Tadaaki Yokoyama

Restoration of SMAD4 by gene therapy reverses the invasive phenotype in pancreatic adenocarcinoma cells.

SMAD4 is a critical cofactor in signal transduction pathways activated in response to transforming growth factor-beta (TGF-β)-related ligands, regulating cell growth and differentiation. The roles played by SMAD4 inactivation in tumours highlighted it as a tumour-suppressor gene. However, restoration of the TGF-β antiproliferative pathway following SMAD4 gene transfer in null-tumour cell lines is controversial. Herein, we report the inhibitory effects of SMAD4 on pancreatic tumour invasion and angiogenesis. Adenoviral transfer of this gene in a panel of SMAD4 homozygous-deleted human pancreatic tumour cell lines restored SMAD4 protein expression and function. Although it did not affect proliferation significantly in vitro, SMAD4 inhibited in vivo tumour growth in immunodeficient mice. In this xenograft setting, differential suppression of tumour growth in vivo was mediated, at least in part, through downregulation of vascular endothelial growth factor and expression of gelatinases. We documented the reduced invasion and angiogenesis histologically and by intravital microscopy, and gained mechanistic insight at the messenger and protein level. Finally, we found a negative reciprocal regulation between SMAD4 and ETS-1. ETS-1 is considered a marker for tumour invasion. Upon SMAD4 deletion, we detected high expression levels of ETS-1 in pancreatic tumour cells, suggesting the shift of the pancreatic tumour toward an invasive phenotype.

Genomic analysis of DUSP6, a dual specificity MAP kinase phosphatase, in pancreatic cancer

DUSP6 (alias PYST1), one of the dual-specificity tyrosine phosphatases, is localized on 12q21, one of the regions of frequent allelic loss in pancreatic cancer. This gene is composed of three exons, and two forms of alternatively spliced transcripts are ubiquitously expressed. Although no mutations were observed in 26 pancreatic cancer cell lines, reduced expressions of the full-length transcripts were observed in some cell lines, which may suggest some role for DUSP6 in pancreatic carcinogenesis.

Association of poor prognosis with loss of 12q, 17p, and 18q, and concordant loss of 6q/17p and 12q/18q in human pancreatic ductal adenocarcinoma

OBJECTIVE:Pancreatic cancer is one of the diseases with the poorest prognosis, but the associated genetic alterations are not yet well understood. The genetic alterations reported to date in pancreatic cancer include frequent mutations of the KRAS, TP53, p16, and SMAD4 genes. Mutation of the TP53 gene was reported to be associated with a poor prognosis. In this study, we analyzed the association of loss of heterozygosity (LOH) with clinicopathological features to attempt to devise effective methods in the future for clinically applying our results to diagnosis and treatment.METHODS:A total of 55 tumors from patients with primary pancreatic ductal carcinomas (34 men and 21 women, mean average age 63.9 yr) in which all the relevant clinical and pathological data were available were analyzed. A total of 46 cases were surgically resected, and nine cases were not. Tumor cells as well as corresponding normal cells were collected by microdissection under a microscope, and DNAs were purified. Allelotype analysis was performed by the PCR-based method, and the results were statistically analyzed.RESULTS:LOH of ≥30% were observed on chromosome arms 17p (47%, 17/36), 9p (45%, 14/31), 18q (43%, 15/35), 12q (34%, 10/29), and 6q (30%, 10/33). LOH of 12q, 17p, and 18q were significantly associated with a poor prognosis. Concordant losses of 6q with 17p and 18q were significantly associated with a poor prognosis. Concordant losses of 6q with 17p and of 12q with 18q were also found.CONCLUSIONS:Because LOH of 12q, 17p, and 18q are significantly associated with a poor prognosis, mutation of the putative tumor suppressor genes on these chromosome arms may play significant roles in the disease progression. Concordant losses of 6q with 17p and of 12q with 18q suggest that protein products of putative tumor suppressor genes on 6q and 12q may function in association with TP53 and SMAD4, respectively.

