Hajime Orita

Selective Inhibition of Fatty Acid Synthase for Lung Cancer Treatment

Purpose: Fatty acid synthase (FAS) is overexpressed in many human cancers and is considered to be a promising target for therapy. However, in vitro use of previous generations of FAS inhibitors has been limited by severe, but reversible, anorexia in treated animals, which is thought to be related to a parallel stimulation of fatty acid oxidation by these agents. This study investigated pharmacologic inhibition of FAS using C93, a rationally designed molecule that inhibits FAS activity without affecting fatty acid oxidation in preclinical models of lung cancer. Experimental Design: Activity of C93 on FAS and fatty acid oxidation was evaluated in cultured non–small cell lung cancer (NSCLC) cells. Antineoplastic activity of the compound, given orally or by i.p. injection, was evaluated in s.c. and orthotopic NSCLC xenografts. Results: Our experiments confirm that C93 effectively inhibits FAS without stimulating fatty acid oxidation in lung cancer cells. More importantly, C93 significantly inhibits the growth of both s.c. and orthotopic xenograft tumors from human NSCLC cell lines without causing anorexia and weight loss in the treated animals. Conclusions: We conclude that inhibition of FAS can be achieved without parallel stimulation of fatty acid oxidation and that inhibition of tumor growth in vivo can be achieved without anorexia and weight loss. Thus, this therapeutic strategy holds promise for clinical treatment of cancers, including non–small cell lung cancer, the leading cause of cancer mortality in the United States and Europe.

Inhibiting Fatty Acid Synthase for Chemoprevention of Chemically Induced Lung Tumors

Purpose: Fatty acid synthase (FAS) is overexpressed in lung cancer, and we have investigated the potential use of FAS inhibitors for chemoprevention of lung cancer. Experimental Design: Expression of FAS was evaluated in preinvasive human lung lesions (bronchial squamous dysplasia and atypical adenomatous hyperplasia) and in murine models of lung tumorigenesis [4-(methylnitrosamino)-I-(3-pyridyl)-1-butanone–induced and urethane-induced lung tumors in A/J mice]. Then, the ability of pharmacologic inhibitors of FAS to prevent development of the murine tumors was investigated. Finally, the effect of the FAS inhibitor treatment of levels of phosphorylated Akt in the murine tumors was evaluated by immunohistochemistry. Results: Immunohistochemical studies show that human bronchial dysplasia and atypical adenomatous hyperplasia express high levels of FAS compared with normal lung tissues, suggesting that FAS might be a target for intervention in lung carcinogenesis. FAS is also expressed at high levels in chemically induced murine lung tumors, and the numbers and sizes of those murine tumors are significantly reduced by treating carcinogen-exposed mice with pharmacologic inhibitors of FAS, C75 and C93. C93 treatment is associated with reduced levels of phosphorylated Akt in tumor tissues, suggesting that inhibition of this signal transduction pathway might be involved in the chemopreventative activity of this compound. Conclusions: We conclude that increased levels of FAS are common in human preinvasive neoplasia of the lung. Based on studies in mouse models, it seems that inhibiting FAS is an effective strategy in preventing and retarding growth of lung tumors that have high expression of this enzyme.

Coix seed extract, A commonly used treatment for cancer in china, inhibits NFκB and protein kinase C signaling

A pharmaceutical grade extract of Coix lachryma-jobi seeds is currently the most commonly used treatment for cancer in China. Although clinical data supports the use of this preparation of a Traditional Chinese Medicine for cancer treatment, biological basis for the activity of this preparation has not been previously established. To address this issue, we first evaluated the anti-neoplastic activity of a Coix extract emulsion in xenografts of MDA-MB-231 breast cancer cells and found that the extract significantly inhibits growth of MDA-MB-231 xenografts in athymic nude mice. Using oligonucleotide microarrays, we determined that Coix seed extract extract also significantly affects gene expression in these cells, including down-regulation of genes (such as COX-2 and matrixmetalloproteinases) that are considered to be important in neoplasia. The specific gene expression changes noted after Coix treatment are characteristic of inhibition of NFκB-dependant transcription, leading us to evaluate how the treatment affects that pathway. An NFκB-dependant reporter assay demonstrated dose-dependant inhibition of NFκB signaling by treatment of cultures with the extract, and immunofluorescent microscopy found that these effects are associated with reduced translocation of the Rel-A/p65 subunit of NFκB to the nucleus. Coix extract also inhibits activity of protein kinase C, a major mediator of signal transduction and activator of NFκB. Thus, this Traditional Chinese Medicine-based cancer treatment affects cellular pathways of recognized importance in neoplasia.

Clinical proteomics identified ATP-dependent RNA helicase DDX39 as a novel biomarker to predict poor prognosis of patients with gastrointestinal stromal tumor.

