Nobuyuki Kamata

Reverse correlation of E-cadherin and snail expression in oral squamous cell carcinoma cells in vitro

The loss of E-cadherin expression has been shown to correlate to the invasion and metastasis of many types of carcinomas. We established E-cadherin positive (HOC719-PE) and negative (HOC719-NE) clones from an oral squamous cell carcinoma (SCC). HOC719-PE cells showed epithelial morphology with E-cadherin expression in the cell membrane, whereas HOC719-NE cells demonstrated fibroblastic morphology without E-cadherin expression. In invasion assay and three dimensional culture, HOC719-NE showed much higher invasive ability than HOC719-PE cells. These cells expressed similar levels of mRNAs for alpha- and beta-catenin. However, HOC719-NE cells, but not HOC719-PE cells, showed strong expression of snail, a transcription factor implicated in the differentiation of epithelial cells into mesenchymal phenotype. This reverse expression of snail and E-cadherin was further observed in other SCC cells including HOC313, and TSU cells that we previously reported to show no expression of E-cadherin protein. These results indicated that the expression of snail has a key role for the acquisition of more invasive and metastatic phenotypes of SCC and the clones we reported here will be useful tools for understanding the mechanism of the transition from epithelial to mesenchymal SCC cells.

Recessive Mutations in the Putative Calcium-Activated Chloride Channel Anoctamin 5 Cause Proximal LGMD2L and Distal MMD3 Muscular Dystrophies

The recently described human anion channel Anoctamin (ANO) protein family comprises at least ten members, many of which have been shown to correspond to calcium-activated chloride channels. To date, the only reported human mutations in this family of genes are dominant mutations in ANO5 (TMEM16E, GDD1) in the rare skeletal disorder gnathodiaphyseal dysplasia. We have identified recessive mutations in ANO5 that result in a proximal limb-girdle muscular dystrophy (LGMD2L) in three French Canadian families and in a distal non-dysferlin Miyoshi myopathy (MMD3) in Dutch and Finnish families. These mutations consist of a splice site, one base pair duplication shared by French Canadian and Dutch cases, and two missense mutations. The splice site and the duplication mutations introduce premature-termination codons and consequently trigger nonsense-mediated mRNA decay, suggesting an underlining loss-of-function mechanism. The LGMD2L phenotype is characterized by proximal weakness, with prominent asymmetrical quadriceps femoris and biceps brachii atrophy. The MMD3 phenotype is associated with distal weakness, of calf muscles in particular. With the use of electron microscopy, multifocal sarcolemmal lesions were observed in both phenotypes. The phenotypic heterogeneity associated with ANO5 mutations is reminiscent of that observed with Dysferlin (DYSF) mutations that can cause both LGMD2B and Miyoshi myopathy (MMD1). In one MMD3-affected individual, defective membrane repair was documented on fibroblasts by membrane-resealing ability assays, as observed in dysferlinopathies. Though the function of the ANO5 protein is still unknown, its putative calcium-activated chloride channel function may lead to important insights into the role of deficient skeletal muscle membrane repair in muscular dystrophies.

The Novel Gene Encoding a Putative Transmembrane Protein Is Mutated in Gnathodiaphyseal Dysplasia (GDD)

Gnathodiaphyseal dysplasia (GDD) is a rare skeletal syndrome characterized by bone fragility, sclerosis of tubular bones, and cemento-osseous lesions of the jawbone. By linkage analysis of a large Japanese family with GDD, we previously mapped the GDD locus to chromosome 11p14.3-15.1. In the critical region determined by recombination mapping, we identified a novel gene (GDD1) that encodes a 913-amino-acid protein containing eight putative transmembrane-spanning domains. Two missense mutations (C356R and C356G) of GDD1 were identified in the two families with GDD (the original Japanese family and a new African American family), and both missense mutations occur at the cysteine residue at amino acid 356, which is evolutionarily conserved among human, mouse, zebrafish, fruit fly, and mosquito. Cellular localization to the endoplasmic reticulum suggests a role for GDD1 in the regulation of intracellular calcium homeostasis.

