Takeshi Hirasawa

Therapeutic strategy targeting the mTOR-HIF-1α-VEGF pathway in ovarian clear cell adenocarcinoma

Malignant tumors usually involve a relatively hypoxic state, which induces overexpression of hypoxia‐inducible factor‐1α (HIF‐1α) to satisfactorily enable the tumor to survive. Thus, inhibition of the mammalian target of rapamycin (mTOR) pathway including HIF‐1α is expected to play a major role in suppression of tumor cell growth, having recently drawn much attention as an anti‐cancer therapeutic strategy for various malignant tumors. In the present study, which compared clear cell adenocarcinoma (CLA) of the ovary with serous adenocarcinoma (SEA), the immunohistochemical expression of mTOR, phosphorylated‐mTOR (p‐mTOR), HIF‐1α, and vascular endothelial growth factor (VEGF) was examined in surgically resected specimens of 29 SEA and 47 CLA. There were no significant differences in expression of mTOR, HIF‐1α and VEGF between SEA and CLA, but it was noted that p‐mTOR expression was more prominent in CLA than SEA. Then, using the cell lines of CLA (RMG‐1 and W3uF), an experimental study was designed to clarify whether tumor suppression due to downregulation of mTOR activity could represent a promising therapeutic strategy for CLA. After treatment of an analogue of rapamycin (everolimus), expression of mTOR, p‐mTOR, HIF‐1α and VEGF was examined on western blot. As a result, although mTOR expression remained unchangeable, expression of p‐mTOR, HIF‐1α and VEGF was shown to be sharply depressed. The same expression alterations were demonstrated in the xenograft model treated with everolimus. In conclusion, mTOR‐targeted therapy through usage of drugs such as everolimus may be more effective for CLA of the ovary because of its significant expression of p‐mTOR.

Granulosa cell tumor with activated mTOR-HIF-1α-VEGF pathway

The DNA‐binding activity of hypoxia‐inducible factor‐1 α (HIF‐1α) has been analyzed for various gynecological tumors. Among the tumors that were studied, there was a finding of a high level of DNA‐binding HIF‐1α activity, although it was limited to one case of adult type granulosa cell tumor (GCT). In this case a 60‐year‐old female had marked immunohistochemical expression of HIF‐1α. The expressions of the mammalian target of rapamycin (mTOR) and phosphorylated‐mTOR (p‐mTOR) were also marked, and vascular endothelial growth factor (VEGF) was moderately expressed. To compare the expression profiles, 11 consecutive cases with adult type GCT were used. All cases showed marked expressions of HIF‐1α and mTOR, but p‐mTOR expression was moderately to markedly observed in four of the 12 cases. VEGF was expressed in all cases in varying degrees. Based on the evidence that downregulation of the mTOR pathway due to treatment with rapamycin (everolimus) would suppress tumor cell growth, an experimental study using the GCT cell line was designed to clarify whether HIF‐1α and VEGF expressions decline. As a result, the expressions of p‐mTOR, HIF‐1α and VEGF were suppressed, but those of mTOR were not. It was concluded that mTOR‐targeted therapy may represent a promising strategy for some GCT with an activated mTOR‐HIF‐1α‐VEGF pathway.

Association of hypoxia-inducible factor-1 expression with histology in epithelial ovarian tumors: a quantitative analysis of HIF-1

BackgroundHypoxia-inducible factor-1 (HIF-1) is an essential transcription factor that mediates cellular and systemic homeostatic responses to reduced oxygen availability in mammals. So far, using immunohistochemistry we have analyzed the association of HIF-1α expression with histological type among epithelial ovarian tumors. In the present study, quantitative analyses of activated HIF-1 level in the nucleus and of accumulated HIF-1α level in the cytoplasm were performed to clarify whether or not the hypoxic state would be correlated to histology, malignancy, and tumor size in epithelial ovarian tumors.MethodHIF-1 level in the nucleus was analyzed using DNA binding assay, and HIF-1α level in the cytoplasm was measured by ELISA for a total of 36 epithelial ovarian tumors as follows: 5 serous adenocarcinomas (SEAs), 7 clear cell adenocarcinomas (CLAs), 7 endometrioid adenocarcinomas (ENAs), 4 mucinous adenocarcinomas (MUAs), 2 mucinous borderline tumors (MBTs), and 11 mucinous adenomas.ResultsHIF-1 level (mg/ml) in the nucleus and HIF-1α level (mg/ml) in the cytoplasm were on average 0.116 and 0.178 for SEAs, 0.328 and 0.306 for CLAs, 0.171 and 0.305 for ENAs, 0.097 and 0.176 for MUAs, 0.224 and 0.180 for mucinous borderline tumors, 0.152 and 0.154 for mucinous adenomas. CLAs showed the highest levels for both of HIF-1 and HIF-1α, while MUAs showed the lowest levels of both. Mucinous adenomas were higher in HIF-1 than MUAs.ConclusionHypoxic state was considered to be closely related to histological type of epithelial ovarian tumors, suggesting that CLAs may be most hypoxic. In the comparison of mucinous tumors, malignancies would not always become most hypoxic. Tumor size may not be strongly associated with hypoxic state.

