Takashi Shida

Slug expression is an independent prognostic parameter for poor survival in colorectal carcinoma patients

Slug, a member of the Snail family of transcription factors, plays a crucial role in the regulation of epithelial-mesenchymal transition (EMT) by suppressing several epithelial markers and adhesion molecules including E-cadherin. Recently, several studies have reported Slug to be expressed in breast carcinoma, oesophageal carcinoma accompanied with shorter survival. In this study, we first investigated expression of Slug mRNA in five colorectal carcinoma cell lines by reverse transcription–polymerase chain reaction. Furthermore, we investigated Slug and E-cadherin expression by immunohistochemistry in 138 patients with colorectal carcinoma. Slug mRNA was clearly expressed in four out of five colorectal carcinoma cell lines. Positive expression of Slug and E-cadherin was observed in 37 and 58% of cases, respectively. The positive expression of Slug was significantly associated with Dukes stage and distant metastasis (P=0.0027 and 0.0007), and the positive expression of Slug had a significant impact on patient overall survival (P<0.0001, log-rank test). Moreover, patients with positive expression of Slug and reduced expression of E-cadherin showed the worst prognosis (P<0.0001, log-rank test). Multivariate analysis indicated that Slug expression was an independent prognostic factor. These results suggest that positive Slug expression in colorectal carcinoma patients may become a significant parameter of poor prognosis.

Distinction of pulmonary large cell neuroendocrine carcinoma from small cell lung carcinoma: a morphological, immunohistochemical, and molecular analysis.

The distinction between pulmonary large cell neuroendocrine carcinoma and small cell carcinoma is difficult in some cases. Some propose that these carcinomas should be classified as one high-grade neuroendocrine carcinoma. We examined biological features of small cell carcinoma (n=23), large cell neuroendocrine carcinoma (n=17), and classic large cell carcinoma (n=12). The average ratio of nuclear diameter of the tumor cells to that of lymphocytes for small cell carcinoma was smaller than that for large cell neuroendocrine carcinoma (P<0.0001). The frequencies of the expressions of CD56, mASH1, TTF-1, and p16 were higher and that of NeuroD was lower in small cell carcinoma than in large cell neuroendocrine carcinoma. The frequency of loss of heterozygosity at 3p was higher in high-grade neuroendocrine carcinomas than in classic large cell carcinoma (P=0.0002). Allelic losses at D5S422 (5q33) were more frequent in small cell carcinoma than in large cell neuroendocrine carcinoma (P=0.0091). Mean fractional regional loss indices of the tumors were 0.38, 0.65, and 0.72 for patients with classic large cell carcinoma, large cell neuroendocrine carcinoma, and small cell carcinoma, respectively (P=0.0003). Five-year overall survivals of patients with classic large cell carcinoma, large cell neuroendocrine carcinoma and small cell carcinoma in stage I were 67, 73, 60%, respectively. Patients with NeuroD expression had better survivals, and those with p63 expression had poorer survivals in large cell neuroendocrine carcinoma. Patients with TTF-1 expression had poorer survivals in small cell carcinoma. Our data suggest that large cell neuroendocrine carcinoma and small cell carcinoma are different morphologically, phenotypically, and genetically, although there are some overlapping features. Although further studies are needed to analyze the biological behavior of high-grade neuroendocrine carcinomas including sensitivity to chemotherapy, the pathological distinction of large cell neuroendocrine carcinoma from small cell carcinoma may be necessary to treat the patients with neuroendocrine tumors.

