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Background: Elevated hyaluronan-mediated motility receptor (RHAMM) has been reported to contribute to disease progression, aggressive phenotype and poor prognosis in multiple cancer types, however, RHAMM's role in ovarian cancer (OC) has not been elucidated. Therefore, we sought to evaluate the role for RHAMM in epithelial OC., Results: Despite little to no expression in normal ovarian surface epithelium, western immunoblotting, immunohistochemical staining and enzyme linked immunosorbent assay showed elevated RHAMM levels in clinical tissue sections, omental metastasis and urine specimens of serous OC patients, as well as in cell lysates. We also found that RHAMM levels increase with increasing grade and stage in serous OC tissues and that RHAMM localizes to the apical cell surface and inclusion cysts. Apical localization of RHAMM suggested protein secretion which was validated by detection of significantly elevated urinary RHAMM levels (p < 0.0001) in OC patients (116.66 pg/mL) compared with normal controls (8.16 pg/mL). Likewise, urinary RHAMM levels decreased following cytoreductive surgery in OC patients suggesting the source of urinary RHAMM from tumor tissue. Lastly, we validated RHAMM levels in OC cell lysate and found at least 12× greater levels compared to normal ovarian surface epithelial cells., Conclusion: This pilot study shows, for the first time, that RHAMM may contribute to OC disease and could potentially be used as a prognostic marker.

Epithelial ovarian cancer is a leading cause of death in gynecological cancers. While several systematic studies have revealed the mutation landscape of serous epithelial ovarian cancer, other non-serous subtypes of the disease have not been explored as extensively. Here we conduct exome sequencing of nine non-serous epithelial ovarian tumors (six endometrioid and three mucinous) and their corresponding normal DNA as well as a tumor-only granulosa cell sample. We integrated the exome data with targeted gene sequencing for 1,321 genes selected for their involvement in cancer from additional 28 non-serous ovarian tumors and compared our results to TCGA ovarian serous cystadenocarcinoma and uterine corpus endometrial carcinomas. Prevalence of TP53 mutations in non-serous was much lower than in serous epithelial OC, whereas the prevalence of PIK3CA, PIK3R1, PTEN, CTNNB1, ARID1A, and KRAS was higher. We confirmed the high prevalence of FOXL2 and KRAS mutations in granulosa cell tumors and in mucinous tumors, respectively. We also identified POLE proofreading domain mutations in three endometrioid ovarian tumors. These results highlight mutational differences between serous and non-serous ovarian cancers, and further distinguish different non-serous subtypes.

Epithelial ovarian cancer (EOC) is the leading cause of gynecological cancer death in women, mainly because it has spread to intraperitoneal tissues such as the omentum in the peritoneal cavity by the time of diagnosis. In the present study, we established in vitro assays, ex vivo omental organ culture system and syngeneic animal tumor models using wild type (WT) and vitamin D receptor (VDR) null mice to investigate the effects of 1α,25-dihydroxyvitamin D3 (1,25D3) and VDR on EOC invasion. Treatment of human EOC cells with 1,25D3 suppressed their migration and invasion in monolayer scratch and transwell assays and ability to colonize the omentum in the ex vivo system, supporting a role for epithelial VDR in interfering with EOC invasion. Furthermore, VDR knockdown in OVCAR3 cells increased their ability to colonize the omentum in the ex vivo system in the absence of 1,25D3, showing a potential ligand-independent suppression of EOC invasion by epithelial VDR. In syngeneic models, ID8 tumors exhibited an increased ability to colonize omenta of VDR null over that of WT mice; pre-treatment of WT, not VDR null, mice with EB1089 reduced ID8 colonization, revealing a role for stromal VDR in suppressing EOC invasion. These studies are the first to demonstrate a role for epithelial and stromal VDR in mediating the activity of 1,25D3 as well as a 1,25D3-independent action of the VDR in suppressing EOC invasion. The data suggest that VDR-based drug discovery may lead to the development of new intervention strategies to improve the survival of patients with EOC at advanced stages. This article is part of a Special Issue entitled "Vitamin D Workshop"., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Background: Early detection of epithelial ovarian cancer (OC) is necessary to overcome the high mortality rate of late stage diagnosis; and, examining the molecular changes that occur at early disease onset may provide new strategies for OC detection. Since the deregulation of inflammatory mediators can contribute to OC development, the purpose of this pilot study was to determine whether elevated urinary levels of Interleukin-1beta (IL-1 beta) are associated with OC and associated clinical parameters., Methods: Urinary and serum levels of IL-1 beta were analyzed by ELISA from a patient cohort consisting of healthy women (N = 10), women with ovarian benign disease (N = 23), women with OC (N = 32), women with other benign gynecological conditions (N = 22), and women with other gynecological cancers (N = 6)., Results: Average urinary IL-1 beta levels tended to be elevated in ovarian benign (1.26 pg/ml) and OC (1.57 pg/ml) patient samples compared to healthy individuals (0.36 pg/ml). Among patients with benign disease, urinary IL-1β levels were statistically higher in patients with benign inflammatory gynecologic disease compared to patients with non-inflammatory benign disease. Interestingly, urinary IL-1 beta levels tended to be 3-6x greater in patients with benign ovarian disease or OC as well as with a concomitant family history of ovarian and/or breast cancer compared to similar patients without a family history of ovarian and/or breast cancer. Lastly, there was a pattern of increased urinary IL-1 beta with increasing body mass index (BMI); patients with a normal BMI averaged urinary IL-1 beta levels of 0.92 pg/ml, overweight BMI averaged urinary IL-1 beta levels of 1.72 pg/ml, and obese BMI averaged urinary IL-1 beta levels of 5.26 pg/ml., Conclusions: This pilot study revealed that urinary levels of IL-1 beta are elevated in patients with epithelial OC supporting the thought that inflammation might be associated with cancer progression. Consequently, further studies of urinary IL-1 beta and the identification of an inflammatory profile specific to OC development may be beneficial to reduce the mortality associated with this disease.

