Research Project

At the Department of Molecular Pathology we conduct studies of molecular pathology on development and progression of cancer. Our primary study subject is gastrointestinal cancer and urinary system tumors.

The following is the summary of the on-going studies.

1) Identification of Cancer Specific Gene by Transcriptome Dissection and Its Implication for Diagnosis & Treatment

Cancer accounts for approximately 30% of all causes of death. The possibility to have cancer is one out of two for men and one out of three for women. Controlling cancer is an extremely important task. The abnormality such as growth factor receptor, cell cycle regulator, cell adhesion molecule, seen in cancer cells determines the biochemical character of cancer cell growth, invasion, as well as metastasis based on the importance of the epigenetic mechanism, being called "Disease of Abnormal Gene Expression". In the genomic medicine era, "Omics" data, such as enormous genome, transcriptome, proteome, as well as metabolic loam, are accumulated. Society demands the aggregated information to be utilized using new techniques and bioinformatics, applied for diagnosis in clinic and prevention.

What we call transcriptome dissection, is to capture various outbreak and progress processes, in gene manifestation change systematically and understand the molecules base of the diseases in greater details. With transcriptome dissection, we are attempting to develop diagnosis, treatment, as well as prevention by targeting new molecules and genes. SAGE (Serial Analysis of Gene Expression) method is a representative cyclopedic gene analytical method, which is lined with DNA microarray. SAGE is superior in quantifiably and reproducibility and can analyze unknown genes. The SAGE method performs a sequence of approximately ten downstream bases (tag) of CATG and analyzes appearance frequency and type of tag resulting in identification of a coding gene. The exact manifestation can be found by counting the appearance frequency. The direct comparison with the SAGE library of approximately 300 types of cell/tissue in SAGEmap of NCBI is possible as well. We conducted transcriptome analysis of gastric cancer and esophageal cancer utilizing this method and completed the SAGE library of the world's one of the largest scales in particular to gastric cancer (GEO Accession no.GSE545: SAGE Hiroshima Gastric Cancer Tissue). We found numbers of genes frequently overexpressed in gastric cancer by comparing those with the SAGE libraries of various normal organs which are indispensable for survival. Thereby, having identified numbers of options of new diagnosis and treatment targets. In regards to these options, we are performing a biological functional analysis, using forced expression system or RNA interference, in addition to expression and secretion in the clinical specimen. By applying these methods, we have found that RegIV and OLFM4 are highly sensitive serodiagnosis markers of gastric cancer, and ADAMTS16 is a new diagnostic and therapeutic target for esophagus squamous cell carcinoma.

Besides SAGE method, to identify efficiently cell surface protein or secreted protein cyclopedically, the CAST (Escherichia coli ampicillin secretion trap) method has been developed. After inserting the cDNA sample targeted for analysis in the vector, (pCAST vector) which incorporated an ampicillin-resistant gene, (-lactamase gene) in which signal sequence is deleted. The principle is that pCAST vector is introduced into the Escherichia coli and an ampicillin resistant cloned are collected. This means that in the resistant clone, a transmembrane domain or signal sequence exists in the genetic sequence in the pCAST. Proceeding with analyzing gastric cancer and prostate cancer, many new diagnostic and therapeutic targets including DSC2 have been identified. Furthermore, obtaining cancer risk information by genetic polymorphism analysis, including cancer-specific genetic SNP is in the scope as well.

The final goal of this study is to obtain results providing cancer clinical study in the form of molecular diagnosis/genetic screening, and identifying new therapeutic targets and establishing the diagnosis system. We have accumulated the experience of building and practicing the routine system of molecular pathological diagnosis for pathology specimen. This is utilizing biopsy based specimen as pathology tissue, possessing a significant driving force to "morpho-pathological genomics", which contains form abnormality from genome transcriptome information. Furthermore, we are convinced that the study which added SNP analysis holds an important key of "Personalized treatment and prevention of cancer based on the evidence" being the direction of cancer genome strategy.

2) Approach to Identification and Analysis of the Cancer Stem Cell in Gastrointestinal Cancer

It has been known for a long time that existence of cancer cells in the cancer tissue is heterogeneous. However, a cell (cancer stem cell) that possesses a stem cell-like cell is presumed, and the connection with self-renewal and anti-cancer agent resistance is drawing attention.

