Toshiki Yajima

E/N-cadherin switch mediates cancer progression via TGF- β -induced epithelial-to-mesenchymal transition in extrahepatic cholangiocarcinoma

Background:Epithelial-to-mesenchymal transition (EMT) is a fundamental process governing not only morphogenesis in multicellular organisms, but also cancer progression. During EMT, epithelial cadherin (E-cadherin) is downregulated while neural cadherin (N-cadherin) is upregulated, referred to as ‘cadherin switch’. This study aimed to investigate whether cadherin switch promotes cancer progression in cholangiocarcinoma (CC).Methods:CC cell lines were examined for migration, invasion, and morphological changes with typical EMT-induced model using recombinant TGF-β1. The changes in E-cadherin and N-cadherin expression were investigated during EMT. We also examined E-cadherin and N-cadherin expression in resected specimens from extrahepatic CC patients (n=38), and the associations with clinicopathological factors and survival rates.Results:TGF-β1 treatment activated cell migration, invasion, and fibroblastic morphological changes, especially in extrahepatic CC HuCCT-1 cells. These changes occurred with E-cadherin downregulation and N-cadherin upregulation, that is, cadherin switch. Patients with low E-cadherin expression had a significantly lower survival rate than patients with high E-cadherin expression (P=0.0059). Patients with decreasing E-cadherin and increasing N-cadherin expression had a significantly lower survival rate than patients with increasing E-cadherin and decreasing N-cadherin expression (P=0.017).Conclusion:Cadherin switch promotes cancer progression via TGF-β-induced EMT in extrahepatic CC, suggesting a target for elucidating the mechanisms of invasion and metastasis in extrahepatic CC.

Transient gene silencing of galectin-3 suppresses pancreatic cancer cell migration and invasion through degradation of β-catenin

Pancreatic cancer is a leading cause of cancer‐related mortality and often has a poor prognosis because of its late diagnosis, aggressive local invasion, early metastasis and poor response to chemotherapy. The chemotherapeutic agent gemcitabine is effective for treating advanced pancreatic cancer, but its efficacy remains less than satisfactory. It is expected that further investigation of pancreatic cancer cell invasion and development of strategies to block this process should improve the disease prognosis. In this study, we tested our hypothesis that galectin‐3 (gal‐3), a multifunctional member of the β‐galactoside‐binding protein family, may regulate pancreatic cancer cell motility and silencing of it inhibit cell motility. Previous studies demonstrated that this protein is associated with tumor cell adhesion, proliferation, differentiation, angiogenesis, apoptosis and metastasis. Here, we used gal‐3 small interfering RNA (siRNA) to silence its expression in various pancreatic cancer cell lines to determine whether gal‐3 regulates cell proliferation, migration and invasion in vitro. We found that silencing gal‐3 reduced cellular migration and invasion, but failed to affect proliferation. In gal‐3 siRNA‐transfected cells, we detected a decrease in β‐catenin expression, an important signal for cancer cell invasion, which was caused by downregulation of phosphorylated Akt and GSK‐3β. We also found that matrix metalloproteinase (MMP)‐2 expression was reduced by gal‐3 silencing. These results indicate that gal‐3‐mediated invasion via MMP‐2 regulated by β‐catenin degradation is initiated by Akt phosphorylation in pancreatic cancer cells. Our results suggest that gal‐3 can be a novel therapeutic target in pancreatic cancer.

FBXW7 Mediates Chemotherapeutic Sensitivity and Prognosis in NSCLCs

Non–small cell lung cancer (NSCLC) is a leading cause of cancer-related deaths worldwide. To improve the prognosis of patients with NSCLCs, new and validated therapeutic targets are critically needed. In this study, we focused on F-box and WD repeat domain containing-7 (FBXW7), an E3 ubiquitin ligase, that regulates the degradation of MCL1, Myc, cyclin E, and TOP2A. Importantly, loss of FBXW7 was associated with increased sensitivity of tumors to a class I–specific histone deacetylase (HDAC) inhibitor, MS-275. Immunohistochemical analysis revealed increased expression of FBXW7 targets, MCL1 and TOP2A, in NSCLC tumors with low expression of FBXW7. Moreover, clinical specimens exhibiting low FBXW7 expression presented with more progressive cancer and significantly shorter cancer-specific survival than patients with high FBXW7 expression. Mechanistic study of NSCLC cell lines with silenced FBXW7 revealed enhanced MS-275 sensitivity and taxol resistance. Interestingly, taxol resistance was eliminated by MS-275 treatment, suggesting the potential of HDAC inhibitors for the treatment of aggressive taxol-resistant NSCLCs that lack FBXW7. Implications: FBXW7 status impacts chemosensitivity and is a prognostic marker in NSCLCs. Visual Overview: http://mcr.aacrjournals.org/content/early/2013/12/19/1541-7786.MCR-13-0341/F1.large.jpg. Mol Cancer Res; 12(1); 32–37. ©2013 AACR.

