Hideo Shigeishi

Expression of receptors for advanced glycation end‐products (RAGE) is closely associated with the invasive and metastatic activity of gastric cancer

The receptor for advanced glycation end‐products (RAGE) is a newly recognized factor regulating cancer cell invasion and metastasis. This study investigated the expression of RAGE in gastric carcinomas and its association with invasion and metastasis. Of eight gastric cancer cell lines examined, seven constitutively expressed RAGE messenger ribonucleic acid (mRNA), MKN45 being the exception. RAGE protein expression of MKN28 cells treated with RAGE antisense S‐oligodeoxynucleotide was nine times less than that of sense S‐oligodeoxynucleotide‐treated cells. Growth of cells under RAGE antisense S‐oligodeoxynucleotide treatment was not different from that seen under sense S‐oligodeoxynucleotide treatment in MKN28 (a cell line producing high levels of RAGE) and MKN45 (a non‐RAGE‐expressing cell line). RAGE antisense S‐oligodeoxynucleotide treatment suppressed the invasive activity of RAGE‐positive MKN28 cells, as estimated by in vitro invasion assay. The number of MKN28 cells invading the type IV collagen‐coated membrane under RAGE antisense S‐oligodeoxynucleotide treatment was significantly lower than under RAGE sense S‐oligodeoxynucleotide treatment (p<0.0001). In contrast, antisense and sense S‐oligodeoxynucleotide‐treated RAGE‐negative MKN45 cells showed no difference. A wound‐healing assay showed that no RAGE antisense S‐oligodeoxynucleotide‐treated MKN28 cells migrated into the scraped area, whereas sense S‐oligodeoxynucleotide‐treated cells showed many budding nests in the scraped area. Immunohistochemistry of gastric carcinoma tissue showed that 62 (65%) of the 96 cases examined were RAGE‐positive and that poorly differentiated adenocarcinomas preferentially expressed RAGE protein (38/42, 90%) (p<0.0001). Strong RAGE immunoreactivity was also correlated with depth of invasion and lymph node metastasis (p<0.0001). RAGE‐positive cancer cells tended to be distributed at the invasive front of primary tumours and were detected in all metastatic foci in lymph nodes. In contrast, a major RAGE ligand, amphoterin, was expressed in 82 (85%) of the 96 cases, regardless of histological type and disease progression. RAGE expression appears to be closely associated with invasion and metastasis in gastric cancer. Copyright

Promoter hypermethylation of MGMT is associated with protein loss in gastric carcinoma

Aberrant methylation of CpG islands within promoter regions is associated with transcriptional inactivation of various tumor suppressor genes in neoplasms. Recently, O6‐methylguanine‐DNA methyltransferase, MGMT, was shown to be hypermethylated in certain carcinomas, resulting in loss of MGMT protein. We studied DNA methylation of CpG islands of the MGMT gene by methylation specific PCR in 26 gastric carcinoma tissues and 8 gastric carcinoma cell lines for comparison with levels of MGMT protein expression. In addition, we examined p53 mutation status in the same tissues by PCR‐SSCP analysis for comparison with MGMT protein expression levels. In total, promoter hypermethylation of the MGMT gene was found in 8 (31%) of the 26 gastric carcinomas with reduced expression of MGMT protein, whereas the hypermethylation was not detected in the 18 carcinomas with non‐reduced MGMT expression. MGMT protein expression levels were associated with promoter hypermethylation of MGMT (p = 0.0001; Mann‐Whitney test); however, MGMT expression was not associated with p53 mutation status (p = 0.461; Mann‐Whitney test). Among in gastric carcinoma cell lines, the TMK‐1 cell line showed loss of the MGMT protein association with promoter hypermethylation and this loss was rectified by treatment with a demethylating agent, 5‐Aza‐2′‐deoxycytidine. Our results suggest that transcriptional inactivation of MGMT by aberrant methylation of the promoter region may participate in carcinogenesis in the stomach.

