Nao Takano

Identification of intragenic methylation in the TUSC1 gene as a novel prognostic marker of hepatocellular carcinoma

Patients with hepatocellular carcinoma (HCC) have a poor prognosis, and novel molecular targets for treating recurrence and progression of the disease along with associated biomarkers are urgently required. In the present study, expression and the regulatory mechanism of TUSC1 (tumor suppressor candidate 1) were investigated to determine if it is a candidate tumor suppressor gene for HCC, which shows repressed transcription that involves aberrant DNA methylation. TUSC1 mRNA expression levels in HCC cell lines and 94 pairs of surgical specimens were determined using quantitative real-time reverse transcription polymerase chain reaction assay. Methylation status of HCC cell lines and clinical samples were analyzed to investigate the regulatory mechanism of TUSC1 transcription and the relationship between the methylation status of the TUSC1 gene and clinicopathological factors. The expression and distribution of the TUSC1 protein in liver tissues were determined using immunohistochemistry. A majority of HCC cell lines (89%) and surgical specimens (84%) demonstrated reduced expression levels of TUSC1 mRNA compared with paired non-cancerous liver tissues. The mean mRNA expression level in HCC was significantly lower than in corresponding non-cancerous liver. In contrast, no significant difference was found in TUSC1 mRNA expression level between adjacent normal and cirrhotic liver tissue from HCC patients. The TUSC1 protein expression pattern in HCC and liver tissues was consistent with TUSC1 mRNA expression. Twenty-nine (31%) of 94 patients showed intragenic hypermethylation of the TUSC1 gene in HCC, and hypermethylation was significantly associated with advanced pathological stage. Subsequently, patients with hypermethylation of the TUSC1 gene had a significantly poorer prognosis than patients without hypermethylation. Our results suggest that TUSC1 is a candidate tumor suppressor gene and intragenic hypermethylation is one of the suppressive mechanisms that regulate TUSC1 transcription in HCC. Intragenic methylation of the TUSC1 gene may serve as a novel prognostic marker of HCC.

Protein tyrosine kinase 7: a hepatocellular carcinoma-related gene detected by triple-combination array

BACKGROUND Hepatocellular carcinoma (HCC) is one of the top five causes of cancer-related deaths worldwide. We developed a novel technique to identify cancer-related genes of HCC as follows: triple-combination array analysis, which combines gene expression profiles, single nucleotide polymorphism arrays, and methylation arrays. MATERIALS AND METHODS Triple-combination array analysis was performed on one HCC sample from a 68-y-old female patient, and one candidate cancer-related gene was selected. Subsequently, we analyzed the identified gene by quantitative real-time reverse-transcriptase polymerase chain reaction (PCR) and methylation-specific PCR in nine HCC cell lines and in samples from 48 HCC patients. Additionally, we evaluated gene expression by immunohistochemistry and Western blotting. RESULTS Using this method, protein tyrosine kinase 7 (PTK7) was detected as a candidate cancer-related gene. PTK7 was revealed to be hypermethylated (methylation value 0.826, range 0-1.0) in cancer tissue, compared with that of adjacent noncancerous tissues (0.047) by methylation array. Of the 48 clinical samples, 30 HCC samples (62.5%) showed PTK7 promoter hypermethylation. Downregulation of PTK7 (expressions in tumor tissues decreased by ≥ 50% compared with the noncancerous tissues) was significantly associated with age >60 y (P = 0.030) and elevation in serum protein induced by vitamin K absence or antagonists-II (P = 0.033). Moreover, patients with downregulation were significantly inferior in overall survival (P < 0.001) than the others. CONCLUSIONS Our data imply that PTK7 acts as a cancer-related gene and may be a potent prognostic marker for HCC. Triple-combination array analysis was once again found to be useful in identifying cancer-related genes.

CCNJ detected by triple combination array analysis as a tumor-related gene of hepatocellular carcinoma

Hepatocellular carcinoma (HCC) has a high likelihood of recurrence and a poor prognosis. To detect cancer-related genes of HCC, we developed a new technique: triple combination array analysis, consisting of a methylation array, a gene expression array and a single nucleotide polymorphism array. A surgical specimen obtained from a 68-year-old female HCC patient was analyzed using triple combination array, which identified cyclin J (CCNJ) as a candidate cancer-related gene of HCC. Subsequently, samples from 85 HCC patients were evaluated for CCNJ promoter hypermethylation and expression status using methylation-specific PCR (MSP) and quantitative reverse transcriptase RT-PCR, respectively. CCNJ was found to be hypermethylated (methylation value, 0.906; range, 0-1.0) in cancer tissue, compared with adjacent non-cancerous tissue (0.112) using a methylation array. MSP revealed that CCNJ was hypermethylated in 67 (78.8%) of the tumor samples. CCNJ expression was significantly decreased in cases with hypermethylation (P<0.0001). Furthermore, cases with both promoter hypermethylation and decreased expression of CCNJ in the tumor tissue had a worse overall survival than the other cases (P=0.0383). In conclusion, our results indicated that CCNJ could be a novel prognostic marker of HCC, and this study indicated that triple combination array analysis was effective in detecting new tumor-related genes and their mechanisms.