Transient silencing of galectin-3 expression promotes both in vitro and in vivo drug-induced apoptosis of human pancreatic carcinoma cells

Pancreatic cancer demonstrates a strong resistance to anticancer drugs, presumably due to its resistance to drug induced apoptosis. Although gemcitabine (GEM) might be partially effective for treating advanced pancreatic cancer, its efficacy is still less than satisfactory. Galectin-3 (gal-3), a member of the β-galactoside-binding protein family, is a multifunctional protein with roles in tumor cell adhesion, proliferation, differentiation, angiogenesis, metastasis, and apoptosis. We have utilized gal-3 small interfering RNA (siRNA) to probe whether gal-3 regulates anticancer drug-induced apoptosis in pancreatic cancer cells. We found that Gal-3 siRNA augmented GEM- and cisplatin-induced apoptosis in pancreatic cancer cell lines in vitro. Mitochondrial depolarization induction was increased in gal-3-silenced cells after GEM treatment, resulting in activation of caspase-9, but not caspase-8. Akt phosphorylation was significantly downregulated in gal-3- silenced cells in association with apoptosis. Moreover, intratumoral administration of gal-3 siRNA increased the GEM sensitivity of tumor xenografts produced by subcutaneous inoculation of pancreatic cancer cells into nude mice. These results suggest that gal-3 might provide a novel therapeutic target in pancreatic cancer.

SIRT6 expression is associated with poor prognosis and chemosensitivity in patients with non‐small cell lung cancer

Despite advances in the development of various therapeutic agents, non‐small cell lung cancer (NSCLC) is associated with a poor prognosis. To improve the prognosis of patients with NSCLC, new therapeutic targets for overcoming drug resistance are required. The process of autophagy is required to support the tumorigenesis and drug resistance of cancer cells. We investigated the clinical significance of SIRT6, a member of the NAD+‐dependent deacetylase family, which regulates a variety of cancer‐related processes, including autophagy.

Phosphoglucose isomerase/autocrine motility factor promotes melanoma cell migration through ERK activation dependent on autocrine production of interleukin-8

It is well known that phosphoglucose isomerase/autocrine motility factor (AMF) promotes cell migration in an autocrine manner in various tumor cells. However, it remains unclear whether certain cytokines modulate the effects of AMF on tumor cell migration. Because interleukin (IL)-8, a proinflammatory cytokine, is produced by melanoma cells and has been correlated with melanoma migration, the migratory ability of melanoma cells induced by AMF may also involve induction of IL-8 expression. In the present study, we assessed whether AMF promotes melanoma cell migration through autocrine production of IL-8. We found that AMF stimulation increased IL-8 production through up-regulation of IL-8 mRNA transcription, especially in biologically early stage melanoma cells. AMF-induced migration of these cells was inhibited by a specific neutralizing antibody against IL-8. The IL-8 production induced by AMF was mediated by the ERK1/2 pathways. These findings suggest that melanoma migration induced by AMF is mediated by autocrine production of IL-8 as a novel downstream modulator of the AMF signaling pathway.

Pulmonary function after pulmonary resection by posterior thoracotomy, anterior thoracotomy or video-assisted surgery

OBJECTIVE Predicted postoperative forced expiratory volume in 1s (ppoFEV1) is estimated in lung cancer patients before pulmonary resection, as well as the clinical stage. This study aims to evaluate ppoFEV1 and ppo-vital capacity (ppoVC) on postoperative day 7 (POD7) and to compare the results following video-assisted thoracic surgery (VATS) and open thoracotomy procedures. METHODS Of the 155 patients who underwent pulmonary resection, 70 had VATS; 30 had muscle-sparing thoracotomy (anterior limiting thoracotomy (AL)); and 55 had postero-lateral thoracotomy (PL). VC and FEV1 were measured on POD7 and compared with the VC and FEV1 before surgery using analysis of covariance (ANCOVA). The ratio of the actual- and the ppoVC and FEV1 was evaluated to identify factors associated with variations in postoperative residual VC/FEV1. RESULTS There were significant differences by analysis of covariance (ANCOVA) in the VC/FEV1 among the three surgical approaches. In the VATS group, the VC ratio and the FEV1 ratio were 96.5% and 94.7%, respectively; they were significantly higher in the VATS group than in the thoracotomy group (AL: 90.4% and 90.1%, respectively; PL: 87.4% and 87.6%, respectively). Non-chronic obstructive pulmonary disease (COPD) and upper lobectomy were also associated with a low VC ratio and FEV1 ratio. CONCLUSION Predicted postoperative pulmonary function might be overestimated in COPD patients or in those undergoing VATS or lower lobectomy.

