Youhei Nagai

MicroRNA-21 Regulates the Proliferation and Invasion in Esophageal Squamous Cell Carcinoma

Purpose: MicroRNAs are ∼22 nucleotide noncoding RNA molecules that posttranscriptionally regulate gene expression. The aim of this study was (a) to determine a role of microRNA-21 in esophageal squamous cell carcinoma and (b) to elucidate the regulation of the programmed cell death 4 (PDCD4) gene by microRNA-21. Experimental Design: MicroRNA-21 expression was investigated in 20 matched normal esophageal epitheliums and esophageal squamous cell carcinomas and seven esophageal squamous cell carcinoma cell lines (TE6, TE8, TE10, TE11, TE12, TE14, KYSE30) by TaqMan quantitative real-time PCR and in situ hybridization. To evaluate the role of microRNA-21, cell proliferation and invasion were analyzed with anti–microRNA-21–transfected cells. In addition, the regulation of PDCD4 by microRNA-21 was elucidated to identify the mechanisms of this regulation. Results: Of 20 paired samples, 18 cancer tissues overexpressed microRNA-21 in comparison with matched normal epitheliums. Specifically, patients with lymph node metastasis or venous invasion showed significantly high expression of microRNA-21. In situ hybridization for microRNA-21 showed strong positive staining in paraffin-embedded esophageal squamous cell carcinoma tissues. All seven esophageal squamous cell carcinoma cell lines also overexpressed microRNA-21, and anti–microRNA-21–transfected cells showed significant reduction in cellular proliferation and invasion. The PDCD4 protein levels in esophageal squamous cell carcinoma cells have an inverse correlation with microRNA-21 expression. Anti–microRNA-21–transfected cells increased PDCD4 protein expression without changing the PDCD4 mRNA level and increased a luciferase-reporter activity containing the PDCD4-3′ untranslated region construct. Conclusions: MicroRNA-21 targets PDCD4 at the posttranscriptional level and regulates cell proliferation and invasion in esophageal squamous cell carcinoma. It may serve as a novel therapeutic target in esophageal squamous cell carcinoma.

Aberrant activation of the mTOR pathway and anti-tumour effect of everolimus on oesophageal squamous cell carcinoma

Background:The mammalian target of rapamycin (mTOR) protein is important for cellular growth and homeostasis. The presence and prognostic significance of inappropriate mTOR activation have been reported for several cancers. Mammalian target of rapamycin inhibitors, such as everolimus (RAD001), are in development and show promise as anti-cancer drugs; however, the therapeutic effect of everolimus on oesophageal squamous cell carcinoma (OSCC) remains unknown.Methods:Phosphorylation of mTOR (p-mTOR) was evaluated in 167 resected OSCC tumours and 5 OSCC cell lines. The effects of everolimus on the OSCC cell lines TE4 and TE11 in vitro and alone or in combination with cisplatin on tumour growth in vivo were evaluated.Results:Mammalian target of rapamycin phosphorylation was detected in 116 tumours (69.5%) and all the 5 OSCC cell lines. Everolimus suppressed p-mTOR downstream pathways, inhibited proliferation and invasion, and induced apoptosis in both TE4 and TE11 cells. In a mouse xenograft model established with TE4 and TE11 cells, everolimus alone or in combination with cisplatin inhibited tumour growth.Conclusion:The mTOR pathway was aberrantly activated in most OSCC tumours. Everolimus had a therapeutic effect both as a single agent and in combination with cisplatin. Everolimus could be a useful anti-cancer drug for patients with OSCC.

Laminin-332 promotes the invasion of oesophageal squamous cell carcinoma via PI3K activation

Laminin-332 is major component of epithelial basement membrane, and has an important role in cell migration and tumour invasion. Recently, the phosphatidylinositol 3-kinase (PI3K) activation induced by laminin-332 during carcinogenesis or tumour invasion has been highlighted in skin squamous cell carcinoma. The expression of laminin-332 in 126 resected oesophageal squamous cell carcinoma (ESCC) specimens was immunohistochemically examined to determine its associations with the clinicopathological characteristics, and the effect of laminin-332 on the invasiveness and the PI3K activation was assessed by in vitro experiments using ESCC cell lines (ESCCs). Sections with immunostaining signals in >30% cancer cells, which were observed in 55 of 126 cases, were judged to be positive for laminin-332. The positivity was significantly correlated with pTNM stage and poor prognosis. Inactivation of the PI3K pathway by laminin-332 blocking antibody suppressed the invasiveness of TE8 cell line, which secreted laminin-332 at high level and had high PI3K activity. The addition of the purified laminin-332 activated the PI3K pathway and increased the invasiveness of TE11 cell line, which secreted laminin-332 at lower level and had low PI3K activity. The deactivation of PI3K pathway using the PI3K inhibitor decreased the invasiveness of ESCCs and the secretion of laminin-332 in vitro. The expression of laminin-332 was one of the prognostic factors of ESCC. Laminin-332 could provide the autocrine positive-feedback loop through PI3K activation, contributing the invasive ability. Therefore, the inhibitor of PI3K pathway might be useful as the anticancer therapies for ESCC.

