Masaki Mandai

Identification of an ovarian clear cell carcinoma gene signature that reflects inherent disease biology and the carcinogenic processes

Ovarian clear cell carcinoma (OCCC) shows unique clinical features including an association with endometriosis and poor prognosis. We previously reported that the contents of endometriotic cysts, especially high concentrations of free iron, are a possible cause of OCCC carcinogenesis through iron-induced persistent oxidative stress. In this study, we conducted gene expression microarray analysis using 38 ovarian cancer cell lines and identified genes commonly expressed in both OCCC cell lines and clinical samples, which comprise an OCCC gene signature. The OCCC signature reproducibly predicts OCCC specimens in other microarray data sets, suggesting that this gene profile reflects the inherent biological characteristics of OCCC. The OCCC signature contains known markers of OCCC, such as hepatocyte nuclear factor-1β (HNF-1β) and versican (VCAN), and other genes that reflect oxidative stress. Expression of OCCC signature genes was induced by treatment of immortalized ovarian surface epithelial cells with the contents of endometriotic cysts, indicating that the OCCC signature is largely dependent on the tumor microenvironment. Induction of OCCC signature genes is at least in part epigenetically regulated, as we found hypomethylation of HNF-1β and VCAN in OCCC cell lines. This genome-wide study indicates that the tumor microenvironment induces specific gene expression profiles that contribute to the development of distinct cancer subtypes.

The activated transforming growth factor‐beta signaling pathway in peritoneal metastases is a potential therapeutic target in ovarian cancer

Peritoneal dissemination including omental metastasis is the most frequent route of metastasis and an important prognostic factor in advanced ovarian cancer. We analyzed the publicly available microarray dataset (GSE2109) using binary regression and found that the transforming growth factor (TGF)‐beta signaling pathway was activated in omental metastases as compared to primary sites of disease. Immunohistochemical analysis of TGF‐beta receptor type 2 and phosphorylated SMAD2 indicated that both were upregulated in omental metastases as compared to primary disease sites. Treatment of the mouse ovarian cancer cell line HM‐1 with recombinant TGF‐β1 promoted invasiveness, cell motility and cell attachment while these were suppressed by treatment with A‐83‐01, an inhibitor of the TGF‐β signaling pathway. Microarray analysis of HM‐1 cells treated with TGF‐β1 and/or A‐83‐01 revealed that A‐83‐01 efficiently inhibited transcriptional changes that are induced by TGF‐β1. Using gene set enrichment analysis, we found that genes upregulated by TGF‐β1 in HM‐1 cells were also significantly upregulated in omental metastases compared to primary sites in the human ovarian cancer dataset, GSE2109 (false discovery rate (FDR) q = 0.086). Therapeutic effects of A‐83‐01 in a mouse model of peritoneal dissemination were examined. Intraperitoneal injection of A‐83‐01 (150 μg given three times weekly) significantly improved survival (p = 0.015). In summary, these results show that the activated TGF‐β signaling pathway in peritoneal metastases is a potential therapeutic target in ovarian cancer.

Ovarian clear cell carcinoma as a stress-responsive cancer: influence of the microenvironment on the carcinogenesis and cancer phenotype.

Although it is well known that ovarian endometriosis occasionally gives rise to ovarian cancers with specific histology such as endometrioid and clear cell carcinomas, its etiology is not fully understood. We have shown that a stressful microenvironment within the endometriotic cyst may lead to cancer development by inducing unique gene expressions, which potentially serves as a molecular marker for treatment modality. In this review, by referring to other articles in this field, we explore how the carcinogenic microenvironment affects the phenotype and gene expression of a cancer, and how we can develop new treatment based on this concept.

Sorafenib efficacy in ovarian clear cell carcinoma revealed by transcriptome profiling

The purpose of this study was to investigate a new modality of therapy against ovarian clear cell carcinoma (OCCC), a chemoresistant subtype of ovarian cancer. Microarray datasets of ovarian cancer cell lines and cancer tissues were analyzed using bioinformatic tools. The gene expression profile of OCCC was similar to that of renal cell carcinoma (RCC). This similarity was at least partially due to hepatocyte nuclear factor 1 pathway activation common to both malignancies. In addition, oncogenic pathway alterations were characteristic of OCCC including hypoxia inducible factor 1 alpha subunit and relatively high Ras activities. Therefore, we predicted that the multi‐kinase inhibitor sorafenib, which is approved for RCC and suppresses Ras activity, would also be effective against OCCC. Orally administered sorafenib (40u2003mg/kg per day) significantly inhibited tumor growth in nude mice when it was given after inoculation with the OCCC cell line RMG‐2 (Pu2003=u20030.002). Furthermore, sorafenib significantly reduced tumor size when it was administered to established RMG‐2 tumors (Pu2003=u20030.0002), while intraperitoneal injection of cisplatin (5u2003mg/kg per week) did not. In conclusion, the prominent anti‐tumor effect of sorafenib against OCCC indicates that sorafenib is a promising candidate drug and supports the need for clinical trials using sorafenib against OCCC. This report demonstrates a method to utilize genome‐wide information to facilitate translational research for treatments against less common subtypes of cancers. (Cancer Sci 2010; 101: 2658–2663)