Stem cell, is a cell that differentiates by division caused by some sort of signals, and has two characteristics of "differentiation ability"; division to produce a cell that possess a specific role in an internal fixed location, and "self-renewal" to reproduce itself, so that the original cell does not deplete every differentiation that takes place, and regenerates.
In the in vivo, only a stem cell and cancer cell have self-renewal ability. However, the difference between the two cells is whether it is a controlled self-renewal, or it is a self-renewal that is uncontrollable. It is thought that canceration of the cell is an acquisition of the uncontrollable self-renewal ability. From this point of view, there is a possibility that stem cell and cancer cell may have common and distinct molecular bases. Additionally, it is not homogeneous with cancer cells and only a special group of cells called cancer stem cells have self-renewal ability amongst the cancer cells. It is considered that this heterogeneous property causes the resistance in treatment. After the identification of cancer stem cell of acute myeloid leukemia (Nature Med, by Bonnet & Dick) in 1997, the existence of cancer stem cell in brain tumor, breast cancer, and various types of solid tumors, including lung cancer was reported. Concerning cancer stem cell marker, CD44+CD24-/lowESA+ is identified in breast cancer, CD44+CD24+ESA+ in pancreatic cancer, CD44, ALDH1, CD133 in brain tumor and colon cancer.

Furthermore, side population cell, a cell having similar character to these cells have been discovered from various cancers and are attracting attention that there may be a possibility that side population cells are cancer stem cells. The characteristic of side population cells in cancer, is that they express an ABC transporter at a high level similar with hematopoietic cells, and it is confirmed that it possesses tolerance to anti-cancer drugs. From the results of the studies of the hepatocellular carcinoma, colon cancer, as well as gastric cancer, side population cells are found to occupy only several percent among total cancer cell population. However, they express ABCG2 and ABCB1 at high levels, and they evidently show strong resistance against doxorubicin, 5-FU, and gemcitabine. Therefore, for effective treatment to eradicate all cancer cells, it is necessary to identify a special cancer cell group called side population cell or cancer stem cell based on the diagnosis and to make it a target of the treatment. Furthermore, the importance of cancer stem cell in metastasis has been pointed out. We have also found that the ALDH1-positive cancer cell, a candidate stem cell marker, is clearly more present in the metastatic lesion than the primary lesion of diffuse-type gastric cancer.

Our goal is to find the application for histo-pathological diagnosis and serodiagnosis by identifying new molecules prescribing characteristics of cancer stem cell, by extracting membrane protein or secreted protein. Also, by understanding the cancer stem cell marker and its characteristics, it will enable us to develop cancer stem cell targeted therapy and drug-resistance evasion therapy.

3) Significance of non-coding RNA in Development and Progression of Gastrointestinal and Urological Cancer

A New control mechanism based on duplex RNA has been elucidated in sequence, such as the discovery of the RNA interference represented by Fire and Mello, who won the Nobel Prize for Medicine, and it has become clear that the non-coding RNA has a lot of functions but not junk. There are approximately 20,000 RNA that encode protein. More non-coding RNAs exist, hence it is called, "the RNA New World". Among the non-coding RNAs, approximately 20 bases micro RNAs (miRNAs) participate in transcription suppression, mRNA degradation, DNA methylate, heterochromatin formation, as well as genome reconfiguration. According to the analysis of the miRNA-binding motif in the gene promoter and 3'-UTR, it indicates that at least 20% of the human genes are regulated by miRNA, and one miRNA controls more than 100 target genes.
The study on the role of miRNA in development and progress of cancer has been pursued enthusiastically. The studies show half of miRNAs are tumor-suppressor genes, as the expression is decreased in cancer, targeting the oncogene, and half of miRNAs function as oncogene as the expression is increased in cancer. The miRNA microarray analysis has been performed actively and the result of the miRNAome analysis in colorectal cancer using miRNA serial analysis of gene expression (combination of direct miRNA cloning and SAGE), has identified many new miRNAs. For gastric cancer, in collaboration with Dr. Croce (Ohio University), several miRNAs have been found to correlate with the clinicopathological parameters and patient's prognosis. However, the challenge is to identify which target gene of miRNA with abnormal expression is crucial. In addition, it has become clear that the miRNA expression is controlled epigenetically by, such as DNA methylation or histone modification, the same way as the ordinal gene. Some of the miRNAs exist in CpG island, and the expression is induced significantly through a chromatin remodeling by de-methylating agent or a histone deacetylase inhibitor. If the miRNA, which targets an important gene of carcinogenesis, is identified, epigenetic treatment through DNA methylation and histone modification may become the new strategy of cancer treatment through miRNA.