Down-regulation of Wnt-4 and up-regulation of Wnt-5a expression by epithelial-mesenchymal transition in human squamous carcinoma cells.

Gene expression of Wnt‐1, 2, 3, 4, 5a, 6 and 7a was analyzed by RT‐PCR in eleven squamous cell carcinoma (SCC) cell lines and compared with that in two normal oral keratinocyte strains. There appeared to be an inverse relationship between Wnt‐4 and Wnt‐5a expressions, i.e., Wnt‐4 was not expressed in HOC719‐NE, HOC313 or TSU cells, while Wnt‐5a was strongly expressed only in these cells. These cell lines showed decreased expression of E‐cadherin and elevated expression of vimentin accompanied with strong expressions of Snail and δEF1, which have been reported to be transrepressors of E‐cadherin and to trigger epithelial‐mesenchymal transition (EMT), suggesting associations of Wnt‐4 with epithelial phenotype and Wnt‐5a with mesenchymal phenotype of SCC cells. To study whether the expressions of these Wnt genes are regulated by EMT, we transfected a Snailexpression vector into A431 and OM1 cells, which express Wnt‐4 but not Wnt‐5a. The stably Snail‐overexpressing clones showed spindle morphology, increased expression of vimentin and decreased expression of E‐cadherin accompanied with augmented expression of δEF1. In these clones, down‐regulation of Wnt‐4 and up‐regulation of Wnt‐5a were clearly observed. These results indicated that Wnt‐4 and Wnt‐5a are oppositely affected by EMT, and down‐regulation of Wnt‐4 and up‐regulation of Wnt‐5a are possible markers of the malignant phenotype of human SCC.

Reg IV is a serum biomarker for gastric cancer patients and predicts response to 5-fluorouracil-based chemotherapy

Regenerating gene family, member 4 (Reg IV), a secreted protein, is overexpressed in several cancers, including gastric cancer (GC). In the present study, we measured Reg IV levels in sera from patients with GC by enzyme-linked immunosorbent assay. We also examined the effect of forced Reg IV expression on the apoptotic susceptibility to 5-fluorouracil (5-FU). Forced expression of Reg IV inhibited 5-FU-induced apoptosis. Induction of Bcl-2 and dihydropyrimidine dehydrogenase was involved in inhibition of apoptosis. Among 36 GC patients treated with a combination chemotherapy of low-dose 5-FU and cisplatin, all 14 Reg IV-positive patients showed no change or disease progression. The serum Reg IV concentration was similar between healthy individuals (mean±s.e., 0.52±0.05 ng/ml) and patients with chronic-active gastritis (0.36±0.09 ng/ml). However, the serum Reg IV concentration in presurgical GC patients was significantly elevated (1.96±0.17 ng/ml), even at stage I. The diagnostic sensitivity of serum Reg IV (36.1%) was superior to that of serum carcinoembryonic antigen (11.5%) or carbohydrate antigen 19-9 (13.1%). These results indicate that expression of Reg IV is a marker for prediction of resistance to 5-FU-based chemotherapy in patients with GC. Serum Reg IV represents a novel biomarker for GC.

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.

Genetic analyses in patients with familial isolated hyperparathyroidism and hyperparathyroidism–jaw tumour syndrome

Background  A subset of familial isolated primary hyperparathyroidism (FIHP) is a variant of hyperparathyroidism–jaw tumour syndrome (HPT‐JT).