FGF10/FGFR2 signal induces cell migration and invasion in pancreatic cancer

Pancreatic cancer has one of the highest mortalities among all malignancies and there is an urgent need for new therapy. This might be achieved by resolving the detailed biological mechanism, and in this study we examined how pancreatic cancer cells develop aggressive properties by focusing on signalling through the fibroblast growth factor (FGF)10 and FGF receptor (FGFR)2, which play important roles in pancreatic organogenesis. Immunostaining of pancreatic cancer tissues showed that FGFR2 was expressed in cancer cells, whereas FGF10 was expressed in stromal cells surrounding the cancer cells. Patients with high FGFR2 expression in cancer cells had a shorter survival time compared to those with low FGFR2 expression. Fibroblast growth factor 10 induced cell migration and invasion of CFPAC-1 and AsPC-1 pancreatic cancer cells through interaction with FGFR2-IIIb, a specific isoform of FGFR2. Fibroblast growth factor 10 also induced expression of mRNA for membrane type 1-matrix metalloproteinase (MT1-MMP) and transforming growth factor (TGF)-β1, and increased secretion of TGF-β1 protein from these cell lines. These data indicate that stromal FGF10 induces migration and invasion in pancreatic cancer cells through interaction with FGFR2, resulting in a poor prognosis. This suggests that FGF10/FGFR2 signalling is a promising target for new molecular therapy against pancreatic cancer.

Fibroblast growth factor 19 expression correlates with tumor progression and poorer prognosis of hepatocellular carcinoma

BackgroundAlthough fibroblast growth factor 19 (FGF19) can promote liver carcinogenesis in mice, its involvement in human hepatocellular carcinoma (HCC) has not been well investigated. FGF19, a member of the FGF family, has unique specificity for its receptor FGFR4. This study aimed to clarify the involvement of FGF19 in the development of HCC.MethodsWe investigated human FGF19 and FGFR4 expression in 40 hepatocellular carcinoma specimens using quantitative real-time reverse transcription polymerase chain reaction (RT-PCR) analysis and immunohistochemistry. Moreover, we examined the expression and the distribution of FGF19 and FGFR4 in 5 hepatocellular carcinoma cell lines (HepG2, HuH7, HLE, HLF, and JHH7) using RT-PCR and immunohistochemistry. To test the role of the FGF19/FGFR4 system in tumor progression, we used recombinant FGF19 protein and small interfering RNA (siRNA) of FGF19 and FGFR4 to regulate their concentrations.ResultsWe found that FGF19 was significantly overexpressed in HCCs as compared with corresponding noncancerous liver tissue (P < 0.05). Univariate and multivariate analyses revealed that the tumor FGF19 mRNA expression was an independent prognostic factor for overall and disease-free survival. Moreover, we found that the FGF19 recombinant protein could increase the proliferation (P < 0.01, n = 12) and invasion (P < 0.01, n = 6) capabilities of human hepatocellular carcinoma cell lines and inhibited their apoptosis (P < 0.01, n = 12). Inversely, decreasing FGF19 and FGFR4 expression by siRNA significantly inhibited proliferation and increased apoptosis in JHH7 cells (P < 0.01, n = 12). The postoperative serum FGF19 levels in HCC patients was significantly lower than the preoperative levels (P < 0.01, n = 29).ConclusionsFGF19 is critically involved in the development of HCCs. Targeting FGF19 inhibition is an attractive potential therapeutic strategy for HCC.

Expression of an activated mammalian target of rapamycin (mTOR) in gastroenteropancreatic neuroendocrine tumors

AimsGastroenteropancreatic neuroendocrine tumors are rare, and the current WHO classification divides this tumor entity into well-differentiated (neuro)endocrine tumors, well-differentiated (neuro)endocrine carcinomas, and poorly differentiated (neuro)endocrine carcinomas. Poorly differentiated (neuro)endocrine carcinoma is extremely aggressive, and no appropriate therapeutic approach has been established. The mammalian target of rapamycin (mTOR), an important regulator of cell proliferation and protein translation, is activated in various malignancies. Recent phase II trial has revealed the efficacy of mTOR inhibitor (RAD001; everolimus) against low-to-intermediate grade neuroendocrine tumors. However, the beneficial role of mTOR inhibitor against poorly neuroendocrine carcinoma remains uncertain. The purpose of the present study was to determine the activation of mTOR in gastropancreatic neuroendocrine tumors, especially in poorly differentiated neuroendocrine carcinomas.MethodsExpression of p-mTOR(Ser2448) was assessed by immunohistochemistry in 20 gastropancreatic neuroendocrine tumors (seven well-differentiated neuroendocrine tumors, four well-differentiated neuroendocrine carcinomas, and nine poorly differentiated neuroendocrine carcinomas). Double immunohistochemistry was performed with p-Akt for patients with high p-mTOR expression.ResultsExpression of mTOR was seen in 9 (45%) of 20 gastroenteropancreatic neuroendocrine tumors. High expression of p-mTOR was seen in 6 (67%) of 9 poorly differentiated neuroendocrine carcinomas which was higher than the expression rate of well-differentiated neuroendocrine tumors and carcinomas, 3 (27%) of 11. All large cell neuroendocrine carcinomas showed high p-mTOR expression. Some tumor cells showed positive staining for p-mTOR co-expressed p-Akt.ConclusionsHigh expression rate of p-mTOR in poorly differentiated neuroendocrine carcinomas (large-cell type) may suggest the potential role of mTOR inhibitors as effective therapeutic agents for this highly malignant disease.