Introduction: Fallopian tube intraepithelial cancer is a postulated precursor of epithelial ovarian carcinomas. As research continues on epithelial ovarian carcinomas' developmental pathways, representative tubal tissue must be procured for diagnostic, biological, and molecular studies without compromising pathological diagnosis., Materials and Methods: Fallopian tube fimbrial epithelia were harvested from postmenopausal women undergoing surgery for non-neoplastic gynecologic lesions (n = 16) and epithelial ovarian carcinomas (n = 6). Cytological imprints and washings were obtained from each fimbria and stained by Diff-Quik and rapid Papanicolaou for general cytomorphology; by Trypan blue for cell viability; and by rapid immunohistochemistry for evaluation of low molecular weight cytokeratin, MIB-1, p53, and high-mobility group A (HMGA2) expression., Results: Benign and malignant tubal imprints harvests yielded means of 3.5 × 10 5 and 1.2 × 10 6 cells/fimbria, respectively, with viabilities higher than 85%. A mean of 2.5 × 10 5 cells/fimbria was obtained from fimbrial washings. The mean DNA, RNA, and protein contents of benign imprints were 2.4, 1.5, and 67 μg/fimbria, respectively. Benign cell populations contained nearly 97% cytokeratin-positive and p53/HMGA2-negative cells, which were dispersed within a watery to proteinaceous material and rare microcalcifications. Fimbrial imprints from serous carcinomas involving the fimbriae exhibited abnormal p53 and HMGA2 expression, high proliferation, and diagnostic criteria of malignancy, including prominent nucleoli and cell crowding., Conclusions: Ex vivo harvest from operative specimens allows for collection of cell populations representative of native fimbrial epithelium and free of significant contaminants. Tubal harvest facilitates triaging of cellular material for basic, clinical, and translational studies on cancer pathobiology and also represents a potential diagnostic adjunct to emerging in vivo high-resolution optical technologies., (Copyright © 2014 American Society of Cytopathology. Published by Elsevier Inc. All rights reserved.)

MutS protein homolog 2 (MSH2) is a key DNA mismatch repair protein. It forms the MSH2-MSH6 (MutSα) and MSH2-MSH3 (MutSβ) heterodimers, which help to ensure genomic integrity. MutSα not only recognizes and repairs mismatched nucleotides but also recognizes DNA adducts induced by DNA-damaging agents, and triggers cell-cycle arrest and apoptosis. Loss or depletion of MutSα from cells leads to microsatellite instability (MSI) and resistance to DNA damage. Although the level of MutSα can be reduced by the ubiquitin-proteasome pathway, the detailed mechanisms of this regulation remain elusive. Here we report that histone deacetylase 6 (HDAC6) sequentially deacetylates and ubiquitinates MSH2, leading to MSH2 degradation. In addition, HDAC6 significantly reduces cellular sensitivity to DNA-damaging agents and decreases cellular DNA mismatch repair activities by downregulation of MSH2. Overall, these findings reveal a mechanism by which proper levels of MutSα are maintained., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Objective: In epithelial ovarian cancer, concordance between results of microsatellite instability (MSI) and immunohistochemical (IHC) testing has not been demonstrated. This study evaluated the association of MSI-high (MSI-H) status with loss of expression (LoE) of mismatch repair (MMR) proteins on IHC and assessed for potential factors affecting the strength of the association., Methods: Tumor specimens from three population-based studies of epithelial ovarian cancer were stained for MMR proteins through manual or automated methods, and results were interpreted by one of two pathologists. Tumor and germline DNA was extracted and MSI testing performed. Multivariable logistic regression models were fitted to predict loss of IHC expression based on MSI status after adjusting for staining method and reading pathologist., Results: Of 834 cases, 564 (67.6%) were concordant; 41 were classified as MSI-H with LoE and 523 as microsatellite stable (MSS) with no LoE. Of the 270 discordant cases, 83 were MSI-H with no LoE and 187 were MSS with LoE. Both IHC staining method and reading pathologist were strongly associated with discordant results., Conclusions: Lack of concordance in the current study may be related to inconsistencies in IHC testing methods and interpretation. Results support the need for validation studies before routine screening of ovarian tumors is implemented in clinical practice for the purpose of identifying Lynch syndrome.

