostic marker and potential target for treatment of HGSC patients., (Published by Elsevier Inc.)

Protein glycosylation perturbations are implicated in a variety of diseases, including cancer. Aberrant glycosylation in cancer is frequently attributed to altered expression of polypeptide GalNAc transferases (GalNAc‑Ts) - enzymes initiating mucin-type O-glycosylation. A previous study from our group demonstrated that one member of this family (GALNT3) is overexpressed in epithelial ovarian cancer (EOC), and GALNT3 expression correlated with shorter progression-free survival (PFS) in EOC patients with advanced disease. As considerable degree of redundancy between members of the GalNAc‑Ts gene family has been frequently observed, we decided to investigate whether other members of this family are essential in EOC progression. In silico analysis based on publically available data was indicative for altered expression of five GalNAc‑Ts (GALNT2, T4, T6, T9 and T14) in ovarian high-grade serous carcinoma (HGSC) samples compared to non-tumoral (control) ovarian tissue. We analyzed protein expression of these GalNAc‑Ts in EOC cells and tumors by western blotting, followed by immunohistochemical (IHC) evaluation of their expression in EOC tumor and control samples using tissue microarrays (TMAs). Western blot analyses were indicative for low expression of GALNT2 and strong expression of GALNT6, T9 and T14 in both EOC cells and tumors. These observations were confirmed by IHC. GALNT2 displayed significantly lower expression, while GALNT6, GALNT9 and GALNT14 showed significantly higher expression in HGSC tumors compared to control tissue. Importantly, GALNT6 and GALNT14 expression correlated with poor prognosis of serous EOC patients. Moreover, our results suggest for overlapping functions of some GalNAc‑Ts, more specifically GALNT3 and GALNT6, in directing EOC progression. Our results are indicative for a possible implication of different members of the GalNAc‑T gene family in modulating EOC progression, and the potential use of GALNT6 and GALNT14 as novel prognostic EOC biomarkers. These data warrant future studies on the role of members of the GalNAc‑Ts gene family in ovarian tumorigenesis.

We aimed to identify the possible role of oxidant-antioxidant status in epithelial ovarian cancer (EOC) by measuring (a) antioxidant enzyme (AOE) activities [total superoxide dismutase (SOD total ), manganese-SOD (Mn-SOD), copper,zinc-SOD (Cu,Zn-SOD), catalase (CAT) and glutathione peroxidase (GPx1)], (b) Mn-SOD protein expression, (c) lipid peroxidation markers [malondialdehyde (MDA), 8-epi-prostaglandin-F2α (8-epi-PGF2α)] and by evaluating the possible correlations between tumor biomarkers, reproductive hormone levels and all measured parameters, comprehensively. The data obtained from the patients with EOC (M, n = 26) evaluated according to the histopathological/clinical characteristics of tumors and compared with data of healthy controls [C tissue (C1) and C blood/urine (C2), n = 30, respectively). Significantly, low activities of tumor SOD total (52%), Mn-SOD (42%), Cu,Zn-SOD (55%); high activities of tumor and erythrocyte CAT (66%, 33% respectively) and tumor GPx1 (60%); high levels of tumor Mn-SOD protein expression; tumor MDA (193%) and urinary 8-epi-PGF2α (179%) were observed in serous EOC tumors (M1, n = 18) compared with controls (P < 0.05). However, higher levels of tumor MDA, Mn-SOD protein expression and urinary 8-epi-PGF2α were observed along with lower tumor CAT activity in poorly differentiated or undifferentiated (grade 3, G 3) versus well or moderately well differentiated (grade 1-2, G 1-2) serous EOC tumors. Obtained data indicate the presence of a severe redox imbalance in EOC and draw attention to the criticial role of AOEs in the pathogenesis of the disease. © 2017 IUBMB Life, 69(10):802-813, 2017., (© 2017 International Union of Biochemistry and Molecular Biology.)

Objective: Lysyl oxidase (LOX) is an enzyme that catalyzes the cross-linking of collagen and elastin in the extracellular matrix, thus controlling the tensile strength of tissues. Along with this primary function, there are evidences supporting a role for LOX in many critical biological functions, including gene expression regulation, cell growth, adhesion and migration. Accordingly, recent studies have supported a pivotal role for LOX in cancer progression and metastasis. The current study aimed at investigating the prognostic significance and the functional role of intracellular LOX in ovarian cancer., Methods: To this end, we analyzed LOX expression by immunohistochemistry in archived tumor material from advanced high grade serous ovarian cancer (HGSOC) patients (n=70) and correlated data with clinicopathological parameters and with response to chemotherapy. In vitro experiments were also used to investigate the functional consequences of LOX expression on behavioral aspects of HGSOC cells., Results: Our results showed that nuclear LOX expression is associated with unfavorable outcome in advanced HGSOC, being an independent prognostic factor for disease recurrence. Besides, high nuclear levels were seen to be associated with resistance to first-line chemotherapy. Through gene expression modulation experiments in HGSOC cell lines, we demonstrate that LOX positively regulates cell proliferation, migration and anchorage-independent growth., Conclusions: Collectively, our data suggest that LOX functions as a tumor promoter in HGSOC and positively regulates several aspects of the metastatic cascade., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Ovarian serous carcinoma (OSC) is the most common epithelial ovarian cancer. Inorganic pyrophosphatase (PPA1) catalyzes the hydrolysis of pyrophosphate to inorganic phosphate, thereby providing extra energy for metabolism. The significance of PPA1 in the prognosis of OSC has not been investigated. Our study aimed to explore the expression and predictive role of PPA1 in OSC progression. We screened the expression of PPA1 protein in OSC tissues from 139 patients by immunohistochemistry, and evaluated its correlation with clinicopathological characteristics. PPA1 was categorized as high expression in 58 OSC cases (41.7%), which was correlated with poor differentiation, positive lymph node (LN) metastasis and advanced FIGO (The International Federation of Gynecology and Obstetrics) stages. Univariate and multivariate analyses identified PPA1 as a novel independent prognostic biomarker in OSC patients; meanwhile, conventional factors such as LN status and FIGO stages also showed statistical significance. Moreover, the expression levels of PPA1 protein were higher in A2780 and OVCAR3 human ovarian cancer cell lines than those in normal ovarian surface epithelial cells. Using these ovarian cancer cell lines, we showed that PPA1 overexpression caused the decrease in the expression level of p53, the tumor suppressor, with the increase in β-catenin level, as determined by Western blot analysis. Conversely, knockdown of PPAI expression was associated with the increase of p53 level and the decreased of β-catenin level. Consistently, the proliferation and invasion capacities of ovarian cancer cells were enhanced upon PPA1 overexpression. In conclusion, PPA1 serves as a potential prognostic biomarker for patients with OSC.

