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| Research: Murakami Yoshinori M.D. Ph.D. |
Research on the Mechanisms of Cancer Development
Genetic alterations in human cancer
Alterations in specific genes, including oncogenes and tumor suppressor
genes, are involved in multistage carcinogenesis of human tumors. Examination
of epidermal growth factor receptor (EGFR) in lung adenocarcinoma (ADC)
revealed that EGFR mutations occurred frequently in non-invasive bronchioloalveolar
carcinoma (60%) as well as its metastasis to the brain. A mutant EGFR (delE746-A750),
but not a wild type EGFR, phosphorylated AKT and STAT3 after serum starvation
in in vitro study. The MYO18B gene was previously isolated as a candidate
lung tumor suppressor in this institute. Yeast two-hybrid screening identified
Sug1, a 19S regulator subunit of the 26S proteasome, as a binding partner
of MYO18B, suggesting that MYO18B is a substrate for proteasomal degradation.
To elucidate the genetic and genomic alterations, oligonucleotide array-based CGH (comparative genomic hybridization) was applied to various human cancers. A homozygous deletion at the DOCK8 locus at chromosome 9p24 was detected in a lung cancer cell line, providing another candidate of lung tumor suppressor. Analysis of nodule in nodule-type hepatocellular carcinoma (HCC) newly identified genetic inactivation of the APC gene in HCC. The analysis of esophageal squamous cell carcinoma (ESCC) revealed that amplification of the CCND1 gene and homozygous deletion of the p16 gene associated with ESCC progression. Somatic mutation of KEAP1, whose product induces phase II enzymes, was also detected in lung cancer in collaboration with other groups.
Non-homologous end joining (NHEJ) repair and homologous recombinational
repair (HRR) are the two main activities to repair the DNA double-strand
break (DSB) in vertebrates. Therefore, a plasmid-based system to assess
both NHEJ and HRR was developed to investigate the role of these repair
activities in human carcinogenesis. The germline polymorphisms and mutations
implicated in cancer susceptibilities or drug responses were analyzed and
novel methods for genomic data analysis were developed in collaboration
with National Cancer Center Hospital and other institutes. A part of the
single nucleotide polymorphism (SNP) data was publicized from a database
(www.gemdbj.nibio.go.jp).
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Epigenetic mechanisms of carcinogenesis
Methylation of CpG islands (CGIs) is deeply involved in human carcinogenesis. Some cancer cells show high incidence of aberrant DNA mehylation, known as the CpG island methylator phenotype (CIMP), and the finding of increased rates of de novo methylation in some cancer cells that lead to induction of dense methylation in CGIs would provide important information in understanding the mechanisms of CIMP. In liver carcinogenesis, epigenetic-instability-dependent HCC, showing frequent epigenetic aberrations but no chromosomal instability, was newly categorized.
Methylation-sensitive-representational difference analysis (MS-RDA) that
was originally developed in this institute identified the peroxiredoxin
2 (PRDX2), a negative regulator of platelet-derived growth factor signaling,
as one of the silenced genes in melanoma. Combinatorial analyses of chemical
genomic screening using a demethylating agent, 5-aza-2’-deoxycytidine,
with expression microarray was also useful to identify genes methylated
and silenced in various cancers. These include the very low density lipoprotein
receptor (VLDLR) and the MTSS1, a metastasis receptor, in gastric cancer
and the UCHL1, a regulator of cellular ubiquitin levels, in colorectal
cancer.
Significance of aberrant DNA methylation in multistage carcinogenesis was
also investigated in organ specific manner. In kidney, the incidence of
methylated CpG islands was significantly higher in non-tumorous renal tissues
from patients with renal cell carcinoma (RCC) than in normal renal tissues
from patients without RCC. Degree of CpG methylation in non-tumorous renal
tissues from RCC patients was correlated with a higher histological grade
of corresponding RCCs. Furthermore, degree of CpG methylation in RCCs was
correlated with various malignant features of RCCs as well as the poor
prognosis of the patients. In pancreas, the incidence and the degree of
methylation in tumor-related genes were significantly higher in pancreatic
duct epithelia with inflammation or pancreatic intraepithelial neoplasias
(PanINs) than in pancreatic duct epithelia without inflammation. Ductal
carcinoma showed frequent methylation of the BRCA1, APC, p16 and TIMP-3
genes, where considerable heterogeneity in the degree of methylation was
observed. In addition, degree of DNA methylation was correlated with expression
level of DNA methyltransferase 1 (DNMT1). These findings indicate that
aberrant DNA methylation is involved in multistage carcinogenesis of the
kidney and pancreas from a precancerous condition to malignant progression.
In human stomach, MS-RDA was applied to intestinal metaplasia (IM) and
the ZIK1, ZNF141, KAL1, and FGF14 genes were identified as methylated and
down-regulated genes in glands with IM. An independent ZIK1 methylation
in physically distant glands in IM suggests that methylation of specific
genes could also play a role in disorders with polyclonal origins.
Methylation analyses of specific tumor suppressor genes revealed that the
tumor suppressor TSLC1/IGSF4, previously identified in this institute,
was inactivated by methylation in 44% of lung ADC, providing a possible
indicator of poor prognosis of the patients. Another candidate tumor suppressor,
DAL-1/4.1B, that binds to TSLC1/IGSF4, was also frequently methylated in
lung ADC as well as RCC. These results coupled with other findings suggest
that the cascade including TSLC1/IGSF4 and DAL-1/4/1B as essential components
is involved in various cancers as well as spermatogenesis.
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Gene expression analysis of human tumors and tumor suppressor cascades.
Expression microarray analyses in combination with laser capture microdissection were performed in various human cancers to categorize tumors into several subtypes or to identify novel tumor markers as well as possible therapeutic targets. Analysis of AML harboring chromosomal translocations revealed that t(8;21)-AML and inv(16)-AML showed very similar profiles in gene expression, suggesting that AML1-MTG8 and CBFb-MYH11 chimeric proteins affect a common cascade of leukemogenesis. Expression profiling of pretreatment biopsy specimens from 33 ESCC patients identified 57 and 120 genes that correlate with short-term and long-term survival, respectively.
An advanced microarray data analysis using a boosted fuzzy classifier could classify ESCC with intramural metastasis, a subtype with poorer prognosis, and identified CDK6 as a possible novel diagnostic marker for ESCC. Gene expression analyses were also performed in surgical specimens from lung ADC, neuroblastoma, soft tissue sarcoma, and gastric cancer to identify profiles specific to each disease. A part of the expression profile data is available at the database (www.gemdbj.nibio.go.jp).
Since altered expression of microRNA (miRNA) would provide another molecular mechanism important in human carcinogenesis, expression profile of miRNA was examined in lung and colon cancers and possible diagnostic and prognostic markers were identified in lung cancer.
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