Over the last few decades, study of cancer in mouse models has gained popularity. Sophisticated genetic manipulation technologies and commercialization of these murine systems have made it possible to generate mice to study human disease. Given the large socio-economic burden of cancer, both on academic research and the health care industry, there is a need for in vivo animal cancer models that can provide a rationale that is translatable to the clinic. Such a bench-to-bedside transition will facilitate a long term robust strategy that is economically feasible and clinically effective to manage cancer. The major hurdles in considering mouse models as a translational platform are the lack of tumor heterogeneity and genetic diversity, which are a hallmark of human cancers. The present review, while critical of these pitfalls, discusses two newly emerging concepts of personalized mouse models called "Mouse Avatars" and Co-clinical Trials. Development of "Mouse Avatars" entails implantation of patient tumor samples in mice for subsequent use in drug efficacy studies. These avatars allow for each patient to have their own tumor growing in an in vivo system, thereby allowing the identification of a personalized therapeutic regimen, eliminating the cost and toxicity associated with non-targeted chemotherapeutic measures. In Co-clinical Trials, genetically engineered mouse models (GEMMs) are used to guide therapy in an ongoing human patient trial. Murine and patient trials are conducted concurrently, and information obtained from the murine system is applied towards future clinical management of the patient's tumor. The concurrent trials allow for a real-time integration of the murine and human tumor data. In combination with several molecular profiling techniques, the "Mouse Avatar" and Co-clinical Trial concepts have the potential to revolutionize the drug development and health care process. The present review outlines the current status, challenges and the future potential of these two new in vivo approaches in the field of personalized oncology., (Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.)

Histone deacetylase 6 (HDAC6) is well known for its ability to promote cell migration through deacetylation of its cytoplasmic substrates such as α-tubulin. However, how HDAC6 itself is regulated to control cell motility remains elusive. Previous studies have shown that one third of extracellular signal-regulated kinase (ERK) is associated with the microtubule cytoskeleton in cells. Yet, no connection between HDAC6 and ERK has been discovered. Here, for the first time, we reveal that ERK binds to and phosphorylates HDAC6 to promote cell migration via deacetylation of α-tubulin. We have identified two novel ERK-mediated phosphorylation sites: threonine 1031 and serine 1035 in HDAC6. Both sites were phosphorylated by ERK1 in vitro, whereas Ser-1035 was phosphorylated in response to the activation of EGFR-Ras-Raf-MEK-ERK signaling pathway in vivo. HDAC6-null mouse embryonic fibroblasts rescued by the nonphosphorylation mimicking mutant displayed significantly reduced cell migration compared with those rescued by the wild type. Consistently, the nonphosphorylation mimicking mutant exerted lower tubulin deacetylase activity in vivo compared with the wild type. These data indicate that ERK/HDAC6-mediated cell motility is through deacetylation of α-tubulin. Overall, our results suggest that HDAC6-mediated cell migration could be governed by EGFR-Ras-Raf-MEK-ERK signaling.

Telomerase is an essential enzyme that counteracts the telomere attrition accompanying DNA replication during cell division. Regulation of the promoter activity of the gene encoding its catalytic subunit, the telomerase reverse transcriptase, is established as the dominant mechanism conferring the high telomerase activity in proliferating cells, such as embryonic stem and cancer cells. This study reveals a new mechanism of telomerase regulation through non-coding small RNA by showing that microRNA-498 (miR-498) induced by 1,25-dihydroxyvitamin D3 (1,25(OH)(2)D(3)) decreases the mRNA expression of the human telomerase reverse transcriptase. MiR-498 was first identified in a microarray analysis as the most induced microRNA by 1,25(OH)(2)D(3) in ovarian cancer cells and subsequently validated by quantitative polymerase chain reaction assays in multiple human cancer types. A functional vitamin D response element was defined in the 5-prime regulatory region of the miR-498 genome, which is occupied by the vitamin D receptor and its coactivators. Further studies showed that miR-498 targeted the 3-prime untranslated region of human telomerase reverse transcriptase mRNA and decreased its expression. The levels of miR-498 expression were decreased in malignant human ovarian tumors as well as human ovarian cancer cell lines. The ability of 1,25(OH)(2)D(3) to decrease human telomerase reverse transcriptase mRNA and to suppress ovarian cancer growth was compromised when miR-498 was depleted using the sponges in cell lines and mouse tumor models. Taken together, our studies define a novel mechanism of telomerase regulation by small non-coding RNAs and identify miR-498 as an important mediator for the anti-tumor activity of 1,25(OH)(2)D(3).

Background: Utility of immunohistochemistry (IHC) for mismatch repair (MMR) protein expression has been demonstrated in colorectal cancer but remains incompletely defined in ovarian cancer. We evaluated MMR protein expression in three population-based samples of epithelial ovarian cancers., Materials and Methods: IHC staining was performed on full-section (FS) or tissue microarray (TMA) slides for MLH1, MSH2, and MSH6 expression., Results: Out of 487 cases, 147 and 340 were performed through FS and TMA, respectively. Overall, Loss of Expression (LoE) of at least one MMR protein was observed in 12.7% based on an expression score of ≤3 (on a scale of 9). Notably, LoE was significantly higher in TMAs (17.9%) compared to FS cases (0.7%) (p<0.001)., Conclusion: A substantial proportion of epithelial ovarian cancers have a loss of MMR protein expression. Protein expression results vary significantly by the tissue sampling methodology utilized, raising concerns about the clinical utility of this test for ovarian tumors.

