Bevacizumab increases the sensitivity of olaparib to homologous recombination-proficient ovarian cancer by suppressing CRY1 via PI3K/AKT pathway.

PARP inhibitors have changed the management of advanced high-grade epithelial ovarian cancer (EOC), especially homologous recombinant (HR)-deficient advanced high-grade EOC. However, the effect of PARP inhibitors on HR-proficient (HRP) EOC is limited. Thus, new therapeutic strategy for HRP EOC is desired. In recent clinical study, the combination of PARP inhibitors with anti-angiogenic agents improved therapeutic efficacy, even in HRP cases. These data suggested that anti-angiogenic agents might potentiate the response to PARP inhibitors in EOC cells. Here, we demonstrated that anti-angiogenic agents, bevacizumab and cediranib, increased the sensitivity of olaparib in HRP EOC cells by suppressing HR activity. Most of the γ-H2AX foci were co-localized with RAD51 foci in control cells. However, most of the RAD51 were decreased in the bevacizumab-treated cells. RNA sequencing showed that bevacizumab decreased the expression of CRY1 under DNA damage stress. CRY1 is one of the transcriptional coregulators associated with circadian rhythm and has recently been reported to regulate the expression of genes required for HR in cancer cells. We found that the anti-angiogenic agents suppressed the increase of CRY1 expression by inhibiting VEGF/VEGFR/PI3K pathway. The suppression of CRY1 expression resulted in decrease of HR activity. In addition, CRY1 inhibition also sensitized EOC cells to olaparib. These data suggested that anti-angiogenic agents and CRY1 inhibitors will be the promising candidate in the combination therapy with PARP inhibitors in HR-proficient EOC.

Paclitaxel sensitizes homologous recombination-proficient ovarian cancer cells to PARP inhibitor via the CDK1/BRCA1 pathway.

OBJECTIVE: An effective treatment strategy for epithelial ovarian cancer (EOC) with homologous recombination (HR)-proficient (HRP) phenotype has not been established, although poly (ADP-ribose) polymerase inhibitors (PARPi) impact the disease course with HR-deficient (HRD) phenotype. Here, we aimed to clarify the cellular effects of paclitaxel (PTX) on the DNA damage response and the therapeutic application of PTX with PARPi in HRP ovarian cancer. METHODS: Two models with different PTX dosing schedules were established in HRP ovarian cancer OVISE cells. Growth inhibition and HR activity were analyzed in these models with or without PARPi. BRCA1 phosphorylation status was examined in OVISE cells by inhibiting CDK1, which was reduced by PTX treatment. CDK1 expression was evaluated in EOC patients treated with PTX-based neoadjuvant chemotherapy. RESULTS: PTX suppressed CDK1 expression resulting in impaired BRCA1 phosphorylation in OVISE cells. The reduced CDK1 activity by PTX could decrease HR activity in response to DNA damage and therefore increase the sensitivity to PARPi. Immunohistochemistry showed that CDK1 expression was attenuated in samples collected after PTX-based chemotherapy compared to those collected before chemotherapy. The decrease in CDK1 expression was greater with dose-dense PTX schedule than with the conventional PTX schedule. CONCULSIONS: PTX could act synergistically with PARPi in HRP ovarian cancer cells, suggesting that the combination of PTX with PARPi may be a novel treatment strategy extending the utility of PARPi to EOC. Our findings provide cules for future translational clinical trials evaluating the efficacy of PTX in combination with PARPi in HRP ovarian cancer.

