Taisuke Mori

Synuclein-γ Is Closely Involved in Perineural Invasion and Distant Metastasis in Mouse Models and Is a Novel Prognostic Factor in Pancreatic Cancer

Purpose: Perineural invasion is associated with the high incidence of local recurrence and a dismal prognosis in pancreatic cancer. We previously reported a novel perineural invasion model and distinguished high– and low–perineural invasion groups in pancreatic cancer cell lines. This study aimed to elucidate the molecular mechanism of perineural invasion. Experimental Design: To identify key biological markers involved in perineural invasion, differentially expressed molecules were investigated by proteomics and transcriptomics. Synuclein-γ emerged as the only up-regulated molecule in high–perineural invasion group by both analyses. The clinical significance and the biological property of synuclein-γ were examined in 62 resected cases of pancreatic cancer and mouse models. Results: Synuclein-γ overexpression was observed in 38 (61%) cases and correlated with major invasive parameters, including perineural invasion and lymph node metastasis (P < 0.05). Multivariate analyses revealed synuclein-γ overexpression as the only independent predictor of diminished overall survival [hazard ratio, 3.4 (95% confidence interval, 1.51-7.51)] and the strongest negative indicator of disease-free survival [2.8 (1.26-6.02)]. In mouse perineural invasion and orthotopic transplantation models, stable synuclein-γ suppression by short hairpin RNA significantly reduced the incidence of perineural invasion (P = 0.009) and liver/lymph node metastasis (P = 0.019 and P = 0.020, respectively) compared with the control. Conclusions: This is the first study to provide in vivo evidence that synuclein-γ is closely involved in perineural invasion/distant metastasis and is a significant prognostic factor in pancreatic cancer. Synuclein-γ may serve as a promising molecular target of early diagnosis and anticancer therapy.

Specific expression of hepatocyte nuclear factor-1β in the ovarian clear cell adenocarcinoma and its application to cytological diagnosis

Ascitic cytological diagnosis is critical, but ovarian adenocarcinoma cells and reactive mesothelial cells can be difficult to distinguish because they usually have atypical cell nuclei and increased nuclear/cytoplasmic ratios. Previous studies using DNA microarrays have demonstrated that hepatocyte nuclear factor‐1β (HNF‐1β) is expressed specifically in clear cell adenocarcinoma (CCC). Thus, in the present study, we investigated the usefulness of HNF‐1β as an immunocytochemical diagnostic marker of CCC in ascitic specimens. We first confirmed that HNF‐1β expression levels were significantly higher in CCC than in non‐CCC (i.e. serous adenocarcinoma, mucinous adenocarcinoma and endometrioid adenocarcinoma) in 55 surgical specimens at both the mRNA (P < 0.05) and protein (P < 0.05) levels by real‐time polymerase chain reaction and immunohistochemistry, respectively. Immunocytochemistry of 60 cytological specimens showed significant positivity in CCC cases whereas all non‐CCC cells, except for three endometrioid adenocaricnoma cases, and mesothelial cells in the background stained negatively for anti‐HNF‐1β antibody (P < 0.05). The sensitivity and specificity were calculated to be 0.955 and 0.921, respectively. Immmunostaining patterns of HNF‐1β on cytological specimens were similar to those observed on histopathological ovarian specimens from the same patients. Double immunohistochemical staining using anti‐HNF‐1β antibody and HBME‐1, a mesothelium‐specific monoclonal antibody, confirmed that anti‐HNF‐1β antibody distinguished CCC cells and mesothelial cells. In conclusion, our findings indicate the specific expression of HNF‐1β in ovarian CCC and possible clinical applications of HNF‐1β immunocytochemical staining for the differential cytopathological diagnosis of CCC from non‐CCC, as well as from mesothelial cells using cytological specimens from ovarian carcinoma patients. (Cancer Sci 2007; 98: 387–391)

