Wei-Fan Chiang

Galectin-1, a novel ligand of neuropilin-1, activates VEGFR-2 signaling and modulates the migration of vascular endothelial cells

Galectin-1 (Gal-1), a homodimeric prototype of the galectins with a single carbohydrate-recognition domain, was recently identified as being overexpressed in tumor-associated capillary endothelial cells. The role of Gal-1 in endothelial cellular functions and the mechanism of action of Gal-1 remain unknown. Neuropilin-1 (NRP1) is a neuronal receptor that mediates repulsive growth cone guidance, and NRP1 functions in endothelial cells as a coreceptor (with vascular endothelial growth factor receptors (VEGFRs)) for VEGF165. In this study, we found that Gal-1 was overexpressed in the tumor-associated endothelial cells of oral squamous cell carcinomas (P<0.001). Gal-1 increased the proliferation and adhesion of endothelial cells, and enhanced cell migration in combination with VEGF165. Surprisingly, Gal-1 selectively bound NRP1 via the carbohydrate-recognition domain, but did not bind VEGFR-1, VEGFR-2 or VEGFR-3. The Gal-1–NRP1 interaction mediated the migration and adhesion of endothelial cells. The binding of Gal-1 to NRP1 enhanced VEGFR-2 phosphorylation and stimulated the activation of the mitogen activated protein (MAP) kinases SAPK1/JNK (stress activated protein kinase-1/c-Jun NH2-terminal kinase). These findings show, for the first time, that Gal-1 can directly bind to NRP1 on endothelial cells, and can promote the NRP1/VEGFR-2-mediated signaling pathway as well as NRP1-mediated biological activities.

Combinational polymorphisms of four DNA repair genes XRCC1, XRCC2, XRCC3, and XRCC4 and their association with oral cancer in Taiwan.

BACKGROUND Many single nucleotide polymorphisms (SNPs) have been found to be associated with oral cancer but the biological interactions through SNPs are seldom addressed. In this study, we focused on the joint effect for SNP combinations of four DNA repair genes, X-ray repair cross-complementing groups (XRCCs) 1-4, involved in major cancer-related pathways. METHODS Single nucleotide polymorphism genotyping was determined using by polymerase chain reaction-restriction fragment length polymorphism in this study (case = 103, control = 98). Different numbers of combinational SNPs with genotypes called the pseudo-haplotypes from these chromosome-wide genes were used to evaluate their joint effect on oral cancer risk. RESULTS Except for XRCC2 rs2040639-AG, none of these SNPs was found to individually contribute to oral cancer risk. However, for two combined SNPs, the proportion of subjects with oral cancer was significantly higher in the pseudo-haplotype with AG-CC genotypes in rs2040639-rs861539 (XRCC2-XRCC3) compared with those with non-AG-CC genotypes. Similarly, the pseudo-haplotype of rs2040639-rs861539-rs2075685 (XRCC2-XRCC3-XRCC4) and rs2040639-rs861539-rs2075685-rs1799782 (XRCCs 1-4) with specific genotype pattern (AG-CC-TG and CT-AG-CC-TG) among three and four combinational SNPs were significantly associated with oral cancer. After controlling for age, gender, smoking, drinking, and betel nut chewing, the estimated odds ratio of oral cancer were 2.45, 5.03, and 10.10 for two, three and four specific SNP combinations, respectively, comparing these specific pseudo-haplotypes to their corresponding non-pseudo-haplotypes. CONCLUSION We have identified the potential combined XRCCs 1-4 SNPs with genotypes that were associated with oral cancer risk and may have an impact on identification of a high-risk population.