The expression of epidermal growth factor (EGF) receptors is common in human carcinomas and the proteins are used as therapeutics targets. In this study, we analyzed the immunoexpression of EGFR, HER2 (EGFR2) and HER3 (EGFR3) in 41 cases of serous borderline ovarian tumors and carcinomas, in relation to the degree of differentiation and tumor stage. The quantification of the results was done using the final staining score (FSS), which took into account the number of labeled cells and the intensity of immunoreactions. For all the receptors, the FSS values corresponding to the high-grade serous carcinomas were significantly superior compared with low-grade carcinomas and borderline tumors. Also, the FSS values associated with advanced stages ovarian tumors were significantly superior compared to those in the initial stages. In this study, we found positive linear correlations between the values associated with the expression of EGF receptors. The relation of EGFR, HER2 and HER3 immunoexpression with the lesion subtype, tumor grade and stage, designates the EGF receptors system as possible therapeutic target in ovarian serous tumors.

Ovarian carcinoma is the leading cause of death from gynecologic malignancies. The oncogenic role of anaplastic lymphoma kinase (ALK) is well characterized in many hematopoietic and solid tumors. ALK expression in ovarian carcinoma has been reported but the exact status of ALK protein and its association with clinicopathologic features requires further investigation. ALK expression was determined by immunohistochemistry in 110 primary ovarian carcinomas, including 85 cases of serous carcinoma and 25 cases of mucinous carcinoma. Fluorescence in situ hybridization (FISH) and real-time reverse transcription polymerase chain reaction (RT-PCR) were used for evaluating ALK translocation in ALK-positive ovarian carcinomas. Among 110 ovarian carcinomas, 23 (20.9%) cases were ALK positive by immunohistochemistry. All ALK-positive cases were ovarian high-grade serous carcinoma. ALK expression was detected in 23/85 (27.1%) ovarian serous carcinoma and 0/25 (0%) in ovarian mucinous carcinoma. None of the 23 ALK IHC-positive cases harbored ALK gene translocations by FISH or RT-PCR. ALK protein expression was associated with patient age, tumor stage, and histologic type. Specifically, the probability of ALK protein expression was significantly higher in high-grade serous carcinomas in older patients (above 50 y) with advanced disease (FIGO stage III and IV) compared with the low-grade serous and mucinous carcinomas in younger patients with relatively early disease. In conclusion, aberrant ALK expression is observed in ovarian serous carcinoma but not in mucinous carcinoma, is independent of gene translocation, and might be associated with progression and prognosis.

Background: Amplified centrosomes are widely recognized as a hallmark of cancer. Although supernumerary centrosomes would be expected to compromise cell viability by yielding multipolar spindles that results in death-inducing aneuploidy, cancer cells suppress multipolarity by clustering their extra centrosomes. Thus, cancer cells, with the aid of clustering mechanisms, maintain pseudobipolar spindle phenotypes that are associated with low-grade aneuploidy, an edge to their survival. KIFC1, a nonessential minus end-directed motor of the kinesin-14 family, is a centrosome clustering molecule, essential for viability of extra centrosome-bearing cancer cells. Given that ovarian cancers robustly display amplified centrosomes, we examined the overexpression of KIFC1 in human ovarian tumors., Results: We found that in clinical epithelial ovarian cancer (EOC) samples, an expression level of KIFC1 was significantly higher when compared to normal tissues. KIFC1 expression also increased with tumor grade. Our In silico analyses showed that higher KIFC1 expression was associated with poor overall survival (OS) in serous ovarian adenocarcinoma (SOC) patients suggesting that an aggressive disease course in ovarian adenocarcinoma patients can be attributed to high KIFC1 levels. Also, gene expression levels of KIFC1 in high-grade serous ovarian carcinoma (HGSOC) highly correlated with expression of genes driving centrosome amplification (CA), as examined in publically-available databases. The pathway analysis results indicated that the genes overexpressed in KIFC1 high group were associated with processes like regulation of the cell cycle and cell proliferation. In addition, when we performed gene set enrichment analysis (GSEA) for identifying the gene ontologies associated to KIFC1 high group, we found that the first 100 genes enriched in KIFC1 high group were from centrosome components, mitotic cell cycle, and microtubule-based processes. Results from in vitro experiments on well-established in vitro models of HGSOC (OVSAHO, KURAMOCHI), OVCAR3 and SKOV3) revealed that they display robust centrosome amplification and expression levels of KIFC1 was directly associated (inversely correlated) to the status of multipolar mitosis. This association of KIFC1 and centrosome amplification with HGSOC might be able to explain the increased aggressiveness in this disease., Conclusion: These findings compellingly underscore that KIFC1 can be a biomarker that predicts an aggressive disease course in ovarian adenocarcinomas.

