Keisuke Miyake

LINE-1 hypomethylation is associated with a poor prognosis among patients with curatively resected esophageal squamous cell carcinoma.

Objective: To investigate the relationship between the long interspersed nucleotide element-1 (L1/LINE-1) methylation level and the disease-free survival and cancer-specific survival in patients with esophageal squamous cell carcinoma (ESCC). Background: Cancer cells exhibit 2 types of deoxyribonucleic acid (DNA) methylation alterations: global DNA hypomethylation and site-specific CpG island promoter hypermethylation. Global DNA hypomethylation plays a role in genomic instability and carcinogenesis. DNA methylation in the LINE-1 repetitive element is a good indicator of the global DNA methylation level. Although the LINE-1 methylation level is attracting interest as a useful marker for predicting cancer prognosis, the prognostic significance of LINE-1 hypomethylaiton in ESCC remains unclear. Methods: Using 217 curatively resected ESCC specimens, we quantified the LINE-1 methylation by utilizing the bisulfite pyrosequencing technology. Promoter methylation levels of MGMT and MLH1 were also evaluated by pyrosequencing. Results: ESCC showed significantly lower LINE-1 methylation levels in comparison with matched normal esophageal mucosa (P < 0.0001; N = 50). LINE-1 hypomethylation was significantly associated with disease-free survival [log-rank P = 0.0008; univariate hazard ratio (HR): 2.32, 95% confidence interval (CI): 1.38–3.84, P = 0.0017; multivariate HR: 1.81, 95% CI: 1.06–3.05, P = 0.031] and cancer-specific survival (log-rank P = 0.0020; univariate HR: 2.21, 95% CI: 1.33–3.60, P = 0.0026; multivariate HR: 1.87, 95% CI: 1.12–3.08, P = 0.018]. MGMT and MLH1 hypermethylation were not associated with patient prognosis. Conclusions: LINE-1 hypomethylation in ESCC is associated with a shorter survival, thus suggesting that it has potential for use as a prognostic biomarker.

Identification of CXCL5/ENA‐78 as a factor involved in the interaction between cholangiocarcinoma cells and cancer‐associated fibroblasts

Knowledge of tumor‐stromal interactions is essential for understanding tumor development. We focused on the interaction between cholangiocarcinoma and cancer‐associated fibroblasts (CAFs) in intrahepatic cholangiocarcinoma and reported their positive interaction in vitro and in vivo. The aim of this study is to identify the key protein involved in the interaction between cholangiocarcinoma cells and CAFs and its role on cholangiocarcinoma progression. Using the conditioning medium from cholangiocarcinoma cells, hepatic stellate cells and coculture of them, Protein‐Chip analysis with SELDI–TOF–MS showed that the peak of an 8,360‐Da protein remarkably increased in the coculture medium. This protein was identified as CXCL5/ENA78, epithelial cell‐derived neutrophil‐activating peptide‐78, by q‐TOF/MS/MS analysis. Two cholangiocarcinoma cell lines, HuCCT1 and RBE, produced CXCL5 that promoted their invasion and migration in an autocrine fashion. These effects of CXCL5 significantly decreased by inhibition of CXC‐receptor 2, which is the receptor for CXCL5. In addition, IL‐1β produced by hepatic stellate cells induced the expression of CXCL5 in cholangiocarcinoma cells. In human tissue samples, a significant correlation was observed between CAFs and CXCL5 produced by cholangiocarcinoma cells in intrahepatic cholangiocarcinoma (p = 0.0044). Furthermore, the high‐CXCL5‐expression group exhibited poor overall survival after curative hepatic resection (p = 0.027). The presence of tumor‐infiltrating neutrophils expressing CD66b was associated with CXCL5 expression in tumor cells (p < 0.0001). These data suggest that CXCL5 is important for the interaction between cholangiocarcinoma and CAFs, and inhibition of tumor‐stromal interactions may be a useful therapeutic approach for cholangiocarcinoma.

Macrophage-derived reactive oxygen species suppress miR-328 targeting CD44 in cancer cells and promote redox adaptation

CD44 is frequently overexpressed in a wide variety of epithelial malignancies including gastrointestinal cancer and causes resistance to currently available treatments. MicroRNAs (miRNAs) are non-coding RNAs that regulate molecular pathways in cancer by targeting various genes. The aim of this study was to investigate the regulation of CD44 expression by miRNAs and to develop new molecular targets in gastrointestinal cancer. We performed miRNA screening in six human gastrointestinal cancer cell lines and identified three candidate miRNAs that could regulate CD44 expression in gastrointestinal cancer. Among these, we focused on miR-328 and examined its functional relevance using growth assays and cytotoxicity assays. CD44 expression was reduced in gastrointestinal cancer cell lines forced to express miR-328, leading to inhibition of cancer cell growth in vitro and in vivo, and impaired resistance to chemotherapeutic drugs and reactive oxygen species (ROS). In contrast, induction of CD44 expression by miR-328 inhibitor led to promotion of cancer cell growth. Furthermore, we revealed that ROS produced by macrophages triggered CD44 expression through suppression of miR-328 in gastric cancer cells. Finally, tumor-infiltrating macrophages (CD68 and CD163) were closely related to both miR-328 downregulation and CD44 upregulation in 63 patients with surgically resected gastric cancer. These findings suggest that macrophages in the tumor microenvironment may cause increased CD44 expression through miR-328 suppression, resulting in tumor progression by enhancing ROS defense. miR-328-CD44 signaling mediated by macrophages may thus represent a potential target for the treatment of gastrointestinal cancer.