The Relationship between the Glucose Transporter Type 1 Expression and 18F-Fluorodeoxyglucose Uptake in Esophageal Squamous Cell Carcinoma

Objective: Glucose transporter type 1 (Glut1) has been reported to be present in several types of carcinomas. The aims of this study are to evaluate Glut1 expression in both primary tumors and metastatic lymph nodes (LNs) of esophageal squamous cell carcinoma (ESCC) and to assess the relationship between Glut1 expression and 18F-fluorodeoxyglucose (FDG) accumulation. Methods: We immunohistochemically examined the expression of Glut1 in 60 surgically resected primary lesions and 95 metastatic LNs of ESCC and classified them into 3 groups. The FDG accumulation was assessed with a positron emission tomography (PET). Results: In the primary tumors, a high Glut1 expression was found to be significantly associated with advanced lesions: depth of tumor (p < 0.01), LN metastasis (p < 0.05) and advanced pathological stage (p < 0.01). The Glut1 expression of the metastatic LNs significantly correlated with that of each primary tumor (p < 0.001). The PET-positive lesions had a larger size and higher Glut1 expression than the PET-negative lesions in both the primary tumors and metastatic LNs. Conclusions: In both the primary tumors and metastatic LNs of ESCC, the Glut1 expression and tumor size correlated with the FDG accumulation and influence the sensitivity of the PET scan.

The role of oxysterol binding protein-related protein 5 in pancreatic cancer

The expression of oxysterol binding protein‐related protein (ORP) 5 is related to invasion and a poor prognosis in pancreatic cancer patients. ORP5 induced the expression of sterol response element binding protein (SREBP) 2 and activated the downstream gene of sterol response element. ChIP using SREBP2 antibody revealed that histone deacetylase 5 (HDAC5) was one of the downstream genes of SREBP2. The effect of HMG‐CoA reductase inhibitors (statins) were analyzed according to the expression level of ORP5. The invasion rate and growth was suppressed in cells that strongly expressed ORP5 in a time‐ and dose‐dependent manner, but had less effect in cells weakly expressing ORP5, suggesting that when the potential of invasion and growth relies on the cholesterol synthesis pathway, it becomes sensitive to HMG‐CoA reductase inhibitor. Furthermore, HDAC inhibitor, tricostatin A, induced the expression of phosphatase and tensin homolog as well when ORP5 was suppressed or the cells were treated with statin. Treatment with both statin and tricostatin A showed a synergistic antitumor effect in cells that highly expressed ORP5. Therefore, in some pancreatic cancers, continuous ORP5 expression enhances the cholesterol synthesis pathway and this signal transduction regulates phosphatase and tensin homolog through HDAC5 expression. This is the first report to detail how the signal transduction of cholesterol synthesis is related to cancer invasion and why statins can suppress invasion and growth.

RPN2 expression predicts response to docetaxel in oesophageal squamous cell carcinoma

Background:Neoadjuvant chemotherapy – often using docetaxel in various combinatorial regimens – is a standard treatment choice for advanced oesophageal squamous cell carcinoma (ESCC) in Japan. However, no useful markers exist that predict docetaxel’s effects on ESCC. Ribophorin II (RPN2) silencing, which reduces glycosylation of P-glycoproteins and decreases membrane localisation, promotes docetaxel-dependent apoptosis. We investigated whether RPN2 expression in ESCC biopsy specimens could be a predictive biomarker in docetaxel-based neoadjuvant chemotherapy.Methods:We evaluated RPN2 expression immunohistochemically in biopsy specimens from 79 patients with node-positive ESCC, who received docetaxel-based adjuvant chemotherapy, and compared clinical and pathological responses between the RPN2-positive and RPN2-negative groups. We also studied susceptibility of RPN2-suppressed ESCC cells to docetaxel.Results:The RPN2-negative group had better clinical and pathological responses to docetaxel than the RPN2-positive group. We also found RPN2 suppression to alter docetaxel susceptibility in vitro.Conclusion:Expression of RPN2 in biopsy specimens could be a useful predictive marker for response to docetaxel-based neoadjuvant chemotherapy in ESCC.

