Mikio Terauchi

Involvement of SDF‐1α/CXCR4 axis in the enhanced peritoneal metastasis of epithelial ovarian carcinoma

Epithelial ovarian carcinoma (EOC) spreads by implantation of tumor cells onto the human peritoneal mesothelial cells (HPMCs) lining the peritoneal cavity. The aim of this study was to determine whether the stromal cell‐derived factor‐1α (SDF‐1α)/CXCR4 axis is involved in the interaction of EOC cells with HPMCs in peritoneal metastasis. Clinically, we first evaluated CXCR4 expression in sections from 36 primary EOCs using immunohistochemistry. We next examined whether SDF‐1α played roles in EOC progression, including in proliferation, cell motility, attachment to HPMCs, and the in vivo development of peritoneal metastasis through CXCR4. Of the 36 carcinomas, 16 cases (44.4%) were positive for CXCR4 immunoexpression. Positive CXCR4 expression significantly predicted poorer overall survival compared with negative expression (p = 0.0069). We found CXCR4 expression in both EOC cells and HPMCs. In contrast, the level of production of SDF‐1α by HPMCs was higher than that by various EOC cells. Functionally, SDF‐1α induced enhanced attachment between ES‐2 cells and HPMCs or extracellular matrix components. The enhancement of adhesion potential by SDF‐1α was inhibited by AMD3100, a CXCR4 antagonist, and by phosphatidylinositol 3 kinase and p44/42 inhibitors. Furthermore, intraperitoneal treatment with AMD3100 resulted in reduced dissemination in nude mice inoculated with ES‐2 cells. The present results suggest that there may be a link between the SDF‐1α/CXCR4 axis and enhanced intraperitoneal dissemination of EOC and that CXCR4 may be a novel target for the treatment of EOC.

Indoleamine 2,3-dioxygenase is a novel prognostic indicator for endometrial cancer

Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolising enzyme inducing immune tolerance. The present study aimed to investigate IDO expression and its prognostic significance in endometrial cancer. Indoleamine 2,3-dioxygenase expression in endometrial cancer tissues (n=80) was immunohistochemically scored as four groups (IDO−, 1+, 2+, and 3+). The high IDO expression (IDO2+ or 3+) in tumour cells was found in 37 (46.3%) of the 80 cases, and was positively correlated with surgical stage, myometrial invasion, lymph-vascular space involvement, and lymph node metastasis, but not with the histological grade. Patients with high IDO expression had significantly impaired overall survival and progression-free survival (PFS) (P=0.002 and P=0.001, respectively) compared to patients with no or weak expression of IDO (IDO− or 1+). The 5-year PFS for IDO−/1+, 2+, and 3+ were 97.7, 72.9, and 36.4%, respectively. Even in patients with early-stage disease (International Federation of Gynecology and Obstetrics I/II, n=64), the PFS for IDO2+/3+ was significantly poor (P=0.001) compared to that for IDO−/1+. On multivariate analysis, IDO expression was an independent prognostic factor for PFS (P=0.020). These results indicated that the high IDO expression was involved in the progression of endometrial cancer and correlated with the impaired clinical outcome, suggesting that IDO is a novel and reliable prognostic indicator for endometrial cancer.

Inverse Correlation between Tumoral Indoleamine 2,3-Dioxygenase Expression and Tumor-Infiltrating Lymphocytes in Endometrial Cancer: Its Association with Disease Progression and Survival