Accumulation of regulatory T cells in sentinel lymph nodes is a prognostic predictor in patients with node-negative breast cancer.

It has been revealed that sentinel lymph nodes (SLNs) from patients with node-negative breast cancer involve RT-PCR detected micrometastases and isolated tumour cells. However, the prognostic significance of the pathologically undetectable micrometastases is still controversial. In this study, we evaluated Foxp3 positive regulatory T cells (Treg) in SLNs as host-side immune marker that has the potential to detect these micrometastases. In the analyses of training set (n=30), elevated Treg was strongly associated with the pathologically undetectable micrometastases. In the analyses of validation set (n=129) in patients with node-negative, relapse-free survival in patients with elevated Treg was significantly shorter than those with lower Treg (p=0.005). Furthermore, in multivariate analyses, elevated Treg was correlated with relapse-free survival (p=0.012). Our data indicate that Treg may increase in the microenvironment of SLNs along with pathologically undetectable micrometastases and is a prognostic predictor in patients with node-negative breast cancer.

Apolipoprotein C-1 maintains cell survival by preventing from apoptosis in pancreatic cancer cells

Pancreatic cancer still remains one of the most lethal diseases and establishment of new therapy is needed. The purpose of this study is to find novel factors involved in pancreatic cancer progression by proteomic approach. We compared pre- and postoperative serum protein profiling obtained from pancreatic cancer patients who had curative pancreatectomy using surface-enhanced laser desorption ionization time-of-flight mass spectrometry. The peak intensity levels of both 6630 and 6420 Da were significantly higher in the preoperative serum than in the postoperative serum (P<0.002). Sequential amino acid analysis identified these proteins to be apolipoprotein C-1 (ApoC-1). The high level of ApoC-1 in preoperative serum significantly correlated with poor prognosis. Furthermore, ApoC-1 was abundantly expressed in pancreas neoplastic epithelium, and was detected in the culture medium of the pancreatic cancer cell line in vitro, which suggests that cancer cells secrete ApoC-1. Inhibition of ApoC-1 expression by short interfering RNA suppressed cell proliferation and induced apoptosis of pancreatic cancer cells. The specific expression of ApoC-1 and its role in preventing from spontaneous apoptosis in pancreatic cancer cells suggest that ApoC-1 contributes to the aggressiveness of pancreatic cancer and will be useful as a new therapeutic target.

Transparent hood attached to the colonoscope: does it really work for all types of colonoscopes?

BackgroundRecently the use of a transparent hood attached to the tip of the colonoscope has been reported to be useful in cecal intubation, especially in difficult colonoscopy cases. However, patient comfort with or without the use of a transparent hood, the type of colonoscope most suitable for this device have not been fully evaluated.MethodsA total of 372 patients undergoing routine colonoscopy were evaluated. No sedatives were used as a principle, and patients with prior abdominal surgery were excluded. A single endoscopist performed all the examinations. Four types of colonoscopes were used: CF-230I and CF-Q240AI as a standard colonoscope, PCF-240I and PCF-P240AI as a small-caliber colonoscope (Olympus, Tokyo, Japan). A disposable transparent hood was used (Olympus, Tokyo, Japan). Patients were randomly divided into four groups; standard colonoscope without transparent hood (96 patients), standard colonoscope with transparent hood (82 patients), small-caliber colonoscope without transparent hood (102 patients), and small-caliber colonoscope with transparent hood (92 patients). Cecal intubation time, rate, modified visual analogue scale (VAS) of patient’s pain, and technical difficulty were evaluated among the four groups.ResultsStandard colonoscope with the transparent hood showed the shortest cecal intubation time, and lowest modified VAS scale of pain and technical difficulty among the four groups. However there was no significant difference with regard to small-caliber colonoscopes with or without the transparent hood.ConclusionsTransparent hood may be a handy and a cost-effective device for reducing pain and simplifying cecal intubation, especially when using a standard adult colonoscope in routine colonoscopy.