Previous studies have shown aberrant expression of miR-214 in human malignancy. Elevated miR-214 is associated with chemoresistance and metastasis. In this study, we identified miR-214 regulation of ovarian cancer stem cell (OCSC) properties by targeting p53/Nanog axis. Enforcing expression of miR-214 increases, whereas knockdown of miR-214 decreases, OCSC population and self-renewal as well as the Nanog level preferentially in wild-type p53 cell lines. Furthermore, we found that p53 is directly repressed by miR-214 and that miR-214 regulates Nanog through p53. Expression of p53 abrogated miR-214-induced OCSC properties. These data suggest the critical role of miR-214 in OCSC via regulation of the p53-Nanog axis and miR-214 as a therapeutic target for ovarian cancer.

Histone deacetylase inhibitors (HDACi) are promising therapeutic agents which are currently used in combination with chemotherapeutic agents in clinical trials for cancer treatment including non-small cell lung cancer (NSCLC). However, the mechanisms underlying their anti-tumor activities remain elusive. Previous studies showed that inhibition of HDAC6 induces DNA damage and sensitizes transformed cells to anti-tumor agents such as etoposide and doxorubicin. Here, we showed that depletion of HDAC6 in two NSCLC cell lines, H292 and A549, sensitized cells to cisplatin, one of the first-line chemotherapeutic agents used to treat NSCLC. We suggested that depletion of HDAC6 increased cisplatin-induced cytotoxicity was due to the enhancement of apoptosis via activating ATR/Chk1 pathway. Furthermore, we showed that HDAC6 protein levels were positively correlated with cisplatin IC(50) in 15 NSCLC cell lines. Lastly, depletion of HDAC6 in H292 xenografts rendered decreased tumor weight and volume and exhibited increased basal apoptosis compared with the controls in a xenograft mouse model. In summary, our findings suggest that HDAC6 is positively associated with cisplatin resistance in NSCLC and reveal HDAC6 as a potential novel therapeutic target for platinum refractory NSCLC.

Background: More than two-thirds of women who undergo surgery for suspected ovarian neoplasm do not have cancer. Our previous results suggest phospholipids as potential biomarkers of ovarian cancer. In this study, we measured the serum levels of multiple phospholipids among women undergoing surgery for suspected ovarian cancer to identify biomarkers that better predict whether an ovarian mass is malignant., Methodology/principal Findings: We obtained serum samples preoperatively from women with suspected ovarian cancer enrolled through a prospective, population-based rapid ascertainment system. Samples were analyzed from all women in whom a diagnosis of epithelial ovarian cancer (EOC) was confirmed and from benign disease cases randomly selected from the remaining (non-EOC) samples. We measured biologically relevant phospholipids using liquid chromatography/electrospray ionization mass spectrometry. We applied a powerful statistical and machine learning approach, Hybrid huberized support vector machine (HH-SVM) to prioritize phospholipids to enter the biomarker models, and used cross-validation to obtain conservative estimates of classification error rates., Results: The HH-SVM model using the measurements of specific combinations of phospholipids supplements clinical CA125 measurement and improves diagnostic accuracy. Specifically, the measurement of phospholipids improved sensitivity (identification of cases with preoperative CA125 levels below 35) among two types of cases in which CA125 performance is historically poor - early stage cases and those of mucinous histology. Measurement of phospholipids improved the identification of early stage cases from 65% (based on CA125) to 82%, and mucinous cases from 44% to 88%., Conclusions/significance: Levels of specific serum phospholipids differ between women with ovarian cancer and those with benign conditions. If validated by independent studies in the future, these biomarkers may serve as an adjunct at the time of clinical presentation, to distinguish between women with ovarian cancer and those with benign conditions with shared symptoms and features.

Separating adenocarcinoma of the lung from non-pulmonary adenocarcinoma or malignant mesothelioma is difficult, especially in cytology specimens. Consequently, it is important to identify markers that may facilitate this distinction. Thyroid transcription factor-1 (TTF-1) is a homeodomain containing transcription factor expressed selectively in the thyroid, lung, and diencephalon. TTF-1 is also expressed in adenocarcinomas of the lung and is widely used as a pulmonary adenocarcinoma marker in surgical specimens. However, the utility of TTF-1 has rarely been investigated in cytology. In this study, we evaluated the expression of TTF-1 in malignant pleural effusions. The primary tumors included 26 pulmonary adenocarcinomas, 26 non-pulmonary adenocarcinomas (13 breast, 5 ovarian, 2 gastric, 2 prostatic, 1 esophageal, 1 colonic, 1 pancreatic and 1 renal) and 4 malignant mesotheliomas. Immunocytochemistry was performed on sections of cell blocks, using a mouse monoclonal TTF-1 antibody (clone 8G7G3/1) and a biotin-streptavidin detection system. Nuclear immunoreactivity for TTF-1 was detected in 19 pulmonary adenocarcinomas. All non-pulmonary adenocarcinomas and malignant mesotheliomas were negative. These data indicate that TTF-1 maintains its sensitivity (73%) and specificity (100%) in cell block preparations and is useful in separating adenocarcinoma of the lung from non-pulmonary adenocarcinoma and malignant mesothelioma in cytology specimens.