Galectin-1-Mediated Tumor Invasion and Metastasis, Up-Regulated Matrix Metalloproteinase Expression, and Reorganized Actin Cytoskeletons

Galectin-1 (Gal-1) is a β-galactose-binding lectin; its expression level has been reported to correlate with tumor progression. Gal-1 is highly expressed in the invasive front of primary tumors and in the cancer cells of metastatic lesions in the lymph nodes of patients with oral squamous cell carcinoma. However, the molecular mechanism of Gal-1 in tumor metastasis is not completely clear. We found that increased Gal-1 expression is closely associated with its high levels of invasion in lung adenocarcinoma and oral squamous cell carcinoma cell lines. Knocking down Gal-1 with small interfering RNA in highly invasive cancer cells reduced their invasion levels. Moreover, the invasion ability of poorly invasive cancer cells was significantly increased after Gal-1 overexpression of Gal-1. Mechanism studies revealed that Gal-1 promoted tumor invasion mainly by up-regulating matrix metalloproteinase (MMP)-9 and MMP-2 and by reorganizing actin cytoskeleton. Gal-1 enhanced the activation of Cdc42, a small GTPase and member of the Rho family, thus increasing the number and length of filopodia on tumor cells. Furthermore, Gal-1-overexpressing cells had higher metastatic abilities in tail vein metastasis assays in vivo. We conclude that Gal-1 is involved in tumor invasion and metastasis by increasing MMP expression and reorganizing cytoskeletons in oral cancers and lung adenocarcinoma. (Mol Cancer Res 2009;7(3):311–8)

miR-24 up-regulation in oral carcinoma: Positive association from clinical and in vitro analysis

MicroRNAs (miRNAs) play important roles in neoplastic process. miR-24 is localized on chromosome 9q22 and 19p13, regions frequently altered in oral squamous cell carcinoma (OSCC). This study showed that miR-24 was up-regulated in OSCC tissues relative to control samples. In addition, the plasma levels of miR-24 in OSCC patients were significantly higher than in control individuals. miR-24 expression was also higher in OSCC cell lines relative to normal oral keratinocytes. Experiments blocking miR-24 and using exogenous miR-24 expression indicated that miR-24 contributes to the growth of OSCC cells and that miR-24 may target p57. This study suggests that miR-24 is involved in the regulation of OSCC growth and that the miR-24s level in plasma may be validatable as a tumor marker for OSCC patients.

Targeting Galectin-1 in Carcinoma-Associated Fibroblasts Inhibits Oral Squamous Cell Carcinoma Metastasis by Downregulating MCP-1/CCL2 Expression

Purpose: Carcinoma-associated fibroblasts (CAFs) in tumor stroma play an important role in tumor progression and have been associated with a poor prognosis in oral squamous cell carcinoma (OSCC). However, how CAFs influence OSCC malignancy and whether normalizing CAFs inhibits cancer progression remain unclear. Experimental Design: The relationship between the expression of Galectin-1 (Gal-1) and alpha-smooth muscle actin (α-SMA, a CAF marker) in OSCC patient samples and primary cultured CAFs was examined by quantitative real-time PCR, Western blotting, and immunofluorescence. To examine the effect of Gal-1 on CAF activation and CAF-mediated tumor invasion and migration in vitro, Gal-1 expression was knocked down by small hairpin RNA. Finally, cancer cells and CAFs were coimplanted into SCID mice to evaluate the effect of Gal-1 on CAF-modulated tumor progression in vivo. Results: Gal-1 expression is positively associated with α-SMA in the stroma of OSCC specimens. Gal-1 knockdown decreases activated CAF characteristics, resulting in a decrease in α-SMA expression and extracellular matrix protein production. Notably, blocking Gal-1 expression significantly inhibits CAF-conditioned medium-induced tumor cell migration and invasion, possibly by reducing the production of monocyte chemotactic protein-1 (MCP-1/CCL2). MCP-1 induces the migration of OSCC cells by binding to the receptor CCR2; adding an MCP-1 antibody to CAF-conditioned medium that inhibits the interaction between MCP-1 and CCR2 abolishes migration. Finally, we found that Gal-1 knockdown in CAFs significantly reduces CAF-augmented tumor growth and metastasis in vivo. Conclusions: Our findings demonstrate that Gal-1 regulates CAF activation and indicate that targeting Gal-1 in CAFs inhibits OSCC metastasis by modulating MCP-1 expression. Clin Cancer Res; 17(6); 1306–16. ©2011 AACR.

miRNA-491-5p and GIT1 Serve as Modulators and Biomarkers for Oral Squamous Cell Carcinoma Invasion and Metastasis