Methylation levels of LINE-1 in primary lesion and matched metastatic lesions of colorectal cancer

Background:LINE-1 methylation level is a surrogate marker of global DNA methylation. LINE-1 methylation in primary colorectal cancers (CRCs) is highly variable and strongly associated with a poor prognosis. However, no study has examined LINE-1 methylation levels of metastatic CRCs in relation to prognosis or assessed the heterogeneity of LINE-1 methylation level within the primary CRCs.Methods:Pyrosequencing was used to quantify LINE-1 methylation level in 42 liver metastases, 26 matched primary tumours, and 6 matched lymph node (LN) metastases. KRAS, BRAF, and PIK3CA mutation status and microsatellite instability (MSI) status were also examined.Results:The distribution of LINE-1 methylation level in liver metastases was as follows: mean, 67.3; range, 37.1–90.1. Primary tumours showed LINE-1 methylation levels similar to those of matched liver and LN metastases. The difference in LINE-1 methylation level between superficial areas and invasive front areas was within 7.0 in all six cases evaluated. Prognostic impact of LINE-1 hypomethylation in liver metastases on overall survival was not observed. The concordance rate was 94% for KRAS, 100% for BRAF, 88% for PIK3CA, and 97% for MSI.Conclusion:Alteration of LINE-1 methylation level may occur in early CRC tumorigenesis, and the LINE-1 methylation level is relatively stable during CRC progression.

CXCL12/CXCR4 activation by cancer‐associated fibroblasts promotes integrin β1 clustering and invasiveness in gastric cancer

Cancer‐associated fibroblasts (CAFs) are reportedly involved in invasion and metastasis in several types of cancer, including gastric cancer (GC), through the stimulation of CXCL12/CXCR4 signaling. However, the mechanisms underlying these tumor‐promoting effects are not well understood, which limits the potential to develop therapeutic targets against CAF‐mediated CXCL12/CXCR4 signaling. CXCL12 expression was analyzed in resected GC tissues from 110 patients by immunohistochemistry (IHC). We established primary cultures of normal fibroblasts (NFs) and CAFs from the GC tissues and examined the functional differences between these primary fibroblasts using co‐culture assays with GC cell lines. We evaluated the efficacy of a CXCR4 antagonist (AMD3100) and a FAK inhibitor (PF‐573,228) on the invasive ability of GC cells. High CXCL12 expression levels were significantly associated with larger tumor size, increased tumor depth, lymphatic invasion and poor prognosis in GC. CXCL12/CXCR4 activation by CAFs mediated integrin β1 clustering at the cell surface and promoted the invasive ability of GC cells. Notably, AMD3100 was more efficient than PF‐573,228 at inhibiting GC cell invasion through the suppression of integrin β1/FAK signaling. These results suggest that CXCL12 derived from CAFs promotes GC cell invasion by enhancing the clustering of integrin β1 in GC cells, resulting in GC progression. Taken together, the inhibition of CXCL12/CXCR4 signaling in GC cells may be a promising therapeutic strategy against GC cell invasion.

LINE-1 Hypomethylation, DNA Copy Number Alterations, and CDK6 Amplification in Esophageal Squamous Cell Carcinoma

Purpose: Global DNA hypomethylation plays a crucial role in genomic instability and carcinogenesis. DNA methylation of the long interspersed nucleotide element-1, L1 (LINE-1) repetitive element is a good indicator of the global DNA methylation level, and is attracting interest as a useful marker for predicting cancer prognosis. Our previous study using more than 200 esophageal squamous cell carcinoma (ESCC) specimens demonstrated the significant relationship between LINE-1 hypomethylation and poor prognosis. However, the mechanism by which LINE-1 hypomethylation affects aggressive tumor behavior has yet to be revealed. Experimental Design: To examine the relationship between LINE-1 hypomethylation and DNA copy number variations, we investigated LINE-1–hypomethylated and LINE-1–hypermethylated ESCC tumors by comparative genomic hybridization array. Results: LINE-1–hypomethylated tumors showed highly frequent genomic gains at various loci containing candidate oncogenes such as CDK6. LINE-1 methylation levels were significantly associated with CDK6 mRNA and CDK6 protein expression levels in ESCC specimens. In our cohort of 129 patients with ESCC, cases with CDK6-positive expression experienced worse clinical outcome compared with those with CDK6-negative expression, supporting the oncogenic role of CDK6 in ESCC. In addition, we found that the prognostic impact of LINE-1 hypomethylation might be attenuated by CDK6 expression. Conclusion: LINE-1 hypomethylation (i.e., global DNA hypomethylation) in ESCC might contribute to the acquisition of aggressive tumor behavior through genomic gains of oncogenes such as CDK6. Clin Cancer Res; 20(5); 1114–24. ©2014 AACR.