1alpha,25-dihydroxyvitamin D3, 1,25(OH)(2)D(3), regulates gene expression through the vitamin D receptor. The present studies identify the epidermal growth factor receptor, EGFR, as a target gene suppressed by 1,25(OH)(2)D(3) in human ovarian cancer cells. The suppression was detected at both mRNA and protein levels in vitamin D-sensitive human ovarian cancer cells. A novel vitamin D response element was identified in intron 1 of the EGFR genome, a known hotspot for its transcriptional regulation. Chromatin immunoprecipitations and reporter gene analyses showed that the intronic DNA element bound to vitamin D receptor and a co-repressor and was functional in mediating transcriptional suppression of EGFR promoter by 1,25(OH)(2)D(3) under stable transfection conditions. Consistent with the EGFR down regulation, 1,25(OH)(2)D(3) suppressed activation of the external signal regulated kinase by epidermal growth factors. Over expression of an active EGFR in vitamin D sensitive ovarian cancer cells caused resistance to 1,25(OH)(2)D(3)-induced growth suppression and diminished the hormonal regulation of cyclin D1, cyclin E, Skp2 and p27, a group of cell cycle regulators that mediate 1,25(OH)(2)D(3)-induced cell cycle arrest at G1-S checkpoint. Taken together, our studies demonstrate that 1,25(OH)(2)D(3) suppresses the response of human ovarian cancer cells to mitogenic growth factors and couple the suppression to the cell cycle arrest at G1-S checkpoint by the hormone., (Copyright © 2011 Elsevier Ireland Ltd. All rights reserved.)

The transcriptional activity of the androgen receptor (AR) is regulated by both ligand binding and post-translational modifications, including acetylation and small ubiquitin-like modifier (SUMO)ylation. Histone deacetylases (HDACs) are known to catalyze the removal of acetyl groups from both histones and non-histone proteins. In this study, we report that HDAC4 binds to and inhibits the activity of the AR. This inhibition was found to depend on the SUMOylation, instead of deacetylation, of the AR. Consistently, HDAC4 increases the level of AR SUMOylation in both whole-cell and cell-free assay systems, raising the possibility that the deacetylase may act as an E3 ligase for AR SUMOylation. Knock down of HDAC4 increases the activity of endogenous AR and androgen induction of prostate-specific antigen expression and prostate cancer cell growth, which is associated with decreased SUMOylation of the receptor. Overall, the studies identify HDAC4 as a positive regulator for AR SUMOylation, revealing a deacetylase-independent mechanism of HDAC action in prostate cancer cells.

Objective: The poor prognosis associated with epithelial ovarian cancer (EOC) is due to the lack of overt early symptoms and the absence of reliable diagnostic screening methods. Since many tumors over express angiogenic regulators, the purpose of this study was to determine whether elevated levels of the angiogenic or angiostatic molecules vascular endothelial growth factor (VEGF), hepatocyte growth factor (HGF), endostatin (ES), and angiostatin (AS) were elevated in plasma and urine from patients with EOC., Methods: VEGF, HGF, ES and AS were assayed by ELISA in samples from pilot cohort consisting of healthy women (N=48; pre-menopausal N=23, post-menopausal N=25), women with benign gynecological disease (N=54), patients with primary peritoneal cancer (PP) (N=2) and EOC (N=35). Wherever possible, parallel serum samples were measured for CA125 levels by ELISA., Results: AS was the angioregulator that independently discriminated EOC patients from healthy individuals. Levels of urinary AS (uAS) from healthy individuals or women with benign gynecological disease averaged 21.4 ng/mL+/-3.7 and 41.5 ng/mL+/-8.8, respectively. In contrast, uAS averaged 115 ng/mL+/-39.2 and 276 ng/mL+/-45.8 from women with Stage I (N=6) and late stage (N=31) EOC, respectively. Furthermore, uAS was elevated in EOC patients regardless of tumor grade, stage, size, histological subtype, creatinine levels, menopausal status, or patient age, but appeared to complement CA125 measurements., Conclusions: Levels of AS are elevated in the urine of patients with EOC and may be of diagnostic and/or prognostic clinical importance. Further studies of uAS as a biomarker for EOC alone or in combination with other markers are warranted., (Copyright 2009 Elsevier Inc. All rights reserved.)

Bing De Ling is a Chinese herbal formula that has been used to treat cancer patients for more than a decade. However, the molecular mechanisms behind its anti-tumor efficacy are still elusive. Here, we show that Bing De Ling inhibits cell proliferation in ovarian cancer epithelial cell lines, OV2008 and C13. It induces G1/S arrest in a p53-dependent manner in that this effect is attenuated in OV2008 cells transfected with dominant-negative p53 plasmid. Moreover, we show that Bing De Ling up-regulates p53 transcriptional activities as well as its downstream target genes, such as p21Cip1, MDM2, and MDMX. In addition, Bing De Ling inhibits MDMX-p53 interaction which may result in stabilization and activation of p53. Collectively, our results suggest that the anti-tumor activity of Bing De Ling may be in part due to activation of p53.

