Yoshihiko Kitajima

Silencing effect of CpG island hypermethylation and histone modifications on O6-methylguanine-DNA methyltransferase (MGMT) gene expression in human cancer.

O6-methylguanine-DNA methyltransferase (MGMT) repairs the cytotoxic and mutagenic O6-alkylguanine produced by alkylating agents such as chemotherapeutic agents and mutagens. Recent studies have shown that in a subset of tumors, MGMT expression is inversely linked to hypermethylation of the CpG island in the promoter region; however, how the epigenetic silencing mechanism works, as it relates to hypermethylation, was still unclear. To understand the mechanism, we examined the detailed methylation status of the whole island with bisulfite-sequencing in 19 MGMT non-expressed cancer cell lines. We found two highly methylated regions in the island. One was upstream of exon 1, including minimal promoter, and the other was downstream, including enhancer. Reporter gene assay showed that methylation of both the upstream and downstream regions suppressed luciferase activity drastically. Chromatin immunoprecipitation assay revealed that histone H3 lysine 9 was hypermethylated throughout the island in the MGMT negative line, whereas acetylation on H3 and H4 and methylation on H3 lysine 4 were at significantly high levels outside the minimal promoter in the MGMT-expressed line. Furthermore, MeCP2 preferentially bound to the CpG-methylated island in the MGMT negative line. Given these results, we propose a model for gene silencing of MGMT that is dependent on the epigenetic state in cancer.

Comparison of multivisceral resection and standard operation for locally advanced Colorectal cancer: Analysis of Prognostic factors for short-term and long-term outcome

PURPOSEThe aim of the present study is to clarify the characteristics of multivisceral resection and to discuss strategies for improving the overall outcome of multivisceral resection for locally advanced colorectal cancer.METHODSThe study included 323 patients who electively underwent curative surgery for pT3–pT4 colorectal carcinoma without distant metastasis. We evaluated the short-term and long-term outcome of multivisceral resection relative to that of the standard operation by means of multivariate analysis of the prognostic factors.RESULTSOf 323 patients, 53 (16.4 percent) received multivisceral resection because of adhesion to other organs. Multivisceral resection was significantly associated with tumor size, depth of invasion, operative blood loss, operation time, and blood transfusion (all: P < 0.0001). Overall morbidity rates were 49.1 percent after multivisceral resection vs. 17.8 percent after the standard operation (P < 0.0001), and postoperative mortality rate was 0 percent in both groups (not significant). Only multivisceral resection (odds ratio, 2.725; 95 percent confidence interval, 1.125–6.623; P = 0.0264) was an independent factor for overall postoperative complications. The survival rate of patients after multivisceral resection was similar to that after the standard operation (5-year rate, 76.6 percent vs. 79.5 percent, P = 0.9347). Lymph node metastasis (hazard ratio, 2.510; 95 percent confidence interval, 1.460–4.315; P = 0.0009) and blood transfusion (hazard ratio, 2.353; 95 percent confidence interval, 1.185–4.651; P = 0.0145) were independently associated with patient survival.CONCLUSIONSFor locally advanced colorectal cancer, the long-term outcome after multivisceral resection is comparable to that after the standard operation. However, it should be recognized that multivisceral resection is associated with higher postoperative morbidity. In addition, a reduction in the incidence of blood transfusion may contribute to improving patient survival.

Tumor-stromal cell interaction under hypoxia increases the invasiveness of pancreatic cancer cells through the hepatocyte growth factor/c-Met pathway.