Interleukin-6 as an enhancer of anti-angiogenic therapy for ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) is a subtype of epithelial ovarian cancer (EOC) that is associated with elevated interleukin-6 (IL-6) expression, resistance to chemotherapy, and increased mortality. Although bevacizumab (Bev) is a widely used anti-angiogenic agent for EOC, the efficacy of Bev and the role of IL-6 in modulating angiogenesis in OCCC are unknown. We performed tube formation assays using human umbilical vein endothelial cells (HUVEC) cultured in OCCC cell-conditioned medium and using cells directly co-cultured with OCCC cells. We observed that IL-6 inhibition significantly mitigated the ability of Bev to impede tube formation in both cases. Furthermore, IL-6 blockade disrupted the anti-angiogenic efficacy of Bev and its concomitant anti-tumor activity. In addition, IL-6 inhibition resulted in a significant increase in angiopoietin-1 (Ang1) secretion and decreased vascular endothelial growth factor (VEGF) expression. Clinical specimens also exhibited this reciprocal relationship between IL-6 and Ang1 expression. Finally, depletion of Ang1 abrogated the effects of IL-6 inhibition on Bev activity, demonstrating that IL-6 supports the anti-angiogenic activity of Bev by suppressing Ang1 expression and promoting dependence on VEGF for angiogenesis. Altogether, our data suggest that OCCC tumors with high IL-6 levels are candidates for Bev therapy.

MicroRNA-34a/IL-6R pathway as a potential therapeutic target for ovarian high-grade serous carcinoma.

Accumulating evidence has indicated that microRNAs play a critical role in the pathogenesis of human cancers. microRNA-34a (miR-34a) has been shown to be a key regulator of tumor suppression by targeting several cancer-related signals, including the interleukin-6 receptor (IL-6R)/Signal Transducers and Activator of Transcription 3 (STAT3) signaling pathway. Previously, we determined that miR-34a expression was frequently reduced in high-grade serous carcinoma (HGSC), the major subtype of epithelial ovarian cancer (EOC). Considering that the IL-6R/STAT3 signaling pathway is upregulated and believed to be a potential therapeutic target in EOC, we investigated the biological significance of reduced miR-34a expression in HGSC with regard to IL-6R signaling. Additionally, we evaluated the viability of miR-34a as a therapeutic application for HGSC both in vitro and in vivo. Accordingly, we found that the ectopic expression of miR-34a significantly reduced tumor proliferation and invasion through downregulation of IL-6R expression, suggesting that reduced miR-34a expression might play an important role in the malignant potential of HGSC through upregulation of the IL-6R/STAT3 signaling pathway. Moreover, we demonstrated that replacement of miR-34a reduced tumorigenicity of HGSC in vivo. Therefore, this study may provide the rationale for miR-34a replacement as a promising therapeutic strategy for HGSC.

Application of Artificial Intelligence for Preoperative Diagnostic and Prognostic Prediction in Epithelial Ovarian Cancer Based on Blood Biomarkers.

PURPOSE: We aimed to develop an ovarian cancer-specific predictive framework for clinical stage, histotype, residual tumor burden, and prognosis using machine learning methods based on multiple biomarkers. EXPERIMENTAL DESIGN: Overall, 334 patients with epithelial ovarian cancer (EOC) and 101 patients with benign ovarian tumors were randomly assigned to "training" and "test" cohorts. Seven supervised machine learning classifiers, including Gradient Boosting Machine (GBM), Support Vector Machine, Random Forest (RF), Conditional RF (CRF), Naïve Bayes, Neural Network, and Elastic Net, were used to derive diagnostic and prognostic information from 32 parameters commonly available from pretreatment peripheral blood tests and age. RESULTS: Machine learning techniques were superior to conventional regression-based analyses in predicting multiple clinical parameters pertaining to EOC. Ensemble methods combining weak decision trees, such as GBM, RF, and CRF, showed the best performance in EOC prediction. The values for the highest accuracy and area under the ROC curve (AUC) for segregating EOC from benign ovarian tumors with RF were 92.4% and 0.968, respectively. The highest accuracy and AUC for predicting clinical stages with RF were 69.0% and 0.760, respectively. High-grade serous and mucinous histotypes of EOC could be preoperatively predicted with RF. An ordinal RF classifier could distinguish complete resection from others. Unsupervised clustering analysis identified subgroups among early-stage EOC patients with significantly worse survival. CONCLUSIONS: Machine learning systems can provide critical diagnostic and prognostic prediction for patients with EOC before initial intervention, and the use of predictive algorithms may facilitate personalized treatment options through pretreatment stratification of patients.

