PATHOLOGY DIVISION

1.PATHOLOGY DIVISION

    Research in the Pathology Division is based on a combination of clinicopathological observations and molecular and cellular biological analyses.

Epigenetic and Genetic Alteration during Multistage Carcinogenesis

    DNA hypomethylation on pericentromeric satellite regions is an early and frequent event associated with heterochromatin instability during human hepatocarcinogenesis. Overexpression of DNMT3b4, a splice variant of DNA methyltransferase DNMT3b, and the ratio of expression of DNMT3b4 to that of DNMT3b3 showed significant correlation with DNA hypomethylation on pericentromeric satellite regions in precancerous conditions and hepatocellular carcinomas (HCCs) ⁽¹⁾. Transfection of human epithelial cells with DNMT3b4 cDNA induced DNA demethylation on satellite 2 ⁽¹⁾. Overexpression of DNMT3b4, which may lack DNA methyltransferase activity and compete with the major active variant DNMT3b3 in the targeting of pericentromeric satellite regions, may result in DNA hypo-methylation on these regions, even in precancerous stages. During the development of breast cancers, DNA hypomethylation on satellite 2 has been associated with the accumulation of a large number of numerical chromosome alterations involving chromosome 16 and the acquisition of aggressive histological features ⁽²⁾.
    Although the first evidence that mutations of the DNMT1 gene possibly causes genome-wide DNA methylation alteration has been presented, mutational inactivation is, if anything, a rare event during carcinogenesis. Progressive increases in DNMT1 expression may be accompanied by hepatocarcinogenesis from the precancerous stage to the malignant progression of HCC and become a biological predictor of both HCC recurrence and a poor prognosis for HCC patients.
    A comprehensive study on multiple chromosomal loci with laser-capture micro-dissection methods revealed different patterns of loss of heterozygosity among different histological subtypes of uterine cervix ⁽³⁾ and ovarian ⁽⁴⁾ cancers. An attempt to identify the target genes within chromosomal amplicons has been made in various cancers ^(5,⁶⁾.

Mechanisms of Cancer Invasion and Metastasis

    An alternative pathway inactivating the E-cadherin-mediated "cell adhesion and invasion-suppressor" system has been identified. A newly identified cell membrane glycoprotein, dysadherin, is strongly expressed in a wide variety of cancer cells ⁽⁷⁾. Transfection of dysadherin cDNA into an HCC cell line resulted in reduced cell-cell adhesiveness based on a Ca²⁺ -dependent cell aggregation assay ⁽⁷⁾. In transfectants, E-cadherin expression markedly reduced at the protein level in inverse proportion to the level of dysadherin ⁽⁷⁾. Dysadherin transfectants formed a markedly higher number of metastatic nodules compared to mock-transfectants in a mouse orthotopic implantation model for intrahepatic metastasis, suggesting the metastasis promoting propensity of dysadherin ⁽⁷⁾.
    Treatment of cancer cells with both a Src family kinase inhibitor ⁽⁸⁾ and a Ras farnesylation inhibitor ⁽⁹⁾ can each activate the functioning of the E-cadherin-mediated cell adhesion system, which is associated with the suppression of metastasis in mouse inoculation models. Selective inhibition of Src or Ras activation may be potentially useful in the prevention of cancer metastasis.
    Laminin-5 is an extracellular matrix protein that plays a key role in cell migration and tumor invasion. The epidermal growth factor receptor gene amplification and protein expression were closely correlated with laminin-5g2 chain expression in oral squamous cell carcinoma cell lines ⁽¹⁰⁾ and resected tongue cancer specimens ⁽¹¹⁾. Co-stimulatory mechanisms may exist that increase the expression of cox-2 and laminin-5g2 chain at the invasive front of lung adenocarcinomas ⁽¹²⁾. Increased cytoplasmic localization of laminin-5g2 chain has been associated with a poor prognosis for the patients with tongue ⁽¹¹⁾ and colon ⁽¹³⁾ cancers.
    Akt is a direct downstream effector of phosphatidylinositol 3-kinase (PI3K). Transfection of kinase-dead Akt and treatment with a PI3K inhibitor each suppressed the anchorage independent growth of cultured HCC cells and intrahepatic metastasis in the mouse orthotopic implantation model, suggesting that the activated PI3K /Akt pathway may play an important role in hematogeneous metastasis ⁽¹⁴⁾.

Molecular Background of Biological Characteristics of Human Cancers

    To elucidate the functions of the mutant b-catenin protein in colon carcinogenesis, the wild or the mutant b-catenin gene of a colorectal cancer cell line was disrupted by somatic gene targeting. There was no significant difference in in vitro and in vivo growth between the two genotypes ⁽¹⁵⁾. This suggests that mutant b-catenin may no more give rise to growth advantage in some colorectal cancers ⁽¹⁵⁾. Mutation of the b-catenin gene was detected in sporadic fundic gland polyps ⁽¹⁶⁾ and craniopharyngiomas of the adamantinomatous type ⁽¹⁷⁾.
    Suppression subtractive hybridization ⁽¹⁸⁾ and differential display analysis ⁽¹⁹⁾ were employed to identify genes expressed in a lesion specific manner. Expression of protein kinase C receptor RACK 1 ⁽¹⁸⁾ and osteopontin ⁽¹⁹⁾ were detected in colon cancers and HCCs showing cancer-stromal interaction, respectively. Reduced expression of galectin-3, a member of the b-galactoside- binding lectin family, was associated with advanced stage, tumor de-differentiation and metastasis in ductal adenocarcinomas of the pancreas ⁽²⁰⁾.

Clinicopathological Studies

    Clinicopathological studies were also conducted to further understand the pathogenesis and promote the diagnosis and treatment of various tumors ^(21-⁴⁸⁾.