Background: Ovarian cancer is the most lethal gynecologic malignancy. The ovarian tumor microenvironment is comprised of tumor cells, surrounding stroma, and circulating lymphocytes, an important component of the immune response, in tumors. Previous reports have shown that the anti-apoptotic protein Bcl-2 is overexpressed in many solid neoplasms, including ovarian cancers, and contributes to neoplastic transformation and drug-resistant disease, resulting in poor clinical outcome. Likewise, studies indicate improved clinical outcome with increased presence of lymphocytes. Therefore, we sought to examine Bcl-2 expression in normal, benign, and cancerous ovarian tissues to determine the potential relationship between epithelial and stromal Bcl-2 expression in conjunction with the presence of lymphocytes for epithelial ovarian tumor progression., Methods: Ovarian tissue sections were classified as normal (n = 2), benign (n = 17) or cancerous (n = 28) and immunohistochemically stained for Bcl-2. Bcl-2 expression was assessed according to cellular localization, extent, and intensity of staining. The number of lymphocyte nests as well as the number of lymphocytes within these nests was counted., Results: While Bcl-2 staining remained cytoplasmic, both percent and intensity of epithelial and stromal Bcl-2 staining decreased with tumor progression. Further, the number of lymphocyte nests dramatically increased with tumor progression., Conclusion: The data suggest alterations in Bcl-2 expression and lymphocyte infiltration correlate with epithelial ovarian cancer progression. Consequently, Bcl-2 expression and lymphocyte status may be important for prognostic outcome or useful targets for therapeutic intervention.

I-kappa-B kinase e (IKBKE; IKKepsilon) has been recently identified as a breast cancer oncogene, and its alteration appears to be an early event in breast cancer development. In this study, we demonstrated that IKKepsilon is frequently overexpressed and activated in human ovarian cancer cell lines and primary tumors. Of 96 ovarian cancer specimens examined, 63 exhibited elevated levels of IKKepsilon. Furthermore, alterations of IKKepsilon were associated with late-stage and high-grade tumors, suggesting a role of IKKepsilon in ovarian tumor progression rather than in tumor initiation. Overall survival in patients with elevated levels of IKKepsilon was significantly lower than patients whose tumors expressed normal levels of IKKepsilon. Moreover, both early and late-stage tumors that overexpressed IKKepsilon conferred a poor prognosis, as compared with those that did not possess elevated IKKepsilon levels. Notably, overexpression of IKKepsilon rendered cells resistant to cisplatin, whereas knockdown of IKKepsilon overcame cisplatin resistance in both A2780CP and C13 cells, which express high levels of endogenous IKKepsilon. Therefore, these data demonstrate for the first time that deregulation of IKKepsilon is a highly recurrent event in human ovarian cancer and could play a pivotal role in tumor progression and cisplatin resistance. IKKepsilon could also serve as a prognostic marker and potential therapeutic target for this malignancy.

Members of the FOXO (forkhead O) class of transcription factors are tumor suppressors that also control aging and organismal life span. Mammalian FOXO degradation is proteasome-mediated, although the ubiquitin E3 ligase for FOXO factors remains to be defined. We show that MDM2 binds to FOXO1 and FOXO3A and promotes their ubiquitination and degradation, a process apparently dependent on FOXO phosphorylation at AKT sites and the E3 ligase activity of MDM2. Binding of MDM2 to FOXO occurs through the region of MDM2 that directs its cellular trafficking and the forkhead box of FOXO1. MDM2 promotes the ubiquitination of FOXO1 in a cell-free system, and its knockdown by small interfering RNA causes accumulation of endogenous FOXO3A protein in cells and enhances the expression of FOXO target genes. In cells stably expressing a temperature-sensitive p53 mutant, activation of p53 by shifting to permissive temperatures leads to MDM2 induction and degradation of endogenous FOXO3A. These data suggest that MDM2 acts as an ubiquitin E3 ligase, downstream of p53, to regulate the degradation of mammalian FOXO factors.

FoxO (mammalian forkhead subclass O) proteins are transcription factors acting downstream of the PTEN (phosphatase and tensin homolog deleted on chromosome 10) tumor suppressor. Their activity is negatively regulated by AKT-mediated phosphorylation. Our previous studies showed that the transcriptional activity of the androgen receptor (AR) was inhibited by PTEN in an AKT-sensitive manner. Here, we report the repression of the activity of the full-length AR and its N-terminal domain by FoxO1 and the participation of FoxO1 in AR inhibition by PTEN. Ectopic expression of active FoxO1 decreased the transcriptional activity of AR as well as androgen-induced cell proliferation and production of prostate-specific antigen. FoxO1 knock down by RNA interference increased the transcriptional activity of the AR in PTEN-intact cells and relieved its inhibition by ectopic PTEN in PTEN-null cells. Mutational analysis revealed that FoxO1 fragment 150-655, which contains the forkhead box and C-terminal activation domain, was required for AR inhibition. Mammalian two-hybrid and glutathione-S-transferase pull-down assays demonstrated that the inhibition of AR activity by PTEN through FoxO1 involved the interference of androgen-induced interaction of the N- and C-termini of the AR and the recruitment of the p160 coactivators to its N terminus and to the androgen response elements of natural AR target genes. These studies reveal new mechanisms for the inhibition of AR activity by PTEN-FoxO axis and establish FoxO proteins as important nuclear factors that mediate the mutual antagonism between AR and PTEN tumor suppressor in prostate cancer cells.

