Masaki Kunizaki

Validation of the histone methyltransferase EZH2 as a therapeutic target for various types of human cancer and as a prognostic marker

The emphasis in anticancer drug discovery has always been on finding a drug with great antitumor potential but few side‐effects. This can be achieved if the drug is specific for a molecular site found only in tumor cells. Here, we find the enhancer of zeste homolog 2 (EZH2) to be highly overexpressed in lung and other cancers, and show that EZH2 is integral to proliferation in cancer cells. Quantitative real‐time PCR analysis revealed higher expression of EZH2 in clinical bladder cancer tissues than in corresponding non‐neoplastic tissues (P < 0.0001), and we confirmed that a wide range of cancers also overexpress EZH2, using cDNA microarray analysis. Immunohistochemical analysis showed positive staining for EZH2 in 14 of 29 cases of bladder cancer, 135 of 292 cases of non‐small‐cell lung cancer (NSCLC), and 214 of 245 cases of colorectal cancer, whereas no significant staining was observed in various normal tissues. We found elevated expression of EZH2 to be associated with poor prognosis for patients with NSCLC (P = 0.0239). In lung and bladder cancer cells overexpressing EZH2, suppression of EZH2 using specific siRNAs inhibited incorporation of BrdU and resulted in significant suppression of cell growth, even though no significant effect was observed in the normal cell strain CCD‐18Co, which has undetectable EZH2. Because EZH2 expression was scarcely detectable in all normal tissues we examined, EZH2 shows promise as a tumor‐specific therapeutic target. Furthermore, as elevated levels of EZH2 are associated with poor prognosis of patients with NSCLC, its overexpression in resected specimens could prove a useful molecular marker, indicating the necessity for a more extensive follow‐up in some lung cancer patients after surgical treatment. (Cancer Sci 2011; 102: 1298–1305)

The Lysine 831 of Vascular Endothelial Growth Factor Receptor 1 Is a Novel Target of Methylation by SMYD3

We previously identified SMYD3 as a histone methyltransferase and showed that its expression was elevated in colorectal, hepatocellular, and breast carcinomas. In the investigation of methyltransferase activity of SMYD3, we have found that vascular endothelial growth factor receptor 1 (VEGFR1) was also methylated by SMYD3. We further identified the methylated residue at VEGFR1 lysine 831, which is located in the kinase domain and is conserved among VEGFR1 orthologues. We also found that the lysine is followed by serine, which is conserved among some of the methylation targets of histone methyltransferases. Furthermore, methylation of VEGFR1 enhanced its kinase activity in cells. These data should be helpful for the profound understanding of the biological role of SMYD3 and regulatory mechanisms of VEGFR1. Additionally our finding may facilitate the development of strategies that may inhibit the progression of cancer cells.

MiRNA-205 modulates cellular invasion and migration via regulating zinc finger E-box binding homeobox 2 expression in esophageal squamous cell carcinoma cells

BackgroundEsophageal squamous cell carcinoma (ESCC) is often diagnosed at later stages until they are incurable. MicroRNA (miR) is a small, non-coding RNA that negatively regulates gene expression mainly via translational repression. Accumulating evidence indicates that deregulation of miR is associated with human malignancies including ESCC. The aim of this study was to identify miR that could be specifically expressed and exert distinct biological actions in ESCC.MethodsTotal RNA was extracted from ESCC cell lines, OE21 and TE10, and a non-malignant human esophageal squamous cell line, Het-1A, and subjected to microarray analysis. Expression levels of miR that showed significant differences between the 2 ESCC and Het-1A cells based on the comprehensive analysis were analyzed by the quantitative reverse transcriptase (RT)-PCR method. Then, functional analyses, including cellular proliferation, apoptosis and Matrigel invasion and the wound healing assay, for the specific miR were conducted. Using ESCC tumor samples and paired surrounding non-cancerous tissue obtained endoscopically, the association with histopathological differentiation was examined with quantitative RT-PCR.ResultsBased on the miR microarray analysis, there were 14 miRs that showed significant differences (more than 2-fold) in expression between the 2 ESCC cells and non-malignant Het-1A. Among the significantly altered miRs, miR-205 expression levels were exclusively higher in 5 ESCC cell lines examined than any other types of malignant cell lines and Het-1A. Thus, miR-205 could be a specific miR in ESCC. Modulation of miR-205 expression by transfection with its precursor or anti-miR-205 inhibitor did not affect ESCC cell proliferation and apoptosis, but miR-205 was found to be involved in cell invasion and migration. Western blot revealed that knockdown of miR-205 expression in ESCC cells substantially enhanced expression of zinc finger E-box binding homeobox 2, accompanied by reduction of E-cadherin, a regulator of epithelial mesenchymal transition. The miR-205 expression levels were not associated with histological differentiation of human ESCC.ConclusionsThese results imply that miR-205 is an ESCC-specific miR that exerts tumor-suppressive activities with EMT inhibition by targeting ZEB2.

