Yasuyuki Sugiyama

Tumor Suppressor miR-22 Determines p53-Dependent Cellular Fate through Post-transcriptional Regulation of p21

Selective activation of p53 target genes in response to various cellular stresses is a critical step in determining the ability to induce cell-cycle arrest or apoptosis. Here we report the identification of the microRNA miR-22 as a p53 target gene that selectively determines the induction of p53-dependent apoptosis by repressing p21. Combinatorial analyses of the AGO2 immunocomplex and gene expression profiles identified p21 as a direct target of miR-22. Induction of p21 was inhibited by miR-22 after exposure to the genotoxic agent Adriamycin (doxorubicin; Bedford Laboratories), sensitizing cells to p53-dependent apoptosis. Interestingly, the activation of miR-22 depended on the intensity of the stresses that induced cells to undergo apoptosis in the presence of p21 suppression. Our findings define an intrinsic molecular switch that determines p53-dependent cellular fate through post-transcriptional regulation of p21.

Development of Gastric Tumors in ApcMin/+ Mice by the Activation of the β-Catenin/Tcf Signaling Pathway

Although several lines of evidence suggest the involvement of the Wnt pathway in the development of gastric cancers, the functional significance of the pathway in gastric carcinogenesis is still poorly defined. To examine the role of the Apc/beta-catenin signaling pathway in the development of gastric cancers, we investigated the gastric mucosa of the Apc(Min/+) mouse, which is a murine model for familial adenomatous polyposis, carrying a germ-line mutation at codon 850 of Apc. We found that aged Apc(Min/+) mice spontaneously develop multiple tumors in the stomach, which are accompanied by loss of heterozygosity of Apc. Such tumors consisted of adenomatous glands with strong nuclear accumulation of beta-catenin. Even a single adenomatous gland already showed nuclear accumulation of beta-catenin, suggesting that Apc/beta-catenin pathway is an initiating event in gastric tumorigenesis in Apc(Min/+) mice. Myc and cyclin D1 expressions, which are transcriptional targets of beta-catenin/Tcf, increased in the adenomatous lesions. Furthermore, beta-catenin/Tcf reporter transgenic mice with Apc(Min) allele showed higher levels of the transcriptional activity of beta-catenin/Tcf in the gastric tumors. We also treated Apc(Min/+) and wild-type mice with N-methyl-N-nitrosourea (MNU), an alkylating agent that induces adenomas and adenocarcinomas in the stomach. Consequently, MNU-treated Apc(Min/+) mice significantly enhanced the tumor development in comparison with Apc(Min/+) mice or MNU-treated wild-type mice. Several gastric tumors in MNU-treated Apc(Min/+) mice showed invasion into the submucosal layer. These results indicate that the Apc/beta-catenin pathway may play an important role in at least subset of gastric carcinomas. In addition, Apc(Min/+) mice combined with MNU could be a useful short-term model to investigate multistage carcinogenesis in the stomach.

Clinicopathological Features and Prognostic Factors of Adenocarcinoma of the Esophagogastric Junction According to Siewert Classification: Experiences at a Single Institution in Japan

BackgroundTreatment strategy for adenocarcinoma of the esophagogastric junction (AEG) remains controversial. The aims of this study are to evaluate results of surgery for AEG, to clarify clinicopathological differences according to the Siewert classification, and to define prognostic factors.MethodsWe retrospectively analyzed 179 consecutive patients with Siewert type I, II, and III AEG who underwent curative (R0) resection at the National Cancer Center Hospital East between January 1993 and December 2008.ResultsPatients with AEG were divided according to tumor: 10 type I (5.6%), 107 type II (59.8%), and 62 type III (34.6%). Larger, deeper tumors and nodal metastasis were more common in type III than type II tumors. No significant differences were seen in 5-year survival rates among the three types: type I (51.4%), type II (51.8%), and type III (62.6%). Multivariate analysis showed that depth of tumor and mediastinal lymph node metastasis were independent prognostic indicators. The recurrence rate for patients with mediastinal lymph node metastasis was 87.5%. The risk factors for mediastinal lymph node metastasis were length of esophageal invasion and histopathological grade.ConclusionsMediastinal lymph node metastasis and tumor depth were significant and independent factors for poor prognosis after R0 resection for AEG. Esophageal invasion and histopathological grade were significant and independent factors for mediastinal lymph node metastasis.

Human lung tumors, patients' peripheral blood lymphocytes and tumor infiltrating lymphocytes propagated in scid mice.

