Yasuhisa Terao

High-Risk Ovarian Cancer Based on 126-Gene Expression Signature Is Uniquely Characterized by Downregulation of Antigen Presentation Pathway

Purpose: High-grade serous ovarian cancers are heterogeneous not only in terms of clinical outcome but also at the molecular level. Our aim was to establish a novel risk classification system based on a gene expression signature for predicting overall survival, leading to suggesting novel therapeutic strategies for high-risk patients. Experimental Design: In this large-scale cross-platform study of six microarray data sets consisting of 1,054 ovarian cancer patients, we developed a gene expression signature for predicting overall survival by applying elastic net and 10-fold cross-validation to a Japanese data set A (n = 260) and evaluated the signature in five other data sets. Subsequently, we investigated differences in the biological characteristics between high- and low-risk ovarian cancer groups. Results: An elastic net analysis identified a 126-gene expression signature for predicting overall survival in patients with ovarian cancer using the Japanese data set A (multivariate analysis, P = 4 × 10−20). We validated its predictive ability with five other data sets using multivariate analysis (Tothills data set, P = 1 × 10−5; Bonomes data set, P = 0.0033; Dressmans data set, P = 0.0016; TCGA data set, P = 0.0027; Japanese data set B, P = 0.021). Through gene ontology and pathway analyses, we identified a significant reduction in expression of immune-response–related genes, especially on the antigen presentation pathway, in high-risk ovarian cancer patients. Conclusions: This risk classification based on the 126-gene expression signature is an accurate predictor of clinical outcome in patients with advanced stage high-grade serous ovarian cancer and has the potential to develop new therapeutic strategies for high-grade serous ovarian cancer patients. Clin Cancer Res; 18(5); 1374–85. ©2012 AACR.

Hemizygous Disruption of Cdc25A Inhibits Cellular Transformation and Mammary Tumorigenesis in Mice

CDC25A phosphatase activates multiple cyclin-dependent kinases (CDK) during cell cycle progression. Inactivation of CDC25A by ubiquitin-mediated degradation is a major mechanism of DNA damage-induced S-G(2) checkpoint. Although increased CDC25A expression has been reported in various human cancer tissues, it remains unclear whether CDC25A activation is a critical rate-limiting step of carcinogenesis. To assess the role for CDC25A in cell cycle control and carcinogenesis, we used a Cdc25A-null mouse strain we recently generated. Whereas Cdc25A(-/-) mice exhibit early embryonic lethality, Cdc25A(+/-) mice show no appreciable developmental defect. Cdc25A(+/-) mouse embryonic fibroblasts (MEF) exhibit normal kinetics of cell cycle progression at early passages, modestly enhanced G(2) checkpoint response to DNA damage, and shortened proliferative life span, compared with wild-type MEFs. Importantly, Cdc25A(+/-) MEFs are significantly resistant to malignant transformation induced by coexpression of H-ras(V12) and a dominant negative p53 mutant. The rate-limiting role for CDC25A in transformation is further supported by decreased transformation efficiency in MCF-10A human mammary epithelial cells stably expressing CDC25A small interfering RNA. Consistently, Cdc25A(+/-) mice show substantially prolonged latency in mammary tumorigenesis induced by MMTV-H-ras or MMTV-neu transgene, whereas MMTV-myc-induced tumorigenesis is not significantly affected by Cdc25A heterozygosity. Mammary tissues of Cdc25A(+/-);MMTV-neu mice before tumor development display less proliferative response to the oncogene with increased tyrosine phosphorylation of CDK1/2, but show no significant change in apoptosis. These results suggest that Cdc25A plays a rate-limiting role in transformation and tumor initiation mediated by ras activation.

Prevalence of Uterine and Adnexal Involvement in Pulmonary Lymphangioleiomyomatosis: A Clinicopathologic Study of 10 Patients

Lymphangioleiomyomatosis (LAM), a systemic disorder affecting almost exclusively young women, is characterized by the abnormal proliferation of smooth muscle-like cells (LAM cells). LAM can occur either in association with the tuberous sclerosis complex (TSC) (TSC-LAM) or without TSC (sporadic LAM). Recent studies have demonstrated that LAM is a neoplasm arising from constitutive activation of the mammalian target of rapamycin signaling pathway dysregulated by a functional loss of TSC genes, but the primary organ of origin remains unclear. Therefore, we performed histologic and immunohistologic analyses of gynecologic organs in 20 patients, half with and the other half without pulmonary LAM, to determine how often LAM involves the uterus. The results showed that 9 of 10 (90%) patients with pulmonary LAM had uterine LAM lesions. In contrast, no patients without pulmonary LAM had so. All uterine LAM lesions were accompanied by LAM lesions in retroperitoneal or pelvic lymph nodes and LAM cell clusters, each enveloped by a monolayer of vascular endothelial growth factor receptor-3–positive lymphatic endothelial cells. Furthermore, when we compared uterine lesions of TSC-LAM with those of sporadic LAM, proliferation of HMB45-positive epithelioid-shaped LAM cells and infiltrates with a tongue-like growth pattern was more prominent in the former, whereas the extent of lymphangiogenesis within the myometrium was greater in the latter. These results indicate that uterine involvement is a common manifestation of LAM, and, possibly, that the uterus or an adjacent locale in the retroperitoneum or pelvic cavity is the primary site of origin of LAM.