Enhanced methyltransferase activity of SMYD3 by the cleavage of its N-terminal region in human cancer cells.

Histone methylation is involved in the regulation of gene expression and DNA replication through alteration of chromatin structure. We earlier showed that SMYD3, a histone H3-lysine 4-specific methyltransferase, is frequently upregulated in human colorectal, liver and breast cancer compared to their matched non-cancerous cells, and that its activity is associated with the growth of these tumors. In the present study, we found that human cancer cells express both the full-length and a cleaved form of SMYD3 protein. Amino acid sequence analysis uncovered that the cleaved form lacks the 34 amino acids in the N-terminal region of the full-length protein. Interestingly, the cleaved protein and mutant protein containing substitutions at glycines 15 and 17, two highly conserved amino acids in the N-terminal region, revealed a higher histone methyltransferase (HMTase) activity compared to the full-length protein. Furthermore, the N-terminal region is responsible for the association with heat shock protein 90α (HSP90α). These data indicate that the N-terminal region plays an important role for the regulation of its methyltransferase activity and suggest that a structural change of the protein through the cleavage of the region or interaction with HSP90α may be involved in the modulation. These findings may help for a better understanding of the mechanisms that modulate the HMTase activity of SMYD3, and contribute to the development of novel anticancer drugs targeting SMYD3 methyltransferase activity.

Inflammation-based scoring is a useful prognostic predictor of pulmonary resection for elderly patients with clinical stage I non-small-cell lung cancer

OBJECTIVES The number of elderly lung cancer patients requiring surgery has been increasing due to the ageing society and less invasive perioperative procedures. Elderly people usually have various comorbidities, but there are few simple and objective tools that can be used to determine prognostic factors for elderly patients with clinical stage I non-small-cell lung cancer (NSCLC). The aim of this retrospective study was to evaluate the prognostic factors of surgically treated, over 80-year old patients with clinical stage I NSCLC. METHODS The preoperative data of 97 over 80-year old patients with clinical stage I NSCLC were collected at Nagasaki University Hospital from 1990 to 2012. As prognostic factors, inflammation-based scoring systems, including the Glasgow Prognostic Score (GPS) determined by serum levels of C-reactive protein and albumin, the neutrophil lymphocyte ratio (NLR) and the platelet lymphocyte ratio (PLR) were evaluated, as well as other clinicopathological factors, including performance status, body mass index, carcinoembryonic antigen, Charlson comorbidity index and type of surgical procedure. RESULTS The median age was 82 (range, 80-93) years. There were 62 (64.0%) clinical stage IA cases and 35 IB cases. Operations included 64 (66.0%) lobectomies, 15 segmentectomies and 18 wedge resections. The pathological stage was I in 76 (78.4%) patients, II in 12 (12.4%), III in 8 (8.2%) and IV in 1 (1.0%). Twelve (12.4%) patients underwent mediastinal lymph node dissection. Overall survival and disease-specific 5-year survival rates were 55.5 and 70.0%, respectively. The average GPS score was 0.4 (0-2). Disease-specific 5-year survival was significantly longer with GPS 0 than with GPS 1-2. (74.2%, 53.7%, respectively, P = 0.03). Overall 5-year survival was significantly longer with GPS 0 than with GPS 1-2. (59.7%, 43.1%, respectively, P = 0.005). Both the NLR (median value = 1.9) and the PLR (median value = 117) were not correlated with disease-specific and overall 5-year survival. On multivariate analysis, pathological stage I (P = 0.01) and GPS 0 (P = 0.04, hazard ratio: 2.13, 95% confidence interval 1.036-4.393) were significant prognostic factors. CONCLUSIONS The preoperative GPS appears to be a useful predictor of overall survival and could be a simple prognostic tool for elderly patients with clinical stage I NSCLC.

Usefulness of sonazoid–ultrasonography during hepatectomy in patients with liver tumors: A preliminary study

To improve diagnostic accuracy of intraoperative ultrasonography (IOUS), we investigated the usefulness of new contrast medium of microbubble agent, Sonazoid as a preliminary study.