PIK3CA and KRAS mutations in cell free circulating DNA are useful markers for monitoring ovarian clear cell carcinoma.

Ovarian clear cell carcinoma (OCCC) exhibits distinct phenotypes, such as resistance to chemotherapy, poor prognosis and an association with endometriosis. Biomarkers and imaging techniques currently in use are not sufficient for reliable diagnosis of this tumor or prediction of therapeutic response. It has recently been reported that analysis of somatic mutations in cell-free circulating DNA (cfDNA) released from tumor tissues can be useful for tumor diagnosis. In the present study, we attempted to detect mutations in PIK3CA and KRAS in cfDNA from OCCC patients using droplet digital PCR (ddPCR). Here we show that we were able to specifically detect PIK3CA-H1047R and KRAS-G12D in cfDNA from OCCC patients and monitor their response to therapy. Furthermore, we found that by cleaving wild-type PIK3CA using the CRISPR/Cas9 system, we were able to improve the sensitivity of the ddPCR method and detect cfDNA harboring PIK3CA-H1047R. Our results suggest that detection of mutations in cfDNA by ddPCR would be useful for the diagnosis of OCCC, and for predicting its recurrence.

Prognostic impact of interleukin-6 expression in stage I ovarian clear cell carcinoma.

OBJECTIVE: Ovarian clear cell carcinoma (OCCC) frequently presents at an early stage. In stage I OCCC, the prognosis differs according to substage. In particular, predictive biomarkers and new treatment strategies are needed for stage IC2/IC3 disease. We investigated tumor biology and prognostic factors for stage I OCCC from a clinicopathological perspective, including the expression of ARID1A and IL-6, which are considered critical for OCCC carcinogenesis. METHODS: A retrospective cohort study of 192 patients with stage I OCCC treated at a single institution was performed. We calculated overall survival (OS) with respect to 12 clinicopathological parameters that included the unique and diverse histological features of OCCC. RESULTS: The estimated 5-year OS rate in patients with all stage I OCCC was 88.9% during a median of 91months of follow-up. The multivariate analysis indicated that substage classification and IL-6 expression status were associated with poor OS (p=0.010 and p=0.027, respectively). Loss of ARID1A expression had no impact on survival; however, it was associated with substage (p=0.001), capsule rupture status (p=0.011), and ascites cytology (p=0.016). No clear association was found between ARID1A and IL-6 expressions. Histological findings, including the presence of endometriosis, adenofibroma, architectural pattern, and tumor cell type, showed no prognostic effects. CONCLUSIONS: Both substage classification and IL-6 expression status may be independent prognostic factors in stage I OCCC. Therefore, IL-6 molecular stratification may be crucial in optimizing therapeutic strategies for early stage OCCC to improve survival.

Genomic consequences of aberrant DNA repair mechanisms stratify ovarian cancer histotypes.

We studied the whole-genome point mutation and structural variation patterns of 133 tumors (59 high-grade serous (HGSC), 35 clear cell (CCOC), 29 endometrioid (ENOC), and 10 adult granulosa cell (GCT)) as a substrate for class discovery in ovarian cancer. Ab initio clustering of integrated point mutation and structural variation signatures identified seven subgroups both between and within histotypes. Prevalence of foldback inversions identified a prognostically significant HGSC group associated with inferior survival. This finding was recapitulated in two independent cohorts (n = 576 cases), transcending BRCA1 and BRCA2 mutation and gene expression features of HGSC. CCOC cancers grouped according to APOBEC deamination (26%) and age-related mutational signatures (40%). ENOCs were divided by cases with microsatellite instability (28%), with a distinct mismatch-repair mutation signature. Taken together, our work establishes the potency of the somatic genome, reflective of diverse DNA repair deficiencies, to stratify ovarian cancers into distinct biological strata within the major histotypes.

MicroRNA Gene Expression Signature Driven by miR-9 Overexpression in Ovarian Clear Cell Carcinoma.