MicroRNAs offer tools to identify and treat invasive cancers. Using highly invasive isogenic oral squamous cell carcinoma (OSCC) cells, established using in vitro and in vivo selection protocols from poorly invasive parental cell populations, we used microarray expression analysis to identify a relative and specific decrease in miR-491-5p in invasive cells. Lower expression of miR-491-5p correlated with poor overall survival of patients with OSCCs. miR-491-5p overexpression in invasive OSCC cells suppressed their migratory behavior in vitro and lung metastatic behavior in vivo. We defined the G-protein-coupled receptor kinase-interacting protein 1 (GIT1)-as a direct target gene for miR-491-5p control. GIT1 overexpression was sufficient to rescue miR-491-5p-mediated inhibition of migration/invasion and lung metastasis. Conversely, GIT1 silencing phenocopied the ability of miR-491-5p to inhibit migration/invasion and metastasis of OSCC cells. Mechanistic investigations indicated that miR-491-5p overexpression or GIT1 attenuation reduced focal adhesions, with a concurrent decrease in steady-state levels of paxillin, phospho-paxillin, phospho-FAK, EGF/EGFR-mediated extracellular signal-regulated kinase (ERK1/2) activation, and MMP2/9 levels and activities. In clinical specimens of OSCCs, GIT1 levels were elevated relative to paired normal tissues and were correlated with lymph node metastasis, with expression levels of miR-491-5p and GIT1 correlated inversely in OSCCs, where they informed tumor grade. Together, our findings identify a functional axis for OSCC invasion that suggests miR-491-5p and GIT1 as biomarkers for prognosis in this cancer.

Serine protease inhibitor (SERPIN) B1 promotes oral cancer cell motility and is over-expressed in invasive oral squamous cell carcinoma

Lymph node metastasis is the hallmark of malignant neoplasms in patients of oral cancer, accounting for the poor diagnosis and reduced 5-year survival rate. Here we sought to identify cell motility-associated proteins of oral cancer by proteomic approach. We compared the proteomes of two oral cancer cells, CAL-27 and SAS, with the highest and the lowest migration potential, respectively, amongst six different oral cancer cell lines. Subsequent identification of differentially expressed proteins by LC-MS/MS and Western analysis revealed that SERPINB1 (serine protease inhibitor, clade B, member1) was highly expressed in CAL-27, the high-motility oral cancer cells. Semi-quantitative and real-time PCR further confirmed differential expression of SERPINB1 in these two cell lines at mRNA level. To verify the motility-promoting function of SERPINB1 in oral cancer cells, we showed that endogenous expression of SERPINB1 correlated positively with cell migration. Moreover, ectopic expression of SERPINB1 in oral cancer cells, SAS, Ca9-22, CAL-27 and HSC-3, increased cell migration by 25%, 52%, 90% and 100%, respectively. Finally, we found that the expression of SERPINB1 was significantly higher in 5 of 8 (62.5%) oral cancer tissues compared with the matched adjacent normal tissues. Besides, immunohistochemical results indicated over-expression of SERPINB1 in clinicopathologically invasive oral squamous cell carcinoma (OSCC) but not in normal oral mucosa (p<0.01). Together, our findings have provided a possible biomarker for oral cancer metastasis.

Surgical outcomes and prognostic factors of oral cancer associated with betel quid chewing and tobacco smoking in Taiwan

Oral squamous cell carcinoma (OSCC) is one of the most common cancers in geographic regions where betel quid (BQ) chewing is prevalent; OSCC is an extremely malignant neoplasm whose prognostic factors are multiple and complex. The purpose of this study was to assess clinicopathological prognostic factors and treatment outcomes in 698 consecutive OSCC patients who had undergone surgery as the primary treatment in an area with a high prevalence of both betel quid chewing and tobacco smoking. The prognostic factors were predicted using Coxs proportional-hazards regression model, and the survival rate was calculated using Kaplan-Meier analysis. The median followup for all patients was 44 months. The 5-year cumulative overall, disease-specific, and locoregional control survival rates were 61%, 62%, and 46%, respectively. Multivariate analysis showed that the lower level of nodal metastasis, advanced stage, tumor thickness >7 mm, and treatment failures were independent risk factors of overall survival. Furthermore, history of alcohol drinking, lower level of nodal metastasis, advanced stage, poor cell differentiation, and treatment failures were independent predictors of poor disease-specific survival. However, we did not find any significant factor that affected locoregional recurrence. Due to the high frequencies of locoregional recurrence and second primary cancer, our findings emphasize that aggressive surgical excision, adjuvant treatments according to clinicopathological prognostic factors and close surveillance are important to the survival of OSCC patients in an area with a high prevalence of betel quid chewing and tobacco smoking.