Dienogest reduces HSD17β1 expression and activity in endometriosis

Endometriosis is an estrogen-dependent disease. Abnormally biosynthesized estrogens in endometriotic tissues induce the growth of the lesion and worsen endometriosis-associated pelvic pain. Dienogest (DNG), a selective progesterone receptor agonist, is widely used to treat endometriosis and efficiently relieves the symptoms. However, its pharmacological action remains unknown. In this study, we elucidated the effect of DNG on enzymes involved in local estrogen metabolism in endometriosis. Surgically obtained specimens of 23 ovarian endometriomas (OE) and their homologous endometrium (EE), ten OE treated with DNG (OE w/D), and 19 normal endometria without endometriosis (NE) were analyzed. Spheroid cultures of stromal cells (SCs) were treated with DNG and progesterone. The expression of aromatase, 17β-hydroxysteroid dehydrogenase 1 (HSD17β1), HSD17β2, HSD17β7, HSD17β12, steroid sulfatase (STS), and estrogen sulfotransferase (EST) was evaluated by real-time quantitative PCR. The activity and protein level of HSD17β1 were measured with an enzyme assay using radiolabeled estrogens and immunohistochemistry respectively. OESCs showed increased expression of aromatase, HSD17β1, STS, and EST, along with decreased HSD17β2 expression, when compared with stromal cells from normal endometria without endometriosis (NESCs) (P<0.01) or stromal cells from homologous endometrium (EESCs) (P<0.01). In OESCs, DNG inhibited HSD17β1 expression and enzyme activity at 10(-7) M (P<0.01). Results of immunohistochemical analysis displayed reduced HSD17β1 staining intensity in OE w/D (P<0.05). In conclusion, DNG exerts comprehensive inhibition of abnormal estrogen production through inhibition of aromatase and HSD17β1, contributing to a therapeutic effect of DNG on endometriosis.

Neoadjuvant weekly carboplatin and paclitaxel followed by radical hysterectomy for locally advanced cervical cancer: long-term results.

Introduction: To determine the long-term effect of neoadjuvant chemotherapy with paclitaxel and carboplatin on a weekly schedule followed by radical surgery for patients with locally advanced cervical cancer. Materials and Methods: Thirty patients with stage IB2 to IIIB uterine cervical cancer were treated with paclitaxel (60 mg/m2) and carboplatin (area under the curve, 2-an area under the time-concentration curve of 2 mg × min/mL based on creatinine clearance) every week for 6 cycles. A radical hysterectomy was performed 6 days after the final administration of neoadjuvant chemotherapy. The patients were followed up, and 5-year progression-free survival (PFS) and overall survival (OS) were evaluated. Results: Of 30 patients, 28 were followed up. The median follow-up period was 55.6 months (range, 26-83 months). An objective response (complete response + partial response) to the treatment was observed in 26 patients (87%; 95% confidence interval, 70%-95%). Two had complete response, 4 had stable disease, and the remaining patients had partial response; progressive disease was not seen in this study. A radical hysterectomy was performed in 28 patients without delay. Thirteen patients with high-risk factors received radiotherapy after surgery. The 5-year PFS and OS rates were 78.6% and 81.8%, respectively. The 5-year PFS and OS for patients with stage IB2 to IIB cervical cancer were 79.2% and 83.1%, respectively, which were comparable with those in the concurrent chemoradiation therapy study previously reported. There was no significant correlation in survival between preoperative staging and cell type, whereas larger initial tumor size and lymph node metastasis tended to be negatively correlated with survival. Conclusions: Neoadjuvant chemotherapy with paclitaxel and carboplatin on a weekly schedule followed by radical surgery for patients with locally advanced cervical cancer is a promising mode of therapy that may improve the prognosis. It would be worthwhile to conduct larger-scale trials for comparison with the results of the chemoradiation therapy study.

A pilot study of docetaxel-carboplatin versus paclitaxel-carboplatin in Japanese patients with epithelial ovarian cancer

BackgroundIt has been reported that a docetaxel-carboplatin combination as first-line chemotherapy for ovarian cancer showed a level of progression-free survival similar to that of paclitaxel-carboplatin while reducing neurotoxicity and improving quality of life. We investigated the recommended doses of docetaxel-carboplatin in Japanese patients with ovarian cancer and conducted a comparative study of docetaxel-carboplatin versus paclitaxel-carboplatin.MethodsThirty-nine patients with ovarian cancer were enrolled in this study and 38 patients were evaluated. We conducted a dose-escalation study using a docetaxel dose of 70 mg/m2 and carboplatin AUC 5 and 6. In the comparative study, patients received either docetaxel 70 mg/m2 and carboplatin AUC 5 or paclitaxel 175 mg/m2 and carboplatin AUC 5. Progression-free survival, survival rate at 2 years, response rate, toxicity, and quality of life were investigated.ResultsIn the dose-finding study, we determined the recommended doses as docetaxel 70 mg/m2 and carboplatin AUC 5. In the comparative study, the two arms showed similar progression-free survival. Grade 4 neutropenia occurred more frequently in the docetaxel-carboplatin group (84.6%) than in the paclitaxel-carboplatin group (43.8%), while sensory neurotoxicity was less frequent in the docetaxel-carboplatin group (53.8%) than in the paclitaxel-carboplatin (68.8%) group. There were significant differences in the quality-of-life data in favor of docetaxel-carboplatin.ConclusionWe determined the recommended doses of docetaxel-carboplatin for Japanese patients with ovarian cancer to be docetaxel 70 mg/m2 and carboplatin AUC 5. In the comparative study, we suggest that the docetaxel-carboplatin combination is effective and well tolerated as first-line chemotherapy for Japanese patients with ovarian cancer.