PAX5 gene as a novel methylation marker that predicts both clinical outcome and cisplatin sensitivity in esophageal squamous cell carcinoma

ABSTRACT Therapeutic strategies for esophageal cancer largely depend on histopathological assessment. To select appropriate treatments of individual patients, we examined the background molecular characteristics of tumor malignancy and sensitivity to multidisciplinary therapy. Seventy-eight surgically-resected esophageal squamous cell carcinoma (ESCC) cases during 2001–2013 were examined. PAX5, a novel gene methylation marker in ESCC, was evaluated in the specimens, as methylation of this gene was identified as an extremely tumor-specific event in squamous cell carcinogenesis of head and neck. PAX5 methylation status was evaluated by quantitative MSP (QMSP) assays. Mean QMSP value was 15.7 (0–136.3) in ESCCs and 0.3 (0–8.6) in adjacent normal tissues (P < 0.001). The 78 cases were divided into high QMSP value (high QMSP, n = 26) and low QMSP value (low QMSP, n = 52). High QMSP cases were significantly associated with downregulated PAX5 expression (P = 0.040), and showed significantly poor recurrence-free survival [Hazard Ratio (HR) = 2.84; P = 0.005; 95% Confidence Interval (CI): 1.39–5.81] and overall survival (HR = 3.23; P = 0.002; 95%CI: 1.52–7.01) in multivariable analyses with histopathological factors. PAX5-knockdown cells exhibited significantly increased cell proliferation and cisplatin resistance. PAX5 gene methylation can predict poor survival outcomes and cisplatin sensitivity in ESCCs and could be a useful diagnostic tool for cancer therapy selection.

High expression of Janus kinase 2 in background normal liver tissue of resected hepatocellular carcinoma is associated with worse prognosis

When assessing hepatocellular carcinoma (HCC), it is important to examine prognostic factors in the background normal liver tissue and consider malignant aspects of the primary lesion. Candidate genes were extracted from the background normal liver samples via multiarray analysis. Control samples, termed supernormal (SN) liver, were obtained from 11 cases of metastatic liver cancer. Corresponding normal (CN) liver tissue was surgically obtained from a typical HCC patient with chronic hepatitis C background for comparison. Expression profile and methylation array demonstrated that Janus kinase 2 (JAK2) gene expression was increased by 2.378‑fold in the CN tissue. Methylation array reported a lower value for CN (0.125) than SN tissues (0.748). We then investigated JAK2 expression by real-time quantitative reverse transcription-polymerase chain reaction in 100 consecutive resected HCC cases. The average expression level of JAK2 (normalized to GAPDH) was significantly lower in CN (9.24±6.43, n=100) than in SN (35.21 ± 21.38, n=11) tissues (P<0.001). As such a result was contrary to our expectation, the case used for array analysis seemed to be a rare incidence. One hundred HCC cases were subsequently divided into two groups based on JAK2 expression in the adjacent normal tissue: one consisting of the upper 70% of cases (n=70) and the other of the remaining 30% (n=30). Higher JAK2 expression in the adjacent tissue demonstrated significant correlation with worse survival (P=0.022). Furthermore, multivariate analysis identified higher JAK2 expression in the background normal liver tissue of HCC as an independent prognostic factor (P=0.032). Our findings suggest that higher JAK2 expression in the background normal liver tissue of HCC may be a good prognostic biomarker for resected HCC.

