Yi-Shing Shieh

Axl promotes cell invasion by inducing MMP-9 activity through activation of NF-|[kappa]|B and Brg-1

Activity of the Axl receptor tyrosine kinase is positively correlated with tumor metastasis; however, its detailed role in the mechanism of tumor invasion is still not completely understood. Here, we show that Axl enhances the expression of matrix metalloproteinase 9 (MMP-9), required for Axl-mediated invasion both in vitro and in vivo. We found that the highly selective MEK1/2 inhibitors U0126 and PD98059, and the expressed dominant-negative form of extracellular signal-regulated kinase (ERK), completely block Axl-mediated MMP-9 activation. In contrast, the phosphatidylinositol 3-kinase inhibitor LY294002 and wortmannin had little effect on activation. Interestingly, however, the Axl ligand Gas6 is not involved in Axl-mediated MMP-9 activation. Mutation of Glu59Axl and Thr77Axl dramatically reduced Gas6–Axl binding but continued to induce MMP-9 activation. In addition, overexpression of Axl-activated ERK and enhanced nuclear factor-κB (NF-κB) transactivation and brahma-related gene-1 (Brg-1) translocation. Exposure to the NF-κB inhibitor silibinin, which inhibits IκBα kinase activity, or overexpression of the dominant-negative mutant IκB and Brg-1 strikingly inhibited Axl-mediated MMP-9 activation. These data indicate that coordination of ERK signaling and NF-κB and Brg-1 activation are indispensable to regulation of Axl-dependent MMP-9 gene transcription. Together with previous data, our results provide a plausible mechanism for Axl-mediated tumor invasion and establish a functional link between the Axl and MMP-9 signaling pathways.

Intratumoral Macrophage Counts Correlate With Tumor Progression in Colorectal Cancer

The role of intratumoral tumor‐associated macrophages (TAMs) in colorectal cancer (CRC) is not clear. We aim to examine the relationships of TAMs and the clinicopathologic features of CRC and the expression of matrix metalloproteinases (MMP)‐2 and MMP‐9.

Epidermal growth factor receptor regulates β-catenin location, stability, and transcriptional activity in oral cancer

BackgroundMany cancerous cells accumulate β-catenin in the nucleus. We examined the role of epidermal growth factor receptor (EGFR) signaling in the accumulation of β-catenin in the nuclei of oral cancer cells.ResultsWe used two strains of cultured oral cancer cells, one with reduced EGFR expression (OECM1 cells) and one with elevated EGFR expression (SAS cells), and measured downstream effects, such as phosphorylation of β-catenin and GSK-3β, association of β-catenin with E-cadherin, and target gene regulation. We also studied the expression of EGFR, β-catenin, and cyclin D1 in 112 samples of oral cancer by immunostaining. Activation of EGFR signaling increased the amount of β-catenin in the nucleus and decreased the amount in the membranes. EGF treatment increased phosphorylation of β-catenin (tyrosine) and GSK-3β(Ser-(9), resulting in a loss of β-catenin association with E-cadherin. TOP-FLASH and FOP-FLASH reporter assays demonstrated that the EGFR signal regulates β-catenin transcriptional activity and mediates cyclin D1 expression. Chromatin immunoprecipitation experiments indicated that the EGFR signal affects chromatin architecture at the regulatory element of cyclin D1, and that the CBP, HDAC1, and Suv39h1 histone/chromatin remodeling complex is involved in this process. Immunostaining showed a significant association between EGFR expression and aberrant accumulation of β-catenin in oral cancer.ConclusionsEGFR signaling regulates β-catenin localization and stability, target gene expression, and tumor progression in oral cancer. Moreover, our data suggest that aberrant accumulation of β-catenin under EGFR activation is a malignancy marker of oral cancer.