Objective: To analyze the expression profile of placental type alkaline phosphatase (PLAP), cancer antigen 125 (CA125), and human epididymis protein 4 (HE4) in serous ovarian cancer and to correlate their expression with the tumor aggressiveness and progression., Methods: Retrospective study considering a tissue microarray of 82 women affected by ovarian serous cancer. Protein expression was assessed by immunohistochemistry on ovarian serous cancer tissue samples. Immunohistochemical staining was semiquantitatively evaluated as H-score., Results: Median H-score values were lower for PLAP, 1 (IQR 0-4) than CA125, 10 (IQR 6-12) or HE4, 8 (IQR 5-12). Even if PLAP was less expressed in the cells of serous ovarian cancer than CA125 or HE4 it was relatively more expressed in the fourth quartile of its H-score distribution among cases with low CA125 or HE4 expression. Furthermore, PLAP and HE4 high expression resulted to be significantly correlated with a better prognosis., Conclusions: PLAP could be an additional marker for early detection of serous ovarian carcinoma, together with the established CA125 and HE4. In addition, PLAP expression is correlated with prognosis, giving, in this way, an additional tool for improving treatment approach.

Ovarian cancer is a silent disease with a poor prognosis that urgently requires new therapeutic strategies. In low-grade ovarian tumours, mutations in the MAP3K BRAF gene constitutively activate the downstream kinase MEK. Here we demonstrate that an additional MAP3K, MAP3K8 (TPL-2/COT), accumulates in high-grade serous ovarian carcinomas (HGSCs) and is a potential prognostic marker for these tumours. By combining analyses on HGSC patient cohorts, ovarian cancer cells and patient-derived xenografts, we demonstrate that MAP3K8 controls cancer cell proliferation and migration by regulating key players in G1/S transition and adhesion dynamics. In addition, we show that the MEK pathway is the main pathway involved in mediating MAP3K8 function, and that MAP3K8 exhibits a reliable predictive value for the effectiveness of MEK inhibitor treatment. Our data highlight key roles for MAP3K8 in HGSC and indicate that MEK inhibitors could be a useful treatment strategy, in combination with conventional chemotherapy, for this disease.

Objective: To reassess the effect cyclooxygenase-2 (COX-2) expression in carcinosarcoma on survival based on mature 5-year survival data., Method: A comparison of 5-year survival of 27 patients with carcinosarcoma according to the presence of COX-2 immunohistochemical staining and staining score was performed., Results: The 5-year survival of those with positive and negative COX-2 staining was statistically not different. However, there was a clear trend for more favorable 5-year survival in patients with a high staining score than in those with a low score, and the difference was of borderline significance (38.5% vs 7.1%; P = 0.06)., Conclusion: In view of the role of COX-2 in carcinogenesis, our finding that COX-2 expression may confer a better survival in patients with carcinosarcoma is intriguing. Larger studies are indicated to elucidate the effect of COX-2 expression on survival in patients with carcinosarcoma because this may have therapeutic implications.

Background: Accumulating studies reveal that aberrant microRNA (miRNA) expression can affect the development of chemotherapy drug resistance by modulating the expression of relevant target proteins. The aim of this study was to investigate the role of miR-133b in the development of drug resistance in ovarian cancer cells., Methods: We examined the levels of miR-133b expression in ovarian carcinoma tissues and the human ovarian carcinoma cell lines (A2780, A2780/DDP and A2780/Taxol, respectively). We determined the cell viability of these cell lines treated with cisplatin or paclitaxel in the presence or absence of miR-133b or anti-miR-133b transfection using the MTT assay. Reverse transcription polymerase chain reaction and Western blotting were used to assess the mRNA and protein expression levels of two drug-resistance-related genes: glutathione S-transferase (GST)-π and multidrug resistance protein 1 (MDR1). The dual-luciferase reporter assay was used to detect the promoter activity of GST-π in the presence and absence of miR-133b., Results: The expression of miR-133b was significantly lower in primary resistant ovarian carcinomas than in the chemotherapy-sensitive carcinomas (P<0.05), and the same results were found in primary resistant ovarian cell lines (A2780/Taxol and A2780/DDP) compared to the chemotherapy-sensitive cell line (A2780; P<0.05). Following miR-133b transfection, four cell lines showed increased sensitivity to paclitaxel and cisplatin, while anti-miR-133b transfection reduced cell sensitivity to paclitaxel and cisplatin. Dual-luciferase reporter assay showed that miR-133b interacted with the 3'-untranslated region of GST-π. Compared to controls, the mRNA and protein levels of MDR1 and GST-π were downregulated after miR-133b transfection and upregulated after anti-miR-133b transfection., Conclusion: MicroRNA-133b may reduce ovarian cancer drug resistance by silencing the expression of the drug-resistance-related proteins, GST-π and MDR1. In future, the combination of miR-133b with chemotherapy agents may prevent the development of drug resistance in ovarian cancers.

Objectives: To compare the molecular profile of a large cohort of uterine papillary serous carcinoma (UPSC) and ovarian serous carcinoma (EOC-S)., Methods: 628 UPSC and 5335 EOC-S tumors were evaluated using a commercial multiplatform profiling service (CARIS Life Sciences, Phoenix, AZ). Specific testing performed included a combination of gene sequencing (Sanger, next generation sequencing), protein expression (IHC) and gene amplification (CISH or FISH)., Results: TP53 was the most commonly mutated gene in both UPSC and EOC-S (76% vs. 69%, P = 0.03). UPSC were more likely to have mutation in PIK3CA (29% vs. 2%, P < 0.001), FBXW7 (12% vs. 1%, P < 0.001), KRAS (9% vs. 5%, P < 0.001) PTEN (7% vs. 1%, P < 0.001), and CTNNB1 (2% vs. 0%, P < 0.001) compared to EOC-S. On the other hand, EOC-S were more likely to harbor mutation in BRCA1 (20% vs. 9% P = 0.17) and BRCA2 (18% vs. 6% P = 0.09). HER2 gene amplification (17% vs. 4%, P < 0.001) and Her2/neu expression (10% vs. 2%, P < 0.001) were more frequent in UPSC than EOC-S, respectively., Conclusion: UPSC have a distinct mutation profile indicating higher activity of PI3K/AKT/mTOR, and MAPK pathways and Her2 expression/amplification but a trend toward lower frequency of alteration in homologous recombination pathway compared to EOC-S. Targeted PI3K/AKT/mTOR inhibitors should be evaluated in UPSC., (© 2015 Wiley Periodicals, Inc.)