Purpose: Tumor escape from host immune systems is a crucial mechanism for disease progression. We recently showed that the immunosuppressive enzyme indoleamine 2,3-dioxygenase (IDO) is a prognostic indicator for endometrial cancer. The purpose of the present study was to investigate the relationship between IDO expression and tumor-infiltrating lymphocytes (TIL) or natural killer (NK) cells and to clarify their prognostic effect in endometrial cancer. Experimental Design: Immunohistochemical staining for IDO expression in endometrial cancer tissues (n = 65) was done. Tumor-infiltrating CD3+ and CD8+ lymphocytes, as well as CD57+ NK cells, were counted in serial tissue sections. Results: High IDO expression in tumor cells was found in 32 of 65 cases and was positively correlated with myometrial invasion, nodal metastasis, and lymph-vascular space involvement. We also found a significant correlation between high IDO expression and reduced numbers of CD3+, CD8+, and CD57+ cells infiltrating into both the tumor epithelium and stroma. Patients with high IDO expression, a low number of stromal CD3 (<60), low intraepithelial CD8 (<25), or low stromal CD8 (<40) had significantly impaired progression-free survival. On multivariate analysis, IDO expression and the number of stromal CD3+ TILs were independent prognostic factors for impaired progression-free survival. Conclusions: Tumoral IDO expression correlated with a reduced number of TILs and NK cells in endometrial cancer, possibly contributing to disease progression and impaired clinical outcome. These findings suggest that targeting IDO to restore host antitumor immunity may be a therapeutic strategy for endometrial cancer.

Expression of Twist increases the risk for recurrence and for poor survival in epithelial ovarian carcinoma patients

Twist is a transcription factor that regulates the expression of tumour suppressors such as E-cadherin. We examined the distribution and expression of Twist in human epithelial ovarian carcinoma (EOC) to examine its clinical significance. Paraffin sections from EOC tissues (n=82) were immunostained with Twist antibody, and the staining intensity was evaluated. The clinicopathological factors examined were age, International Federation of Gynecology and Obstetrics staging, histological type, tumour grade, preoperative value of CA125, peritoneal cytology, volume of ascites and residual tumour after cytoreductive surgery. Overall survival (OS) and progression-free survival (PFS) were evaluated using the Kaplan–Meier method, and multivariate analysis was performed using the Cox proportional hazard analysis. Of the 82 carcinomas, 49 (59.8%) cases were negative for Twist immunoexpression, and 33 (40.2%) were positive immunoexpression. When categorized into negative vs positive expression, Twist was not associated with any of the clinicopathological parameters examined. However, positive Twist expression significantly predicted poorer OS and PFS when compared with negative expression (P<0.0001). In the multivariate analyses, positive Twist expression was the only independent prognostic factor for survival in this study (P<0.0001). Positive Twist expression seems to be a useful marker in patients with EOC likely to have an unfavourable clinical outcome.

Inhibition of APN/CD13 leads to suppressed progressive potential in ovarian carcinoma cells.

BackgroundAminopeptidase N (APN/CD13), a 150-kDa metalloprotease, is a multifunctional cell surface aminopeptidase with ubiquitous expression. Recent studies have suggested that APN/CD13 plays an important role in tumor progression of several human malignancies. In the current study, we investigated the role of APN/CD13 in ovarian carcinoma (OVCA) progression.MethodsWe first examined the expression of APN/CD13 at the protein level in a variety of OVCA cell lines and tissues. We subsequently investigated whether there was a correlation between APN/CD13 expression and invasive potential of various OVCA cell lines. Moreover, we investigated the function of APN/CD13 in OVCA cells using bestatin, an APN/CD13 inhibitor, or transfection of siRNA for APN/CD13.ResultsWe confirmed that APN/CD13 was expressed in OVCA tissues and cell lines to various extents. There was a positive correlation between APN/CD13 expression and migratory potential in various OVCA cell lines with accordingly enhanced secretion of endogenous MMP-2. Subsequently, we found a significant decrease in the proliferative and migratory abilities of OVCA cells after the addition of bestatin or the inhibition of APN/CD13 expression by siRNA. Furthermore, in an animal model, daily intraperitoneal administration of bestatin after inoculation of OVCA cells resulted in a decrease of peritoneal dissemination and in prolonged survival of nude mice.ConclusionThe current data indicate the possible involvement of APN/CD13 in the development of OVCA, and suggest that clinical use of bestatin may contribute to better prognosis for ovarian carcinoma patients.