The hypoxic environment in tumor is reported to play an important role in pancreatic cancer progression. The interaction between stromal and cancer cells also contributes to the malignant behavior of pancreatic cancer. In the present study, we investigated whether hypoxic stimulation affects stromal as well as pancreatic cancer cells. Our findings demonstrated that hypoxia remarkably elevated the HIF‐1α expression in both pancreatic cancer (PK8) and fibroblast cells (MRC5). Hypoxic stimulation accelerated the invasive activity of PK8 cells, and invasiveness was thus further accelerated when the hypoxic PK8 cells were cultured with conditioned medium prepared from hypoxic MRC5 cells (hypoxic conditioned medium). MMP‐2, MMP‐7, MT1‐MMP and c‐Met expressions were increased in PK8 cells under hypoxia. Hypoxic stimulation also increased the hepatocyte growth factor (HGF) secretion from MRC5 cells, which led to an elevation of c‐Met phosphorylation in PK8 cells. Conversely, the elevated cancer invasion, MMP activity and c‐Met phosphorylation of PK8 cells were reduced by the removal of HGF from hypoxic conditioned medium. In immunohistochemical study, the HIF‐1α expression was observed in surrounding stromal as well as pancreatic cancer cells, thus indicating hypoxia exists in both of cancer and stromal cells. Moreover, the stromal HGF expression was found to significantly correlate with not only the stromal HIF‐1α expression but also the c‐Met expression in cancer cells. These results indicate that the hypoxic environment within stromal as well as cancer cells activates the HGF/c‐Met system, thereby contributing to the aggressive invasive features of pancreatic cancer.

Induction of hepatocyte growth factor activator gene expression under hypoxia activates the hepatocyte growth factor/c-Met system via hypoxia inducible factor-1 in pancreatic cancer

Hepatocyte growth facor activator (HGFA) is a serine protease that converts hepatocyte growth factor (HGF) into its active form. Our previous study demonstrated that tumor–stromal interaction under hypoxia augments the aggressive invasive features of pancreatic cancer line PK8 through activated HGF/c‐Met signaling. The present study investigated whether or not hypoxia increases HGFA expression in PK8 cells and promotes the processing of HGF, and leads to c‐Met activation. Moreover, HGFA promoter assays were performed to define whether hypoxia inducible factor‐1 alpha (HIF‐1α) directly activates the HGFA promoter in a hypoxia‐dependent fashion. As a result, hypoxia induced the HGFA mRNA and protein expression in PK8 and the elevation under hypoxia was inhibited by the transfection of HIF‐1α siRNA, thus indicating HIF‐1α‐dependent induction of HGFA. The transfection of siRNA against HGFA to PK8 cells suppressed the conversion to the active HGF, which is secreted from fibroblast MRC5. Furthermore, the phosphorylation of c‐Met and cancer invasion of PK8 cells were decreased by the transfection of HGFA siRNA under hypoxia. Using the luciferase reporter system, HIF‐1α was shown to transactivate the HGFA promoter under hypoxia. These experiments demonstrated for the first time that HGFA is a novel HIF‐1 target gene. Under hypoxia, HGFA might be overexpressed and secreted from pancreatic cancer cells, which contributes to accelerate processing of HGF from fibroblast, resulting in the activation of the c‐Met pathway. HGF/HGFA/c‐Met recruited between cancer‐stromal fibroblasts is activated under hypoxic conditions and therefore might play a central role in the aggressive invasion of pancreatic cancer. (Cancer Sci 2008; 99: 1341–1347)

The Hypoxic Environment in Tumor-Stromal Cells Accelerates Pancreatic Cancer Progression via the Activation of Paracrine Hepatocyte Growth Factor/c-Met Signaling

BackgroundPancreatic cancer is one of the representative solid tumors, in which the hypoxic microenvironment plays a crucial role in malignant progression. We previously demonstrated that tumor-stromal interaction under hypoxia enhances the invasiveness of pancreatic cancer cells through hepatocyte growth factor (HGF)/c-Met signaling.MethodsWe investigated the immunohistochemical expression of hypoxia inducible factor-1α (HIF-1α) c-Met, and HGF in both cancer and stromal cells using 41 pancreatic cancer tissue specimens, and tried to identify any correlations with the clinical features and survival.ResultsPositive staining for HIF-1α was observed in both pancreatic cancer and the surrounding stromal cells in more than 30% of the cases, and it significantly correlated with lymph node metastasis (P < .05). A significant correlation was observed between the expression of HIF-1α and HGF in stromal cells (P < .05). In addition, the c-Met expression in cancer cells was found to significantly correlate with the HGF expression in not only cancer but also stromal cells. The disease-free survival rates of the patients with HIF-1α in cancer, stromal, c-Met in cancer, and an HGF expression in stromal cells was significantly worse than those without such expressions (P < .05).ConclusionsThese data suggest that the HGF/c-Met signaling via HIF-1α ?may therefore negatively affect the prognosis in patients with pancreatic cancer, and targeting tumor stroma under hypoxia might thus be potentially useful as a novel therapy for this cancer.