Previous studies have identified microRNA (miRNA) involvement in human cancers. This study aimed to elucidate potential clinical and biological associations of ovarian cancer-related miRNA gene expression profiles in high-grade serous carcinoma (HGSC) and ovarian clear cell carcinoma (OCCC). Accordingly, we investigated 27 patients with ovarian cancer (12 HGSC and 15 OCCC cases) using quantitative real-time reverse transcription polymerase chain reaction to determine the cancer-related miRNA expressions. Gene Cluster 3.0 was used for hierarchical clustering analysis, and differentially expressed miRNAs between HGSC and OCCC were identified by the class comparison analysis using BRB-ArrayTools. An unsupervised hierarchical clustering analysis identified two distinct miRNA expression clusters, with histological subtype-related significant differences in the associations between clusters and clinicopathological features. A comparison of miRNA expression in HGSCs and OCCCs identified five miRNAs (miR-132, miR-9, miR-126, miR-34a, and miR-21), with OCCCs demonstrating a statistically higher expression. Further investigation of the biological significance of miR-9 overexpression in OCCC revealed that miR-9 inhibition reduced the cell invasion ability and upregulated E-cadherin expression. Using a luciferase reporter assay, we further demonstrated the direct binding of miR-9 to E-cadherin. Global cancer-related miRNA expression analysis identified statistically unique profiles that could discriminate ovarian cancer histotypes. In OCCC, miR-9 overexpression may affect pathogenesis by targeting E-cadherin, thereby inducing an epithelial-mesenchymal transition. Therefore, miR-9 may be a promising therapeutic target strategy for OCCC.

Antitumor effects of interleukin-6 (IL-6)/interleukin-6 receptor (IL-6R) signaling pathway inhibition in clear cell carcinoma of the ovary.

Among epithelial ovarian cancers, clear cell carcinoma of the ovary (CCC) has unique clinical and molecular characteristics that include chemoresistance resulting in poor prognosis. It was shown that CCC recently was characterized by specific upregulation of the IL-6/IL-6R-signal transducer and activator of transcription 3 (Stat3) signaling pathway. In this study, we aim to clarify whether IL-6/IL-6R mediated signaling pathway could have clinical relations with CCC and to evaluate inhibitory effects of the pathway on CCC carcinogenesis. A total of 84 CCC cases collected from primary surgical specimens were evaluated by the immunohistochemical analysis for IL-6R and phosphorylated Stat3 (pStat3), and we found that high IL-6R expression correlated with poor patient survival both by the univariate and multivariate analyses, suggesting that IL-6/IL-6R signaling pathway could be implicated in the progression of CCC. We further investigated the effects of IL-6/IL-6R mediated signaling pathway inhibition either by IL-6R small interfering RNA (siRNA) approach or humanized anti-human IL-6R antibody (tocilizumab) in CCC. Inhibition of endogenous IL-6R including tocilizumab in CCC cells did reduce cell invasion ability and restored their response to cytotoxic reagent. These data suggest that IL-6/IL-6R signaling pathway could act on CCC cells to enhance invasion and chemoresistance and, therefore, targeting IL-6/IL-6R mediated signaling pathway could be a promising therapeutic strategy for CCC.

Profiling of actionable gene alterations in ovarian cancer by targeted deep sequencing.

To construct a profile of therapeutically actionable gene alterations in the major histological types of ovarian cancer, 72 Japanese patients with surgically resected ovarian cancers were selected from an original cohort consisting of 267 patients who had not received pre-treatment before surgery. Somatic mutations and copy number alterations at 740 hotspots in 46 cancer-related genes were detected by deep sequencing of genomic DNAs obtained from snap-frozen tumor tissues using a next generation sequencer. The alterations were verified by Sanger sequencing and quantitative genomic PCR. Mutations and/or copy number aberrations which will make tumors respond to molecular targeting drugs were detected in nine genes of 35/72 (48.6%) patients; PIK3CA (25.0%), KRAS (13.9%), ERBB2 (4.3%), PTEN (2.8%), RB1 (2.8%), CDKN2A (2.8%), AKT1 (1.4%), CTNNB1 (1.4%) and NRAS (1.4%). These mutations tended to occur in a mutually exclusive manner. Non-serous histological type tumors showed such actionable gene alterations frequently (32/47; 68.1%). Therefore, ovarian cancers, particularly of non-serous types, frequently carry gene aberrations that link to therapy using molecular targeting drugs.