Possible involvement of TWIST in enhanced peritoneal metastasis of epithelial ovarian carcinoma

Loss of E-cadherin triggers peritoneal dissemination, leading to an adverse prognosis for most patients with epithelial ovarian carcinoma (EOC). Because TWIST mainly regulates the epithelial-to-mesenchymal transition and is one of the E-cadherin repressors, we investigated the possibility that TWIST expression affects peritoneal metastasis of EOC using siRNA technique. In the present study, we showed a correlation between TWIST expression and EOC cellular morphology. Furthermore, we demonstrated that the suppression of TWIST expression in EOC cells (HEY) alters the cellular morphology from a fibroblastic and motile phenotype to an epithelial phenotype, and inhibits the adhesion of these cells to mesothelial monolayers. To investigate the mechanism by which down-regulation of TWIST leads to inhibition of adhesion to mesothelial cells (MCs), expression of adhesion molecules (CD29, CD44 and CD54) were observed. Moreover, matrix metalloproteinase 2 and membrane type 1 matrix metalloproteinase, important markers associated with invasive and metastatic potential, were remarkably reduced. This findings suggests that reduced expression of TWIST suppresses the multistep process of peritoneal dissemination (detachment from the primary lesion, adhesion to MCs and invasion of MCs) and may be a potential therapeutic target for the treatment of this carcinoma.

Aminopeptidase N (APN)/CD13 inhibitor, Ubenimex, enhances radiation sensitivity in human cervical cancer.

BackgroundRadiotherapy can be used to treat all stages of cervical cancer. For improving local control via radiotherapy, it is important to use additional antitumor agents. Aminopeptidase N (APN)/CD13, a 150-kDa metalloproteinase, is a multifunctional cell surface aminopeptidase with ubiquitous expression. Recent studies have suggested that APN/CD13 plays an important role in tumor progression in several human malignancies.MethodsWe investigated whether the suppression of APN/CD13 using Ubenimex, an inhibitor of APN/CD13 activity, may affect tumor radiosensitivity in cervical cancer cells both in vitro and in vivo. Cell surface APN/CD13 activity in HeLa cells was calculated using alanine-p-nitroanilido as a substrate. For colony formation assays, single-dose radiation and/or Ubenimex were administered to each dish of HeLa cells, and these dishes were cultured for 14 days. Molecular changes of apoptosis were determined by Western blot. Apoptosis was evaluated by Annexin-V PI staining (flow cytometry analysis) and the Tunel method. Moreover, we investigated the effect of combining Ubenimex and low-dose radiation on tumor growth using nude mice.ResultsWe demonstrated that Ubenimex enhanced the effectiveness of radiotherapy, acting as a radiosensitizer both in vitro and in vivo. In colony formation assays, a significant decline in clonogenic survival was observed in Ubenimex-treated cells. Mice treated with a combination of radiation and Ubenimex showed a significant prolongation of the tumor-doubling time compared with the control, Ubenimex, or radiation-alone groups. We also showed that ubenimex enhanced radiation-induced apoptosis in vitro and in vivo.ConclusionAlthough further studies are needed, this report suggests that Ubeniemx acts as a radiosensitizer in cervical cancer treatment, and that the inhibition of APN/CD13 activity may represent a new approach for improving the therapeutic efficacy of radiotherapy for uterine cervical cancer.

Involvement of aminopeptidase N in enhanced chemosensitivity to paclitaxel in ovarian carcinoma in vitro and in vivo

Aminopeptidase N (APN/CD13), a 150‐kDa metalloproteinase, is a multifunctional cell surface aminopeptidase with ubiquitous expression. Recent studies have suggested that APN/CD13 plays an important role in tumor progression in several human malignancies. In the current study, we investigated the role of APN/CD13 in paclitaxel (PAC)‐resistance of ovarian carcinoma (OVCA) cells. We first examined the correlation between APN/CD13 expression and IC50 values of PAC in a variety of OVCA cell lines. Next we investigated whether suppression of APN/CD13 using bestatin, an inhibitor of APN/CD13 activity or the siRNA technique influenced PAC‐sensitivity in ES‐2 cells, which highly express APN/CD13. Moreover, we investigated the effect of bestatin on peritoneal metastasis using nude mice. We found a negative correlation between APN/CD13 expression and chemosensitivity to PAC in various carcinoma cell lines. Subsequently, we found a significant increase in PAC‐sensitivity of APN/CD13 expressing OVCA cells by suppression of this enzyme, using the addition of bestatin or the siRNA technique. Furthermore, in a peritoneal metastasis model using nude mice, combination treatment with PAC and bestatin caused a synergistic increase of survival time compared with PAC alone treatment. (mean survival time: 37.7 ± 7.0 s and 27.1 ± 6.6 days, respectively). The present findings showed that APN/CD13 may be involved in decreased sensitivity to PAC in OVCA cells and that the mechanism of this effect involves its enzyme activity at least in part. APN/CD13 may be a therapeutic target for the treatment of OVCA in combination with chemotherapy.