The comprehensive assessment of local immune status of ovarian cancer by the clustering of multiple immune factors

The aim of this study was to evaluate the local immune status of human ovarian cancers by the comprehensive analysis of tumor-infiltrating immune cells and immunosuppressive factors, and to elucidate the local immunity in clinical course. The numbers of CD1α+, CD4+, CD8+, CD57+, forkhead box P3+ and programmed cell death-1+ cells were counted, and the intensity of immunosuppressive factors, such as programmed cell death-1 ligand (PD-L)1, PD-L2, cyclooxygenase (COX)-1, COX-2 and transforming growth factor β1, were evaluated in 70 ovarian cancer specimens stained by immunohistochemistry. Then hierarchical clustering of these parameters showed the four clusters into ovarian cancer cases. Cluster 1, which had significantly better prognosis than the others, was characterized by high infiltration of CD4+ and CD8+ cells. In conclusion the comprehensive analysis of local immune status led to subdivide ovarian cancers into groups with better or worse prognoses and may guide precise understanding of the local immunity.

Oct4 expression in immature teratoma of the ovary: Relevance to histologic grade and degree of differentiation

Immature teratoma of the ovary is an uncommon tumor comprising 1% of ovarian malignancies. The amount of immature neuroepithelium in the teratoma is an important prognostic factor. Although the current grading system based on this criterion is widely accepted, the biological significance of immature neuroepithelium is poorly understood. In this study, we used immunohistochemistry to evaluate the expression of Oct4 (also known as Oct3 or POU5F1), a transcription factor expressed in primordial germ cells and embryonic stem cells, along with that of PAX6, a transcription factor contributing to neurogenesis, and CD56, a known marker for tumors of neural origin, in 18 cases of pure immature teratoma of the ovary. Oct4 was expressed in the immature neuroepithelium of all 7 grade-3 cases and 2 grade-2 cases. It was not expressed in 4 grade-2 cases and all 5 grade-1 cases. These tumor cells lacked CD30 or &agr;-fetoprotein expression, which supported the diagnosis of pure immature teratoma. PAX6 was expressed in the immature neuroepithelium of all immature teratomas, but not in Oct4-positive cells. CD56 was expressed in neural components of various maturities including PAX6-positive immature neuroepithelium, but not in Oct4-positive cells. These data suggest that the expression of these markers probably reflects the differentiation status of neural tissue in immature teratomas. The finding that Oct4 expression was exclusively detected in immature neuroepithelium of high-grade immature teratomas indicates that Oct4 might serve as a promising biomarker for the diagnosis of highly malignant cases of immature teratoma.

Activated Local Immunity by CC Chemokine Ligand 19‐Transduced Embryonic Endothelial Progenitor Cells Suppresses Metastasis of Murine Ovarian Cancer

Although tumor microenvironments play a key role in successful tumor immunotherapy, effective manipulation of local immunity is difficult because of the lack of an appropriate target system. It is well known that bone marrow‐derived endothelial progenitor cells (EPCs) are actively recruited during tumor angiogenesis. Using this feature, we attempted to establish a novel therapeutic modality that targets tumor vessels of multiple metastases using embryonic endothelial progenitor cells (eEPCs) transduced with an immune‐activating gene. The eEPCs were retrovirally transduced with the mouse CC chemokine ligand 19 (CCL19) gene, a lymphocyte‐migrating chemokine. The mouse ovarian cancer cell line OV2944‐HM‐1 (HM‐1) was inoculated subcutaneously into B6C3F1 mice, along with CCL19‐tranduced eEPCs (eEPC‐CCL19), resulting in immunologic activity and tumor‐inhibitory effects. In this model, eEPC‐CCL19 showed tumor repression accompanied by increased tumor‐infiltrating CD8+ lymphocytes compared with the control group. In contrast, no tumor repression was observed when the same experiment was done in immunodeficient (SCID) mice, suggesting a crucial role of T‐cell function in this system. Next, we established a lung metastasis model by injecting HM‐1 cells or B16 melanoma cells via the tail vein. Subsequent intravenous injection of eEPC‐CCL19 leads to a decrease in the number of lung metastasis and prolonged survival. Antitumor effects were also observed in a peritoneal dissemination model using HM‐1. These results suggest that systemic delivery of an immune‐activating signal using EPCs can alter the tumor immune microenvironment and lead to a therapeutic effect, which may provide a novel strategy for targeting multiple metastases of various malignancies. STEM CELLS 2010;28:164–173

