Shigehiro Ono

Histone Acetylation and Gastrointestinal Carcinogenesis

Abstract: The importance of altered histone acetylation in gastrointestinal carcinogenesis, especially in relation to invasion and metastasis, is described. Histone acetylation and chromatin remodeling linked with CpG island methylation play a major role in epigenetic regulation of gene expression. Acetylation of histones through an imbalance of histone acetyltransferases and deacetylases disrupts nucleosome structure, which leads to DNA relaxation and subsequent increase in accessibility to transcription factors. The expression of acetylated histone H4 is reduced in a majority of gastric and colorectal cancers, indicating the low level of global histone acetylation in tumor cells. Moreover, reduced histone acetylation is significantly associated with depth of tumor invasion and nodal metastasis of gastrointestinal cancers. A histone deacetylase inhibitor, trichostatin A (TSA), induces growth arrest and apoptosis and suppresses invasion of cancer cells. Treatment with TSA, which is followed by increased histone acetylation in the promoters, induces the expression of many genes that are suppressors of invasion and metastasis, including tissue inhibitors of metalloproteinase and nm23H1/H2, in addition to negative cell cycle regulators and apoptosis‐related molecules. Our approach, serial analysis of gene expression (SAGE), enabled us to identify a gene that is a novel candidate for a metastasis suppressor, whose expression is induced by histone acetylation. These findings suggest that, by modifying gene expression, histone deacetylation may participate not only in tumorigenesis but also in invasion and metastasis. Therefore, histone acetylation should be a promising target for cancer therapy, especially against invasive and metastatic disease, but also for cancer prevention.

Effect of trichostatin A on cell growth and expression of cell cycle- and apoptosis-related molecules in human gastric and oral carcinoma cell lines

The effect of trichostatin A (TSA), histone deacetylase inhibitor, on cell growth and the mechanism of growth modulation was examined in 8 gastric and 3 oral carcinoma cell lines which included 9‐cis‐retinoic acid resistant (MKN‐7 and Ho‐1‐N‐1) and IFN‐β resistant cell lines (MKN‐7, ‐28 and ‐45). TSA inhibited growth in all cell lines examined. Apoptotic cell death was confirmed by apoptotic ladder formation and induction of a cleaved form (85 kDa) of poly (ADP‐ribose) polymerase (PARP) induction. TSA enhanced the protein expression of p21WAF1, CREB‐binding protein, cyclinE, cyclin A, Bak and Bax, while it reduced the expression of E2F‐1, E2F‐4, HDAC1, p53 and hyperphosphorylated form of Rb. Furthermore, TSA induced morphological changes, such as elongation of cytoplasm and cell‐to‐cell detachment, in gastric and oral carcinoma cell lines. These results suggest that TSA may inhibit cell growth and induce apoptosis of gastric and oral carcinoma cells through modulation of the expression of cell cycle regulators and apoptosis‐regulating proteins. Int. J. Cancer 88:992–997, 2000.

ΔNp63α-dependent expression of Id-3 distinctively suppresses the invasiveness of human squamous cell carcinoma

p63 is a member of the p53 family and ΔNp63α is the dominant‐expressing isoform of p63 in basal layer of normal stratified epithelium and human squamous cell carcinoma (SCC) cells. We have previously reported that down‐regulation of p63 was accompanied with epithelial‐to‐mesenchymal transition (EMT) by Snail‐expressing SCC cells, in which re‐expression of ΔNp63α diminished their invasiveness (Higashikawa K, Yoneda S, Tobiume K, Taki M, Shigeishi H, Kamata N. Snail‐induced down‐regulation of ΔNp63α acquires invasive phenotype of human squamous cell carcinoma. Cancer Res 2007;67:9207–13). In this study, we found that ΔNp63α positively regulated inhibitor of differentiation‐3 (Id‐3) expression. Id is a dominant negative regulator of E2A which is a transcriptional repressor of E‐cadherin. Enforced expression of Id‐3 was incapable of invoking E‐cadherin expression in the SCC cells with EMT phenotype, whereas it significantly impaired their invasiveness with down‐regulation of matrix‐metalloproteinase‐2 (MMP‐2) expression. Reporter gene assay revealed that the Ets‐1‐induced MMP‐2 promoter activity was suppressed by the Id‐3, while the Id‐3‐dependent E‐cadherin promoter activity was remarkably reduced in the presence of Snail. Furthermore, knockdown of p63 in SCC cells significantly decreased Id‐3 expression, in which up‐regulation of MMP‐2 expression was concomitant with the acquired invasiveness. These findings propose a particular role of the off‐signaling of the ΔNp63α‐Id‐3 axis incident to Snail‐mediated EMT for the MMP‐2‐dependent invasiveness in SCC cells.