Deficient expression ofO 6-Methylguanine-DNA methyltransferase combined with mismatch-repair proteins hMLH1 and hMSH2 is related to poor prognosis in human biliary tract carcinoma

BackgroundO6-Methylguanine-DNA methyltransferase (MGMT) is a DNA repair enzyme that transfers methyl groups fromO6-methylguanine to itself. Alkylation of DNA at theO6 position of guanine is an important step in the induction of mutations in the organism by alkylating agents. TheO6-methyl G:T mismatch is recognized by the mismatch-repair (MMR) pathway. The biliary duct is highly exposed to alkylating agents because of its anatomical location.MethodsWe examined 39 surgically resected gallbladder carcinomas and 35 extrahepatic bile duct carcinomas and evaluated the expression of MGMT and MMR protein (hMLH1 and hMSH2) by immunohistochemical staining.ResultsMGMT-negative staining was detected in 59.0% of gallbladder carcinoma specimens and 60.0% of extrahepatic bile duct carcinoma specimens. In gallbladder carcinoma, hMLH1- and hMSH2-negative staining was observed in 51.3% and 59.0%, respectively, whereas in extrahepatic bile duct carcinoma, the respective values were 57.1% and 65.7%. MGMT-negative staining correlated with hepatic invasion in gallbladder carcinoma and with poor prognosis in both types of tumor. Furthermore, a combined MGMT and MMR status was shown to be a more significant prognostic biomarker in both tumor types.ConclusionsCombined MGMT and MMR is a possible prognostic marker that probably reflects an accumulation of genetic mutations.

Silencing of imprinted CDKN1C gene expression is associated with loss of CpG and histone H3 lysine 9 methylation at DMR-LIT1 in esophageal cancer

The putative tumor suppressor CDKN1C is an imprinted gene at 11p15.5, a well-known imprinted region often deleted in tumors. The absence of somatic mutations and the frequent diminished expression in tumors would suggest that CDKN1C expression is regulated epigenetically. It has been, however, controversial whether the diminution is caused by imprinting disruption of the CDKN1C/LIT1 domain or by promoter hypermethylation of CDKN1C itself. To clarify this, we investigated the CpG methylation index of the CDKN1C promoter and the differentially methylated region of the LIT1 CpG island (differentially methylated region (DMR)-LIT1), an imprinting control region of the domain, and CDKN1C expression in esophageal cancer cell lines. CDKN1C expression was diminished in 10 of 17 lines and statistically correlated with the loss of methylation at DMR-LIT1 in all but three. However, there was no statistical correlation between CDKN1C promoter MI and CDKN1C expression. Furthermore, loss of CpG methylation was associated with loss of histone H3 lysine 9 (H3K9) methylation at DMR-LIT1. Histone modifications at CDKN1C promoter were not correlated with CDKN1C expression. The data suggested that the diminished CDKN1C expression is associated with the loss of methylation of CpG and H3K9 at DMR-LIT1, not by its own promoter CpG methylation, and is involved in esophageal cancer, implying that DMR-LIT1 epigenetically regulates CDKN1C expression not through histone modifications at CDKN1C promoter, but through that of DMR-LIT1.