Studies of the pathogenesis of human tumors and the evaluation of new therapeutic modalities have been limited by the lack of a suitable experimental animal model. Currently very little is known regarding patients’ immune response to their tumors and the consequences of this response to tumor growth. Further, the evaluation of novel tumor therapies has been largely restricted to animal tumor models which may not accurately reflect the efficacy of such therapies for human tumors (Bankert et al. 1989). While athymic homozygous nu/nu mice have been used extensively for the heterotransplantation of human tumors (Fogh and Trempe 1975), their use in evaluating therapies for human cancer is limited by their ability to respond to thymic independent antigens, by the presence of immunoglobulin in their circulation and tissues and by phenotypic and karyotypic changes in the tumor that have been observed during tumor growth in nude mice. We have previously reported (Reddy et al. 1987) that xenografts of human lung tumors can be successfully propagated in the B cell and T cell deficient mouse mutant strain, CB-17 scid (described by Bosma et al. 1983). Several characteristics of the scid mouse make it particularly attractive as a potential model to evaluate new therapeutic approaches to the treatment of human cancers. In this report we summarize our findings that scid mice support the growth of human tumors and we establish here that this mouse is a viable model with which to evaluate the antibody directed delivery of cytotoxic agents to human lung tumors.

Accumulated phosphatidylcholine (16:0/16:1) in human colorectal cancer; possible involvement of LPCAT4

The identification of cancer biomarkers is critical for target‐linked cancer therapy. The overall level of phosphatidylcholine (PC) is elevated in colorectal cancer (CRC). To investigate which species of PC is overexpressed in colorectal cancer, an imaging mass spectrometry was performed using a panel of non‐neoplastic mucosal and CRC tissues. In the present study, we identified a novel biomarker, PC(16:0/16:1), in CRC using imaging mass spectrometry. Specifically, elevated levels of PC(16:0/16:1) expression were observed in the more advanced stage of CRC. Our data further showed that PC(16:0/16:1) was specifically localized in the cancer region when examined using imaging mass spectrometry. Notably, because the ratio of PC(16:0/16:1) to lyso‐PC(16:0) was higher in CRC, we postulated that lyso‐PC acyltransferase (LPCAT) activity is elevated in CRC. In an in vitro analysis, we showed that LPCAT4 is involved in the deregulation of PC(16:0/16:1) in CRC. In an immunohistochemical analysis, LPCAT4 was shown to be overexpressed in CRC. These data indicate the potential usefulness of PC(16:0/16:1) for the clinical diagnosis of CRC and implicate LPCAT4 in the elevated expression of PC(16:0/16:1) in CRC.

Factors predicting mortality in emergency abdominal surgery in the elderly

ObjectiveThis study aimed to investigate clinical features of abdominal emergency surgery in elderly patients, and to determine factors predicting mortality in these patients.MethodsThe study population included 94 patients aged 80 years or older who underwent emergency surgery for acute abdominal diseases between 2000 and 2010. Thirty-six patients (38.3%) were male and fifty-eight patients (61.7%) were female (mean age, 85.6 years). Main outcome measures included background of the patient’s physical condition (concomitant medical disease, and performance status), cause of disease, morbidity and mortality, and disease scoring system (APACHE II, and POSSUM). Prognostic factors affecting mortality of the patient were also evaluated by univariate analysis using Fisher’s exact test and Mann–Whitney U–test, and by multivariate analysis using multiple logistic regression analysis.ResultsOf the 94 patients, 71 (75.5%) had a co-existing medical disease; most patients had hypertension (46.8%). The most frequent surgical indications were acute cholecystitis in 23 patients (24.5%), followed by intestinal obstruction in 18 patients (19.1%). Forty-one patients (43.6%) had complications during hospital stay; the most frequent were surgical site infection (SSI) in 21 patients (22.3%) and pneumonia in 12 patients (12.8%). Fifteen patients died (overall mortality, 16%) within 1 month after operation. The most common causes of death were sepsis related to pan-peritonitis in 5 patients (5.3%), and pneumonia in 4 patients (4.3%). Multiple logistic regression analysis showed that time from onset of symptoms to hospital admission and the POSSUM scoring system could be prognostic factors for mortality.ConclusionsMortality in elderly patients who underwent emergency surgery for acute abdominal disease can be predicted using the disease scoring system (POSSUM) and on the basis of delay in hospital admission.

Potential responders to FOLFOX therapy for colorectal cancer by Random Forests analysis

Background:Molecular characterisation using gene-expression profiling will undoubtedly improve the prediction of treatment responses, and ultimately, the clinical outcome of cancer patients.Methods:To establish the procedures to identify responders to FOLFOX therapy, 83 colorectal cancer (CRC) patients including 42 responders and 41 non-responders were divided into training (54 patients) and test (29 patients) sets. Using Random Forests (RF) algorithm in the training set, predictor genes for FOLFOX therapy were identified, which were applied to test samples and sensitivity, specificity, and out-of-bag classification accuracy were calculated.Results:In the training set, 22 of 27 responders (81.4% sensitivity) and 23 of 27 non-responders (85.1% specificity) were correctly classified. To improve the prediction model, we removed the outliers determined by RF, and the model could correctly classify 21 of 23 responders (91.3%) and 22 of 23 non-responders (95.6%) in the training set, and 80.0% sensitivity and 92.8% specificity, with an accuracy of 69.2% in 29 independent test samples.Conclusion:Random Forests on gene-expression data for CRC patients was effectively able to stratify responders to FOLFOX therapy with high accuracy, and use of pharmacogenomics in anticancer therapy is the first step in planning personalised therapy.