GPR54 Is a Target for Suppression of Metastasis in Endometrial Cancer

Invasion into deep myometrium and/or lymphovascular space is a well-known risk factor for endometrial cancer metastasis, resulting in poor prognosis. It is therefore clinically important to identify novel molecules that suppress tumor invasion. Reduced expression of the metastasis suppressor, kisspeptin (KISS1), and its endogenous receptor, GPR54, has been reported in several cancers, but the significance of the KISS1/GPR54 axis in endometrial cancer metastasis has not been clarified. Metastin-10 is the minimal bioactive sequence of genetic products of KISS1. Clinicopathological analysis of 92 endometrial cancers revealed overall survival is improved in cancers with high expression of GPR54 (P < 0.05) and that GPR54 expression is associated with known prognostic factors including FIGO stage, grade, and deep myometrial invasion. Through RNAi and microarray analyses, metastin-10 was predicted to suppress metastasis of GPR54-expressing endometrial cancers in vivo. Methylation analysis revealed GPR54 is epigenetically regulated. Metastin-GPR54 axis function was restored following treatment with the DNA hypomethylating agent 5-aza-DC. These data suggest that metastin-10 may be effective at inhibiting the metastatic spread of endometrial cancers in combination with demethylating agents to induce GPR54 expression. Mol Cancer Ther; 10(4); 580–90. ©2011 AACR.

Minimal deviation mucinous adenocarcinoma (‘adenoma malignum’) of the uterine corpus

Primary mucinous adenocarcinomas of the uterine corpus are typically low grade and frequently associated with endometrial hyperplasia and/or ordinary endometrioid adenocarcinoma, but may appear as a heterogeneous group of neoplasms. A case is described of a 56‐year‐old postmenopausal woman who presented with mucinous vaginal discharge. Imaging demonstrated thickened myometrium due to adenomyosis. Serum CA19‐9 levels were elevated to 486u2003U/mL. Microscopic examination of hysterectomy specimens indicated highly differentiated mucinous adenocarcinoma diffusely infiltrating the portion of adenomyosis of the corpus. In some areas endometrial glands of adenomyosis were replaced by benign‐looking mucinous metaplasia. The uterine cervix showed no abnormalities. HIK1083 and MUC6 immunohistochemistry indicated a gastric phenotype of the tumor, as seen in cases of prototypical minimal deviation adenocarcinoma (MDA) of the cervix. In summary, mucinous endometrial adenocarcinoma rarely shows features similar to MDA of the cervix. This case provokes a discussion on diagnostic and management strategy, and histogenesis of mucinous neoplasm of the endometrium.

Suppression of metastatic murine ovarian cancer cells by transduced embryonic progenitor cells.

Ovarian cancer is the leading cause of death among gynecological malignancies. Chemotherapy alone is not sufficient to achieve long-term survival of the patient with advanced stage ovarian cancer. Although cancer immune therapy has long been expected as a new modality for ovarian cancer, very few trials have been clinically successful. One of the reasons for the failure in practical immune therapy is the immune-suppressive cancer microenvironment. We have reported that immune-suppressive molecules including PD-L1, Cox or ULBP-2 are expressed in human ovarian cancer, and they suppress local tumor immunity by disturbing CD8+T cell infiltration. Thus, we attempted to develop an immune therapy that can target multiple metastatic foci and increase CD8+T cell infiltration by altering local tumor environment. Endothelial progenitor cells (EPC) were transduced with the chemokine CCL19. When injected intravenously, this “immune-stimulatory EPC” was incorporated efficiently into local tumor vessels, and exerted an anti-tumor effect in a subcutaneous tumor model, a lung metastasis model and a peritoneal dissemination model. The anti-tumor effect was not observed when immunodeficient mice were used for the experiment, suggesting that the effect is mediated by immune cells. These results suggest that EPC are ideal carriers with which to deliver immune-stimulatory signals to multiple remote metastases. Alteration of local immune environment by this method may be used in the future for individualized cancer immune therapy.