Ovarian cancer, the most lethal gynaecological cancer, is characterised by the shedding of epithelial cells from the ovarian surface, followed by metastasis and implantation onto the peritoneal surfaces of abdominal organs. Our proteomic studies investigating the interactions between peritoneal (LP-9) and ovarian cancer (OVCAR-5) cells found transketolase (TKT) to be regulated in the co-culture system. This study characterized TKT expression in advanced stage (III/IV) serous ovarian cancers (n = 125 primary and n = 54 peritoneal metastases), normal ovaries (n = 6) and benign serous cystadenomas (n = 10) by immunohistochemistry. In addition, we also evaluated the function of TKT in ovarian cancer cells in vitro. Nuclear TKT was present in all primary serous ovarian cancer tissues examined (median 82.0 %, range 16.5-100 %) and was significantly increased in peritoneal metastases compared with matching primary cancers (P = 0.01, Wilcoxon Rank test). Kaplan-Meier survival and Cox regression analyses showed that high nuclear TKT positivity in peritoneal metastases (>94 %) was significantly associated with reduced overall survival (P = 0.006) and a 2.8 fold increased risk of ovarian cancer death (95 % CI 1.29-5.90, P = 0.009). Knockdown of TKT by siRNAs significantly reduced SKOV-3 cell proliferation but had no effect on their motility or invasion. Oxythiamine, an inhibitor of TKT activity, significantly inhibited the proliferation of four ovarian cancer cell lines (OV-90, SKOV-3, OVCAR-3 and OVCAR-5) and primary serous ovarian cancer cells isolated from patient ascites. In conclusion, these findings indicate that TKT plays an important role in the proliferation of metastatic ovarian cancer cells and could be used as novel therapeutic target for advanced disease.

High-grade serous ovarian cancer (HGSC) is among the most lethal forms of cancer in women. Excessive genomic rearrangements, which are expected to create fusion oncogenes, are the hallmark of this cancer. Here we report a cancer-specific gene fusion between BCAM, a membrane adhesion molecule, and AKT2, a key kinase in the PI3K signaling pathway. This fusion is present in 7% of the 60 patient cancers tested, a significant frequency considering the highly heterogeneous nature of this malignancy. Further, we provide direct evidence that BCAM-AKT2 is translated into an in-frame fusion protein in the patient's tumor. The resulting AKT2 fusion kinase is membrane-associated, constitutively phosphorylated, and activated as a functional kinase in cells. Unlike endogenous AKT2, whose activity is tightly regulated by external stimuli, BCAM-AKT2 escapes the regulation from external stimuli. Moreover, a BCAM-AKT2 fusion gene generated via chromosomal translocation using the CRISPR/Cas9 system leads to focus formation in both OVCAR8 and HEK-293T cell lines, suggesting that BCAM-AKT2 is oncogenic. Together, the results indicate that BCAM-AKT2 expression is a new mechanism of AKT2 kinase activation in HGSC. BCAM-AKT2 is the only fusion gene in HGSC that is proven to translate an aberrant yet functional kinase fusion protein with oncogenic properties. This recurrent genomic alteration is a potential therapeutic target and marker of a clinically relevant subtype for tailored therapy of HGSC.

Objective: Focal adhesion kinase (FAK) is overexpressed in serous ovarian cancer. Loss of merlin, a product of the neurofibromatosis 2 tumor suppressor gene, is being evaluated as a biomarker for FAK inhibitor sensitivity in mesothelioma. Connections between merlin and FAK in ovarian cancer remain undefined., Methods: Nine human and two murine ovarian cancer cell lines were analyzed for growth in the presence of a small molecule FAK inhibitor (PF-271, also termed VS-6062) from 0.1 to 1 μM for 72 h. Merlin was evaluated by immunoblotting and immunostaining of a human ovarian tumor tissue array. Growth of cells was analyzed in an orthotopic tumor model and evaluated in vitro after stable shRNA-mediated merlin knockdown., Results: Greater than 50% inhibition of OVCAR8, HEY, and ID8-IP ovarian carcinoma cell growth occurred with 0.1 μM PF-271 in anchorage-independent (p<0.001) but not in adherent culture conditions. PF-271-mediated reduction in FAK Y397 phosphorylation occurred independently of growth inhibition. Suspended growth of OVCAR3, OVCAR10, IGROV1, IGROV1-IP, SKOV3, SKOV3-IP, A2780, and 5009-MOVCAR was not affected by 0.1 μM PF-271. Merlin expression did not correlate with serous ovarian tumor grade or stage. PF-271 (30 mg/kg, BID) did not inhibit 5009-MOVCAR tumor growth and merlin knockdown in SKOV3-IP and OVCAR10 cells did not alter suspended cell growth upon PF-271 addition., Conclusions: Differential responsiveness to FAK inhibitor treatment was observed. Intrinsic low merlin protein level correlated with PF-271-mediated anchorage-independent growth inhibition, but reduction in merlin expression did not induce sensitivity to FAK inhibition. Merlin levels may be useful for patient stratification in FAK inhibitor trials., (Copyright © 2014 Elsevier Inc. All rights reserved.)

"Warburg effect" emphasizes that malignant cells exhibit active glycolysis even under aerobic conditions. Hexokinase 2 (HK2) is a key glycolytic enzyme that helps to exhibit a "Warburg effect". In the present study, the main aim was to detect the expression of HK2 in epithelial ovarian tumor tissues. Immunohistochemistry and qRT-PCR were used to examine the expression of HK2 in different epithelial ovarian tissues. The expression of HK2 in ovarian cancer tissues was significantly higher than that in normal ovarian, benign, and borderline tumors both in protein (p < 0.001) and mRNA (p < 0.05) levels. HK2 expression was significantly higher in Stage III/IV compared to Stage III (p < 0.001). Expression of HK2 in poorly-differentiated carcinoma was higher than that in well-differentiated carcinoma (p = 0.008). The level of HK2 was higher in serous groups than in non-serous groups in both protein (p = 0.008) and mRNA (p < 0.05) level. Collectively, HK2 is highly expressed in epithelial ovarian cancer, especially in serous groups. Its expression is related with clinical stage and histological differentiation.