Abstract 3816: Correlation between worse prognosis and higher expression of the JAK2 gene in corresponding non-neoplastic tissue in patients with hepatocellular carcinoma, extracted by multiarray analysis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA [Background & Aim] Hepatocellular carcinoma (HCC) often recurs in the liver after surgery removing the primary lesion. Multi-centric occurrence (MO) is more common than intrahepatic metastases (IM). Then, it is insufficient to predict the prognosis by considering factors only in resected primary tumor. Factors in the background liver will also play important roles in prognosis. [Material & Methods] Our control samples, termed super normal (SN) liver, were taken from normal tissues of 11 cases of metastatic liver cancer. We selected a corresponding non-neoplastic (CN) tissue of a typical HCC case with chronic hepatitis C as a sample to compare. Then, expression profiling and methylation arrays were performed for comparing the SN and the CN. We identified genes showing differences in gene expression and the rate of methylation. Prognosis was predicted for 100 cases of HCC based on gene expression. [Results] The expression profiling showed that expression of Janus kinase 2 (JAK2) gene was increased 2.378 in CN, and methylation array analysis showed a lower value for CN (0.13) than SN (0.75). We then studied JAK2 gene expression by quantitative real time RT-PCR. Unfortunately, the average expression value of JAK2 (JAK2 x103/GAPDH) had decreased in matching normal tissue (9.24±7.46) relative to SN (35.21±21.38). Only one case was expressed higher than SN average in CN. We divided the cases into two groups. Group A showed higher 70% of JAK2 expression cases (70 cases) and group B showed lower 30% of expression cases (30 cases). Then, the group A had a significant correlations with worth prognosis (p=0.017) by univariate analysis and showed independent worth prognostic factor (p = 0.032) by multivariate analysis. [Discussion & Conclusion] It has been reported that JAK2 has an important role in reproduction of normal hepatocyte, and maintenance of liver functions in the super normal liver. While, in HCC tissues, STAT3-JAK2 pathway was reported as an oncogenic factor in proliferation and progression. In this study, the expression of the JAK2 gene in the background liver in HCC cases also had an important meaning and likely to be a good biomarker. When assessing cases of HCC, as well as considering malignant factor of the primary cancer lesion, it is important to extract prognostic factors from background liver tissue. Citation Format: Shuji Nomoto, Mitsuhiro Hishida, Yoshikuni Inokawa, Nao Takano, Mitsuro Kanda, Yasuhiro Kodera. Correlation between worse prognosis and higher expression of the JAK2 gene in corresponding non-neoplastic tissue in patients with hepatocellular carcinoma, extracted by multiarray analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3816. doi:10.1158/1538-7445.AM2014-3816

Abstract 2870: ZGPAT gene expression in non-tumor hepatocellular carcinoma tissue is a likely biomarker for survival risk

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA [Background & Aim] Hepatocellular carcinoma (HCC) often recurs after surgery and multicentric occurrence is more common than intrahepatic metastasis. We therefore examined factors in non-tumor liver tissue in HCC. [Material & Methods] Control samples, termed super normal liver tissues (SN), were taken from 11 cases of metastatic liver cancer. One of corresponding samples of normal tissues (CN) was selected for comparison from a typical specimen with chronic hepatitis C and HCC. We performed expression profiling and methylation arrays on these samples, identified genes that differed between SN and CN. We analyzed the identified genes using quantitative real-time reverse-transcriptase polymerase chain reaction on SN from 11 cases of metastatic liver cancer and CN from 109 cases of HCC. [Results] We found the zinc finger, CCCH-type with G patch domain (ZGPAT) gene to have expression decreased 4.198-fold in CN, and higher methylation status in CN (0.718), than in SN (0.360). The average expression value of the ZGPAT gene (ZGPAT value ×103 / GAPDH) decreased in CN (9.94 ± 10.68) relative to SN (28.55 ± 22.05). Compared with other patients, those with strongly reduced ZGPAT expression (< 1.5) in their non-tumor liver samples had significantly shorter overall survival (P = 0.024), but not recurrence-free survival (P = 0.515). [Discussion & Conclusion] Our results indicate that expression of the ZGPAT gene in non-tumor liver tissue is a good biomarker for the risk of HCC development. ZGPAT gene is reportedly a transcription repressor that negatively regulates expression of epidermal growth factor receptor, which in turn, mediates cell proliferation, survival, migration. In treating HCC, detection of both prognostic factors from non-tumor liver tissue and malignant factors of the primary cancer lesion is important. Citation Format: Mitsuhiro Hishida, Shuji Nomoto, Yoshikuni Inokawa, Nao Takano, Mitsuro Kanda, Naoki Iwata, Chie Tanaka, Daisuke Kobayashi, Yoko Nishikawa, Suguru Yamada, Goro Nakayama, Tsutomu Fujii, Hiroyuki Sugimoto, Masahiko Koike, Michitaka Fujiwara, Yasuhiro Kodera. ZGPAT gene expression in non-tumor hepatocellular carcinoma tissue is a likely biomarker for survival risk. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2870. doi:10.1158/1538-7445.AM2014-2870