Gene expression profiling to identify genes associated with high-invasiveness in human squamous cell carcinoma with epithelial-to-mesenchymal transition

The process of epithelial-to-mesenchymal transition (EMT) involves the acquisition of high-invasiveness by tumor. Snail represses target genes and induces EMT. In this study, we defined the signatures of gene expressions by cDNA microarray analyses in both human squamous cell carcinoma (SCC) cell lines with spontaneous EMT and with Snail-induced EMT, which exhibited high-invasive behavior in vitro. Of the 17,000 cDNA probes, 61 genes were found differentially expressed with >2- or <0.5-fold ratio and shared among the EMT phenotype cell lines, indicating candidates for invasion-associated genes regulated by Snail. Category analysis showed that these genes were mainly classified as development/differentiation, metabolism, apoptosis, angiogenesis and cell adhesion. These data illustrated that Snail regulates various molecular pathways for the establishment of EMT and the acquisition of high-invasiveness in SCC cells, yielding insight into the progression of SCC.

Expression of cell-cycle-regulating transcription factor E2F-1 in colorectal carcinomas

The expression of E2F-1 in human colorectal carcinomas was examined immunohistochemically, and the correlation of E2F-1 expression with clinicopathological findings and with the expression of p27Kip1 was analyzed to elucidate the role of E2F-1 in the development and progression of colorectal carcinomas. In nonneoplastic mucosa, a small number of epithelial cells in the proliferative zone were weakly positive for E2F-1. Weak expression of E2F-1 was detected in many adenoma cells. Most of the colorectal carcinomas expressed E2F-1 at various levels, and strong expression of E2F-1 was detected in 56% (49/88) of the cases. There was no correlation between the expression of E2F-1 and any clinicopathological parameters such as tumor stage, depth of tumor invasion and lymph node metastasis. Reduced expression of p27Kip1 was confirmed to be significantly correlated with deep tumor invasion and presence of metastasis. No correlation was evident between overexpression of E2F-1 and reduced p27Kip1 expression.

Snail promotes Cyr61 secretion to prime collective cell migration and form invasive tumor nests in squamous cell carcinoma

We previously identified genes associated with Snail-mediated squamous cell carcinoma (SCC) invasiveness, in which we observed significant elevation of Cyr61 expression. In this study, SCC cell lines overexpressing Cyr61 exhibited constitutive activation of Rho A and upregulated invasiveness without the disruption of homophilic cell attachment. Humoral Cyr61 enhanced further production of endogenous Cyr61 by SCC cells, which stimulated collective cell migration and the development of an invasive tumor nest. We propose a Cyr61-dependent model for the development of invasive SCC nest, whereby a subset of tumor cells that highly produce Cyr61 may direct other tumor cells to undergo collective cell migration, resulting in a formation of primary SCC mass.

Effect of 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)- quinolinone (vesnarinone) on the growth of gastric cancer cell lines.