Interweaving MicroRNAs and Proinflammatory Cytokines in Gastric Mucosa with Reference to H. pylori Infection

Using endoscopic biopsies, gastric mucosal expression levels of interleukin (IL)-1β, IL-6, IL-8, and tumor necrosis factor-alpha (TNF-α) messenger RNA (mRNA) and microRNAs (miRNAs) that were differentially expressed in association with Helicobacter pylori were assessed by quantitative reverse-transcriptase polymerase chain reaction. Among the H. pylori-positive mucosa, 17 out of 29 miRNAs had significant correlations with at least one of the four proinflammatory cytokines in expression. Among the 17 miRNAs, 15 were associated with the degree of neutrophil infiltration and, more prominently, the degree of mononuclear cell infiltration, according to the updated Sydney system. Persistent H. pylori infection may affect the mucosal expression profiles of miRNAs via chronic inflammation mediated by proinflammatory cytokines. There were significant positive correlations between certain miRNAs including the microRNA-200 family and IL-1β, IL-6, or TNF-α mRNA in H. pylori-negative gastric mucosa. Underscoring the causal association between miRNAs and proinflammatory cytokines may provide insights into the pathogenesis of H. pylori-associated gastritis linking to gastric carcinogenesis.

Short-term outcomes of laparoscopic surgery for colorectal cancer in oldest-old patients.

Background/Aims: Oldest-old patients generally have several comorbidities, and laparoscopic-assisted colectomy (LAC) has not been performed on these patients. However, the surgical technique of LAC has improved, and its indications have been extended. The aim of this study was to evaluate the safety and effectiveness of LAC for patients over 85 years old. Methods: Fifty-eight patients over 85 years old who underwent colectomy were retrospectively analyzed. The patients were divided into two groups (LAC group n = 15; open surgery group (Open group) n = 43), and clinicopathological features, surgical characteristics, and outcomes were compared. Results: There were no significant differences in clinical background characteristics between the groups. The LAC group had longer operation time and greater lymph node dissection (both p < 0.01). Postoperatively, the use of analgesics (p = 0.01) was less and the start of oral liquid intake (p = 0.03) was faster in the LAC group. Postoperative complications occurred in 3 patients (20%) in the LAC group and 13 patients (30%) in the Open group (p = 0.66); delirium (n = 6) and sub-ileus (n = 4) developed only in the Open group. Conclusion: After LAC, elderly patients tended to have less postoperative pain and started oral liquid intake earlier. LAC can be safe and effective, preventing postoperative complications that occur specifically in oldest-old patients.

Does the placement of a cystic duct tube after a hepatic resection help reduce the incidence of post-operative bile leak?

BACKGROUND In this retrospective study, the effects of cystic duct (C) tube use on the incidence of post-hepatectomy bile leak were assessed. METHODS The subjects were 550 patients who underwent a hepatectomy during 1990-2011, with (n = 83) and without (n = 467) C tube drainage. The use of a C tube was based on the surgeons choice. RESULTS Bile leakage was observed in 44 (8%) patients, and its incidence post-operatively correlated with intrahepatic cholangiocarcinoma, parenchymal transection with forceps fracture and tie, a major hepatectomy, prolonged surgery and excessive blood loss (P < 0.050) but not with the use of a C tube. The incidence of an intra-abdominal infection was higher and the hospital stay was longer in the leak (49 days) than non-leak group (21 days, P < 0.001). ISGLS grade B and C bile leak post-hemi-hepatectomy and extended-hepatectomy were more frequent in the non-C than C tube group (P = 0.016). The duration of hospitalization was not different between the two groups; however, 7 patients in the non-C tube group had prolonged hospitalization (> 60 days) compared with none in the C tube group (P = 0.454). CONCLUSION The usefulness of the C tube in preventing post-hepatectomy bile leak could not be confirmed; however, both bile leak requiring clinical management and long hospitalization after a major hepatectomy could be reduced with C tube use.

Intraductal papillary growth of liver metastasis originating from colon carcinoma in the bile duct: Report of a case

Morphologically, liver metastases from colorectal carcinoma usually form as nodular tumor masses, whereas intraductal papillary growth in the bile duct is rare. A 65-year-old man underwent right hemicolectomy for advanced colon carcinoma, and histology of the primary carcinoma confirmed moderately differentiated adenocarcinoma with subserosal invasion, no vascular infiltration, and no lymph node metastasis. A liver tumor was found in the right paramedian Glisson pedicle and intraductal growth of cholangiocarcinoma was seen on imaging. We performed right hepatectomy and macroscopically, the resected specimen contained a growth in the bile duct lumen similar to cholangiocarcinoma. Histological examination revealed intraductal papillary proliferation of well-differentiated adenocarcinoma without vascular infiltration or lymph node metastasis in the hepatic hilum. Immunohistochemical staining revealed that the tumor cells were negative for cytokeratin-7 and positive for cytokeratin-20. Based on these findings, liver metastasis from colon carcinoma was diagnosed. Liver metastasis from colorectal carcinoma rarely arises as intraductal papillary growth in the bile duct, but the possibility of liver metastases with unusual morphology must be borne in mind for patients with a history of carcinoma in the digestive tract.