Immunohistochemical analysis of phosphorylated epidermal growth factor receptor might provide a surrogate marker of EGFR mutation

PURPOSE Overexpression of EGFR is found in several malignancies including lung cancers. Recently, EGFR mutation has been shown to correlate with responsiveness to tyrosine kinase inhibitors (TKI). Although antibodies against phophorylated EGFR have been used in vitro, phosphorylated EGFR has yet not been examined well in resected non-small cell lung cancers (NSCLCs). EXPERIMENTAL DESIGN We studied the immunohistochemistry of anti-EGFR and phosphorylated EGFR in 97 resected NSCLCs, examined the relationship with EGFR mutation, and performed quantitative RT-PCR of the EGFR gene in the TaqMan assay. RESULT EGFR mutation was seen in 27% of 97 NSCLCs and 37% of 70 adenocarcinomas. EGFR was stained in 60% of 97 NSCLCs. Phosphorylation of tyrosine 845 (pY845) and 1068 (pY1068) was positive in 49% and 48%, respectively. The observed correlation with EGFR mutation and pY845 or pY1068 was statistically significant (P=0.0001 for pY845, P<0.0001 for pY1068, chi square test), although phospho-EGFR status was not associated with a particular mutation type. pY1068-positive tumors also correlated with female, light smoker, and adenocarcinoma histology, but not with mRNA expression. Moreover, patients with pY1068-positive tumors showed prolonged survival (P=0.0093, log-rank test). CONCLUSION It is possible that immunohistochemistry of phosphorylated EGFR can substitute for EGFR mutation analysis. Further investigation is necessary to determine whether phospho-EGFR immunohistochemistry predicts response to TKIs and survival benefit.

Donor CD4 T Cells Are Critical in Allogeneic Stem Cell Transplantation against Murine Solid Tumor

Nonmyeloablative allogeneic stem cell transplantation (SCT) has been used for various malignancies, although detailed mechanisms of antitumor effects remain unclear. We showed that a nonmyeloablative allogeneic SCT regimen, which consists of mixed chimerism induced by an injection of donor spleen and bone marrow cells followed by cyclophosphamide treatment and a donor lymphocyte infusion (DLI), exerted antitumor effects on established murine bladder tumor, MBT-2. An expansion of donor CD4 T cells accompanied by transient but vigorous IFN-gamma production was detected shortly after DLI. In vivo neutralization of IFN-gamma or depletion of CD4 T cells from DLI abolished the antitumor effects, indicating an indispensable role of donor CD4 T cells producing IFN-gamma. Donor as well as host CD8 T cells accumulated in the tumor region with time. Importantly, depletion of CD8 T cells from DLI did not reverse the suppression of tumor growth, indicating that CD4 T cells play a more essential role in mediating early antitumor effects. Furthermore, tumor-specific response of host CD8 T cells was suggested. These results not only provide the first evidence of nonmyeloablative allogeneic SCT for the treatment of bladder tumor but also elucidate detailed mechanisms of antitumor effects provoked by DLI.

Video-Assisted Thoracoscopic Surgery after Preoperative CT-Guided Lipiodol Marking of Small or Impalpable Pulmonary Nodules.

PURPOSE Small pulmonary lesions that include ground-glass attenuation have been increasingly discovered because of progressive imaging diagnostic technologies. Despite the detection of such small lesions, sometimes it is quite difficult to localize them because of their size or considerable depth from the visceral pleura. In the present study, we examined the usefulness of computed tomography-guided lipiodol marking for thoracoscopic resection of impalpable pulmonary nodules. METHODS Fifty-six patients with an undiagnosed peripheral lesion(s) of the lung who had undergone preoperative computed tomography-guided lipiodol marking followed by video-assisted thoracoscopic surgery were studied. RESULTS All of the nodules were successfully marked by computed tomography-guided lipiodol marking, and all except for one case were localized by means of intraoperative fluoroscopy as clear spots. With regard to complications, pneumothorax occurred in 21 patients (37.5%), and only one patient required transient drainage. Although hemorrhaging in the lung parenchyma and hemosputum occurred in nine patients (16.1%) and one patient (1.8%), respectively, no patients were in serious condition. No intra- or postoperative mortality or morbidity was observed. CONCLUSION Preoperative computed tomography-guided lipiodol marking of small or impalpable pulmonary nodules is a safe and useful procedure for thoracoscopic resection of the lung.