Vesnarinone (OPC-8212; 3,4-dihydro-6-[4-(3,4-dimethoxybenzoyl)-1-piperazinyl]-2(1H)-quinolinone ) is a synthetic oral cardiotonic agent that has been used for the treatment of patients with congestive heart failure. Six days of treatment with 30 microg/ml of vesnarinone induced 20-80% growth inhibitions in five out of six gastric carcinoma cell lines examined. Cell cycle analysis revealed that the vesnarinone-sensitive TMK-1 gastric cancer cell line exhibited a significant G0-G1 arrest without evidence of apoptotic cell death induction after 48 h of treatment. Interestingly, this phenomenon was preceded by a marked reduction in the expression of cyclin A, D1 and E as well as cyclin-dependent kinase 2 (CDK2). On the other hand, no significant change was observed in the expression of p21(Waf1/Cip1), p27Kip1 nor various growth factors and their receptor genes. Overall these results indicate that vesnarinone inhibits the growth of gastric cancer cells by down-regulating G1 cyclins and CDK2 to induce G0-G1 arrest through a pathway different from that of cyclin inactivation by p21(Waf1/Cip1) or p27Kip1.

Autocrine galectin-1 promotes collective cell migration of squamous cell carcinoma cells through up-regulation of distinct integrins.

We found that high galectin-1 (Gal-1) mRNA levels were associated with invasive squamous cell carcinoma (SCC) cells that expressed Snail, an epithelial-to-mesenchymal transition (EMT) regulator. Both Gal-1 overexpression and soluble Gal-1 treatment accelerated invasion and collective cell migration, along with activation of cdc42 and Rac. Soluble Gal-1 activated c-Jun N-terminal kinase to increase expression levels of integrins α2 and β5, which were essential for Gal-1 dependent collective cell migration and invasiveness. Soluble Gal-1 also increased the incidence of EMT in Snail-expressing SCC cells; these were a minor population with an EMT phenotype under growing conditions. Our findings indicate that soluble Gal-1 promotes invasiveness through enhancing collective cell migration and increasing the incidence of EMT.

AKT primes snail‐induced EMT concomitantly with the collective migration of squamous cell carcinoma cells

In this study, we found that wounding of a confluent monolayer of squamous cell carcinoma (SCC) cells induced epithelial–mesenchymal transition (EMT) specifically at the edge of the wound. This process required the combined stimulation of TGFβ, TNFα, and PDGF‐D. Such a combined cytokine treatment of confluent monolayers of the cells upregulated the expression levels of Snail and Slug via PI3K. The PI3K downstream effector, AKT, was dispensable for the upregulation of Snail and Slug, but essential for enabling EMT in response to upregulation of Snail and Slug. J. Cell. Biochem. 114: 2039–2049, 2013.

PGE2 targets squamous cell carcinoma cell with the activated epidermal growth factor receptor family for survival against 5-fluorouracil through NR4A2 induction

We found a linear correlation between the Prostaglandin E(2) (PGE(2)) amount and the NR4A2 expression in oral squamous cell carcinoma (SCC) tissues through a statistical analysis among 41 clinical cases. In SCC cell lines, PGE(2) receptor (EP) ligation by exogenous PGE(2) promoted the NR4A2 expression in the cAMP/protein kinase A (PKA)-dependent manner. The process required a nature of SCC cell represented by constitutive activated epidermal growth factor receptor (EGFR) family. Targeted inactivation of the EGFRs interfered the PGE(2)-dependent NR4A2 expression. The PGE(2)-dependent NR4A2 induction is essential for the resistance to anti-cancer drug-induced apoptosis especially in SCC cells which showed constitutive EGFRs activity via autocrine epiregulin, a ligand for EGFRs. Conversely, SCC cells which lack epiregulin expression in their nature could gain the ability to promote the NR4A2 expression in response to PGE(2) and attain the resistance to anti-cancer drug-induced apoptosis under the existence of exogenous epiregulin. These findings suggest that susceptibility of SCC to anti-cancer drug could be compromised when PGE(2) was delivered in the microenvironment of SCC cells supported by constitutive EGFR family activities as their nature.