Snail-Induced Down-Regulation of ΔNp63α Acquires Invasive Phenotype of Human Squamous Cell Carcinoma

p63 is a member of the p53 family and regulates crucial events in the formation of epithelial structures, but the role of p63 in tumor is unclear. We found that Snail-induced epithelial-to-mesenchymal transition (EMT) is accompanied by down-regulation of p63 in human squamous cell carcinomas (SCC). DeltaNp63alpha is the predominantly expressed p63 isoform in SCC cells. DeltaNp63 promoter activity required a CAAT/enhancer binding protein (C/EBP) binding element and was reduced remarkably by Snail. Down-regulation of DeltaNp63alpha and reduction of C/EBPalpha were observed in EMT phenotype cells, which exhibited invasive activity in vitro. p63 knockdown in cells enhanced invasive activity in the presence of E-cadherin. Conversely, forced expression of DeltaNp63alpha blocked invasive activity of cells with the EMT phenotype. These findings indicate that Snail down-regulates DeltaNp63alpha, leading to acquisition of the invasive phenotype by SCC. The invasive activity caused by down-regulation of DeltaNp63alpha does not require down-regulation of E-cadherin.

Influence of factors related to implant stability detected by wireless resonance frequency analysis device

Resonance frequency analysis (RFA) was introduced as a method for measuring implant stability more than a decade ago. Implant stability quotient (ISQ) values obtained using a recently introduced wireless RFA device have made it possible to evaluate stability in a non-invasive technique; however, there are few studies of the factors that affect ISQ values determined using this device. The aim of the present study was to evaluate the association between ISQ values determined by wireless RFA and various factors related to dental implant stability using a pig cortical bone model. Dental implants (Replace) Select Tapered implants) with a length of 10 mm were placed into pig cortical bone samples, then, ISQ values were determined using wireless RFA under various conditions (probe orientation, diameter of implant, insertion torque and peri-implant bone loss). The results of this study showed that ISQ values were not affected by the direction of the probe from parallel to perpendicular to the long axis of the pig bone or to the smart peg. In addition, the diameter of the implant did not have a significant effect on the measured ISQ values. Statistically significant correlations were found between insertion torque and ISQ values (Spearmans test, P < 0.05), and lower ISQ values were observed for deeper peri-implant vertical defects (Mann-Whitney U-test, P < 0.05). A wireless RFA device appears to be useful for measuring implant stability within the limits of the present in vitro study.

RHAMM/ERK interaction induces proliferative activities of cementifying fibroma cells through a mechanism based on the CD44–EGFR

We have previously established immortalized cells (HCF) from cementifying fibroma of the jaw bone. Here, we found that the receptor for hyaluronan (HA)-mediated motility (RHAMM) and epiregulin, a ligand for the epidermal growth factor receptor (EGFR), were highly expressed in HCF cells in comparison with osteoblasts by conducting a microarray analysis. The cell growth of HCF cells was significantly decreased by the knockdown of RHAMM using small interfering RNA (siRNA). RHAMM was associated with extracellular signal-regulated kinase (ERK) and essential for ERK phosphorylation. HCF cells had characteristic growth mechanisms in which epiregulin functions in an extracellular autocrine loop. Interestingly, exogenous HA induced the phosphorylation of EGFR, which was mainly dependent on CD44. The results raise the novel idea that the EGFR may activate Raf–MEK–ERK signaling in response to the binding of HA to CD44. Moreover, RHAMM was able to associate with TPX2 in the nucleus and was required for HA-induced activation of the Aurora A kinase. The results suggest that RHAMM has a predominant role in the cell cycle in HCF. Here, we report the new machinery by which RHAMM/ERK interaction induces the proliferative activity of cementifying fibroma cells via a specific signaling pathway through the CD44–EGFR axis.