The significance of aberrant CHFR methylation for clinical response to microtubule inhibitors in gastric cancer

BackgroundWe studied the correlations between CHFR (checkpoint with FHA and RING finger) gene methylation and responses to microtubule inhibitors (MI) in gastric cancer.MethodsWe examined 9 gastric cancer cell lines and 46 gastric cancer specimens from patients who underwent surgical resection. Promoter methylation was determined by methylation-specific polymerase chain reaction (MSP). CHFR mRNA expression was estimated by quantitative reverse transcription–PCR. The MI-induced growth inhibition was assayed by a standard MTT method.ResultsCHFR expression was silenced by aberrant promoter methylation in 3 of 9 gastric cancer cell lines. The level of CHFR mRNA expression was closely correlated with IC50 in the MI-treated cells (R = 0.889, P = 0.005). In 46 patients with gastric cancers, 24 (52%) presented aberrant CHFR methylation. Among them, 12 patients had received treatment with MI because of advanced-stage tumor or tumor recurrence after surgery. The responders to the MI treatment were 29% in patients with CHFR methylation and 20% in those without the methylation. However, 6 (86%) of 7 patients with methylated CHFR tumor showed some regression or no progression, whereas 4 (80%) of 5 patients with unmethylated CHFR tumor manifested progressive deterioration.ConclusionsThese observations indicated that CHFR methylation may be a clinically useful approach to predict the responsiveness of gastric cancers to treatment with MI.

Tumor Progression Through Epigenetic Gene Silencing of O6−Methylguanine-DNA Methyltransferase in Human Biliary Tract Cancers

ABSTRACTBackgroundWe previously demonstrated in an immunohistochemical study that reduced expression of O6−methylguanine-DNA methyltransferase (MGMT) correlated with a poorer prognosis in patients with biliary tract cancers. The purpose of this study was to clarify how MGMT deficiency leads to a poor outcome in biliary tract cancer. Thus, we examined epigenetic (promoter methylation) and genetic (gene mutation) alterations in biliary tract cancer.MethodsWe examined 37 biliary tract cancer specimens from patients who underwent surgical resection. Promoter methylation was determined by one-step or two-step methylation-specific polymerase chain reaction. Gene mutation was identified by direct sequencing. The expression of MGMT protein in paraffin-embedded tissue was examined by immunohistochemistry.ResultsFrequencies of promoter methylation were 70% for p16/INK4a, 49% for MGMT, 46% for hMLH1, 41% for E-cadherin, and 32% for DAPK genes. MGMT methylation status was closely correlated with the MGMT protein expression determined by immunohistochemistry (P < .001). Although this was not statistically significant, biliary tract cancer tumors with MGMT methylation expressed multigene methylation more frequently than tumors without MGMT methylation (P = .071). A total of 33 mutations were identified in 4 cancer-related genes: p53, K-ras, β-catenin, and p16/INK4a genes. The most common mutation was GC to AT transitions (58%), which were significantly associated with MGMT promoter methylation (P = .011). These findings suggest that loss of MGMT expression by promoter methylation results in accumulation of GC to AT gene mutations.ConclusionsReduced MGMT expression may increase the malignant potential of biliary tract cancer through both epigenetic and genetic mechanisms.

The Critical Impact of HIF-1a on Gastric Cancer Biology.

Hypoxia inducible factor-1 (HIF-1) monitors the cellular response to the oxygen levels in solid tumors. Under hypoxia conditions, HIF-1α protein is stabilized and forms a heterodimer with the HIF-1β subunit. The HIF-1 complex activates the transcription of numerous target genes in order to adapt the hypoxic environment in human cancer cells. In gastric cancer patients, HIF-1α activation following extended hypoxia strongly correlates with an aggressive tumor phenotype and a poor prognosis. HIF-1α activation has been also reported to occur via hypoxia-independent mechanisms such as PI3K/AKT/mTOR signaling and ROS production. This article argues for the critical roles of HIF-1α in glucose metabolism, carcinogenesis, angiogenesis, invasion, metastasis, cell survival and chemoresistance, focusing on gastric cancer.