DNA methylation and histone modification regulate silencing of epithelial cell adhesion molecule for tumor invasion and progression

Epithelial cell adhesion molecule (Ep-CAM) is believed to have a critical role in carcinogenesis and cell proliferation. However, the association of Ep-CAM with cancer invasion and progression is less clear. We found that Ep-CAM was highly expressed on low-invasive cells compared with highly invasive cells. Forced expression of Ep-CAM decreased cancer invasiveness, and silencing Ep-CAM expression elevated cancer invasiveness. Ep-CAM expression was associated with promoter methylation. Treatment with a demethylating agent, and/or the histone deacetylase inhibitor reactivated Ep-CAM expression in Ep-CAM-negative cells and inhibited cancer invasiveness. Using a promoter–reporter construct, we demonstrated methylation of the promoter fragment drive Ep-CAM-silenced transcription. Additionally, silenced Ep-CAM gene in cancer cells was enriched for hypermethylated histone 3 lysine 9. When unmethylated and active, this promoter was associated with acetylated histone 3 lysine 9. Furthermore, we observed an increased association of Ep-CAM promoter with repression components as tumor invasiveness increased. In cancer tissues, Ep-CAM expression significantly correlated with tumor progression and associated with promoter methylation. Our data support the idea that modulation of Ep-CAM plays a pivotal role in tumor invasion and progression. Moreover, aberrant DNA methylation of Ep-CAM is implicated in enhancing invasive/metastatic proclivity of tumors.

Plasma protein growth arrest-specific 6 levels are associated with altered glucose tolerance, inflammation, and endothelial dysfunction.

OBJECTIVE Plasma protein growth arrest–specific 6 (Gas6) is important to the inflammatory process and is involved in the development of diabetic renal and vascular complications. We set out to determine whether plasma Gas6 levels are associated with altered glucose tolerance, insulin sensitivity, inflammation, and endothelial dysfunction. RESEARCH DESIGN AND METHODS A total of 278 adults, including 96 with normal glucose tolerance (NGT), 82 with impaired glucose tolerance (IGT), and 100 with type 2 diabetes were recruited. Plasma Gas6 concentration and biochemical, proinflammatory, and endothelial variables were determined. Insulin sensitivity was examined by homeostasis model assessment. RESULTS Plasma Gas6 concentration was significantly lower among patients with type 2 diabetes compared with subjects with NGT (P < 0.001). The plasma Gas6 value was inversely correlated with fasting glucose, tumor necrosis factor (TNF)-α, interleukin (IL)-6, and vascular cell adhesion molecule (VCAM)-1. In multivariate logistic regression analysis, after adjustment for established diabetes risk factors, higher plasma Gas6 concentrations were significantly associated with a decreased risk of type 2 diabetes. Moreover, the association became slightly stronger after further adjustment for TNF-α, IL-6, high-sensitive C-reactive protein, E-selectin, intercellular adhesion molecule-1, and VCAM-1. CONCLUSIONS Plasma Gas6 is associated with altered glucose tolerance, inflammation, and endothelial dysfunction. It also may represent a novel independent risk factor of type 2 diabetes and a potential surrogate marker of inflammation and endothelial dysfunction.

CITED2 functions as a molecular switch of cytokine-induced proliferation and quiescence.

Transforming growth factor-α (TGF-α)-induced proliferation and transforming growth factor-β (TGF-β)-mediated quiescence are intricately balanced in normal lung-tissue homeostasis but are deregulated during neoplastic progression of lung cancer. Here, we show that Cbp/p300-interacting transactivator with Glu/Asp-rich carboxy-terminal domain 2 (CITED2), a novel MYC-interacting transcriptional modulator, responds to TGF-α induction and TGF-β suppression to orchestrate cellular proliferation and quiescence, respectively. Upon TGF-α induction, CITED2 was induced by MYC and further modulated MYC-mediated transcription in a feed-forward manner. CITED2 recruited p300 to promote MYC-p300-mediated transactivation of E2F3, leading to increased G1/S cell cycle progression. Moreover, CITED2 inhibited cellular quiescence by enhancing MYC-mediated suppression of p21CIP1. CITED2 interacted with histone deacetylase 1 (HDAC1) and potentiated MYC–HDAC1 complex formation. TGF-β stimulation provoked downregulation of CITED2, which abrogated MYC-HDAC1-mediated p21CIP1 suppression, causing cellular quiescence. Ectopic CITED2 expression enhanced tumor growth in nude mice; furthermore, CITED2 knockdown caused tumor shrinkage and increased overall host mouse survival rates. Expression of CITED2/MYC/E2F3/p21CIP1 signaling molecules was associated with poor prognosis of lung cancer patients. Thus, CITED2 functions as a molecular switch of TGF-α and TGF-β-induced growth control, and MYC-CITED2 signaling axis provides a new index for predicting clinical outcome.

The involvement of promoter methylation and DNA methyltransferase-1 in the regulation of EpCAM expression in oral squamous cell carcinoma.

