The present study is aimed at identifying potential candidate genes as prognostic markers in human oral tongue squamous cell carcinoma (SCC) by large scale gene expression profiling. The gene expression profile of patients (n=37) with oral tongue SCC were analyzed using Affymetrix HG_U95Av2 high-density oligonucleotide arrays. Patients (n=20) from which there were available tumor and matched normal mucosa were grouped into stage (early vs. late) and nodal disease (node positive vs. node negative) subgroups and genes differentially expressed in tumor vs. normal and between the subgroups were identified. Three genes, GLUT3, HSAL2, and PACE4, were selected for their potential biological significance in a larger cohort of 49 patients via quantitative real-time RT-PCR. Hierarchical clustering analyses failed to show significant segregation of patients. In patients (n=20) with available tumor and matched normal mucosa, 77 genes were found to be differentially expressed (P< 0.05) in the tongue tumor samples compared to their matched normal controls. Among the 45 over-expressed genes, MMP-1 encoding interstitial collagenase showed the highest level of increase (average: 34.18 folds). Using the criterion of two-fold or greater as overexpression, 30.6%, 24.5% and 26.5% of patients showed high levels of GLUT3, HSAL2 and PACE4, respectively. Univariate analyses demonstrated that GLUT3 over-expression correlated with depth of invasion (P<0.0001), tumor size (P=0.024), pathological stage (P=0.009) and recurrence (P=0.038). HSAL2 was positively associated with depth of invasion (P=0.015) and advanced T stage (P=0.047). In survival studies, only GLUT3 showed a prognostic value with disease-free (P=0.049), relapse-free (P=0.002) and overall survival (P=0.003). PACE4mRNA expression failed to show correlation with any of the relevant parameters. The characterization of genes identified to be significant predictors of prognosis by oligonucleotide microarray and further validation by real-time RT-PCR offers a powerful strategy for identification of novel targets for prognostication and treatment of oral tongue carcinoma.

The mechanisms by which c‐erbB‐dependent signaling contribute to the invasive potential of HNSCC remain to be fully elucidated. We have previously shown that c‐erbB autocrine and/or paracrine stimulation upregulates MMP‐9 but has no effect on the related gelatinase, MMP‐2. BTC, a major c‐erbB ligand, has the ability to efficiently activate all c‐erbB receptors and to bind directly to EGFR and c‐erbB‐4. BTC is commonly expressed in HNSCC cells and exerts the most potent effects in terms of MMP induction relative to other c‐erbB ligands so far tested. In the present study, we explored the contribution of major downstream events triggered by BTC/c‐erbB receptor signaling to the regulation of MMP‐9 and in vitro invasiveness of HNSCC cells. In human HNSCC cell lines, SIHN‐006 and Detroit‐562, BTC treatment resulted in rapid tyrosine phosphorylation of all c‐erbB receptors whereas both endogenous MMP‐9 and BTC‐stimulated MMP‐9 were predominantly mediated via EGFR. BTC induced ERK1/2, JNK/SAPK and Akt phosphorylation with differing kinetics but not p38 kinase. The BTC‐dependent activation of JNK and PI3K/Akt pathways occurred predominantly via EGFR, whereas activation of the MEK‐1/ERK pathway occurred via all 4 c‐erbB receptors, although again predominantly via EGFR. Selective inhibition of ERK/MAPK (by PD98059 or U0126) and PI3K (by LY294002 or wortmannin) led to marked reduction of both basal and BTC‐induced MMP‐9 activity and invasive ability of HNSCC cells. In contrast, inhibition of p38 kinase with SB203580 produced no such effects. A specific inhibitor of NF‐κB, BAY 11‐7085, also blocked the stimulatory effect of BTC. No remarkable inhibition of MMP‐9 and invasion was observed on targeting other cellular activities, such as PKA, PKC and PLC‐γ. Taken together, our data show that BTC induces MMP‐9 production and invasion primarily through activation of EGFR, MAPK and PI3K/Akt in HNSCC cells.

Breast cancer is the most common cancer in females and the leading cause of cancer death in women worldwide. Angiogenesis, the formation of new blood vessels, plays an important role in the development and progression of breast cancer. Vascular endothelial growth factor A (VEGFA), the key modulator of angiogenesis, is highly expressed in cancer tissue and correlates with its more aggressive features. Polymorphisms of VEGFA alter the levels of expression and subsequently influence the susceptibility and aggressiveness of breast cancer. Assessment of VEGFA polymorphisms may be used for the identification of patients suitable for anti-VEGFA therapy.

Matrix metalloproteinases (MMPs) play an important role in several steps of cancer development. A single guanine insertion polymorphism (2G) in the MMP1 promoter sequence at −1,607 creates an Ets binding site and thus results in enhancing transcriptional activity. This study aimed to evaluate the contribution of this 2G polymorphism on susceptibility and aggressiveness of HNSCC. A panel of HNSCC cell lines and peritumoral fibroblasts were examined for the MMP1 genotypes and expression levels. Genomic DNA was extracted from peripheral blood of 300 patients with newly diagnosed HNSCC and from 300 age- and gender-matched cancer-free controls. Genotyping was carried out using a PCR-RFLP assay. The levels of MMP1 mRNA expression were evaluated by the quantitative RT-PCR and a correlation with different genotype was determined. Odds ratio (OR) for cancer risk were calculated using multivariate logistic regression. In addition, a correlation between the 2G/2G genotype and clinicopathological parameters was examined. Eleven out of 18 HNSCC cell lines showed the 2G/2G genotype (61%) and only 1 cell line had the 1G/1G genotype (5.6%). Cell lines with the 2G/2G genotype expressed significantly higher mean MMP1 mRNA level than those with other genotypes. In clinical model, subjects carrying the homozygous 2G/2G genotype had a higher risk of head and neck cancer compared with subjects with other genotypes (adjusted OR: 2.28; 95% CI: 1.58–3.27), controlling for major confounders. A correlation between promoter polymorphisms and the levels of MMP1 expression in cancer tissues was found, and this 2G/2G genotype was correlated with the adverse clinicopathological parameters. Finally, the highest level of MMP1 enhancement was demonstrated in the coculture of tumor cells and peritumoral fibroblasts of 2G homozygotes. These findings suggest that the presence of 2G polymorphism at the MMP1 promoter is associated with the development and progression of HNSCC.