Somatic copy number alterations associated with Japanese or endometriosis in ovarian clear cell adenocarcinoma.

When compared with other epithelial ovarian cancers, the clinical characteristics of ovarian clear cell adenocarcinoma (CCC) include 1) a higher incidence among Japanese, 2) an association with endometriosis, 3) poor prognosis in advanced stages, and 4) a higher incidence of thrombosis as a complication. We used high resolution comparative genomic hybridization (CGH) to identify somatic copy number alterations (SCNAs) associated with each of these clinical characteristics of CCC. The Human Genome CGH 244A Oligo Microarray was used to examine 144 samples obtained from 120 Japanese, 15 Korean, and nine German patients with CCC. The entire 8q chromosome (minimum corrected p-value: q = 0.0001) and chromosome 20q13.2 including the ZNF217 locus (q = 0.0078) were amplified significantly more in Japanese than in Korean or German samples. This copy number amplification of the ZNF217 gene was confirmed by quantitative real-time polymerase chain reaction (Q-PCR). ZNF217 RNA levels were also higher in Japanese tumor samples than in non-Japanese samples (P = 0.027). Moreover, endometriosis was associated with amplification of EGFR gene (q = 0.047), which was again confirmed by Q-PCR and correlated with EGFR RNA expression. However, no SCNAs were significantly associated with prognosis or thrombosis. These results indicated that there may be an association between CCC and ZNF217 amplification among Japanese patients as well as between endometriosis and EGFR gene amplifications.

MicroRNA-21 is a candidate driver gene for 17q23-25 amplification in ovarian clear cell carcinoma.

BACKGROUND: Epithelial ovarian cancer (EOC) is the most common cause of gynecological malignancy-related mortality. Ovarian clear cell carcinoma (CCC) has unique clinical characteristics and behaviors that differ from other histological types of EOC, including a frequent association with endometriosis and a highly chemoresistant nature, resulting in poor prognosis. However, factors underlying its malignant behavior are still poorly understood. Aberrant expression of microRNAs has been shown to be involved in oncogenesis, and microRNA-21 (miR-21) is frequently overexpressed in many types of cancers. The aim of this study was to investigate the role of miR-21 in 17q23-25 amplification associated with CCC oncogenesis. METHODS: We identified 17q23-25 copy number aberrations among 28 primary CCC tumors by using a comparative genomic hybridization method. Next, we measured expression levels of the candidate target genes, miR-21 and PPM1D, for 17q23-25 amplification by real-time RT-PCR analysis and compared those data with copy number status and clinicopathological features. In addition, immunohistochemical analysis of PTEN (a potential target of miR-21) was performed using the same primary CCC cases. We investigated the biological significance of miR-21 overexpression in CCC using a loss-of-function antisense approach. RESULTS: 17q23-25 amplification with both miR-21 overexpression and PTEN protein loss was detected in 4/28 CCC cases (14.2%). The patients with 17q23-25 amplification had significantly shorter progression-free and overall survival than those without 17q23-25 amplification (log-rank test: p = 0.0496; p = 0.0469, respectively). A significant correlation was observed between miR-21 overexpression and endometriosis. Both PTEN mRNA and PTEN protein expression were increased by miR-21 knockdown in CCC cells. We also confirmed that miR-21 directly bound to the 3'-untranslated region of PTEN mRNA using a dual-luciferase reporter assay. CONCLUSIONS: MiR-21 is a possible driver gene other than PPM1D for 17q23-25 amplification in CCC. Aberrant expression of miR-21 by chromosomal amplification might play an important role in CCC carcinogenesis through the regulation of the PTEN tumor suppressor gene.

Cytokine gene expression signature in ovarian clear cell carcinoma.