Epithelial cell adhesion molecule (EpCAM) is important for cell proliferation and differentiation but mechanisms regulating their expression are unclear. Because EpCAM may play a role in carcinogenesis, we investigated the clinicopathologic significance of its expression in oral squamous cell carcinoma (OSCC) and the involvement of DNA methylation machinery in regulation of EpCAM expression during tumorigenesis. Immunohistochemical staining for EpCAM expression and DNA methyltransferase-1 (DNMT1) was done in 112 OSCC cases. Tumor genomic DNA was extracted and EpCAM promoter methylation was examined by methylation-specific polymerase chain reaction in 72 OSCC specimens. Immunoreactivity and methylation were correlated with clinicopathologic features. EpCAM expression was undetectable in normal epithelium; high expression was observed in 51% (57/112) of OSCC. Heterogeneity and plasticity of EpCAM expression was observed during tumor development. Allele methylation was found in 51% (37/72) of OSCC cases analyzed. EpCAM expression was associated with promoter methylation (p = 0.008). However, EpCAM expression and promoter methylation did not correlate with clinicopathologic OSCC variables. DNMT1 expression was occasionally observed in basal cells of normal epithelium; high expression was observed in 47% (53/112) of OSCC. DNMT1 did not correlate with EpCAM expression or methylation status. High DNMT1 expression correlated with tumor size (p < 0.0001) histologic differentiation (p = 0.012) and clinical stage (p < 0.0001) of OSCC. EpCAM expression increased during development of OSCC. EpCAM promoter methylation is associated with EpCAM expression levels in OSCC, suggesting an epigenetically mediated regulation of EpCAM expression. Increased DNMT1 protein expression may be involved in histogenesis and progression of OSCC.

DNA methyltransferase 1 expression and promoter methylation of E-cadherin in mucoepidermoid carcinoma

Loss of E‐cadherin expression is found frequently in many types of human malignancies, including mucoepidermoid carcinoma (MEC). CpG methylation in the promoter region has proven important in the regulation of gene expression implicated in malignant transformation. DNA methyltransferases (DNMTs) are the major enzymes involved in establishing genomic methylation patterns. The current study was designed to test the hypothesis that CpG methylation of the promoter region of the E‐cadherin gene may inactivate its expression and to examine DNMT 1 (DNMT1) protein expression in MEC.

Continued root formation after replantation and root canal treatment in an avulsed immature permanent tooth: a case report

This case report describes the continued root formation following replantation and conventional root canal therapy of a traumatically avulsed open-apex tooth with suppurative apical periodontitis. A 7-year-old male patient had an avulsed upper left central incisor (tooth 21) replanted approximately 50 min after traumatic avulsion. A root canal procedure was initiated due to pulp necrosis and periapical abscess detected in the follow-up period. After endodontic treatment with calcium hydroxide (Ca(OH)(2)) dressing, a normal root length developed including an apical segment beyond the hard tissue barrier. Regeneration of the root occurred without pathology or ankylosis at 1-year of follow up.

The growth arrest-specific 6 (Gas6) gene polymorphism c.834+7G>A is associated with type 2 diabetes

AIMS The plasma protein growth arrest-specific 6 (Gas6) is important to the inflammatory process and involved in the development of diabetic renal and vascular complications. Recently, Gas6 protein also represents a novel independent risk factor of type 2 diabetes. We further investigated the association of c.843+7G>A Gas6 polymorphism and type 2 diabetes. METHODS A total of 278 adults, including 96 with normal glucose tolerance (NGT), 82 with impaired glucose tolerance (IGT), and 100 with type 2 diabetes were recruited. All subjects were genotyped for c.843+7G>A Gas6 polymorphism. RESULTS Plasma Gas6 concentrations were significantly lower among patients with type 2 diabetes compared to subjects with IGT and NGT. Subjects with Gas6 c.843+7AA genotype had higher Gas6 levels and lower glucose values than GG genotype. The AA genotype and A allele were less frequent in patients with type 2 diabetes compared with NGT subjects. In univariate analysis, the AA genotype was found to be associated with a decreased risk for type 2 diabetes. Moreover, the association was even stronger after adjustment for established diabetes risk factors. CONCLUSIONS The Gas6 c.843+7AA genotype and A allele are less prevalent in type 2 diabetes, which may have a protective role for type 2 diabetes.