Small-molecule inhibitors of PARP1/2, such as olaparib, have been proposed to serve as a synthetic lethal therapy for cancers that harbor BRCA1 or BRCA2 mutations. Indeed, in clinical trials, PARP1/2 inhibitors elicit sustained antitumor responses in patients with germline BRCA gene mutations. In hypothesizing that additional genetic determinants might direct use of these drugs, we conducted a genome-wide synthetic lethal screen for candidate olaparib sensitivity genes. In support of this hypothesis, the set of identified genes included known determinants of olaparib sensitivity, such as BRCA1, RAD51, and Fanconi's anemia susceptibility genes. In addition, the set included genes implicated in established networks of DNA repair, DNA cohesion, and chromatin remodeling, none of which were known previously to confer sensitivity to PARP1/2 inhibition. Notably, integration of the list of candidate sensitivity genes with data from tumor DNA sequencing studies identified CDK12 deficiency as a clinically relevant biomarker of PARP1/2 inhibitor sensitivity. In models of high-grade serous ovarian cancer (HGS-OVCa), CDK12 attenuation was sufficient to confer sensitivity to PARP1/2 inhibition, suppression of DNA repair via homologous recombination, and reduced expression of BRCA1. As one of only nine genes known to be significantly mutated in HGS-OVCa, CDK12 has properties that should confirm interest in its use as a biomarker, particularly in ongoing clinical trials of PARP1/2 inhibitors and other agents that trigger replication fork arrest.

Ovarian cancer is a clinically and molecularly heterogeneous disease. The driving forces behind this variability are unknown. Here, we report wide variation in the expression of the DNA cytosine deaminase APOBEC3B, with elevated expression in the majority of ovarian cancer cell lines (three SDs above the mean of normal ovarian surface epithelial cells) and high-grade primary ovarian cancers. APOBEC3B is active in the nucleus of several ovarian cancer cell lines and elicits a biochemical preference for deamination of cytosines in 5'-TC dinucleotides. Importantly, examination of whole-genome sequence from 16 ovarian cancers reveals that APOBEC3B expression correlates with total mutation load as well as elevated levels of transversion mutations. In particular, high APOBEC3B expression correlates with C-to-A and C-to-G transversion mutations within 5'-TC dinucleotide motifs in early-stage high-grade serous ovarian cancer genomes, suggesting that APOBEC3B-catalyzed genomic uracil lesions are further processed by downstream DNA "repair" enzymes including error-prone translesion polymerases. These data identify a potential role for APOBEC3B in serous ovarian cancer genomic instability., (©2013 AACR.)

Objective: To evaluate c-erbB2 gene amplification in a series of primary uterine serous carcinoma (USC) cell lines. To assess the efficacy of AZD8055, a novel dual mTORC1/2 inhibitor against primary HER2/neu amplified vs HER2/neu not amplified USC cell lines., Methods: Twenty-two primary USC cell lines were evaluated for c-erbB2 oncogene amplification by FISH assays. In vitro sensitivity to AZD8055 was evaluated by flow-cytometry-based viability and proliferation assays. Cell cycle profile and downstream cellular responses to AZD8055 were assessed by measuring the DNA content of cells and by phosphorylation of the S6 protein by flow-cytometry., Results: Nine of 22 (40.9%) USC cell lines demonstrated c-erbB2 gene amplification by FISH. AZD8055 caused a strong differential growth inhibition in USC cell lines, with high HER-2/neu-expressors demonstrating significantly higher sensitivity when compared to low HER-2/neu-expressors (AZD-8055 IC50 mean±SEM=0.27±0.05μM in c-erbB2 amplified versus 1.67±0.68μM in c-erbB2 not amplified tumors, P=0.03). AZD8055 growth-inhibition was associated with a significant and dose-dependent increase in the percentage of cells blocked in the G0/G1 cell cycle phase and a dose-dependent decline in pS6 levels in both c-erbB2 amplified vs c-erbB2 not amplified USC cell lines., Conclusions: AZD8055 may represent a novel targeted therapeutic agent in patients harboring advanced/recurrent/refractory USC. c-erbB2 gene amplification may represent a biomarker to identify USC patients who may benefit most from the use of AZD8055., (© 2013.)

Objective: This study analyzed the clinicopathological correlation between ovarian cancer (OC) and RECQL1 DNA helicase to assess its therapeutic potential., Methods: Surgically resected OC from 118 retrospective cases, for which paraffin blocks and all clinical data were complete, were used in this study. RECQL1 and Ki-67 immunostaining were performed on sections to correlate RECQL1 staining with subtype and patient survival. Ten OC and two normal cell lines were then examined for RECQL1 expression and were treated with siRNA against RECQL1 to assess its effect on cell proliferation., Results: Of the 118 cases of adenocarcinoma (50, serous; 26, endometrioid; 21, clear cell; 15, mucinous; 6, other histology), 104 (90%) showed varying levels of RECQL1 expression in the nuclei of OC cells. The Cox hazards model confirmed that diffuse and strong staining of RECQL1 was correlated with histological type. However, RECQL1 expression did not correlate with overall patient survival or FIGO stage. In vitro, RECQL1 expression was exceptionally high in rapidly growing OC cell lines, as compared with normal cells. Using a time-course analysis of RECQL1-siRNA transfection, we observed a significant inhibition in cell proliferation., Conclusions: RECQL1 DNA helicase is a marker of highly proliferative cells. RECQL1-siRNA may offer a new therapeutic strategy against various subtypes of OC, including platinum-resistant cancers, or in recurrent cancers that gain platinum resistance.

