Koichiro Higashikawa

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.

Nodal Lymphangiogenesis and Metastasis Role of Tumor-Induced Lymphatic Vessel Activation in Extramammary Paget's Disease

Nodal lymphangiogenesis promotes distant lymph node (LN) metastasis in experimental cancer models. However, the role of nodal lymphangiogenesis in distant metastasis and in the overall survival of cancer patients remains unknown. Therefore, we investigated mechanisms that might facilitate regional and distant LN metastasis in extramammary Pagets disease (EMPD). We retrospectively analyzed the impact of tumor-induced lymphatic vessel activation on the survival of 116 patients, the largest cohort with EMPD studied to date. Nodal lymphangiogenesis was significantly increased in metastatic, compared with tumor-free, LNs (P = 0.022). Increased lymphatic invasion within regional LNs was significantly associated with distant metastasis in LN (P = 0.047) and organs (P = 0.003). Thus, invasion within regional LNs is a powerful indicator of systemic tumor spread and reduced patient survival in EMPD (P = 0.0004). Lymphatic vessels associated with tumors expressed stromal cell-derived factor-1 (SDF-1), whereas CXCR4 was expressed on invasive Paget cells undergoing epithelial-mesenchymal transition (EMT)-like process. A431 cells overexpressing Snail expressed increased levels of CXCR4 in the presence of transforming growth factor-beta1. Haptotactic migration assays confirmed that Snail-induced EMT-like process promotes tumor cell motility via the CXCR4-SDF-1 axis. Sinusoidal lymphatic endothelial cells and macrophages expressed SDF-1 in subcapsular sinuses of lymph nodes before Paget cell arrival. Our findings reveal that EMT-related features likely promote lymphatic metastasis of EMPD by activating the CXCR4-SDF-1 axis.

Evaluation of CD44 transcription variants in human digestive tract carcinomas and normal tissues

We generated DNA probes for CD44 variable region exons II (v6) to 14 (v9) and also for intronic sequences and examined the expression of aberrant CD44 transcripts in digestive tract carcinomas, colorectal adenomas and intestinal metaplasia of the stomach. The study used the reverse transcription‐polymerase chain reaction/Southern blot technique. Among the probes generated, the CD44 intron 9 probe was the best for distinguishing cancer tissue from normal tissue in adenocarcinomas of the colorectum or stomach. All the colorectal adenocarcinomas revealed overexpression of CD44 variants containing the intron 9 sequence compared with the corresponding normal colorectal mucosa. The overexpression of such abnormal CD44 variants was observed from an early stage in colorectal cancer and did not correlate with nodal or distant metastatic status. In intestinal metaplasia of the stomach, aberrant CD44 transcripts with characteristic 2 or 3 peaks containing intron 9 were observed. On the other hand, in oral or esophageal squamous cell carcinomas and corresponding normal squamous epithelia, overexpression of CD44 variants with variable exons and retained intron 9 sequence was frequently observed in both cancer tissue and corresponding normal mucosa. Examination of the expression of CD44 variants in a screening panel of normal tissues from the whole body revealed that overexpression of transcripts containing exon 11 to 14 as well as of the intron 9 sequence was constantly observed in tissues with squamous epithelia like skin, oral mucosa, esophagus and uterine cervix. Expression of these variants was also found in urinary bladder, respiratory tract, pancreas and salivary glands. Our results overall indicate that detecting the overexpression of abnormal CD44 transcripts, especially ones containing the intron 9 sequence, could be a powerful indicator for the presence of adenocarcinomas in the digestive tract. However, it is not applicable for the diagnosis of malignancies originating from squamous epithelia.

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.

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.

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.

A Case of SAPHO Syndrome With Diffuse Sclerosing Osteomyelitis of the Mandible Treated Successfully With Prednisolone and Bisphosphonate

*Department of Oral and Maxillofacial Surgery, Division of Cervico Gnathostomatology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. †Department of Oral and Maxillofacial Surgery, Division of Cervico Gnathostomatology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. ‡Department of Oral and Maxillofacial Surgery, Division of Cervico Gnathostomatology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. §Department of Oral and Maxillofacial Surgery, Division of Cervico Gnathostomatology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. Department of Oral and Maxillofacial Surgery, Division of Cervico Gnathostomatology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. ¶Department of Clinical Immunology and Rheumatology, Hiroshima University Hospital, Hiroshima, Japan. #Department of Dermatology, Division of Molecular Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. **Department of Oral and Maxillofacial Surgery, Division of Cervico Gnathostomatology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. ††Department of Surgery, Division of Clinical Medical Science, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. ‡‡Department of Oral and Maxillofacial Surgery, Division of Cervico Gnathostomatology, Graduate School of Biomedical Sciences, Hiroshima University, Hiroshima, Japan. Address correspondence and reprint requests to Dr Shigeishi: Department of Oral and Maxillofacial Surgery, Division of CervicoGnathostomatology, Graduate School of Biomedical Sciences, Hirod

Overexpression of receptor for hyaluronan-mediated motility (RHAMM) in MC3T3-E1 cells induces proliferation and differentiation through phosphorylation of ERK1/2

Receptor for hyaluronan (HA)-mediated motility (RHAMM) was first described as a soluble HA binding protein released by sub-confluent migrating cells. We previously found that RHAMM was highly expressed and plays an important role in proliferation in the human cementifying fibroma (HCF) cell line, which we previously established. HCF is a benign fibro-osseous neoplasm of the jaw and is composed of fibrous tissue containing varying amounts of mineralized material. However, the pathogenesis of HCF is not clear. In this paper, we examined the roles of RHAMM in osteoblastic cells. We generated RHAMM-overexpressing MC3T3-E1 cells and examined the cell proliferation and differentiation of osteoblastic cells. In MC3T3-E1 cells, overexpressing RHAMM was located intracellular and activated ERK1/2. Interestingly, the ERK1/2 activated by RHAMM overexpression promoted cell proliferation and suppressed the differentiation of osteoblastic cells. Our findings strongly suggest that RHAMM may play a key role in the osteoblastic differentiation process. The rupture of balance from differentiation to proliferation induced by RHAMM overexpression may link to the pathogenesis of bone neoplasms such as HCF.

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.