Additionally, It becomes clear that miRNA is secreted outside the cell through the microvesicle called exosome, and is stable in the extracellular space and in the body fluid. This secretion type miRNA is thought, just like the paracrine growth factor/cytokine, that there are possibilities to mutually control the interaction of the cancer cell and stromal cell and participate in cell growth, invasion, angiogenesis and epithelial-mesenchymal transition (EMT). Based on these analyses, we are paying attention that a new page is being opened in understanding cancer biology.

Our task is to clearly understand the mechanism of the development and progression of esophago-gastric cancer from the miRNA point of view, through comprehensive expression analysis and epigenetics that our department excels in. We hope to discover the complete picture of the molecular bases of carcinogenesis, by adding the miRNA point of view, to the so far accumulated genetic abnormality and miRNA abnormality. Furthermore, our final goal is to apply the acquired knowledge to the development of novel diagnosis and treatment. We believe the significance will be determining the direction of future cancer molecular pathology in the practical medical care.

The transcribed-ultra conserved regions (T-UCRs) are a novel class of non-coding RNAs (ncRNAs), which are absolutely conserved (100%) between the orthologous regions of the human, rat and mouse genomes. Several T-UCRs show differential expressions in cancers and might be involved in cancer development and progression. We identified that the expression of Uc.158+A was silenced by DNA methylation of its promoter regions both in gastric cancer. miR-153 directly regulates Uc.416+A expression, which could contribute to cell proliferation through the regulation of its target genes. Up-regulated T-CURs such as U.63+, Uc.416+A, were found to be caidiate marker for liquid biopsy. In prostate cancer, Uc.416+A in the blood was significantly higher in cases with metastases, with castration resistance, and with docetaxel resistance. We also found that Uc.249+A, Uc.266+A, etc. might regulate cancer stem cell properties of colorectal cancer. Our approach would be instrumental to pick out T-UCRs having crucial role in cancer biology that can be applied to future diagnosis and treatment.

4) Basic and Applied Research of Gastrointestinal and Urological Cancers using Organoid System

Research on stem cell niches has progressed and from one tissue stem cell in a 3D culture system it is now possible to develop organoids, "mini-organ", that phenocopy structure and function of originated tissues and organs. Stomach stem cells form organoids if media contain EGF, Noggin, R-spondin, Wnt-3A and FGF10. Using the CRISPR-Cas9 genome editing system, specific genetic alterations can be introduced into normal tissue-derived organoid. Using this method, we aim to uncover absolute driver mutations involved in gastric cancer with different mucin phenotypes. We are also trying to demonstrate key genetic aberrations in serrated pathway of colorectal carcinogenesis.

Organoids can also be established from clinical cancer samples. By examining drug sensitivity using cancer organoid, it is possible to select the optimal anticancer drug for each patient. Furthermore, it can be used for screening of new drugs using cancer organoid library.

Organoid is an excellent model that covers from basic stem cell / cancer research to application for personalized medicine.

5) Epigenetic Abnormality in Cancer: The Aberrant Gene Expression Caused by DNA Methylation and Histone Modification

By the epigenetic change that does not have abnormality in the gene itself, the structural change of the chromatin by the modification such as methylation of CpG's in the promoter region and acetylation or the methylation of the core histone holds the key to transcriptional control, which is also called the main switch of the transcription. The "laxity " of the chromatin structure, due to the histone acetylation, the binding of transcription factors, such as E2F-1, to the DNA is considered to promote transcription. The acetylation of the histone is controlled by histone acetylase (HAT) and histone deacetylase (HDAC) such as p300/CBP. The methyl-CpG binding protein (MeCP) recruits histone deacetylase (HDAC) and produces chromatin as an inactivated form for transcription. Furthermore, it is known that histone becomes the code of gene expression, called Histone code. Depending on the location where lysin (K) in histone is methylated, a different functional molecule is recruited. As a result, the transcription is activated or suppressed. It has been also found that methylation of histone controls other histone modification (acetylation and phosphorylation).