Case of uterine cervical carcinosarcoma

Carcinosarcoma (CS) is a rare neoplasm that is called a mixed epithelial and mesenchymal malignancy. CS of the uterine cervix is much less common than its counterparts in the uterine corpus. A 61‐year‐old, gravida 2, para 2 woman, who had undergone menopause 16 years prior to the presentation, was diagnosed with CS of the uterine cervix. A semiradical hysterectomy was carried out on the diagnosis of stage Ib1 cervical cancer. The patient underwent whole pelvic 45 Gy radiation as a postoperative additional treatment, but she died from multiple organ failure by metastasis 17 months after the operation. The tumor protruded from the cervix to the vagina and measured 4.5 × 3.0 cm. Histologically, the tumor was characterized as a squamous cell carcinoma and mesenchymal malignancy, represented by osteosarcomatous components. The stroma was largely composed of atypical spindle‐shaped cells, which were immunohistochemically demonstrated to be of epithelial origin. Uterine cervical CS is one of the aggressive malignancies, and squamous cell carcinomas are common epithelial counterparts of cervical CS as well as adenocarcinomas.

An Up-to-Date Anti-Cancer Treatment Strategy Focusing on HIF-1α Suppression: Its Application for Refractory Ovarian Cancer

Hypoxia inducible factor-1α (HIF-1α) predominantly determines the transcriptional activity of HIF-1, which induces the certain genetic expressions to participate in the proliferation and progression of the tumor. It is supposed that HIF-1α is also an extremely important factor in cancer treatment. Based on the results of our recent analyses using ovarian tumors, which indicated the close association of HIF-1α expression with the acquisition of malignancy and the characterization of histology, we further investigated the possibility of a new strategy of cancer therapy that targeted HIF-1α inhibition in the ovarian carcinoma. The cell line HUOCA-II, which originates from the refractory ovarian clear cell adenocarcinoma, was treated with rapamycin. The inhibitory effect of HIF-1α was analyzed by immunohistochemistry and western blotting. It was demonstrated that inhibition of HIF-1α and vascular endothelial growth factor (VEGF) expressions would lead to the down-regulation of tumor cell proliferation. Interestingly, there was little or no change in GLUT-1 expression by rapamycin administration. Thus, the inhibition of GLUT-1 may also be a key for the new strategy of cancer therapy as well as HIF-1α and VEGF.

Cytologic Study of Ascites and the Endometrium in Ovarian Carcinoma

OBJECTIVE To ascertain the usefulness of endometrial cytology with ovarian cancers when examining extension of the disease and to analyze significant factors associated with migration of ovarian cancer cells into the uterine cavity. STUDY DESIGN Cytologic results on ascites and the endometrium were analyzed in 87 patients with primary ovarian cancer in the absence of metastasis to the endometrium or cervicovagina. RESULTS Positive results for cytology were found in 62/87 of ascites cases (71.3%) and in 20/87 endometrium cases (23.0%). The 15 cases (15/62 or 24.2%) positive for ascitic and endometrial cytology, divided clinically into stage III (6 cases) and stage IV (9 cases), were classified histologically as serous, 7 cases; mucinous, 2 cases; clear cell, 4 cases; endometrioid, 1 case; and unclassified, 1 case. Half the clear cell carcinomas (4/8 or 50.0%) were positive in the ascites and endometrium. The ascitic volume at surgery exceeded 500 mL in 9/15 cases (60.0%). CONCLUSION Papillae with basement membrane material in the cores may be structurally associated with migration of ovarian cancer cells into the uterine cavity, especially in clear cell carcinomas. Cytologic positivity of the endometrium and ascites significantly correlated with ascitic volume.

Immunohistochemical expression of somatostatin type 2A receptor in neuroendocrine carcinoma of uterine cervix

IntroductionSomatostatin and its analogues inhibit the growth of various kinds of endocrine and exocrine cells via the somatostatin receptor (SSTR). Expression of SSTR2A has been reported in many well-differentiated neuroendocrine tumors and small numbers of poorly differentiated neuroendocrine carcinomas. SSTR2A-positive neuroendocrine tumors may be treatable by SS analogues. Uterine cervical neuroendocrine carcinoma is generally associated with poor prognosis.Materials and methodsWe examined the expression of SSTR2A and other neuroendocrine markers [neural cell adhesion molecule (NCAM), chromogranin A, and synaptophysin] by immunohistochemical methods in seven neuroendocrine tumors of the uterine cervix: five small cell carcinomas and two large cell neuroendocrine carcinomas.ResultsSSTR2A was expressed in one small cell carcinoma and two neuroendocrine large cell carcinomas. The cell membrane of cells in these carcinomas showed strong immunohistochemical reactivity for SSTR2A. SSTR2A-positive cases are also frequently positive for the expression of chromogranin A. Treatment with SSTR2A may be effective for endocrine tumors. The results indicate the usefulness of SSTR2A analogs for the treatment of some uterine neuroendocrine carcinomas.