A case of thoracoscopically resected benign esophageal schwannoma with high uptake on FDG-PET

We report a rare case of esophageal schwannoma with high uptake of radiotracer on [18F]fluoro-2-deoxy-d-glucose positron emission tomography (FDG-PET). A 37-year-old woman underwent surgery for sarcoma of the uterus and received general PET examination for postoperative follow-up, which revealed high uptake at the thoracic esophagus. Esophagography, esophageal endoscopy, endoscopic ultrasonography, and computed tomography (CT) scan confirmed a submucosal tumor, about 20 mm in diameter, in the right wall of the upper thoracic esophagus. We performed thoracoscopic enucleation with the preoperative diagnosis of esophageal leiomyoma or gastrointestinal stromal tumor (GIST). Histopathological examination showed a trabecular arrangement of spindle cells and no evidence of mitosis. Immunohistochemical studies were positive for S-100 protein and negative for c-kit, whereby we diagnosed a benign esophageal schwannoma. Even if benign, schwannomas often show high uptake of FDG, so that the evaluation of whether the tumor is malignant should be made by histological diagnosis. Thoracoscopic resection is less invasive and a clinically useful approach to treat such submucosal tumors of the esophagus.

Heterogeneous prognoses of patients with tumors invaded within muscularis propria according to tumor depth in the layers of the muscularis propria

BackgroundThe muscularis propria of the stomach is histologically divided into three layers; namely, the innermost oblique, the inner circular, and the outer longitudinal layers. In patients with gastric cancer the depth of tumor invasion has been reported to correlate with lymph node metastasis and prognosis. However, it is unclear whether the depth of tumor invasion in the muscularis propria has an effect on lymph node metastasis and prognosis.MethodsFifty-nine gastric cancer patients with muscularis propria invasion were analyzed retrospectively. These patients were divided into two groups, the inner group, with invasion up to the inner circular layer; and the outer group, with invasion beyond the inner circular layer. The relationships between tumor invasion and clinicopathological factors and survival were evaluated.ResultsOf the 59 patients, 34 were classified as the inner group, and 25 were classified as the outer group. The inner group had a significantly lower probability of lymph node metastasis (P = 0.0053) and a significantly better overall cancer-specific survival (P = 0.017) than the outer group.ConclusionGastric cancers with muscularis propria invasion had heterogeneous prognoses according to the tumor depth in the muscularis propria layers.

Estimation of Physiologic Ability and Surgical Stress (E-PASS system) in patients with esophageal squamous cell carcinoma undergoing resection

BackgroundThe Estimation of Physiologic Ability and Surgical Stress (E-PASS) scoring system was designed on the hypothesis that the balance between the patient’s physiological reverse capacity and the surgical stress of operation was important in the development of postoperative complications. The aim of this study was to evaluate whether the E-PASS scoring system could predict the occurrence of complications after elective esophagectomy for esophageal cancer, including salvage esophagectomy.MethodsE-PASS predictor equations were applied retrospectively to 142 patients who had undergone esophagectomy for esophageal squamous cell carcinoma [110 patients without preoperative chemoradiotherapy (CRT), 15 patients with neoadjuvant CRT followed by planned esophagectomy, and 17 patients with definitive CRT followed by salvage esophagectomy]. The incidence rates of postoperative complications were compared with the preoperative risk score (PRS), surgical stress score (SSS), and comprehensive risk score (CRS) of the E-PASS scoring system.ResultsOf the 142 patients, 43 (30%) had postoperative complications. The incidence of postoperative complications increased as the CRS score increased. Patients with a CRS > 1.0 were at a particularly high risk of postoperative complications. Definitive CRT increased the incidence of postoperative complications, and the SSS and CRS of patients with definitive CRT were significantly higher than those of patients without preoperative CRT or with neoadjuvant CRT.ConclusionsThe E-PASS scoring system was useful in predicting complications after elective esophagectomy for esophageal cancer. The SSS of the E-PASS scoring system could also reflect the maximum invasiveness of salvage esophagectomy.

Mutation analysis of Rad18 in human cancer cell lines and non small cell lung cancer tissues

BackgroundGenetic instability is known as a cause of oncogenesis. Though Rad18 is reported to function in a post replication mismatch repair system, the relation between the status of Rad18 and human tumorigenesis has not been described so far.MethodsMutation analysis of 34 human cancer cell lines and 32 non small cell lung cancer (NSCLC) tissues were performed by RT-PCR SSCP. Expression level of Rad18 was measured by real time RT-PCR. Stable transfectant was constructed for in vitro study.ResultsNo mutation was found in both cancer cell lines and NSCLC tissues. A single nucleotide polymorphism (SNP) at codon 302 was detected in 51.5% of the cell lines and 62.5% of NSCLC tissues. Interestingly, Rad18 was homozygously deleted in a pulmonary adenocarcinoma cell line PC3. Furthermore, there was no difference in the expression level of wild type Rad18 and Rad18 with SNP. The growth, cell morphology, sensitivity to anti-cancer drugs and in vitro DNA repair activity between wild type Rad18 and Rad18 with SNP revealed to have no difference in vitro.ConclusionThough the frequency of SNP was tended to be higher in NSCLC patients than healthy volunteers (57.7%), as the difference was not significant, we have concluded that there is no relation between Rad18 SNP and lung cancer development.