p15INK4a, hMLH1, BRCA1, Rb, E-cadherin, and RASSF1 are known as the representing genes that are inactivated by DNA methylation, and participate in canceration through dysregulation of cell cycle or the genetic. We have also found inactivation of many tumor suppressor genes such as MGMT, RIZ1, and PINX1 by DNA methylation and histone deacetylation in gastric cancer. Particularly, with the decreasing level of histone H3 acetylation in the p21 promoter and down-regulation of p21 expression, having been the first case in the world proving chromatin structure and the relation with the onset of gene control, using tissue samples.

Additionally, the association between methylation of 12 representing genes and clinicopathological paramaters of gastric cancer was examined, we discovered that gastric cancer with hypermethylation (more than five genes are methylated), independent from the CpG island methylator phenotype, is biologically high-grade malignancy.

We have cyclopedically captured series of genes whose expression levels are altered by the acetylated state of the histone in gastric cancer cell lies, and identified novel genes which are strongly controlled by histone modification. By understanding the function of these genes, we believe that as long as it concerns the cancer development and progress, it will lead to new therapeutic strategy.

6) Molecular Mechanism of the Solid Cancer Development Induced by Radiation Damage and the Relation with a Host Factor.

The usage of radiation has increased remarkably with nuclear energy, medical care, as well as in industry and the influence on health of radiation has become a serious issue. The use of radiation in the medical care; radiation is not only used for diagnosis, but is also the primary pillar of treatment for cancer. However, the influence as an external factor of the carcinogenesis is a world-wide concern. Japan, the nation that having been the victim of atomic bombing, is expected to release academic information to the world on the increased usage of radiation and contributes to security and relief in radiation usage. We elucidate a molecular base of the radiation-induced carcinogenesis and proceed forward with the study for the purpose of presenting the result for clinical applications, such as risk evaluation and treatment development.

It is known that the molecular mechanism of cancer development is related to various genetic and epigenetic abnormalities. The A-bomb survivors can be considered as the important model of studying carcinogenesis caused by radiation. We have produced the oligoDNA custom array, which includes cancer-specific genes extracted by our SAGE analysis and genes related to DNA damage response and repair. Then analyzed the gene expression profiles in fresh frozen tissues of gastric cancer in A-bomb survivors and controls, and specifically extracted the gene clusters differentially expressed. As a marker for diagnosis, we have identified the expression of versican and osteonectin in the gastric cancer stroma. The expression of Reg IV in the gastric cancer was high-frequency in the A-bomb survivor group. In addition, we are also conducting the examination of the macrophage in the cancer stroma. Furthermore, we analyzed the expression of microRNAs using a formalin-fixed paraffin-embedded tissue of gastric cancer of the A-bomb survivor, stored for a period of time. The expression levels of certain microRNAs differ between A-bomb survivor group and the control group. Moreover, by utilizing the results of future study, we are considering showing concrete prevention of radiation damage, caused by occupation radiation exposure and an application method to diagnosis and treatment of cancer.

In addition, the study group of the Public Welfare Labor Science Research Grants (the third-term comprehensive control research for cancer) that Prof. Yasui represents, is researching the cyclopedic DNA methylation analysis on solid cancer and radiation-related secondary cancer, microRNA expression analysis, and gene variation analysis, which occurred in A-bomb survivors, in cooperation with the Radiation Effects Research Foundation, the National Institute of radiological Sciences, the Hiroshima University Research Institute for Radiation Biology and Medicine, and Cancer Institute (Japanese Foundation for Cancer Research). We are also conducting studies concerning analysis and risk evaluation of individual differences of hereditary cancer-causing sensitivities of solid cancer caused by radiation, and from the scope of elucidation of the carcinogenesis mechanism from a genome obstacle and occurrence caused by radiation, as well as studying sensitivity to chemotherapy.