Anti-tumor effect of estrogen-related receptor alpha knockdown on uterine endometrial cancer

Estrogen-related receptor (ERR)α presents structural similarities with estrogen receptor (ER)α. However, it is an orphan receptor not binding to naturally occurring estrogens. This study was designed to investigate the role of ERRα in endometrial cancer progression. Immunohistochemistry analysis on 50 specimens from patients with endometrial cancer showed that ERRα was expressed in all examined tissues and the elevated expression levels of ERRα were associated with advanced clinical stages and serous histological type (p < 0.01 for each). ERRα knockdown with siRNA suppressed angiogenesis via VEGF and cell proliferation in vitro (p < 0.01). Cell cycle and apoptosis assays using flow cytometry and western blot revealed that ERRα knockdown induced cell cycle arrest during the mitotic phase followed by apoptosis initiated by caspase-3. Additionally, ERRα knockdown sensitized cells to paclitaxel. A significant reduction of tumor growth and angiogenesis was also observed in ERRα knockdown xenografts (p < 0.01). These findings indicate that ERRα may serve as a novel molecular target for the treatment of endometrial cancer.

Loss of AF-6/afadin induces cell invasion, suppresses the formation of glandular structures and might be a predictive marker of resistance to chemotherapy in endometrial cancer

BackgroundAF-6/afadin plays an important role in the formation of adherence junctions. In breast and colon cancer, loss of AF-6/afadin induces cell migration and cell invasion. We aimed to elucidate the role of AF-6/afadin in human endometrial cancer.MethodsMorphology and AF-6/afadin expression in endometrial cancer cell lines was investigated by 3-dimensional culture. We used Matrigel invasion assay to demonstrate AF-6/afadin knockdown induced invasive capability. Cell proliferation assay was performed to estimate chemoresistance to doxorubicin, paclitaxel and cisplatin induced by AF-6/afadin knockdown. The associations between AF-6/afadin expression and clinicopathological status were determined by immunohistochemical analysis in endometrial cancer tissues. Informed consent was obtained from all patients before the study.ResultsThe majority of cell clumps in 3-dimensional cultures of Ishikawa cells that strongly expressed AF-6/afadin showed round gland-like structures. In contrast, the cell clumps in 3-dimensional cultures of HEC1A and AN3CA cells—both weakly expressing AF-6/afadin—showed irregular gland-like structures and disorganized colonies with no gland-like structures, respectively. AF-6/afadin knockdown resulted in reduced number of gland-like structures in 3-dimensional cultures and enhancement of cell invasion and phosphorylation of ERK1/2 and Src in the highly AF-6/afadin-expressing endometrial cancer cell line. Inhibitors of MAPK/ERK kinase (MEK) (U0126) and Src (SU6656) suppressed the AF-6/afadin knockdown-induced invasive capability. AF-6/afadin knockdown induced chemoresistance to doxorubicin, paclitaxel and cisplatin in Ishikawa cells, not in HEC1A. Immunohistochemical analysis showed that AF-6/afadin expression was significantly associated with myometrial invasion and high histological grade.ConclusionsAF-6/afadin regulates cell morphology and invasiveness. Invasive capability is partly regulated through the ERK and Src pathway. The inhibitors to these pathways might be molecular-targeted drugs which suppress myometrial invasion in endometrial cancer. AF-6/afadin could be a useful selection marker for fertility-sparing therapy for patients with atypical hyperplasia or grade 1 endometrioid adenocarcinoma with no myometrial invasion. AF-6/afadin knockdown induced chemoresistance especially to cisplatin. Therefore, loss of AF-6/afadin might be a predictive marker of chemoresistance to cisplatin.

Peroxisome proliferator-activated receptor gamma, coactivator 1α enhances local estrogen biosynthesis by stimulating aromatase activity in endometriosis.