Gastrointestinal stromal tumor (GIST) is the most common mesenchymal malignancy of the gastrointestinal tract, comprising a wide spectrum from a curable disorder to highly malignant disease. GIST is characterized by tyrosine kinase mutations, and molecular targeting therapies against these abnormal enzymes require prognostic biomarkers. To identify candidate prognostic biomarkers, we examined proteomic features corresponding to metastasis after surgery. Using two-dimensional difference gel electrophoresis with a large format gel, we compared the primary tumor tissues of GIST patients free of metastasis for two years after surgery (eight cases) with those of patients who developed metastasis within one year after surgery (nine cases). We found the intensities of 38 protein spots to differ significantly between the two groups. Mass spectrometric protein identification revealed that these corresponded to 25 unique genes. Immunohistochemical validation demonstrated ATP-dependent RNA helicase DDX39 to be significantly associated with metastasis and poor clinical outcomes in a group of 72 GIST patients. In conclusion, we have established a novel prognostic utility of ATP-dependent RNA helicase DDX39 in GIST.ATP-dependent RNA helicase DDX39, a novel biomarker for GIST likely to be associated with metastatic disease, can identify patients likely to benefit from new therapeutic strategies such as tyrosine kinase inhibitors.

Pfetin as a prognostic biomarker for gastrointestinal stromal tumor: validation study in multiple clinical facilities.

OBJECTIVE The aim of this study is to confirm the prognostic value of pfetin in gastrointestinal stromal tumor patients. We recently reported the utility of pfetin, a novel prognostic biomarker in gastrointestinal stromal tumor. Gastrointestinal stromal tumor spans a wide spectrum from cases with curable disease to those with fatal tumors due to metastasis and recurrence. There is no biomarker predicting metastasis and/or recurrence of gastrointestinal stromal tumor though imatinib mesylate can improve recurrence-free survival. METHODS Pfetin expression was examined in 40 gastrointestinal stromal tumor patients from the Juntendo University Shizuoka Hospital using immunohistochemistry. Correlations between immunohistochemical findings and clinicopathologic parameters were examined. The pfetin expression results were integrated with the clinicopathologic data in a total of 299 cases including our 40 new gastrointestinal stromal tumor cases and 259 others with previously reported data. RESULTS Immunohistochemical study demonstrated the disease-free survival rate to be 93.75% for pfetin-positive and 25.0% for pfetin-negative patients among the 40 cases from the Juntendo University Shizuoka Hospital (P= 0.0006). When all 299 cases were included, the disease-free survival rate was 92.44% for pfetin-positive and 60.81% for pfetin-negative patients (P< 0.0001). Both uni- and multivariate analyses revealed that, among the clinicopathologic parameters examined, only pfetin expression was an independent prognostic factor (P< 0.05). CONCLUSIONS These results confirm the possible clinical utility of pfetin as a prognostic biomarker for gastrointestinal stromal tumor. Pfetin appears to be a novel clinically applicable prognostic factor, which may be useful for deciding whether to administer imatinib mesylate or not.

High levels of fatty acid synthase expression in esophageal cancers represent a potential target for therapy.

Fatty acid synthase (FAS) is overexpressed in many human cancers and is considered to be a promising target for therapy. To investigate the expression of this candidate target in esophageal cancer, we evaluated expression of FAS protein in 22 cases of esophageal squamous cancer, 79 cases of esophageal adenocarcinoma, and 16 cases of Barretts esophagus with high-grade dysplasia - a lesion thought to represent a pre-invasive precursor to esophageal cancer. Using immunohistochemistry, we found significantly higher levels of FAS expression in 77 % of the squamous cancers, 96% of the adenocarcinomas, and 94% of the Barretts lesions with high-grade dysplasia, when compared to levels in normal esophageal epithelium and non-dysplastic Barrett mucosa. To evaluate the potential for inhibiting this enzyme as a treatment of esophageal cancer, we treated mice bearing xenografts of the Colo680N esophageal squamous cell carcinoma cell line using C93, a rationally designed molecule that inhibits FAS activity. In these experiments, C93 significantly inhibited the growth of orthotopic xenograft tumors without causing anorexia and weight loss in the treated animals. We conclude that, similar to several other common types of human cancer, FAS is expressed at very high levels in esophageal cancer, and growth of these cancers can be inhibited by pharmacological agents that target this enzyme. Moreover, this high expression of FAS is also seen in high-risk, pre-invasive lesions of the esophagus, leading us to propose considering FAS-inhibitors for purposes of esophageal cancer chemoprevention.