Fatty-acid-binding protein 5 promotes cell proliferation and invasion in oral squamous cell carcinoma.

BACKGROUND Oral squamous cell carcinoma (OSCC) is one of the most malignant neoplasms worldwide, and the molecular mechanism of oral tumorigenesis is still unclear. Fatty-acid-binding protein 5 (FABP5) was found in our previous study to be upregulated in oral squamous cell carcinomas by proteomic analysis. The implications of FABP5 overexpression in oral cancer progression have not yet been elucidated. MATERIALS AND METHODS In this study, the recombinant adeno-associated virus vectors were used to deliver and increase the expression of FABP5 in human OSCC cell lines. U6 promoter-driven short-hairpin RNA (shRNA)-triggered RNA interference was used to block FABP5 gene expression. Transwell Matrigel invasion assay, MTS cell proliferation assay, reverse transcription-polymerase chain reaction, Western blot, and gelatin zymography analysis were used to investigate the effects of FABP5 on cell invasion, growth, and matrix metalloproteinase (MMP) production. RESULTS Overexpression of FABP5 in oral cancer cells increased cell proliferation and invasiveness by increasing the expression of MMP-9. Silencing FABP5 with shRNA significantly suppressed cell proliferation, MMP-9 activities, and invasiveness. CONCLUSION Our study provides the first evidence that FABP5 expression modulated MMP-9 production and the invasive behavior of oral cancer cells and suggests that FABP5 may provide novel targets for therapeutic intervention.

Role of SIRT1 in regulation of epithelial-to-mesenchymal transition in oral squamous cell carcinoma metastasis

BackgroundThe epithelial-to-mesenchymal transition (EMT) process results in a loss of cell-cell adhesion, increased cell mobility, and is crucial for enabling the metastasis of cancer cells. Recently, the enzyme SIRT1 has been implicated in a variety of physiological processes; however, its role in regulating oral cancer metastasis and EMT is not fully elucidated. Here, we propose a mechanism by which the enzyme sirtuin1 (SIRT1) regulates the EMT process in oral cancer by deacetylating Smad4 and repressing the effect of TGF-β signaling on matrix metalloproteinase-7 (MMP7).MethodsThe roles of SIRT1 in tumor cell migration/invasion and metastasis to the lungs were investigated using the Boyden chamber assay and orthotopic injections, respectively. RNA interference was used to knockdown either SIRT1 or Smad4 expression in oral squamous cell carcinoma (OSCC) cell lines. Immunoblotting, zymographic assays, and co-immunoprecipitation were used to examine the effects of SIRT1 overexpression on MMP7 expression and activity, as well as on SIRT1/ Smad4 interaction.ResultsWe found that compared with normal human oral keratinocytes (HOKs), SIRT1 was underexpressed in OSCC cells, and also in oral cancer tissues obtained from 14 of 21 OSCC patients compared with expression in their matched normal tissues. Overexpression of SIRT1 inhibited migration of OSCC cells in vitro, as well as their metastasis to the lung in vivo. Furthermore, up-regulation of SIRT1 in metastatic OSCCs significantly inhibited the migration and invasion abilities of OSCC cells, while concomitantly increasing the expression of E-cadherin, and decreasing the expressions of mesenchymal markers. We also identified Smad4, a TGF-β-activated transcription factor, as a direct target protein for SIRT1. Overexpression of SIRT1 in OSCC cells led to decreased levels of acetylated Smad4, and inhibition of TGF-β-induced signaling. By associating and deacetylating Smad4, SIRT1 enzyme can influence MMP7 expression, MMP enzyme activity, and consequently, cell migration, invasion, and tumor metastasis in OSCCs.ConclusionsThese findings provide a valuable insight into the potential role of the SIRT1 enzyme in regulating cell migration and invasion in oral squamous cell carcinoma. Our findings suggest the SIRT1/Smad4/MMP7 pathway as a target for oral cancer driven by EMT.