Abstract 4717: Detection of the Cyclin J (CCNJ) as a new cancer-related gene in human hepatocellular carcinoma by using a method of triple combination array analysis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background: Hepatocellular carcinoma (HCC) has a high rate of recurrence and a poor prognosis. To detect genes correlated with HCC, we have developed a new technique, triple combination array analysis, consisting of methylation array, gene expression array and single nucleotide polymorphism (SNP) array analysis. Methods: A surgical specimen obtained from a 68-year-old female HCC patient was analyzed using triple combination array analysis, which identified a candidate cancer-related gene of HCC. Subsequently, samples from nine cell lines and 48 HCC patients were evaluated for their methylation and expression status of the identified gene using methylation-specific polymerase chain reaction (MSP) and quantitative real-time reverse transcriptase PCR, respectively. Results: Using the triple combination array analysis, cyclin J (CCNJ) was detected as a candidate cancer-related gene. CCNJ was located on chromosome 10q24.1 and the copy number, using SNP chip array, revealed no loss of heterozygosity. According to expression array results, The expression of CCNJ in tumor tissue decreased at two points of the expression array chip, and the decreased values of the chip were −1.3 and −2.3. CCNJ was found to be hypermethylated (methylation value 0.906, range 0-1.0) in cancer tissue compared with adjacent normal tissue (0.112) using the methylation array. Using MSP, hypermethylation of the promoter region of CCNJ was shown to occur in 37 (77.1%) tumor samples. CCNJ expression was significantly decreased in cases with methylation (p<0.0001). Furthermore, HCC patients with methylated CCNJ had a significantly worse prognosis for recurrence-free survival (p=0.0353) and tended to have a worse prognosis for overall survival than those with unmethylated CCNJ (p=0.0838). Conclusion: The present study indicates that triple combination array analysis is an effective method to detect novel genes related to HCC. We suggest that CCNJ acts as a cancer-related gene in HCC. Citation Format: Nao Takano, Shuji Nomoto, Mitsuhiro Hishida, Yoshikuni Inokawa, Masamichi Hayashi, Mitsuro Kanda, Yukiyasu Okamura, Yoko Nishikawa, Chie Tanaka, Daisuke Kobayashi, Suguru Yamada, Goro Nakayama, Tsutomu Fujii, Hiroyuki Sugimoto, Masahiko Koike, Michitaka Fujii, Shin Takeda, Yasuhiro Kodera. Detection of the Cyclin J (CCNJ) as a new cancer-related gene in human hepatocellular carcinoma by using a method of triple combination array analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 4717. doi:10.1158/1538-7445.AM2014-4717

Abstract 3822: Correlation between worse prognosis and lower expression of the TPPP gene in patients with hepatocellular carcinoma, detected by multiarray analysis

Proceedings: AACR Annual Meeting 2014; April 5-9, 2014; San Diego, CA Background. Hepatocellular carcinoma (HCC) patients who undergo liver resection often suffer multicentric occurrence after surgery. Therefore, understanding carcinogenesis in background liver is important. Methods. Control liver samples (super normal [SN]), were taken from 11 cases of metastatic liver cancer. We selected corresponding normal tissue from typical cases of HCC with chronic hepatitis C (corresponding normal [CN]) for comparison. Expression profiling and methylation arrays were performed to compare SN and CN. Array data showed that the tubulin polymerization promoting protein (TPPP) gene showed differences in both array analyses. The relationship between clinicopathological factors of 179 HCC patients and TPPP expression was investigated. Results. The expression array showed that the TPPP gene was decreased by 2.236-fold (log2 ratio) in CN compared with SN. The methylation array showed a higher value of methylation for CN (0.730) than SN (0.384). We studied TPPP gene expression by real-time reverse transcription PCR. When compared expression level of TPPP (TPPP value ×103/GAPDH), among SN (n=11), normal liver tissue of HCC patients (n=179), and tumor tissue of HCC (n=179), there were significant differences (median: 116, 4.60, and 2.63, respectively). The group with lower TPPP expression in tumors compared with normal tissue showed a worse prognosis of overall survival than that in the increased expression group (P=0.0317). Strongly reduced TPPP expression in tumor tissue compared with normal tissue (ratio <0.3, n=57) was detected as an independent prognostic factor by multivariate analysis (P=0.0310). Conclusions. Expression of the TPPP gene in tumor and non-cancerous liver tissue in HCC patients is significantly lower compared with SN. HCC patients with this low TPPP expression show a poor prognosis. This indicates that this gene is a new candidate of tumor suppressor genes in HCC. Low TPPP expression in liver tissue might be a predictor of HCC. Citation Format: Yoshikuni Inokawa, Shuji Nomoto, Mitsuhiro Hishida, Nao Takano, Mitsuro Kanda, Michitaka Fujiwara, Masahiko Koike, Hiroyuki Sugimoto, Tsutomu Fujii, Goro Nakayama, Suguru Yamada, Chie Tanaka, Daisuke Kobayashi, Naoki Iwata, Yasuhiro Kodera. Correlation between worse prognosis and lower expression of the TPPP gene in patients with hepatocellular carcinoma, detected by multiarray analysis. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3822. doi:10.1158/1538-7445.AM2014-3822