Overexpression of the p53-inducible brain-specific angiogenesis inhibitor 1 suppresses efficiently tumour angiogenesis.

The brain-specific angiogenesis inhibitor 1 gene has been isolated in an attempt to find fragments with p53 “functional” binding sites. As reported herein and by others, brain-specific angiogenesis inhibitor 1 expression is present in some normal tissues, but is reduced or lost in tumour tissues. Such data and its particular structure prompted the hypothesis that brain-specific angiogenesis inhibitor 1 may act as a mediator in the local angiogenesis balance. We herein demonstrate that brain-specific angiogenesis inhibitor 1 over-expression suppresses tumour angiogenesis, delaying significantly the human tumour growth in immunodeficient mice. The inhibitory effect of brain-specific angiogenesis inhibitor 1 was documented using our intravital microscopy system, strongly implicating brain-specific angiogenesis inhibitor 1 as a mediator in the control of tumour angiogenesis. In contrast, in vitro tumour cell proliferation was not inhibited by brain-specific angiogenesis inhibitor 1 transfection, whereas some level of cytotoxicity was assessed for endothelial cells. Immunohistochemical analysis of tumour samples confirmed a reduction in the microvessel density index in brain-specific angiogenesis inhibitor 1-overexpressing tumours. At messenger level, moderate changes could be detected, involving the down-regulation of vascular endothelial growth factor and collagenase-1 expression. Furthermore, brain-specific angiogenesis inhibitor 1 expression that was lost in a selection of human cancer cell lines could be restored by wild-type p53 adenoviral transfection. Brain-specific angiogenesis inhibitor 1 should be considered for gene therapy and development of efficient drugs based on endogenous antiangiogenic molecules.

Identification of three commonly deleted regions on chromosome arm 6q in human pancreatic cancer.

Pancreatic cancer has one of the poorest prognoses among malignant diseases. To understand its molecular mechanisms, we studied allelic losses on the long arm of chromosome 6. Using 55 paired DNAs of tumors and their corresponding normal tissues and 30 microsatellite markers that spanned the entire 6q chromosome arm, we found three distinct regions of common allelic loss: region A, a less than 500‐kb region bordered by D6S449 and D6S283 on 6q21 with a loss of heterozygosity (LOH) frequency of 69% (38/55); region B, a 7‐cM region bordered by D6S292 and D6S308 on 6q23–q24 with a LOH frequency of 60% (33/55); and region C, a 13‐cM region bordered by D6S305 and D6S264 with a LOH frequency of 51% (28/55). We further focused on region A and constructed a physical map using yeast artificial chromosome (YAC) clones, their derived cosmid clones, and bacterial artificial chromosome (BAC) clones. Region A was completely covered by three overlapping BAC clones. Our results in the present study should shed light on the cloning and characterization of tumor suppressor genes in pancreatic carcinogenesis. Genes Chromosomes Cancer 25:60–64, 1999.

Isolation and Characterization of the Human Gene Homologous to the Drosophila Headcase (hdc) Gene in Chromosome Bands 6q23-q24, a Region of Common Deletion in Human Pancreatic Cancer

Deletions of the long arm of chromosome 6 (6q) are one of the most common chromosomal abnormalities in multiple human malignancies. Previously, we have identified three commonly deleted regions on 6q (6q21, 6q23-q24, and 6q26) in pancreatic cancer by loss of heterozygosity studies, suggesting the presence of one or more tumor suppressor genes on this chromosome arm. Using a combination of database search and cDNA library screening, we successfully isolated a transcript from 6q24. This mRNA encodes a protein consisting of 543 amino acids with homology to the Drosophila headcase (hdc) gene and, thus, is designated as hHDC. Northern analysis identified a ubiquitously expressed 5.6-kb transcript. Seventeen (81%) of 21 pancreatic cancer cell lines and four (80%) of five renal cell carcinoma cell lines showed low level expression, suggesting that the hHDC gene may play an important role in some human cancers.