The expression of NeuroD and mASH1 in the gastroenteropancreatic neuroendocrine tumors

Gastroenteropancreatic neuroendocrine tumors are uncommon and their tumor biology has not been well elucidated to date. Currently the WHO classification is widely used for the diagnosis and distinction of this tumor entity, which is sometimes cumbersome. Although neuroendocrine tumor markers do exist (ie chromograninA, synaptopyhsin, etc), sensitive and specific markers that accurately predict tumor growth and tumor behavior are still absent. In the present study, we assessed the expression of transcription factors (NeuroD and mASH1) essential for the normal fetal neuronal development in 33 gastroenteropancreatic neuroendocrine tumor patients (12 well-differentiated neuroendocrine tumors, 7 well-differentiated neuroendocrine carcinomas, and 14 poorly differentiated neuroendocrine carcinomas). NeuroD was less expressed in poorly differentiated neuroendocrine carcinoma (small-cell type) compared to well-differentiated neuroendocrine tumor (carcinoid) by reverse transcription–polymerase chain reaction. Immunohistochemical staining revealed that mASH1 was highly (sensitivity of 71%) and specifically (specificity of 95%) expressed in poorly differentiated neuroendocrine carcinoma. High NeuroD expression was seen in all well-differentiated neuroendocrine carcinoma and tumor (carcinoid) patients. Low NeuroD expression was seen in 36% (5 of 14) of poorly differentiated neuroendocrine carcinoma patients, which was associated with significant shorter overall survival. The expression pattern of these transcription factors may represent the biological and pathophysiological difference of gastroenteropancreatic neuroendocrine tumors and may become a new marker for the distinction of gastroenteropancreatic neuroendocrine tumors.

Sonic hedgehog-Gli1 signaling pathway might become an effective therapeutic target in gastrointestinal neuroendocrine carcinomas

Gastrointestinal neuroendocrine carcinomas (NECs) are extremely aggressive and poorly prognostic. We showed previously that human achaete-scute homologue gene 1 (hASH1), a basic helix-loop-helix transcription factor regulated by Notch, was aberrantly expressed in NECs. To date, no effective therapeutic strategies for NECs have been investigated. Notch, Wnt and Hedgehog (Hh) signalings are important for stem cell self-renewal and carcinogenesis in the gastrointestinal epithelium. In this study, we showed that Hh signaling was clearly upregulated in NECs in Gli1-dependent manner. Specific therapeutic effects of cyclopamine on NECs were also demonstrated. RT-PCR showed that among eight frozen samples (three NECs, one carcinoid tumor, three adenocarcinomas and one normal mucosa), the band intensities of Gli1 were the strongest in NECs, moderately strong in a carcinoid tumor, very weak in adenocarcinomas and undetectable in a normal mucosa. In real-time RT-PCR, the expression levels of Gli1 in NECs were 108.4, 28.6 and 16.3 times higher than that in an adenocarcinoma. In immunohistochemistry using 25 paraffin-embedded tissues, all twelve NECs and three of six carcinoid tumors showed positive stainings for Gli1, whereas six of seven adenocarcinomas were negative. In vitro, RT-PCR showed that NEC cell lines expressed Gli1 mRNA significantly. Administration of cyclopamine suppressed cell proliferation and invasion, and induced apoptosis in NECs. In cyclopamine-treated NECs, downregulation of Gli1, Ptch1, Snail and hASH1, and upregulation of E-cadherin were demonstrated at mRNA levels. Such effects were not observed in a Gli1-negative colonic adenocarcinoma cell line or in control alkaloid-treated NECs. Hh signaling may play a crucial role in the pathophysiology of NECs. Blockade of Hh pathway using cyclopamine or its synthetic derivatives might open an effective therapeutic strategy to NECs, not only by suppressing tumor viability but also by altering tumor cell nature.