Hepatocyte growth factor modulates motility and invasiveness of ovarian carcinomas via Ras-mediated pathway

Hepatocyte growth factor (HGF) is a multifunctional growth factor which has pleiotrophic biological effects on epithelial cells such as proliferation, motogenesis, invasiveness and morphogenesis. Peritoneal dissemination is critical for the progression of ovarian cancer, and our study revealed that HGF induces migration and invasion of ovarian cancer cells. We also demonstrated that HGF stimulates autophosphorylation of its receptor, followed by activation of the Ras-MAP (mitogen-activated peptide) kinase cascade. Moreover, infection of ovarian cancer cells with Ras dominant-negative adenovirus reduced the HGF-induced motogenic and invasive activities. Additionally, both MEK and PI3-kinase pathways downstream of Ras were involved in HGF-stimulated ovarian cancer cell invasiveness.

The inhibitory effect of salinomycin on the proliferation, migration and invasion of human endometrial cancer stem-like cells

GOALS We previously demonstrated that side-population (SP) cells in human endometrial cancer cells (Hec1 cells) and in rat endometrial cells expressing oncogenic human K-Ras protein (RK12V cells) have features of cancer stem cells (CSCs). Hec1-SP cells showed enhanced migration and the potential to differentiate into the mesenchymal cell lineage. In this study, we analyzed the association of the epithelial-mesenchymal transition (EMT) with the properties of these endometrial CSCs. We also assessed the effects of salinomycin (a compound with EMT-specific toxicity) on the proliferative capacity, migration and invasiveness of these endometrial CSCs using Hec1-SP cells. METHOD We performed microarray expression analysis to screen for up-regulated genes in CSCs using a set of RK12V-SP cells and -non-SP(NSP) cells and used the Metacore package to identify the Gene GO pathway MAPs involved in the up-regulated genes. To analyze their association with EMT, the expression of several EMT associated genes in Hec1-SP cells was investigated by real time PCR and compared with that in Hec1-NSP cells. We assessed the expression of BAX, BCL2, LEF1, cyclinD and fibronectin by real time PCR. We also evaluated the viabilities, migration and invasive activities, and tumorigenicities of these SP cells and NSP cells in the presence or absence of salinomycin. RESULTS We demonstrated that i) EMT processes were observed in both RK12V-SP cells and Hec1-SP cells, ii) the level of fibronectin was enhanced in Hec1-SP cells and salinomycin reduced the level of fibronectin expression, iii) salinomycin induced apoptosis and inhibited Wnt signaling, and iv) salinomycin inhibited the proliferation, migration, invasiveness and tumorigenicity of these SP cells. CONCLUSION This is the first report of an inhibitory effect of salinomycin on the properties of endometrial CSCs.

Randomized phase III trial of irinotecan plus cisplatin compared with paclitaxel plus carboplatin as first-line chemotherapy for ovarian clear cell carcinoma: JGOG3017/GCIG trial

PURPOSE Clear cell carcinoma (CCC) is a rare histologic subtype that demonstrates poor outcomes in epithelial ovarian cancer. The Japanese Gynecologic Oncology Group conducted the first randomized phase III, CCC-specific clinical trial that compared irinotecan and cisplatin (CPT-P) with paclitaxel plus carboplatin (TC) in patients with CCC. PATIENTS AND METHODS Six hundred sixty-seven patients with stage I to IV CCC of the ovary were randomly assigned to receive irinotecan 60 mg/m(2) on days 1, 8, and 15 plus cisplatin 60 mg/m(2) on day 1 (CPT-P group) every 4 weeks for six cycles or paclitaxel 175 mg/m(2) plus carboplatin area under the curve 6.0 mg/mL/min on day 1 every 3 weeks for six cycles (TC group). The primary end point was progression-free survival. Secondary end points were overall survival, overall response rate, and adverse events. RESULTS Six hundred nineteen patients were clinically and pathologically eligible for evaluation. With a median follow-up of 44.3 months, 2-year progression-free survival rates were 73.0% in the CPT-P group and 77.6% in TC group (hazard ratio, 1.17; 95% CI, 0.87 to 1.58; P = .85). Two-year overall survival rates were 85.5% with CPT-P and 87.4% with TC (hazard ratio, 1.13; 95% CI, 0.80 to 1.61; one-sided P = .76). Grade 3/4 anorexia, diarrhea, nausea, vomiting, and febrile neutropenia occurred more frequently with CPT-P, whereas grade 3/4 leukopenia, neutropenia, thrombocytopenia, peripheral sensory neuropathy, and joint pain occurred more frequently with TC. CONCLUSION No significant survival benefit was found for CPT-P. Both regimens were well tolerated, but the toxicity profiles differed significantly. Treatment with existing anticancer agents has limitations to improving the prognosis of CCC.

Growth-associated gene expression profiles by microarray analysis of trophoblast of molar pregnancies and normal villi.