Objective(s): The poor prognosis associated with ovarian cancer is due to the lack of overt early symptoms and the absence of reliable diagnostic screening methods. Since many tumors overexpress anti-apoptotic proteins, the purpose of this study was to determine whether elevated levels of the anti-apoptotic protein Bcl-2 were present in urine from patients with ovarian cancer., Methods: Bcl-2 was assayed by ELISA in urine samples from two cohorts consisting of a total of 77 healthy women, 161 women with benign gynecologic disease and 150 women with ovarian cancer, 13 with early and 137 with late stage disease, respectively. Wherever possible, parallel serum samples were measured for CA125 levels by ELISA., Results: Urinary levels of Bcl-2 from healthy individuals or women with benign disease averaged 0.59 ng/ml+/-0.61 and 1.12 ng/ml+/-0.79, respectively. In contrast, urinary levels of Bcl-2 averaged 2.60 ng/ml+/-2.23 and 3.58 ng/ml+/-1.55 from women with early (N=13) and late (N=137) stage ovarian cancer. Further, urinary levels of Bcl-2 were elevated in ovarian cancer patients regardless of tumor grade, stage, size, histologic subtype, creatinine levels or patient age, but appeared to complement CA125 measurements., Conclusion(s): Levels of Bcl-2 are elevated in the urine of patients with ovarian cancer and may be of diagnostic and/or prognostic clinical importance. Further studies of urinary Bcl-2 as a biomarker for ovarian cancer alone or in combination with other markers are warranted.

While semaphorins and their receptors appear to play a role in tumor carcinogenesis, little is known about the role of semaphorin 3F (S3F) in epithelial ovarian cancer (EOC) development. Therefore, we sought to determine the clinical relationship between S3F and its receptors, neuropilin-2 (NP-2) and neuropilin-1 (NP-1) with EOC progression. We analyzed the immunohistological expression of S3F, NP-2, and NP-1 in clinical specimens of normal ovaries (N), benign cystadenomas (Cy), well-differentiated adenocarcinomas (WD), poorly-differentiated adenocarcinomas (PD), inclusion cysts (IC), paraovarian cysts (PC), and fallopian tubes (FT). Tissue sections were evaluated for staining intensity and percentage of immunoreactive epithelia. We found that expression of S3F and NP-2 decreased while NP-1 expression increased with EOC progression. Interestingly, we also found elevated expression of S3F, NP-2, and NP-1 in epithelia of ICs, PCs, and FT. Our findings indicate that loss or deregulation of semaphorin signaling may play an important role in EOC development.

MicroRNAs (miRNA) represent a novel class of genes that function as negative regulators of gene expression. Recently, miRNAs have been implicated in several cancers. However, aberrant miRNA expression and its clinicopathologic significance in human ovarian cancer have not been well documented. Here, we show that several miRNAs are altered in human ovarian cancer, with the most significantly deregulated miRNAs being miR-214, miR-199a*, miR-200a, miR-100, miR-125b, and let-7 cluster. Further, we show the frequent deregulation of miR-214, miR-199a*, miR-200a, and miR-100 in ovarian cancers. Significantly, miR-214 induces cell survival and cisplatin resistance through targeting the 3'-untranslated region (UTR) of the PTEN, which leads to down-regulation of PTEN protein and activation of Akt pathway. Inhibition of Akt using Akt inhibitor, API-2/triciribine, or introduction of PTEN cDNA lacking 3'-UTR largely abrogates miR-214-induced cell survival. These findings indicate that deregulation of miRNAs is a recurrent event in human ovarian cancer and that miR-214 induces cell survival and cisplatin resistance primarily through targeting the PTEN/Akt pathway.

SIRT1 is the closest mammalian homologue of yeast SIR2, an important ageing regulator that prolongs lifespan in response to caloric restriction. Despite its importance, the mechanisms that regulate SIRT1 activity are unclear. Our study identifies a novel post-translational modification of SIRT1, namely sumoylation at Lys 734. In vitro sumoylation of SIRT1 increased its deacetylase activity. Conversely, mutation of SIRT1 at Lys 734 or desumoylation by SENP1, a nuclear desumoylase, reduced its deacetylase activity. Stress-inducing agents promoted the association of SIRT1 with SENP1 and cells depleted of SENP1 (but not of SENP1 and SIRT1) were more resistant to stress-induced apoptosis than control cells. We suggest that stress-inducing agents counteract the anti-apoptotic activity of SIRT1 by recruiting SENP1 to SIRT1, which results in the desumoylation and inactivation of SIRT1 and the consequent acetylation and activation of apoptotic proteins.

Background: Ascorbyl stearate (Asc-S) is a synthetic ester of ascorbic acid that has been shown to significantly reduce the mutagenic effects of alkylating agents and hepatocarcinogenesis in vivo. We have previously demonstrated that Asc-S inhibits ovarian carcinoma cell proliferation through modulation of the cell cycle. This study was designed to further elucidate the mechanisms underlying such regulation., Materials and Methods: Wild type p53-expressing cell lines (Ov2008 and C13) were used to evaluate the contributions of p53 to Asc-S-induced G2/M arrest. Cell cycle analysis was performed by flow cytometry. Variation of p53, p21, and GADD45 was evaluated by Western blot and RT-PCR. Knockdown of endogenous p53 was achieved by siRNA., Results: The expression of p53 downstream genes, p21 and GADD45 was upregulated whereas 14-3-3sigma was unaffected. Phosphorylation of Cdc2 at residue tyrosine-15 was also induced by Asc-S treatment. However, pSilencer-p53-siRNA only partially rescued the Asc-S induced G2/M arrest., Conclusion: These data show that the anti-proliferative activity of Asc-S on ovarian cancer cells is due in part to G2/M arrest modulated by a p53-dependent pathway.

