Research Summary

Major Research Accomplishments

Through molecular pathological analyses of gastrointestinal and urological cancers, we have clarified diversity of genetic and molecular abnormalities in carcinogenic process, superiority of the SAGE and CAST methods to identify novel diagnostic and therapeutic target, importance of microRNA control and cancer stem cells, and participation of genetic polymorphism in cancer development and progression.

Molecular pathogenesis of gastrointestinal cancer

Cancer develops as a result of accumulation of various endogenous and exogenous causes. Dietary factors and Helicobacter pylori (H. pylori) infection are important exogenous causes for gastric cancer, while many genetic polymorphisms are found to be associated with predisposition to cancer development. Multiple genetic and epigenetic alterations occur in the course of carcinogenesis and progression of gastric cancer. Some of the genetic and epigenetic changes differ depending on the histological type or mucin phenotype. DNA methylation, histone modification and microRNAs play crucial roles in cellular process through regulating gene expression and participate deeply in epigenetic alterations in gastrointestinal cancer.

Transcriptome dissection of gastrointestinal and prostatic cancers by SAGE and CAST method

In the comprehensive gene expression analysis of esophageal and gastric cancers by SAGE (serial analysis of gene expression) method, we have identified many candidate genes for novel biomarkers and therapeutic targets. We have created one of the largest gastric cancer SAGE libraries in the world and the sequence data are publicly available at SAGEmap (GEO accession number GSE 545, SAGE Hiroshima gastric cancer tissue).
By comparing SAGE libraries of gastric cancer with those of 14 normal tissues including brain, lung, heart, liver, kidney, etc. in the SAGEmap database in combination with quantitative RT-PCR, nine genes (APIN, TRAG3, CYP2W1, MIA, MMP-10, DKK4, OLFM4, Reg IV, and HORMAD1) were found to have gastric cancer-specific expression. CLDN18 (encoding claudin-18) was identified to be down-regulated in more than a half of gastric cancers. In esophageal cancer, NRD1 and ADAMTS16 were highly expressed in comparison with the normal mucosa.
The gene expression profiles by CAST method followed by qRT-PCR using tissue samples revealed that gastric cancer overexpressed DSC2, TSPAN8, TM9SF3, and ZDHHC14, while prostate cancer overexpressed PRL1, STEAP1, ADAM9, CDON, NBL1 and TMEM50B.

Significance of newly-identified genes as diagnostic and therapeutic targets

Expression study in clinical samples and functional analysis using cancer cell lines clarified significance of the newly-identified genes as diagnostic and therapeutic targets.
REGIV increased cell proliferation through EGFR activation, inhibited apoptosis, promoted peritoneal metastasis in mouse model, and participated in 5-FU-resistance. Both REGIV and OLFM4 are a secreted protein and their serum levels can be measured by ELISA. In patients even with stage I gastric cancer, the sensitivity of serum OLFM4 (25%) and REGIV (35%) was superior to that of CA19-9 (5%) or CEA (3%). Furthermore, in patients with stage I GC, the combination of OLFM4 and REGIV elevated the diagnostic sensitivity to 52% (Patent no. 5467256). On the other hand, in the esophageal squamous cell carcinoma, ADAMTS16 was found to be promising serum tumor marker and novel therapeutic target (Patient no. 5055543) . NRD1 also showed a similar trend.
Among the genes identified in gastric cancer analysis by CAST method, TM9SF3 and TSPAN8 are involved in the proliferation and invasion of cancer cells and correlates with a poor prognosis. In prostate cancer, CDON is involved in cancer invasion and 5-FU-resistance. The expression of NBL1 is restricted in prostate and the expression decreases with the progression of cancer.

Genetic and molecular abnormalities and histo-pathology

Gastric cancers are histologically classified into gdifferentiatedh and gundifferentiatedh types, or gintestinalh and gdiffuseh types based on glandular structure. In addition, gastric cancers are phenotypically classified into G (gastric) type and I (intestinal) type on the basis of the mucin expression. Increased expression of OLFM4, TSPAN8, miR-486 is associated with gastric type, while overexpression of REGIV, CDX2, DSC2, MDR1, HOXA10 and down-regulation of CLDN18 are characteristics in intestinal type. PLUNC is a novel and useful marker for differentiating gastric hepatoid adenocarcinoma from hepatocellular carcinoma.
In colorectal cancers, CLDN18 expression correlates with significantly poor survival in colorectal cancer patients and is associated with the gastric phenotype with MUC5AC-positive and CDX2-negative, although it occupied only 4% of colorectal cancers.

microRNA expression in cancers of the stomach and kidney

From the analysis microRNA expression profiles of gastric cancers by microRNA microarray, miR-125b, miR-199a, and miR-100 were the most important microRNAs involved in progression. Low expression of let-7g and miR-433 and high expression of miR-214 are associated with unfavourable outcome of the patients. miR-148a contributes to the maintenance of homeostasis in normal stomach tissue and inactivation of miR-148a by methylation plays an important role in gastric cancer invasion by regulating MMP7 expression. miR-143 expression is higher in scirrhous type gastric cancers than in non-scirrhous type, while miR-143 is expressed by stromal fibroblasts but not by cancer cells. miR-143 enhanced collagen type III expression in cancer-associated fibroblasts through activation of TGF-ƒÀ/SMAD signaling. miR-143 is an independent prognostic factor.
In renal cell carcinomas, levels of miR-155 and miR-486 are increased in clear cell carcinoma compared with normal kidney tissue. Low expression of miR-155 and increased expression of miR-486 represent valuable markers of poor clinical outcomes in patients with stage III and IV renal cell carcinoma.

Role of cancer stem cells in gastric cancer

Cancer stem cells (CSCs) in tumors possess the ability to sustain tumor self-renewal, initiate tumor progression, and also contribute to cancer metastasis and chemo-radiation-resistance. CD44 expression and CD133 expression are independent predictors of survival in patients with gastric cancer. ALDH1 positivity is significantly higher in lymph node metastasis than in the primary tumor of diffuse-type gastric cancer. Katsuno and Miyazono, et al. (University of Tokyo) reported that targeting REGIV in ALDH1+ CICs may provide a novel strategy in the treatment of diffuse-type gastric carcinoma (J Pathol: 2012). From the comprehensive analysis of gene expression in side population cells of gastric cancer and in ROS-decreased cells, we have identified candidates of new cancer stem cell markers.

Genetic polymorphisms and gastric cancer risk

Genetic polymorphisms are deeply involved in development and progression of gastric cancer. Our case-control studies as well as case-case-studies demonstrated that single nucleotide polymorphisms of HER2, HIF-1ƒ¿, EGF, of MMP-1, MMP-9, CDH1, etc. are significantly associated with gastric cancer risk or its progression. In collaboration with the National Cancer Research Center Research Institute, we have reported an association between genetic polymorphisms of PSCA and MUC1 and the risk of developing gastric poorly differentiated adenocarcinoma.