Histone H3 acetylation is associated with reduced p21WAF1/CIP1 expression by gastric carcinoma

Histone acetylation appears to play an important role in transcriptional regulation. Inactivation of chromatin by histone deacetylation is involved in the transcriptional repression of several tumour suppressor genes, including p21WAF1/CIP1. However, the in vivo status of histone acetylation in human cancers, including gastric carcinoma, is not well understood. This study shows that histone H3 in the p21WAF1/CIP1 promoter region is hypoacetylated and that this hypoacetylation is associated with reduced p21WAF1/CIP1 expression in gastric carcinoma specimens. Chromatin immunoprecipitation assays revealed that histone H3 was hypoacetylated in the p21WAF1/CIP1 promoter and coding regions in 10 (34.5%) and 10 (34.5%) of 29 gastric carcinoma specimens, respectively. Hypoacetylation of histone H4 in the p21WAF1/CIP1 promoter and coding regions was observed in 6 (20.7%) and 16 (55.2%) of 29 gastric carcinoma specimens, respectively. p21WAF1/CIP1 mRNA levels were associated with histone H3 acetylation status in the p21WAF1/CIP1 promoter region (p = 0.047) but not p53 mutation status (p = 0.460). In gastric carcinoma cell lines, expression of p21WAF1/CIP1 protein was induced by trichostatin A, a histone deacetylase inhibitor. This induction was associated with hyperacetylation of histone H3 in the p21WAF1/CIP1 promoter region. Hyperacetylation of histone H4 in the p21WAF1/CIP1 promoter region did not appear to be associated with increased expression. Induction of p21WAF1/CIP1 protein expression was associated with hyperacetylation of histones H3 and H4 in the p21WAF1/CIP1 coding region. Expression of a dominant‐negative mutant of p53 reduced expression of p21WAF1/CIP1 protein. Histone H4 acetylation in both the promoter and coding regions of the p21WAF1/CIP1 gene in cells expressing dominant‐negative p53 was less than half of that in cells expressing wild‐type p53, whereas histone H3 acetylation in both the promoter and coding regions was slightly reduced (by approximately 20%) in cells expressing the dominant‐negative p53. These findings provide evidence that alteration of histone acetylation occurs in human cancer tissue specimens such as those from gastric carcinoma. Copyright

The FGFR1 inhibitor PD173074 induces mesenchymal–epithelial transition through the transcription factor AP-1

Background:Epithelial–mesenchymal transition (EMT) is a crucial process in cancer progression that provides cancer cells with the ability to escape from the primary focus, invade stromal tissues and migrate to distant regions. Cell lines that lack E-cadherin show increased tumorigenesis and metastasis, and the expression levels of E-cadherin and Snail correlate inversely with the prognosis of patients suffering from breast cancer or oral squamous cell carcinoma (OSCC). Moreover, recent studies have shown that most EMT cases are regulated by soluble growth factors or cytokines. Among these factors, fibroblast growth factors (FGFs) execute diverse functions by binding to and activating members of the FGF receptor (FGFR) family, including FGFR1–4. Fibroblast growth factor receptor 1 is an oncoprotein that is involved in tumorigenesis, and PD173074 is known to be a selective inhibitor of FGFR1. However, the roles of FGFR1 and FGFR1 inhibitors have not yet been examined in detail.Methods:Here, we investigated the expression of FGFR1 in head and neck squamous cell carcinoma (HNSCC) and the role of the FGFR1 inhibitor PD173074 in carcinogenesis and the EMT process.Results:Fibroblast growth factor receptor 1 was highly expressed in 54% of HNSCC cases and was significantly correlated with malignant behaviours. Nuclear FGFR1 expression was also observed and correlated well with histological differentiation, the pattern of invasion and abundant nuclear polymorphism. Fibroblast growth factor receptor 1 was also overexpressed in EMT cell lines compared with non-EMT cell lines. Furthermore, treatment of HOC313 cells with PD173074 suppressed cellular proliferation and invasion and reduced ERK1/2 and p38 activation. These cells also demonstrated morphological changes, transforming from spindle- to cobble stone-like in shape. In addition, the expression levels of certain matrix metalloproteinases (MMPs), whose genes contain activator protein-1 (AP-1) promoter sites, as well as Snail1 and Snail2 were reduced following PD173074 treatment.Conclusion:Taken together, these data suggest that PD173074 inhibits the MAPK pathway, which regulates the activity of AP-1 and induces MET. Furthermore, this induction of MET likely suppresses cancer cell growth and invasion.

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.