Gene Expression Network Analysis of ETV1 Reveals KCTD10 as a Novel Prognostic Biomarker in Gastrointestinal Stromal Tumor

Background Prognostic biomarkers are required for risk stratification therapy in the patients with gastrointestinal stromal tumor (GIST). In this study, we aimed to identify prognostic biomarkers in GIST. We assessed the prognostic value of E twenty-six variant 1 (ETV1), a recently identified transcription factor unique to GIST. We also examined the clinical utility and functions of its downstream gene, potassium channel tetramerization domain containing protein 10 (KCTD10). Methods The levels of ETV1 and KCTD10 were evaluated immunohistochemically in 112 patients with GIST treated at two hospitals. The functional properties of KCTD10 were examined by gene silencing assay in cultured GIST cells. Results Immunohistochemistry revealed that ETV1 expression in GIST had no prognostic significance. In contrast, the disease-free survival rate was 88.5% in patients with KCTD10-positive tumors and 55.8% in those with KCTD10-negative tumors (p <0.0001). KCTD10 was an independent prognostic factor (p <0.05). In the low-risk classification group, KCTD10 was significantly associated with favorable prognosis (p = 0.0008). Gene silencing of KCTD10 increased cell proliferation and invasion, suggesting that KCTD10 has a tumor-suppressive function. Conclusions The GIST-specific transcription factor ETV1 may have no prognostic potential, whereas its downstream gene KCTD10 is associated with a favorable prognosis. Our study indicated the novel prognostic utility of KCTD10 in GIST, and suggested its tumor-suppressive effects on GIST cells. Further validation studies of KCTD10 for clinical applications, and functional verification of KCTD10 for better understanding of molecular basis of malignant phenotypes are worth challenging in GIST.

Direct hemoperfusion with polymyxin B immobilized fiber for abdominal sepsis in Europe

Since direct hemoperfusion with polymyxin B immobilized fiber (PMX-DHP) received its product certification for use in Europe in 1998, several prospective randomized controlled trials (RCTs) have been conducted in European countries. The first RCT, performed in six European academic medical centers in 2005, concluded that PMX-DHP is associated with improved hemodynamic status and cardiac function. Subsequently, a meta-analysis of PMX-DHP was presented in Italy in 2007. This systematic review found positive effects of PMX-DHP on mean arterial pressure and dopamine/ dobutamine use, PaO2/FiO2 ratio, endotoxin removal, and mortality. However, like most trials on extracorporeal therapies, none of the studies was double-blinded. The EUPHAS study, a multicenter RCT performed in ten Italian intensive care units in 2009, found that PMX-DHP improved 28-day survival, blood pressure, vasopressor requirement, and degree of organ failure. However, investigators in Belgium and Canada pointed out that there was no statistical difference in 28-day survival. Two more RCTs, the ABDO-MIX and EUPHRATES studies, the primary end points of which are 28-day mortality, were started in Europe and the United States at the end of 2010. We are hoping that these RCTs will resolve this issue.

Adult intussusception caused by an inverted meckel diverticulum.

Adult intussusception caused by an inverted Meckel diverticulum is rare. We report a 55-year-old Japanese man with intussusception. He was admitted to our hospital with vomiting and abdominal pain. The abdomen was hard with tenderness and muscle guarding. Computed tomography scanning demonstrated a typical inhomogeneous target-shaped mass in the right abdomen. We diagnosed intussusception and performed emergency surgery. At laparotomy, ileocolic intussusception was observed and the ileocecal segment was resected. The surgical specimen comprised an 84 cm segment of resected ileocecum with an elongated polypoid lesion measuring 11 × 2 cm within the ileal lumen. Histopathological examination demonstrated that the polypoid lesion was an inverted Meckel diverticulum. Postoperatively, the patient made an uneventful recovery.

Expression of Akt and Mdm2 in human esophageal squamous cell carcinoma.

The Akt-Mdm2 pathway plays an important role in carcinogenesis in a variety of malignant tumors. However, the Akt-Mdm2 pathway in esophageal squamous cell carcinoma (ESCC) has not been fully studied. We investigated the proteins and mRNA expression of Akt and Mdm2 to elucidate the roles of these proteins in ESCC. We also examined the effect of Akt knockdown on Mdm2 expression in ESCC cells. ESCC tissue samples were obtained from 23 individuals who underwent surgical resection with no preoperative treatment. Akt1-3 and Mdm2 gene and protein expression were analyzed. The effect of siRNA-mediated Akt knockdown on Mdm2 expression was also studied, using ESCC cell lines. Akt1 and Mdm2 immunoreactivity was detected in 77.8 and 66.7% of tumor specimen from ESCC patients, respectively. Akt1 and Mdm2 mRNA expressions were correlated and significantly elevated in tumor tissue (p<0.0001 and p<0.05, respectively). The siRNA-targeted reduction of each Akt isoform reduced Mdm2 protein expression. The overexpression of Akt1 and Mdm2 may be related to esophageal carcinogenesis. Furthermore, Akt expression regulates Mdm2 expression, which may in turn regulate the function of wild-type p53. These results may provide the basis for future preventative or clinical therapies for esophageal cancer.