Summary: Ovarian cancers with BRCA mutations rely on the alternative DNA repair mechanism of the poly(adenosine diphosphate-ribose) polymerases (PARP)-dependent base excision repair pathway, with a better overall survival and response to chemotherapy, than BRCA1-proficient cases. This can be enhanced further by using PARP inhibitors. Rate of PARP cleavage may have an independent role from BRCA in contributing to response to chemotherapy. We hypothesize that, regardless of BRCA profile, high expression of PARP1 is associated with poor disease outcome and could be used as a biomarker to identify cases that may have a better response to PARP inhibitors. The expressions of BRCA1, PARP1 in its intact and cleaved (C-PARP1) forms were immunohistochemically semiquantified in 174 sporadic high-grade serous carcinoma patients. Association with clinicopathologic variables and survival was analyzed. PARP1 expression was negatively associated with overall survival and progression-free survival in those patients with low BRCA1 profile (P = .04). Analysis of the combined expression of PARP1 and BRCA1 revealed that high expression of PARP1 is associated with poor survival when combined with either high or low BRCA expression. This was reinforced by multivariate analysis showing PARP1 (P = .034) as an independent prognostic factor. A trend toward worse survival was noted with low levels of C-PARP. PARP1 may have an independent role in response to chemotherapy separate from BRCA gene mutation and partly due to reduced PARP cleavage. An approach to exploit PARP expression as a beneficial biomarker to identify patients suitable for PARP inhibitor therapy is suggested., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Cancer stem-like cells (CSCs)/cancer-initiating cells (CICs) are defined as a small population of cancer cells that have high tumorigenicity. Furthermore, CSCs/CICs are resistant to several cancer therapies, and CSCs/CICs are therefore thought to be responsible for cancer recurrence after treatment and distant metastasis. In epithelial ovarian cancer (EOC) cases, disease recurrence after chemotherapy is frequently observed, suggesting ovarian CSCs/CICs are involved. There are four major histological subtypes in EOC, and serous adenocarcinoma and clear cell adenocarcinoma are high-grade malignancies. We therefore analyzed ovarian CSCs/CICs from ovarian carcinoma cell lines (serous adenocarcinoma and clear cell adenocarcinoma) and primary ovarian cancer cells in this study. We isolated ovarian CSCs/CICs as an aldehyde dehydrogenase 1 high (ALDH1(high)) population from 6 EOC cell lines (3 serous adenocarcinomas and 3 clear cell adenocarcinomas) by the ALDEFLUOR assay. ALDH1(high) cells showed greater sphere-forming ability, higher tumorigenicity and greater invasive capability, indicating that ovarian CSCs/CICs are enriched in ALDH1(high) cells. ALDH1(high) cells could also be isolated from 8 of 11 primary ovarian carcinoma samples. Immunohistochemical staining revealed that higher ALDH1 expression levels in ovary cancer cases are related to poorer prognosis in both serous adenocarcinoma cases and clear cell adenocarcinoma cases. Taken together, the results indicate that ALDH1 is a marker for ovarian CSCs/CICs and that the expression level of ALDH1 might be a novel biomarker for prediction of poor prognosis.

Objective: Expression of the mitochondrial protein, nicotinamide adenine dinucleotide dehydrogenase (ubiquinone) Fe-S protein 3 (NDUFS3), down-regulates in some cancers including breast and kidney. This study evaluates NDUFS3 expression and its clinical significance in human serous ovarian adenocarcinoma., Methods: 30 ovarian normal epithelium, 30 benign serous adenoma, and 100 serous carcinoma tissues were collected, and the expression of NDUFS3 protein and messenger RNA was evaluated by immunohistochemistry, Western blot, and real-time reverse transcription-polymerase chain reaction, respectively. Relationships between NDUFS3 protein expression and clinicopathologic parameters and disease-free and overall survival were also studied., Results: The expression of NDUFS3 messenger RNA and protein was significantly decreased in serous ovarian adenocarcinoma compared with normal ovarian epithelium and benign serous adenoma (both P < 0.001). Reduced NDUFS3 immunostaining correlated with advanced Federation of Gynecology and Obstetrics stage (P = 0.002) and suboptimal residual disease after primary surgery (P = 0.021). Reduced NDUFS3 expression also correlated with shorter disease-free (P = 0.002) and overall survival (P = 0.004)., Conclusions: NDUFS3 expression is down-regulated in serous ovarian adenocarcinoma, suggesting that NDUFS3 may contribute to the development of serous ovarian cancer.

Accumulating epidemiological evidence shows that obesity is associated with an increased risk of several types of adult cancers, including endometrial cancer. Chronic hyperinsulinemia, a typical hallmark of diabetes, is one of the leading factors responsible for the obesity-cancer connection. Numerous cellular and circulating factors are involved in the biochemical chain of events leading from hyperinsulinemia and insulin resistance to increased cancer risk and, eventually, tumor development. Metformin is an oral anti-diabetic drug of the biguanide family used for treatment of type 2 diabetes. Recently, metformin was shown to exhibit anti-proliferative effects in ovarian and Type I endometrial cancer, although the mechanisms responsible for this non-classical metformin action remain unclear. The insulin-like growth factors (IGFs) play a prominent role in cancer biology and their mechanisms of action are tightly interconnected with the insulin signaling pathways. Given the cross-talk between the insulin and IGF signaling pathways, the aim of this study was to examine the hypothesis that the anti-proliferative actions of metformin in uterine serous carcinoma (USC) are potentially mediated via suppression of the IGF-I receptor (IGF-IR) pathway. Our results show that metformin interacts with the IGF pathway, and induces apoptosis and inhibition of proliferation and migration of USC cell lines with both wild type and mutant p53. Taken together, our results suggest that metformin therapy could be a novel and attractive therapeutic approach for human USC, a highly aggressive variant of endometrial cancer.