Human Microbiome Fusobacterium Nucleatum in Esophageal Cancer Tissue Is Associated with Prognosis.

Purpose: Fusobacterium nucleatum (F. nucleatum) is a component of the human microbiome that primarily inhabits the oral cavity. It causes periodontal disease and has also been implicated in the development of human cancers. Although there are several reports of the relationship between F. nucleatum and the clinical outcome in human cancers, its prognostic significance in esophageal cancer remains unclear. Experimental Design: We quantified F. nucleatum DNA in 325 resected esophageal cancer specimens by qPCR. Significant pathways in F. nucleatum–positive esophageal cancer tissues were identified by Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis using microarray data. Results: Esophageal cancer tissues contained significantly more F. nucleatum DNA than matched normal esophageal mucosa (P = 0.021; n = 60). F. nucleatum DNA was detected in 74 of 325 cases (23%). F. nucleatum DNA positivity was significantly associated with tumor stage, but not with sex, age, performance status, tobacco use, alcohol use, histology, tumor location, or preoperative treatment. F. nucleatum DNA positivity was also significantly associated with cancer-specific survival [log-rank P = 0.0039; univariate HR = 2.01; 95% confidence interval (CI), 1.22–3.23; P = 0.0068; multivariate HR = 1.78; 95% CI, 1.06–2.94; P = 0.031]. The top-ranked KEGG pathway in F. nucleatum–positive tissues was “cytokine–cytokine receptor interaction.” A significant relationship between F. nucleatum and the chemokine CCL20 was validated by IHC. Conclusions: F. nucleatum in esophageal cancer tissues was associated with shorter survival, suggesting a potential role as a prognostic biomarker. F. nucleatum might also contribute to aggressive tumor behavior through activation of chemokines, such as CCL20. Clin Cancer Res; 22(22); 5574–81. ©2016 AACR.

Cystine/glutamic acid transporter is a novel marker for predicting poor survival in patients with hepatocellular carcinoma

Cystine/glutamic acid transporter (xCT) plays a role in tumor progression by regulating the redox status in several types of cancers. To demonstrate the importance of xCT expression for predicting the prognosis of hepatocellular carcinoma (HCC), we analyzed xCT gene expression in 130 paired HCC and non-cancerous tissues. xCT protein expression was confirmed using 7 HCC cell lines and samples from human subjects. xCT mRNA expression was detected in 34 (26%) tumor tissues. Expression of xCT was higher in HCC tissues compared to the corresponding normal tissues according to quantitative reverse transcriptase-polymerase chain reaction findings (P<0.0001). Patients in the group presenting with xCT mRNA expression showed poorer overall and disease-free survival than did those with an absence of xCT mRNA (P=0.0130 and 0.0416, respectively). xCT mRNA expression proved to be an independent factor for poor prognosis in a multivariate analysis of overall survival (hazard ratio, 1.68; 95% CI, 1.03-2.92). We observed xCT protein expression in both the HCC cell lines and in human tissue samples. In conclusion, the findings of the present study suggest that xCT is useful as a predictive marker for patient prognosis and that it may be a novel therapeutic target for HCC.

Primary neuroendocrine tumor in the liver treated by hepatectomy: report of a case.

A 49-year-old woman was admitted to our hospital under suspicion of an enlarging hepatic tumor, which had been previously diagnosed to be a cavernous hemangioma. Computed tomography revealed three enhanced tumors, one measuring 15 cm in diameter within the right lobe of the liver and two intrahepatic metastases in Couinaud’s hepatic segments 3 and 5. We diagnosed the patient to have primary liver cancer, and suspected a combined liver tumor preoperatively. We performed a right trisectionectomy with radiofrequency ablation of the intrahepatic metastasis in S3. According to the immunohistochemical findings of the resected specimen and the findings of postoperative imaging studies, the tumor was diagnosed to be a primary neuroendocrine tumor in the liver. The patient is presently alive without recurrence at 33 months after the operation.

Noncoding RNA expression aberration is associated with cancer progression and is a potential biomarker in esophageal squamous cell carcinoma

Esophageal cancer is one of the most common cancers worldwide. Esophageal squamous cell carcinoma (ESCC) is the major histological type of esophageal cancer in Eastern Asian countries. Several types of noncoding RNAs (ncRNAs) function as key epigenetic regulators of gene expression and are implicated in various physiological processes. Unambiguous evidence indicates that dysregulation of ncRNAs is deeply implicated in carcinogenesis, cancer progression and metastases of various cancers, including ESCC. The current review summarizes recent findings on the ncRNA-mediated mechanisms underlying the characteristic behaviors of ESCC that will help support the development of biomarkers and the design of novel therapeutic strategies.