Cloning and characterization of the human UDP-N-acetylglucosamine : α-1,3-D-mannoside β-1,4-N-acetylglucosaminyltransferase IV-homologue (hGnT-IV-H) gene

AbstractA novel human gene determining a polypeptide product of 478 residues with an estimated molecular mass of 55kDa, which has significant homology and structural similarity to Bos UDP-N-acetylglucosamine: α-1,3-D-mannoside β-1,4-N-acetylglucosaminyltransferase (GnT-IV), was cloned from the commonly deleted region in pancreatic cancer at 12q21. The gene is composed of at least six exons, and the last three exons cover the open reading frame. Different sized transcripts, 3.8-kb in the heart, brain, and fetal brain and 2.8-kb and 1.7-kb in the testis were observed by Northern blot analysis. By reverse transcription-polymerase chain reaction, expression was also observed in the adult brain, liver, and adrenal gland, but not in pancreas. Because of its significant homology and structural similarity to Bos GnT-IV, it is potentially the gene encoding human GnT-IV or its homologue, which had been one of two genes remaining to be cloned in the human GnT family.

Genomic analysis of the thymine-DNA glycosylase (TDG) gene on 12q22-q24.1 in human pancreatic ductal adenocarcinoma.

SummaryBackground. Recently, a human G/T-specific TDG gene was identified. This protein acts in a system correcting G/T mispairs to G/C pairs. TDG was mapped to chromosome bands 12q22–q24.1, one of the regions frequently lost in pancreatic cancer. Therefore, there is the possibility that the TDG gene on 12q is one of the genes responsible for pancreatic ductal carcinogenesis. Methods. Nucleotide sequences of the entire coding region of the TDG gene were analyzed in 21 human pancreatic cancer cell lines. mRNA expression of the TDG gene was also analyzed by Northern hybridization in several human tissues and 21 human pancreatic cancer cell lines. Results. Decreased levels of mRNA expression were detected in the pancreatic cancer cell lines, but no somatic mutations were observed.Conclusion. Abnormality of the thymine-DNA glycosylase (TDG) gene on 12q22–q24.1 appears to play a limited role in pancreatic ductal carcinogenesis.

The role of chromosome 18 abnormalities in the progression of pancreatic adenocarcinoma.

Abstract: To date, the events that mediate tumor progression in pancreatic cancer are still poorly understood. Cytogenetic, allelotype, and somatic cell hybrid studies in human pancreatic adenocarcinoma have suggested that chromosome 18 may carry tumor suppressor genes (TSGs), including SMAD4. We previously identified that LOH of 18q at the SMAD4 locus, along with LOHs on 17p and 12q, positively associated with poor prognoses of pancreatic cancer patients. However, restoration of the SMAD4 gene did not suppress in vitro proliferation of pancreatic cancer cells that harbored homozygous deletion of this gene. An intraductal papillary mucinous neoplasm (IPMN ) is thought to be one of the premalignant lesions of the pancreas that progresses to carcinoma. Although there were frequent LOH (7/14, 50%) at the SMAD4 locus in IPMN samples, SMAD4 protein was observed immunohistochemically in tumor cells, and no mutations of the SMAD4 gene were observed, suggesting that it is the existence of a TSG in 18q, other than SMAD4, that suppresses cell growth. To functionally assess the activity of chromosome 18 in pancreatic cancer, we transferred a normal copy of the chromosome into pancreatic ductal carcinoma cells with and without completely inactivated SMAD4. In this study, in vitro growth of the hybrid cells was significantly suppressed compared with the parental cells, regardless of the initial SMAD4 status. To estimate the metastatic ability of the hybrids, we used a lung colonization model. At the end of the experiment, there was significant suppression of the number of surface metastases developing in mice injected with hybrids in comparison with those injected with parental cells. To identify and characterize genes that are involved in the progression of pancreatic cancer, we used micro-array expression analysis employing a 20k oligo-array system. It was revealed that there was increased expression of 4 genes relating to apoptosis in the 18 chromosome hybrids cells compared with the parental cells. We are now analyzing the function of these genes.