Cytokine expression in a tumor microenvironment can impact both host defense against the tumor and tumor cell survival. In this study, we sought to clarify whether the cytokine gene expression profile could have clinical associations with ovarian cancer. We analyzed the expression of 16 cytokine genes (IL-1α, IL-1ß, IL-2, IL-4, IL-5, IL-8, IL-10, IL-12p35, IL-12p40, IL-15, IFN-γ, TNF-α, IL-6, HLA-DRA, HLA-DPA1 and CSF1) in 50 ovarian carcinomas. Hierarchical clustering analysis of these tumors was carried out using Cluster software and differentially expressed genes were examined between clear cell carcinoma (CCC) and other subtypes. Following this examination we evaluated the biological significance of IL-6 knockdown in CCC. Unsupervised hierarchical clustering analysis of cytokine gene expression revealed two distinct clusters. The relationship between the two clusters and clinical parameters showed statistically significant differences in CCC compared to other histologies. CCC showed a dominant Th-2 cytokine expression pattern driven largely by IL-6 expression. Inhibition of IL-6 in CCC cells suppressed Stat3 signaling and rendered cells sensitive to cytotoxic agents. The unique cytokine expression pattern found in CCC may be involved in the pathogenesis of this subtype. In particular, high IL-6 expression appears likely to be driven by the tumor cells, fueling an autocrine pathway involving IL-6 expression and Stat3 activation and may influence survival when exposed to cytotoxic chemotherapy. Modulation of IL-6 expression or its related signaling pathway may be a promising strategy of treatment for CCC.

Pilot study of CD147 protein expression in epithelial ovarian cancer using monoclonal antibody 12C3.

AIM: CD147 is a membrane glycoprotein that is expressed in various cancer cells and is involved in tumor invasion and metastasis by inducing stromal fibroblastic cells to produce matrix metalloproteinases. This study was carried out to evaluate the correlation between CD147 expression and various clinicopathologic parameters, including histological grade and prognosis in a small sample set of human ovarian cancer patients. MATERIAL AND METHODS: Paraffin-embedded surgical tissue samples from 25 patients with ovarian serous and endometrioid adenocarcinoma were stained with anti-CD147 antibody (monoclonal antibody 12C3: MoAb 12C3) for immunohistochemical analysis. RESULTS: CD147 protein was expressed in 84.0% (21 of 25 cases) of cancerous lesions, but not in normal lesions. CD147 expression by ovarian cancer cells was inversely correlated with overall survival. There was no correlation between CD147 expression and histological grade. CONCLUSIONS: These results suggest that measurement of CD147 expression may enhance the understanding of the pathophysiology of epithelial ovarian cancer.

Short-term serum deprivation confers sensitivity to taxanes in platinum-resistant human ovarian cancer cells.

BACKGROUND: Based on the evidences showing that serum deprivation provokes apoptosis in a variety of cells, we have investigated the effect of serum deprivation on drug sensitivity. METHODS: After human ovarian cancer cells were preincubated in 0.5 % serum containing medium for 12 hours, cellular drug sensitivities were determined by colony-forming assay. RESULTS: Serum deprivation treatment resulted in significant increase in paclitaxel sensitivity by factors of mean ± SD, 148.6 ± 28.1 and 10.1 ± 1.0 (n = 3; P < 0.001) fold in platinum-resistant C13 and CP70 cells, respectively. Similarly, serum deprivation induced significant docetaxel sensitivity in these cell lines. However, no enhancement effect of serum deprivation was observed in platinum-sensitive 2008 and A2780 cells. Serum deprivation did not have any effect on the sensitivities to cisplatin, vincristin, and doxorubicin in all of these cells. More than 7-fold increase of apoptotic cells were observed in C13 or CP70 cells when they were treated by serum deprivation followed by paclitaxel compared with the treatment of either serum deprivation or paclitaxel alone. Confocal laser microscopy using rhodamine 123 and flow cytometric analysis with 3,3'-dihexyloxacarbocyanine iodide revealed that serum deprivation decreased mitochondrial membrane potential in C13 or CP70 cells, whereas no change was observed in 2008 and A2780 cells. This indicates that serum deprivation induced depolarization specifically in platinum-resistant cells. Electron microscopy revealed that serum deprivation caused regeneration of mitochondrial matrix structure in C13 or CP70 cells where mitochondria were usually destructed and disappeared. DISCUSSIONS: These results indicate that serum deprivation confers taxane hypersensitivity specifically in platinum-resistant cells by recovering their impaired mitochondrial functions. The evidence might be clinically beneficial for the development of new chemotherapeutic technology, particularly for the patients with platinum-resistant ovarian cancer.