Objective: ErbB4 is a member of the ErbB subfamily of receptor tyrosine kinases with a poorly understood biological role in ovarian cancer. Here, we have addressed the expression, subcellular localization, and prognostic relevance of ErbB4 and its alternatively spliced isoforms in serous ovarian adenocarcinoma., Methods: A tissue microarray including 482 samples was analyzed by immunohistochemistry, and a series of 198 samples by isoform-specific real-time RT-PCR. The data were statistically analyzed for associations with clinicopathological markers and survival. The functional effect of expressing the relevant ErbB4 isoforms in ovarian cancer cells was addressed by measuring colony formation in soft agar., Results: While ErbB4 immunoreactivity was present in 90% of the samples, total ErbB4 protein expression was not significantly associated with prognostic markers. However, real-time RT-PCR analysis of serous ovarian cancer samples indicated the presence of two alternatively spliced cytoplasmic isoforms of ERBB4, CYT-1 and CYT-2, previously demonstrated to mediate significantly different cellular activities. Expression of CYT-1, but not of CYT-2, was significantly associated with tumor grade (P=0.014) and poor overall survival (P=0.0028). CYT-1 expression was also an independent prognostic factor (P=0.021) in multivariate analysis of survival. Consistent with a biological effect specific for the one isoform, overexpression of ErbB4 CYT-1, but not of ErbB4 CYT-2, increased anchorage-independent growth of ovarian adenocarcinoma cells in vitro., Conclusions: These results suggest that expression of a specific ErbB4 isoform, CYT-1, is associated with poor survival and enhanced growth in serous ovarian cancer., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Objective: Lithium chloride (LiCl) has been shown to demonstrate anticancer properties at supratherapeutic doses. This study was designed to determine whether LiCl, as a single agent or in combination with cytotoxic agents, reduces ovarian cancer cell growth and metabolic activity at clinically achievable levels., Methods: We studied the effects of LiCl on 2 high-grade serous ovarian cancer cell lines, SKOV3 and OVCA 433, and primary cultures developed from ascitic fluid collected from patients with metastatic high-grade serous ovarian cancer. We assessed proliferation and metabolism using cell cycle analysis, MTT assays, and cellular proliferation and clonogenic potential assays., Results: Treatment with 1 mM LiCl had no effect on the cell cycle distribution or metabolic activity of the SKOV3 and OVCA 433 cell lines. Combination treatment with cisplatin or paclitaxel led to statistically significant decreases in metabolic activity in the OVCA 433 cell line and 50% of cultures investigated. The decreased metabolic activity was not, however, associated with decreased cell growth or clonogenic potential., Conclusions: Combination treatment with LiCl and cytotoxic agents at physiologically achievable drug concentrations reduces ovarian cancer cell metabolism but does not appear to affect cellular proliferation. The potential for combined lithium/cytoxic therapies appears to be limited based on our analysis of both established cell lines and short-term ovarian cancer cultures.

Objective: The aim was to evaluate antitumor activity of the combination of ixabepilone and sunitinib in pre-clinical models of chemotherapy naïve and refractory epithelial ovarian tumors, and to investigate the mechanism of synergy of such drug combination., Methods: HOVTAX2 cell line was derived from a metastatic serous papillary epithelial ovarian tumor (EOC) and a paclitaxel-resistant derivative was established. Dose response curves for ixabepilone and sunitinib were generated and synergy was determined using combination indexes. The molecular mechanism of antitumor synergy was examined using shRNA silencing., Results: The combination of ixabepilone and sunitinib demonstrated robust antitumor synergy in naïve and paclitaxel-resistant HOVTAX2 cell lines due to increased apoptosis. The GTPase, RhoB, was synergistically upregulated in cells treated with ixabepilone and sunitinib. Using shRNA, RhoB was demonstrated to mediate antitumor synergy. These results were validated in two other EOC cell lines., Conclusions: Ixabepilone plus sunitinib demonstrated antitumor synergy via RhoB in naïve and paclitaxel-resistant cells resulting in apoptosis. This study demonstrates a novel mechanism of action leading to antitumor synergy and provides 'proof-of-principle' for combining molecular targeted agents with cytotoxic chemotherapy to improve antitumor efficacy. RhoB could be envisioned as an early biomarker of response to therapy in a planned Phase II clinical trial to assess the efficacy of ixabepilone combined with a receptor tyrosine kinase inhibitor such as sunitinib. To the best of our knowledge, this is the first demonstration of antitumor synergy between these two classes of drugs in EOC and the pivotal role of RhoB in this synergy., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objective: Xanthine oxidoreductase (XOR) is a key enzyme in the degradation of DNA, RNA and high-energy phosphates. In the human cancers previously studied, down-regulated XOR identifies patients with unfavorable prognosis. We assessed the clinical relevance of XOR expression in serous ovarian cancer., Methods: XOR protein was determined in tissue microarrays from 474 patients with serous ovarian cancer and analyzed with respect to clinical parameters and survival., Results: XOR was down regulated in 64% of the tumors as compared to the corresponding normal tissue. Decreased XOR was associated with a poorly differentiated tumor and an abnormal p53 expression, but not with age at diagnosis, FIGO stage, Ki-67 or tumor size. XOR expression was associated with outcome, and the five year ovarian cancer specific survival in patients with strong XOR expression was 59% compared to 44% in those with moderate (hazard ratio, HR; 1.44; P=0.0083) and 26% in patients with lack of XOR (HR, 2.07; P=0.0003). This was also true in patients whose tumors were highly differentiated (HR, 3.67; P=0.008) and in patients with a small (<1cm) residual tumor (HR, 2.62; P=0.017), and in patients whose tumors show a low Ki-67 protein expression (HR, 3.79; P<0.0001). In multivariate survival analysis, absence of XOR emerged as an independent prognostic factor (HR, 1.82; P=0.015)., Conclusions: Decreased XOR is associated with poorer prognosis in patients with serous ovarian cancer especially in those with an otherwise more favorable prognostic profile., (Copyright © 2011. Published by Elsevier Inc.)