Overexpression of Indoleamine 2,3-Dioxygenase in Human Endometrial Carcinoma Cells Induces Rapid Tumor Growth in a Mouse Xenograft Model

Purpose: Indoleamine 2,3-dioxygenase (IDO) is a tryptophan-catabolizing enzyme that induces immune tolerance in mice. Our prior study showed that high tumoral IDO expression in endometrial cancer tissues correlates with disease progression and impaired patient survival. The purpose of the present study was to clarify the functional role of IDO in human endometrial cancer cells and to investigate the therapeutic potential of IDO inhibitors. Experimental Design: IDO cDNA was transfected into the human endometrial carcinoma cell line AMEC, resulting in the establishment of stable clones of IDO-overexpressing AMEC cells (AMEC-IDO). AMEC-IDO cells were characterized in vitro as well as in vivo using a mouse xenograft model. Results: There was no significant difference in in vitro cell proliferation, migration, or chemosensitivity to paclitaxel between AMEC-IDO and control vector–transfected cells (AMEC-pcDNA). However, in vivo tumor growth was markedly enhanced in AMEC-IDO–xenografted nude mice when compared with AMEC-pcDNA–xenografted mice. Splenic natural killer (NK) cell counts in AMEC-IDO–xenografted mice were significantly decreased when compared with control mice. Furthermore, conditioned medium obtained from AMEC-IDO cell cultures markedly reduced the NK lysis activity of nude mice. Finally, oral administration of the IDO inhibitor 1-methyl-d-tryptophan in combination with paclitaxel in AMEC-IDO–xenografted mice strongly potentiated the antitumor effect of paclitaxel, resulting in significantly prolonged survival. Conclusions: This is the first evidence showing that IDO overexpression in human cancer cells contributes to tumor progression in vivo with suppression of NK cells. Our data suggest that targeting IDO may be a novel therapeutic strategy for endometrial cancer.

Taxol Resistance Among the Different Histological Subtypes of Ovarian Cancer May Be Associated With the Expression of Class III β-Tubulin

Summary: The prognostic significance of histology has been well established in epithelial ovarian cancer (EOC). Clear cell and mucinous histologies are especially generally accepted to result in an adverse outcome because of their poor chemotherapy response. Previous reports suggested that class III &bgr;-tubulin induced taxol resistance in association with a reduced effect on microtubule dynamic instability. Thus, this study aimed to evaluate class III &bgr;-tubulin expression and examine whether the protein level of class III &bgr;-tubulin was correlated with the histological difference in chemosensitivity. Class III &bgr;-tubulin expression in EOC tissues (n = 80) was immunohistochemically scored into 4 groups (−, ±, +, ++). High-level (+, ++) class III &bgr;-tubulin expression was detected in 30 of 35 clear cell carcinomas, in 8 of 10 mucinous carcinomas, 5 of 11 endometrioid carcinomas, and 5 of 24 serous carcinomas. Nineteen patients were evaluable for response. In 5 responders, high-level class III &bgr;-tubulin expression was not detected. On the other hand, it was detected in 10 of 14 nonresponders. In some ovarian cancer cell lines, we evaluated class III &bgr;-tubulin expression by Western blot analysis. Class III &bgr;-tubulin expression in nonserous carcinoma tended to be higher than that in serous carcinoma. Taxol-resistant SKOV cells showed high-level class III &bgr;-tubulin expression compared with wild-type SKOV cells. Taxol sensitivity differing among histological subtypes in EOC is associated with the expression of class III &bgr;-tubulin.