Low intrinsic running capacity is associated with reduced skeletal muscle substrate oxidation and lower mitochondrial content in white skeletal muscle.

Chronic metabolic diseases develop from the complex interaction of environmental and genetic factors, although the extent to which each contributes to these disorders is unknown. Here, we test the hypothesis that artificial selection for low intrinsic aerobic running capacity is associated with reduced skeletal muscle metabolism and impaired metabolic health. Rat models for low- (LCR) and high- (HCR) intrinsic running capacity were derived from genetically heterogeneous N:NIH stock for 20 generations. Artificial selection produced a 530% difference in running capacity between LCR/HCR, which was associated with significant functional differences in glucose and lipid handling by skeletal muscle, as assessed by hindlimb perfusion. LCR had reduced rates of skeletal muscle glucose uptake (∼30%; P = 0.04), glucose oxidation (∼50%; P = 0.04), and lipid oxidation (∼40%; P = 0.02). Artificial selection for low aerobic capacity was also linked with reduced molecular signaling, decreased muscle glycogen, and triglyceride storage, and a lower mitochondrial content in skeletal muscle, with the most profound changes to these parameters evident in white rather than red muscle. We show that a low intrinsic aerobic running capacity confers reduced insulin sensitivity in skeletal muscle and is associated with impaired markers of metabolic health compared with high intrinsic running capacity. Furthermore, selection for high running capacity, in the absence of exercise training, endows increased skeletal muscle insulin sensitivity and oxidative capacity in specifically white muscle rather than red muscle. These data provide evidence that differences in white muscle may have a role in the divergent aerobic capacity observed in this generation of LCR/HCR.

Cyclin D1 predicts the prognosis of advanced serous ovarian cancer.

We previously reported that cyclin E (CCNE1) amplification is strongly associated with resistance to treatment in serous ovarian cancer by high-resolution oligonucleotide copy number analysis. Dysregulation of cell cycle control has been implicated as the key event in human oncogenesis, and aberrant expression of G1-S phase-related genes in particular has been reported in epithelial ovarian cancer (EOC). Nevertheless, there are conflicting results concerning the prognostic values of these abnormalities in EOC. This study focused on advanced serous EOC cases and investigated the association between the expression of G1-S phase-regulatory proteins and clinicopathological parameters. The utility of these proteins as prognostic factors was assessed, and whether these targets reflect chemoresistance of advanced serous EOC was investigated. A total of 66 patients treated by primary surgery were evaluated in this study. Immunohistochemical analysis for cyclin D1, pRb, p16, p53, p27(Kip1), p21(Waf1/Cip1) and cyclin E was performed on formalin-fixed tissue sections collected from primary surgical specimens. The correlations between the expression of these proteins and the clinicopathological parameters, including progression-free survival (PFS), overall survival (OS) and chemosensitivity, were examined. Upon univariate analysis, overexpression of cyclin D1 was positively correlated with reduced PFS (p=0.00062) and OS (p=0.00037). Reduced expression of p27(Kip1) was associated with shorter OS (p=0.064). Upon multivariate analysis, overexpression of cyclin D1 (p=0.0019), reduced expression of p27(Kip1) (p=0.042) and residual tumor volume (p=0.0092) were identified as independent predictors of OS. Overexpression of cyclin D1 (p=0.011) as well as residual tumor volume (p=0.006) were significantly associated with first-line chemosensitivity. In advanced serous EOC, overexpression of cyclin D1 contributed largely to poor prognosis, and this may have been in part mediated by chemoresistance. Cyclin D1 is a possible target for overcoming the refractory nature of advanced serous EOC.