Objective: Both vascular endothelial growth factor (VEGF) and lysophosphatidic acid (LPA) are secreted by ovarian cancer cells and are known to promote cancer cell growth though the exact mechanism(s) are not completely understood. Since telomerase, a ribonucleprotein expressed in 95% of ovarian cancers, plays an important role in cellular immortalization, growth, and tumor progression, we examined whether telomerase is a molecular target of LPA and VEGF in ovarian cancer., Methods: Telomerase-positive ovarian carcinoma cell lines PA-1, SW 626, and one telomerase-negative, non-tumorigenic SV40 large-T antigen-transfected human ovarian surface epithelial (IOSE) cell line, FHIOSE 118, derived from normal ovarian surface epithelium were cultured with and without VEGF and LPA for 4 h and 24 h, respectively. Telomerase PCR-ELISA, RT-PCR, VEGF ELISA and luciferase assays were performed to determine the effect of VEGF and LPA on telomerase activity in ovarian cancer cells. Western blot analyses were used to examine the signaling pathway involved in telomerase regulation by VEGF and LPA., Results: We report that: (1) both VEGF and LPA upregulate telomerase activity; (2) LPA induction of telomerase activity is VEGF-dependent; (3) VEGF and LPA induction of telomerase activity is ERK 1/2-dependent; and (4) Sp1 binding sites within the proximal 976- to 378-bp regions of the hTERT promoter are essential for VEGF- and LPA-induced hTERT promoter activity., Conclusion: Consequently, these data show the novel finding that VEGF can regulate telomerase activity in non-endothelial cells and that telomerase appears to be a novel molecular target of LPA.

The purpose of this study was to determine the ultrastructure of the blood-brain barrier (BBB) and blood-spinal cord barrier (BSCB) in G93A SOD1 mice modeling ALS at different stages of disease. Electron microscope examination of brainstem, cervical and lumbar spinal cords was performed in ALS mice at early and late stages of disease. Our results show disorganized mitochondrial cristae and degenerating mitochondria in endothelial cells and neuropil, swollen astrocyte foot processes, swollen and degenerating capillary endothelial cells, astrocytes and motor neurons and extensive extracellular edema. In spite of progressive extracellular edema in neural tissue, capillary endothelial cell tight junctions appeared to remain intact in early and late symptomatic animals. Results show that disruption of BBB and BSCB was evident in areas of motor neuron degeneration in G93A mice at both early and late stages of disease. Capillary rupture was observed in brainstem in early symptomatic G93A mice. Capillary ultrastructure revealed that endothelial cell membrane and/or basement membrane damage occurred, followed by vascular leakage.

The serine protease HtrA2/Omi is released from the mitochondria into the cytosol following apoptosis stimuli, leading to the programmed cell death in caspase-dependent and -independent manners. The function of HtrA2/Omi closely relates to its protease activity, which is required for cleavage of its substrate such as the members of the X-linked inhibitor of apoptotic protein family. However, the regulation of HtrA2/Omi by signaling molecule has not been documented. Here we report that serine/threonine kinases Akt1 and Akt2 phosphorylate mitochondria-released HtrA2/Omi on serine 212 in vivo and in vitro, which results in attenuation of its serine protease activity and pro-apoptotic function. Abolishing HtrA2/Omi phosphorylation by Akt through mutation of serine 212 to alanine (HtrA2/Omi-S212A) retains its serine protease activity and induces more apoptosis as compared with wild-type HtrA2/Omi. Conversely, HtrA2/Omi-S212D, a mutant mimicking phosphorylation, lost the protease activity and failed to induce the programmed cell death. Furthermore, the phosphorylated HtrA2/Omi fails to cleave X-linked inhibitor of apoptotic protein without interfering with their complex formation. In addition, Akt inhibits the release of HtrA2/Omi from the mitochondria into the cytoplasm in response to cisplatin treatment. These data reveal for the first time that HtrA2/Omi is directly regulated by Akt and provide a mechanism by which Akt induces cell survival at post-mitochondrial level.

Mutations of the neurofibromatosis 2 (NF2) tumor suppressor gene have frequently been detected not only in schwannomas and other central nervous system tumors of NF2 patients but also in their sporadic counterparts and malignant tumors unrelated to the NF2 syndrome such as malignant mesothelioma, indicating a broader role for the NF2 gene in human tumorigenesis. However, the mechanisms by which the NF2 product, merlin or schwannomin, is regulated and controls cell proliferation remain elusive. Here, we identify a novel GTP-binding protein, dubbed NGB (referring to NF2-associated GTP binding protein), which binds to merlin. NGB is highly conserved between Saccharomyces cerevisiae, Caenorhabditis elegans, and human cells, and its GTP-binding region is very similar to those found in R-ras and Rap2. However, ectopic expression of NGB inhibits cell growth, cell aggregation, and tumorigenicity in tumorigenic schwanomma cells. Down-regulation and infrequent mutation of NGB were detected in human glioma cell lines and primary tumors. The interaction of NGB with merlin impairs the turnover of merlin, yet merlin does not affect the GTPase nor GTP-binding activity of NGB. Finally, the tumor suppressor functions of NGB require merlin and are linked to its ability to suppress cyclin D1 expression. Collectively, these findings indicate that NGB is a tumor suppressor that regulates and requires merlin to suppress cell proliferation.