Fully sialylated alpha-chain of complement 4–binding protein: Diagnostic utility for ovarian clear cell carcinoma

Objective. While a certain fraction of endometriomas can develop de novo epithelial ovarian cancer (EOC) such as clear cell carcinoma (OCCC), there is currently no useful biomarker available for early detection of OCCC from endometriomas. The aim of this study was to describe the diagnostic utility of a novel biomarker for EOC especially for OCCC to distinguish from endometrioma. Methods. More than 100,000 glycan structures of serum glycoproteins obtained from 134 pretreatment all stage EOC patients (including 45 OCCCs) and 159 non-cancer control women (including 36 endometriomas) were explored for a mass spectrum approach. Diagnostic accuracy of identified biomarker was compared to the one of CA-125 by comparing area under curve (AUC) and positive/negative predictive values (PPV and NPV). Results. A2160, a fully-sialylated alpha-chain of complement 4-binding protein, was identified as a candidate target marker. A2160 was significantly elevated in all stages of OCCC compared to with endometriomas. Diagnostic accuracy of A2160 (cutoff 1.6 U/mL) to distinguish early stage OCCC from endometrioma is significantly higher than that of CA-125 (cutoff 35 IU/L): AUC for A2160 versus CA-125,0.92 versus 0.67; PPV 95% versus 64%; and NPV 85% versus 58%. In addition, fully-sialylated glycans had a higher accuracy for diagnosing EOC as compared to partially-sialylated glycans of alpha-chain of complement 4-binding protein. Conclusion. Our study suggested that A2160 may be a useful biomarker to distinguish early-stage OCCC from endometrioma. This new biomarker can be potentially applied for the monitoring of endometrioma patients, making possible the early diagnosis of OCCC.

Alterations of hypoxia-induced factor signaling pathway due to mammalian target of rapamycin (mTOR) suppression in ovarian clear cell adenocarcinoma: in vivo and in vitro explorations for clinical trial.

Objectives Before setting into the clinical trial using a combination of mammalian target of rapamycin (mTOR) inhibitors (rapamycin and everolimus) and other anticancer drugs, this study was conducted to confirm the efficacy of the new therapeutic strategy for ovarian clear cell adenocarcinoma (CCA), which targeted mTOR–hypoxia-induced factor (HIF) signal transduction system. Materials and Methods Using the cultured cells of CCA and animal models, alteration of mTOR-HIF cofactors and cell proliferation under the mTOR inhibitor–treated condition were analyzed. Results Mammalian target of rapamycin–HIF cofactors were inhibited dependent on concentration by mTOR inhibitor, resulting in suppression of the cultured CCA proliferation. However, von Hippel-Lindau was up-regulated at the messenger RNA level. In the nude mice with subcutaneously implanted CCA cells, apoptosis and necrosis were detected especially around the center of the tumors in the mTOR inhibitor–treated group more conspicuously than in the nontreated group. In the assessment of combination therapy with other antitumor agents, a combined treatment with mTOR inhibitor and chemotherapeutic agents caused a significant decrease in tumor size compared to the chemotherapeutic agents–only group. Conclusions Treatment by mTOR inhibitor is expected to down-regulate the cell proliferation of the CCA as a new therapeutic strategy.

Analysis of mTOR Inhibition-Involved Pathway in Ovarian Clear Cell Adenocarcinoma

This study was designed to clarify the mechanism of the mammalian target of rapamycin (mTOR)-hypoxia inducible factor-1 (HIF-1) pathway using the cultured cell strain derived from human ovarian clear cell adenocarcinoma (CCA). Everolimus (a derivative of rapamycin)-treated cells and non-treated cells did not show any difference in mTOR expression. But, phosphorylated-mTOR (p-mTOR) expression significantly decreased in the treated cells, and mTOR-related factors such as phosphorylated-4E-BP1 (p-4E-BP1), HIF-1α, and vascular endothelial growth factor (VEGF) in the downstream region of mTOR revealed a marked decrease in expression. The analysis of influences of the drug on the HIF-1α degradation system showed an increase in von-Hippel Lindau (VHL) expression in the treated cells. Increase of cleaved caspase-3, one of key factors involved in apoptosis, was also shown in the treated cells. In the next step, using nude mice implanted with RMG-1 cells, a decrease in tumor size was demonstrated in 4 of the 7 mice which were orally administered with everolimus. As a result, it was suggested that everolimus administration would be helpful as an anti-tumor therapy for CCA not only via down-regulation of p-mTOR but also degradation of HIF-1α by VHL and induction of apoptosis by cleaved caspase-3.