CONTEXT Endometriosis is an estrogen-dependent disease, and estrogen is overproduced by abnormally elevated aromatase in endometriotic tissues. Peroxisome proliferator-activated receptor gamma, coactivator 1α (PGC-1α) is a transcriptional coactivator-modulating steroid hormone. OBJECTIVE To investigate the effect of PGC-1α on aromatase activity in endometriosis. DESIGN Specimens from ovarian endometrioma (OE), endometrium with endometriosis (EE), and normal endometrium (NE) were analyzed for PGC-1α and aromatase expression. PGC-1α-dependent changes in aromatase expression in primary cultured stromal cells (SCs) were identified using luciferase and enzymatic assays, exon I-specific RT-PCR, and real-time PCR. Environmental stimulus-induced changes in PGC-1α were also examined. RESULTS PGC-1α was more highly expressed in OE than in EE and NE (P < .01). In OE, PGC-1α was coexpressed with aromatase, and their mRNA expressions were also correlated (r = 0.56, P = .02). PGC-1α was recruited to the nuclear receptor half-site between PI.3 and PII in the aromatase promoter. PGC-1α overexpression enhanced aromatase promoter activity (P < .01), mRNA expression (P < .05), and enzymatic activity (P < .01) in SCs from OE, but not in SCs from EE or NE. The levels of PI.3, PII, and exon II mRNA increased and transcriptional enhancement was abolished by mutation of the PGC-1α-interacting site. PGC-1α expression was enhanced in SCs from OE by tumor necrosis factor (TNF)-α (P < .05) but not by hypoxia or 17β-estradiol. CONCLUSIONS PGC-1α stimulated by TNF-α regulates aromatase expression and activity to promote local estrogen biosynthesis in OE, suggesting that PGC-1α is a promising candidate for novel targeted therapies in endometriosis treatment.

Intravenous paclitaxel is specifically retained in human gynecologic carcinoma tissues in vivo.

Paclitaxel and carboplatin are commonly used and well-tolerated agents for gynecologic malignancies. The persistence of platinum in human tissues for 14 days and the long-term retention of platinum in tissues for up to 17 months have been reported. Paclitaxel remains in human uterine cervical cancer tissues for 6 days. These findings prompted us to determine the retention of paclitaxel and carboplatin in human uterine cervical carcinoma, endometrial carcinoma, ovarian carcinoma, and pelvic lymph nodes to establish baseline parameters and guide the development of more effective treatment interventions. Thirty patients with uterine or ovarian carcinomas were treated with intravenous weekly paclitaxel-carboplatin chemotherapy before surgery. The concentrations of these agents in carcinoma tissue, normal cervical, myometrial and ovarian tissues, and pelvic lymph nodes were measured 5 days after the final administration. Paclitaxel was specifically retained in cervical, endometrial, and ovarian carcinoma tissues but was not detected in lymph nodes. In contrast to paclitaxel, carboplatin was readily detectable with similar levels in all tumor-associated and normal host tissues. In addition, a low paclitaxel concentration in cervical carcinoma tissue was significantly associated with short progression-free survival and overall survival. Further studies are needed to clarify the tissue distribution of anticancer drugs in humans and promote optimal treatment strategies enhancing paclitaxel lymphatic targeting.

Exacerbation of endometriosis due to regulatory T cell dysfunction.

Context: Endometriosis is a chronic inflammatory disease associated with altered immune response to endometrial cells facilitating the implantation and proliferation of ectopic endometrial tissues. Although regulatory T (Treg) cells play a key role in T cell‐mediated immune response and development of immune disorders, their significance in endometriosis remains to be elucidated. Recently, CD4+CD45RA‐ forkhead box protein 3 (Foxp3)hi T cells, activated Treg cells, have been identified as a functionally true suppressive population of Treg cells. Objective: To investigate the role of Treg cells in endometriosis. Design: Three Treg cell fractions (resting Treg cells, activated Treg cells, and non‐Treg cells) were examined using flow cytometry in the endometrioma, endometrium, peritoneal fluid, and peripheral blood obtained from women with (n = 27) and without (n = 28) endometriosis. A mouse model of endometriosis was made in Foxp3tm3Ayr/J (Foxp3DTR) C57BL/6 Treg cell‐depleted mice (n = 28). Results: In women with endometrioma, the proportion of activated Treg cells in the endometrioma and the endometrium, but not in the peritoneal fluid or peripheral blood, was significantly decreased compared with that in women without endometriosis. In Foxp3DTR/diphtheria toxin mice, the number and weight of endometriotic lesions, inflammatory cytokine levels and angiogenetic factors were significantly increased compared with those in control mice. Conclusions: Treg cell deficiency exaggerates local inflammation and angiogenesis and simultaneously facilitates the attachment and growth of endometrial implants. The findings provide an insight into dysregulated immune response for the pathogenesis and development.