Characterization of two independent, exposure-time dependent paclitaxel-resistant human ovarian carcinoma cell lines.

This experiment was conducted to address the question of whether acquired paclitaxel resistance is dependent upon whether it is given as a single brief exposure or as a long-term exposure. PX2 and PX24 were established from 2008 human ovarian cancer cells by 2-h single exposure or 24-h continuous exposure to paclitaxel. PX2 acquired paclitaxel resistance faster than PX24 by twofold. Drug resistant pattern was exposure-time dependent. In 2-h exposure, PX2 showed 53.86 ± 4.96 (mean ± standard deviation [SD]) fold paclitaxel resistance while PX24 showed 9.51 ± 1.01 fold resistance (P = 0.002). In 24-h exposure, PX2 showed 2.31 ± 0.3 fold paclitaxel resistance while PX24 showed 28.17 ± 0.98 fold resistance (P = 0.040). PX2 and PX24 acquired cross-resistance to docetaxel and SN38 and the resistance degrees were significantly higher in PX2 than PX24. They displayed approximately twofold cisplatin collateral sensitivity. PX24 also displayed sensitivity to other platinum drugs, oxaliplatin and ZD0473, whereas PX2 acquired significant resistance to both of them. Although differential tubulin-isotype expressions were noted among 2008, PX2 and PX24, they were not significant. In electron microscopy, prominent, densely stained lysosomes were observed more in the resistant cells than 2008. Two independent, exposure-time dependent paclitaxel-resistant human ovarian carcinoma cell lines were established. Understanding the characteristics of the differential resistance pattern could be clinically beneficial for the selection of second line chemotherapy for relapsed ovarian cancer.

Mesenchymal to epithelial transition in the human ovarian surface epithelium focusing on inclusion cysts.

Most ovarian cancers arise from the mesothelial surface lining of the ovaries or from invaginations of this lining into the superficial ovarian cortex that form cortical inclusion cysts. Thus, these cysts are thought to be precursor lesions of ovarian carcinoma. Epithelial-mesenchymal transition, which is a transcriptional program for inducing maintenance of the mesenchymal phenotype, acts in tumor progression and metastasis. Little is known about the mechanisms involved in mesenchymal-epithelial transition (MET). We aimed to characterize the human ovarian surface epithelium (OSE) and inclusion cysts by immunohistochemical analysis to examine whether MET occurs during inclusion cyst formation in the OSE. We used specimens from 9 endometrial cancer patients who had undergone hysterectomy and bilateral salpingo-oophorectomy. Immunohistochemical analysis was performed in 10 normal ovaries containing 92 inclusion cysts and in 4 normal tubes to examine the expression of antigen markers including calretinin, podoplanin, D2-40, thrombomodulin, HBME-1, vimentin, EMA, WT1, CA125, MOC31, TAG-72, Ber-EP4 and E-cadherin. The positive staining rates for mesothelial markers in normal OSE were 100% (10/10) for calretinin, 80% (8/10) for podoplanin, 80% (8/10) for D2-40, 70% (7/10) for thrombomodulin, 100% (10/10) for HBME-1, 100% (10/10) for vimentin. The positive staining rates for epithelial markers in tubal epithelium were 100% (4/4) for HBME-1, 100% (4/4) for vimentin, 100% (4/4) for EMA, 75% (3/4) for TAG-72, 100% (4/4) for Ber-EP4. Inclusion cysts showed positive staining for both markers with an incidence of 51% (47/92) for HBME-1, 44% (41/92) for vimentin, 65% (60/92) for TAG-72, 88% (81/92) for Ber-EP4. The OSE showed the characteristics of both mesenchymal and epithelium cells. In contrast, inclusion cysts gained epithelial characteristics, but lost mesenchymal characteristics. These findings support that MET occurs during the inclusion cyst formation from OSE.