1.PATHOLOGY DIVISION



	Research in the Pathology Division is based on a combination of clinicopathological observations and molecular and cellular biological analyses. Epigenetic and Genetic Alteration during Multistage Carcinogenesis DNA hypomethylation on pericentromeric satellite regions is an early and frequent event associated with heterochromatin instability during human hepatocarcinogenesis. Overexpression of DNMT3b4, a splice variant of DNA methyltransferase DNMT3b, and the ratio of expression of DNMT3b4 to that of DNMT3b3 showed significant correlation with DNA hypomethylation on pericentromeric satellite regions in precancerous conditions and hepatocellular carcinomas (HCCs) ⁽¹⁾. Transfection of human epithelial cells with DNMT3b4 cDNA induced DNA demethylation on satellite 2 ⁽¹⁾. Overexpression of DNMT3b4, which may lack DNA methyltransferase activity and compete with the major active variant DNMT3b3 in the targeting of pericentromeric satellite regions, may result in DNA hypo-methylation on these regions, even in precancerous stages. During the development of breast cancers, DNA hypomethylation on satellite 2 has been associated with the accumulation of a large number of numerical chromosome alterations involving chromosome 16 and the acquisition of aggressive histological features ⁽²⁾. Although the first evidence that mutations of the DNMT1 gene possibly causes genome-wide DNA methylation alteration has been presented, mutational inactivation is, if anything, a rare event during carcinogenesis. Progressive increases in DNMT1 expression may be accompanied by hepatocarcinogenesis from the precancerous stage to the malignant progression of HCC and become a biological predictor of both HCC recurrence and a poor prognosis for HCC patients. A comprehensive study on multiple chromosomal loci with laser-capture micro-dissection methods revealed different patterns of loss of heterozygosity among different histological subtypes of uterine cervix ⁽³⁾ and ovarian ⁽⁴⁾ cancers. An attempt to identify the target genes within chromosomal amplicons has been made in various cancers ^(5,⁶⁾. Mechanisms of Cancer Invasion and Metastasis An alternative pathway inactivating the E-cadherin-mediated "cell adhesion and invasion-suppressor" system has been identified. A newly identified cell membrane glycoprotein, dysadherin, is strongly expressed in a wide variety of cancer cells ⁽⁷⁾. Transfection of dysadherin cDNA into an HCC cell line resulted in reduced cell-cell adhesiveness based on a Ca²⁺ -dependent cell aggregation assay ⁽⁷⁾. In transfectants, E-cadherin expression markedly reduced at the protein level in inverse proportion to the level of dysadherin ⁽⁷⁾. Dysadherin transfectants formed a markedly higher number of metastatic nodules compared to mock-transfectants in a mouse orthotopic implantation model for intrahepatic metastasis, suggesting the metastasis promoting propensity of dysadherin ⁽⁷⁾. Treatment of cancer cells with both a Src family kinase inhibitor ⁽⁸⁾ and a Ras farnesylation inhibitor ⁽⁹⁾ can each activate the functioning of the E-cadherin-mediated cell adhesion system, which is associated with the suppression of metastasis in mouse inoculation models. Selective inhibition of Src or Ras activation may be potentially useful in the prevention of cancer metastasis. Laminin-5 is an extracellular matrix protein that plays a key role in cell migration and tumor invasion. The epidermal growth factor receptor gene amplification and protein expression were closely correlated with laminin-5g2 chain expression in oral squamous cell carcinoma cell lines ⁽¹⁰⁾ and resected tongue cancer specimens ⁽¹¹⁾. Co-stimulatory mechanisms may exist that increase the expression of cox-2 and laminin-5g2 chain at the invasive front of lung adenocarcinomas ⁽¹²⁾. Increased cytoplasmic localization of laminin-5g2 chain has been associated with a poor prognosis for the patients with tongue ⁽¹¹⁾ and colon ⁽¹³⁾ cancers. Akt is a direct downstream effector of phosphatidylinositol 3-kinase (PI3K). Transfection of kinase-dead Akt and treatment with a PI3K inhibitor each suppressed the anchorage independent growth of cultured HCC cells and intrahepatic metastasis in the mouse orthotopic implantation model, suggesting that the activated PI3K /Akt pathway may play an important role in hematogeneous metastasis ⁽¹⁴⁾. Molecular Background of Biological Characteristics of Human Cancers To elucidate the functions of the mutant b-catenin protein in colon carcinogenesis, the wild or the mutant b-catenin gene of a colorectal cancer cell line was disrupted by somatic gene targeting. There was no significant difference in in vitro and in vivo growth between the two genotypes ⁽¹⁵⁾. This suggests that mutant b-catenin may no more give rise to growth advantage in some colorectal cancers ⁽¹⁵⁾. Mutation of the b-catenin gene was detected in sporadic fundic gland polyps ⁽¹⁶⁾ and craniopharyngiomas of the adamantinomatous type ⁽¹⁷⁾. Suppression subtractive hybridization ⁽¹⁸⁾ and differential display analysis ⁽¹⁹⁾ were employed to identify genes expressed in a lesion specific manner. Expression of protein kinase C receptor RACK 1 ⁽¹⁸⁾ and osteopontin ⁽¹⁹⁾ were detected in colon cancers and HCCs showing cancer-stromal interaction, respectively. Reduced expression of galectin-3, a member of the b-galactoside- binding lectin family, was associated with advanced stage, tumor de-differentiation and metastasis in ductal adenocarcinomas of the pancreas ⁽²⁰⁾. Clinicopathological Studies Clinicopathological studies were also conducted to further understand the pathogenesis and promote the diagnosis and treatment of various tumors ^(21-⁴⁸⁾.