Prognostic Significance of the Metastatic Lymph Node Ratio in Gastric Cancer Patients

BackgroundLymph node metastasis is considered one of the most important prognostic factors in gastric cancer. However, the optimal system for accurate staging of lymph node metastasis for patients with gastric cancer remains controversial. This study was designed to investigate the prognostic significance of the metastatic lymph node ratio (MLR), which is calculated by dividing the number of metastatic lymph nodes by the total number of nodes harvested from patients with gastric cancer.MethodsWe retrospectively analyzed the clinical data of 186 consecutive patients diagnosed with gastric cancer who underwent curative gastrectomy at our hospital. The lymph node status was classified according to three systems: the International Union Against Cancer/American Joint Committee on Cancer (UICC/AJCC) system; the Japanese Gastric Cancer Association (JGCA) system; and an MLR-based system (MLR0: 0, MLR1: 0.01–0.19, MLR2: ≥0.2). The influence of the MLR on patient survival was determined using univariate Kaplan-Meier survival analysis, the generalized Wilcoxon test, and analysis with the multivariate Cox proportional hazards model.ResultsThe 5-year survival rate of the patients with MLR0, MLR1, and MLR2 was 88.6%, 59.4%, and 13.4%, respectively. In addition to the MLR, the UICC/AJCC N category, JGCA n category, tumor stage (pT category), and tumor diameter significantly influenced the 5-year survival rate, as determined by univariate analysis. Multivariate analyses revealed that of the three factors used to stage lymph node involvement, MLR was the most significant prognostic factor.ConclusionsThe MLR is an important and easy-to-assess prognostic factor that should be considered for staging lymph node metastasis in patients with gastric cancer.

CD40-CD40 ligand (CD154) engagement is required but not sufficient for modulating MHC class I, ICAM-1 and Fas expression and proliferation of human non-small cell lung tumors.

To determine the possible functional significance of CD40 expression on human non‐small cell lung carcinomas and to assess the potential of CD40 as a therapeutic target, 18 lung tumor cell lines were established from biopsy tissues and were monitored for phenotypic changes on the cell surface and alterations in tumor cell proliferation after the ligation of CD40 with a trimeric fusion protein complex of CD40 ligand (CD40Lt). CD40 cross‐linking resulted in up to a 6‐fold increase in the surface expression of major histocompatibility complex (MHC) class I, Fas and intracellular adhesion molecule (ICAM)‐1 in a subset of tumors expressing the highest levels of CD40. Suppression of tumor proliferation was seen after the ligation of CD40 on CD40Lt‐responsive cell lines. The suppression was dose dependent, reversible and resulted from a delay of the tumor cells entering S‐phase. No change in the cell phenotype or in proliferation were observed in CD40‐negative tumors or in tumors expressing moderate‐to‐low levels of CD40 after incubation with CD40Lt. CD40‐negative tumors transfected with the CD40 gene expressed high levels of CD40 on their surface, but were also unresponsive to CD40Lt cross‐linking of CD40. Our data establish that CD40 is required (but not sufficient) for transducing a signal that results in phenotypic changes in human lung tumors and suppression in their proliferation. We conclude that CD40 on non‐small cell lung tumors may represent a potential therapeutic target, but only on a subset of the CD40+ tumors.

Human inflammatory cells within the tumor microenvironment of lung tumor xenografts mediate tumor growth suppression in situ that depends on and is augmented by interleukin-12

The human tumor microenvironment includes a mixture of tumor cells, inflammatory cells, fibroblasts, and endothelial cells, all of which are tethered to an extracellular matrix. It has been difficult to study the dynamic interactions of these cells in human tumors in situ for obvious ethical and logistical considerations that prohibit experimental manipulations of tumors while still in patients. Fresh tissue from human lung tumor biopsy implanted into SCID mice was shown to remain viable, and the histologic appearance of the tumor microenvironment was maintained in the tumor xenografts for at least 3 months. In this study, the authors established that the inflammatory cells within human tumor xenografts can suppress tumor growth, and that this suppression is a result, in part, of endogenously produced interleukin-12 (IL-12) because IL-12 neutralizing antibodies enhance the growth of the tumor xenografts. The tumor-inhibitory activity of the inflammatory leukocytes is also enhanced by the local and sustained release of human recombinant IL-12 into the tumor microenvironment from cytokine-loaded biodegradable microspheres. Neither the anti–IL-12 neutralizing antibody nor the delivery of exogenous IL-12 from microspheres had any effect on tumor xenografts in the absence of the inflammatory leukocytes. In conclusion, the inflammatory cells within the tumor microenvironment of human lung tumor xenografts are functional and can suppress tumor growth, and the dynamic effects of the inflammatory cells can be modulated by exogenous cytokines.