Expression of Bub1 Gene Correlates with Tumor Proliferating Activity in Human Gastric Carcinomas

Bub1 plays an important role at the spindle assembly checkpoint to prevent cell cycle progression following spindle damage. We examined the expression of human Bub1 mRNA in 20 gastric carcinoma tissues and corresponding nonneoplastic mucosas by reverse transcriptase-polymerase chain reaction and analyzed the relation with proliferative activity monitored by the expression of proliferating cell nuclear antigen (PCNA) on Western blotting as well as Ki-67 labeling index by immunohistochemistry. Increased expression of Bub1 mRNA was detected in 8 (40%) of the gastric carcinomas in comparison with their nonneoplastic counterparts, while 4 (20%) expressed Bub1 at lower levels. The expression of Bub1 mRNA was confirmed by in situ hybridization. The expression levels of Bub1 mRNA were well correlated with the levels of PCNA protein in 16 (80%) gastric carcinoma cases. The examination of Ki-67 labeling indices proved the close correlation between the expression levels of Bub1 and proliferating activity. These findings suggest that mRNA expression of human Bub1 gene is closely associated with the tumor-proliferating activity. Since genetic alterations of human Bub1 rarely occur in gastrointestinal cancers, the functional machinery of Bub1 to prevent cell cycle progression into anaphase might be well preserved in gastric carcinomas even with high proliferative activity.

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.

Maintenance of stem cell self‐renewal in head and neck cancers requires actions of GSK3β influenced by CD44 and RHAMM

Cells sorted from head and neck cancers on the basis of their high expression of CD44 have high potency for tumor initiation. These cells are also involved in epithelial to mesenchymal transition (EMT) and we have previously reported that cancer stem cells (CSCs) exist as two biologically distinct phenotypes. Both phenotypes are CD44high but one is also ESAhigh and maintains epithelial characteristics, the other is ESAlow, has mesenchymal characteristics and is migratory. Examining CD44‐regulated signal pathways in these cells we show that CD44, and also RHAMM, act to inhibit phosphorylation of glycogen synthase kinase 3β (GSK3β). We show that inhibitory phosphorylation reduces the formation of both “tumor spheres” and “holoclone” colonies, functional indicators of stemness. GSK3β inhibition also reduces the expression of stem cell markers such as Oct4, Sox2, and Nanog and upregulates expression of the differentiation markers Calgranulin B and Involucrin in the CD44high/ESAhigh cell fraction. Transition of CSCs out of EMT and back to the epithelial CSC phenotype is induced by GSK3β knockdown. These results indicate that GSK3β plays a central role in determining and maintaining the phenotypes and behavior of CSCs in vitro and are likely to be involved in controlling the growth and spread of tumors in vivo.

Regulation of CXCL9/10/11 in Oral Keratinocytes and Fibroblasts

Th1 and Th2 cytokines such as interferon-γ (IFN-γ ) , tumor necrosis factor- α (TNF-α ), and IL-4 are expressed in T-cell-mediated inflammation in the oral cavity. We tested the hypothesis that those cytokines may act on CXCR3-agonistic chemokines, T-cell recruiting factors, and on neighboring cells, including oral keratinocytes and fibroblasts. Human immortalized oral keratinocytes (RT7) and fibroblasts (GT1) after 24-hour stimulation with IFN-γ showed increased mRNA levels of CXCL9 (600- and 700-fold), CXCL10 (10,000- and 150-fold), and CXCL11 (5000- and 300-fold), respectively. In contrast, TNF-α caused an increase in CXCL9 (300-fold), CXCL10 (2000-fold), and CXCL11 (2000-fold) mRNA levels in GT1, but not RT7 cells, at 24 hrs. IL-4 reinforced the promotion of CXCL9, CXCL10, and CXCL11 expression by IFN-γ in RT7 cells, whereas IL-4 inhibited the increased levels by IFN-γ and TNF-α in GT1 cells. Thus, IFN-γ , TNF-α , and IL-4 appear cooperatively to regulate CXCR3-agonistic chemokines in oral keratinocytes and fibroblasts in T-cell-mediated oral inflammation sites.

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.