Unlabelled: Effective oncoprotein-targeted therapies have not yet been developed for ovarian cancer. To explore the role of PI3 kinase/AKT signaling in this disease, we performed a genetic and functional analysis of ovarian cancer cell lines and tumors. PI3K pathway alterations were common in both, but the spectrum of mutational changes differed. Genetic activation of the pathway was necessary, but not sufficient, to confer sensitivity to selective inhibition of AKT and cells with RAS pathway alterations or RB1 loss were resistant to AKT inhibition, whether or not they had coexistent PI3K/AKT pathway activation. Inhibition of AKT1 caused growth arrest in a subset of ovarian cell lines, but not in those with AKT3 expression, which required pan-AKT inhibition. Thus, a subset of ovarian tumors are sensitive to AKT inhibition, but the genetic heterogeneity of the disease suggests that effective treatment with AKT pathway inhibitors will require a detailed molecular analysis of each patient's tumor., Significance: A subset of ovarian cancers exhibits AKT pathway activation and is sensitive to selective AKT inhibition. Ovarian tumors exhibit significant genetic heterogeneity and thus an individualized approach based on real-time, detailed genomic and proteomic characterization of individual tumors will be required for the successful application of PI3K/AKT pathway inhibitors in this disease.

Background: Ovarian cancer is the number one killer among all the gynecological cancers. We undertook association study to identify potential alterations in the genomic DNA of a DNA repair gene, DNA polymerase beta (polβ), involved in base excision repair (BER), in ovarian carcinomas of patients from Haldia, India. Mutations, splice variants have been reported earlier in different tumors other than ovarian tumors., Aim: In this study we explored the possibility of association of any mutation of pol beta (Exon 8) with prognosis in 152 ovarian cancer samples., Results: Alteration in the exon 8 region (Exon 8:468, AgC; 15.1%) was noted among fifty seven polymorphism positive samples. Alteration in the intervening sequence 8 (IVS8, -25, AgC; 3.9%) was also noted. All alterations are heterozygous in nature., Conclusions: We found no significant association among the samples from serous type, stage IV, and the polβ mutations (P ≤ 0.01). Only a slight tendency of association was evident between IVS8, -25, A to C; and stage III. Further analysis with a larger number of samples is needed.

Background: Most high-grade pelvic serous carcinomas (HGPSCs) arise from fallopian tube epithelium (FTE). To date, few markers have been shown to characterize FTE transformation. Stathmin 1 (STMN1) is a candidate oncogene whose activity is influenced by p53, p27Kip1 (p27), and PI3K/Akt pathway activation. As a microtubule destabilizing protein, STMN1 regulates cytoskeletal dynamics, cell cycle progression, mitosis, and cell migration. This study examines the expression of STMN1 and its negative regulator p27 along the morphologic continuum from normal FTE to invasive carcinoma., Methods: STMN1 and p27 expression were examined by immunohistochemistry (IHC) in benign (n=12) and malignant (n=13) fallopian tubes containing normal epithelium, morphologically benign putative precursor lesions ("p53 signatures"), potential transitional precursor lesions ("proliferative p53 signatures"), tubal intraepithelial carcinoma (TIC), and/or invasive serous carcinoma. STMN1 expression was further assessed in 131 late-stage HGPSCs diagnosed as primary ovarian and in 6 ovarian cancer cell lines by IHC and Western blot, respectively., Results: STMN1 expression was absent in benign FTE and infrequently detected in p53 signatures. However, it was weakly expressed in proliferative p53 signatures and robustly induced upon progression to TIC and invasive carcinoma, typically accompanied by decreased p27 levels. STMN1 was expressed in >80% of high-grade serous ovarian carcinomas and cell lines., Conclusions: STMN1 is a novel marker of early serous carcinoma that may play a role in FTE tumor initiation. Our data are consistent with a model by which STMN1 overexpression, resulting from loss of p27-mediated regulation, may potentiate aberrant cell proliferation, migration, and/or loss of polarity during early tumorigenesis., (Copyright © 2011 Elsevier Inc. All rights reserved.)

Objectives: Secretory leukocyte protease inhibitor (SLPI) is amplified in serous ovarian cancer. We have dissected its function, showing it is a survival factor for ovarian cancer and promotes tumorigenesis and paclitaxel-resistance. We hypothesized that the protease inhibitory function was responsible for modulating SLPI's invasive capacity., Methods: Stable HEYA8 ovarian cancer transfectants expressing vector, wild type SLPI, and protease inhibitor null (F-)SLPI were examined in vitro and in xenografts. Invasion, enzyme activity, and MMP production and function assays were applied. SLPI and MMP immunoexpression was graded on tissue microarray and clinical samples. Statistical comparisons used unpaired t test and ANOVA, where appropriate., Results: SLPI and F-SLPI cells caused greater parenchymal and peritoneal dissemination over control cells in xenografts and invasion assays (p<0.001). MMP-9 protease activity was increased in SLPI and F-SLPI cells over control. SLPI, but not F-SLPI, inhibited plasmin activity, necessary for MMP-9 activation and release, and inhibited activation of MMP-9. However, paradoxically, both induced quantitative MMP-9 transcription (p<0.05) and protein (p<0.008), yielding an increased net MMP-9 activity in the face of plasmin inhibition. SLPI and MMP-9 expression were strongly correlated in serous ovarian cancers (r(2)=0.986) and a set of ovarian cancers (p<0.02). SLPI expression was greater in serous than endometrioid ovarian cancers (p=0.04)., Conclusions: SLPI stimulates ovarian cancer invasion, modulated in part by its serine protease inhibitory activity attenuating MMP-9 release. However, SLPI induction of MMP-9, independent of protease inhibition activity, is greater yielding a net pro-invasive behavior. These findings further support SLPI as a molecular target for ovarian cancer., (Published by Elsevier Inc.)