We used microarray analysis to investigate expression profiles of 589 known genes committed to cell growth control to characterize regulatory circuitry for cell proliferation in complete moles (CMs). CMs are characterized by hyperplastic trophoblast and have a high propensity to give rise to choriocarcinoma. Characteristic alterations in gene expression profiles were observed when compared with normal villi. Fifty-seven genes were significantly up-regulated in CMs and involved the Ras-Map kinase 3, Jak-STAT5, and Wnt signal pathways, implicating growth factor or cytokine-mediated signal pathways in the trophoblastic hyperplasia of CMs. Several genes associated with anti-apoptosis, cell structuring, and/or cell attachment were also up-regulated in CMs. In contrast, relatively fewer genes were down-regulated and these involved IGFBPs, versican, interleukin-1, tumor necrosis factor receptor, CD44, and RAD52. Genes identified in this study may elucidate regulation mechanisms of trophoblastic proliferation and mechanisms causing a pathological phenotype in CMs.

Uterine angiosarcoma associated with lymphangioleiomyomatosis in a patient with tuberous sclerosis complex: an autopsy case report with immunohistochemical and genetic analysis.

A 41-year-old woman carrying a germline tuberous sclerosis complex 2 (TSC2) mutation, whose regular medical follow-up for tuberous sclerosis complex and tuberous sclerosis complex-associated lymphangioleiomyomatosis had continued for 2 years, had uterine angiosarcoma concomitant with uterine lymphangioleiomyomatosis. Immunohistochemically, the uterine angiosarcoma cells showed an extremely skewed lymphatic differentiation; they were diffusely immunopositive for CD31 but negative for other vascular endothelial markers including factor VIII and CD34 yet strongly immunopositive for lymphatic endothelial markers including D2-40 and Prox-1. Loss of heterozygosity analysis demonstrated that not only lymphangioleiomyomatosis and renal angiomyolipoma but also the uterine angiosarcoma had loss of heterozygosity on TSC2. Furthermore, direct sequencing revealed a TP53 mutation in the uterine angiosarcoma. Collectively, the findings suggest that combined dysfunction of the p53 and TSC2 tumor suppressor proteins may contribute to the development of uterine angiosarcoma in this rare clinical setting.

Relevance of ER to the Development of Endometrial Hyperplasia and Adenocarcinoma.

Estrogen has an important role in both the etiology and treatment of hormone-dependent endometrial cancers, although the mechanism remains elusive. To define the role of estrogen-mediated signaling we investigated the biological significance of estrogen receptors (ER) in NIH3T3 cell transformation via the [12Val] K-Ras mutant. This mutant enhanced the steady state level and transcriptional activity of ER. In addition, overexpression of both wild type K-Ras and ER transformed NIH3T3 cells. Co-expression of the progesterone receptor (PR) with mutant K-Ras led to suppression of tumorigenicity and inhibition of ER activation. The antisense oligomers complementary to ER suppressed proliferation and transformed phenotypes of K12V cells. These observations support the importance of ER in Ras-mediated cell transformation.To address whether ER activation is also important in the development of human endometrial cancers, we investigated ER and PR expression levels in premalignant and malignant endometrial lesions. The results suggested the implication of ER abundance in endometrial hyperplasias, though modulation of PR expression by ER was retained. Gl adenocarcinoma also expressed higher levels of ER while PR modulation by ER was abrogated. These data implied the importance of ER activities in endometrial hyperplasia and Gl adenocarcinoma development.

Whole-genome sequencing revealed novel prognostic biomarkers and promising targets for therapy of ovarian clear cell carcinoma

Background:Ovarian clear cell carcinoma (OCCC) is mostly resistant to standard chemotherapy that results in poor patient survival. To understand the genetic background of these tumours, we performed whole-genome sequencing of OCCC tumours.Methods:Tumour tissue samples and matched blood samples were obtained from 55 Japanese women diagnosed with OCCC. Whole-genome sequencing was performed using the Illumina HiSeq platform according to standard protocols.Results:Alterations to the switch/sucrose non-fermentable (SWI/SNF) subunit, the phosphatidylinositol-3-kinase (PI3K)/Akt signalling pathway, and the receptor tyrosine kinase (RTK)/Ras signalling pathway were found in 51%, 42%, and 29% of OCCC tumours, respectively. The 3-year overall survival (OS) rate for patients with an activated PI3K/Akt signalling pathway was significantly higher than that for those with inactive pathway (91 vs 40%, hazard ratio 0.24 (95% confidence interval (CI) 0.10–0.56), P=0.0010). Similarly, the OS was significantly higher in patients with the activated RTK/Ras signalling pathway than in those with the inactive pathway (91 vs 53%, hazard ratio 0.35 (95% CI 0.13–0.94), P=0.0373). Multivariable analysis revealed that activation of the PI3K/Akt and RTK/Ras signalling pathways was an independent prognostic factor for patients with OCCC.Conclusions:The PI3K/Akt and RTK/Ras signalling pathways may be potential prognostic biomarkers for OCCC patients. Furthermore, our whole-genome sequencing data highlight important pathways for molecular and biological characterisations and potential therapeutic targeting in OCCC.