To identify putative biomarkers in squamous cell carcinoma (SCC), a survey of parallel chromosomal alterations and gene expression studies in 10 SCC cell lines were performed using array-comparative genomic hybridization (CGH) and oligo-microarray techniques. The most frequent changes were gains of 11q13.1-13.3 and losses of 18q12.1-23 in SCC. Furthermore, the expression levels of the sets of genes at both these loci in SCC were measured using microarray analysis. By combining the array-CGH with the microarray data, 10 genes at 11q13.1-13.3 and 6 genes at 18q12.1-23 whose expression correlated with chromosomal alterations were identified. To verify the expression levels of the identified genes, we used expression analysis data derived from our earlier study of clinical specimens. In clinical samples, six genes (GAL, GSTP1, MRPL11, MRPL21, SF3B2, and YIF1A) at 11q13.1-13.3 and one gene (GALR1) at 18q23 showed a significant difference between normal and tumor samples. GAL, coding for the neuropeptide galanin, and GALR1, a galanin receptor, were identified as candidate genes of oncogenesis in SCC. The expression levels of GAL, GALR1, GALR2, and GALR3 were confirmed by real-time PCR. The expression ratio between GAL and GALR1 showed a significant negative correlation. GALR1 is a G-protein-coupled receptor that activates GTP-binding proteins to trigger signaling cascades such as the mitogen-activated protein kinase pathway, and is a well-established mitogenic pathway. This further supports the hypothesis that the genes involved in the GAL signaling cascade are candidates for regulation of oncogenesis in SCC.

Distant metastasis is a major factor associated with poor prognosis in head and neck squamous cell carcinomas (HNSCC), but little is known of its molecular mechanisms. New markers that predict clinical outcome, in particular the ability of primary tumors to develop metastatic tumors, are urgently needed. Based on a genomewide gene expression analysis using clinical specimens of HNSCC, we narrowed our focus to the analysis of the neurotensin (NTS) and neurotensin receptor 1 (NTSR1) oncogenic signal pathways. Kaplan-Meier curves and log rank tests revealed that high mRNA expression levels of NTS and NTSR1 had a significant adverse effect on metastasis-free survival rate, suggesting a contribution of this pathway in HNSCC cancer progression. In HNSCC cells, which expressed NTSR1, a NTS agonist promoted cellular invasion, migration and induction of several mRNAs, such as interleukin 8 and matrix metalloproteinase 1 transcripts. In addition, knock down of NTSR1 expression with small interfering RNAs resulted in reduction of cellular invasion and migration in HNSCC cell lines. Our findings suggest a critical role for the NTS and NTSR1 oncogenic pathways in invasion and migration of HNSCC cells during the metastatic process. Our study raises the possibility that NTS and NTSR1 could be a useful predictive marker of poor prognosis in patients with HNSCC and a molecular therapeutic target in antimetastatic strategies for HNSCCs. ' 2008 Wiley-Liss, Inc.Key words: head and neck squamous cell carcinomas; microarray; neurotensin; neurotensin receptor 1 Head and neck squamous cell carcinomas (HNSCC) represent 6% of all cancers. 1 Despite of considerable advances in surgery, radiotherapy and chemotherapy, the overall 5-year survival rate for patients with this type of cancer is among the lowest of all major cancer types and has not improved dramatically during the last decade. Local tumor recurrence and distant metastasis after conventional therapy appear to be major contributing factors for restricted survival of HNSCC patients. 1 In particular, distant metastasis represents a major challenge in the therapeutic management of cancer patients. About 60% of patients have microscopic or clinically evident metastases at the time of diagnosis of primary tumors. 2 It is likely that the metastatic rate will be higher in HNSCC because many patients are at an advanced stage of the disease by the time of diagnosis and treatment. Currently, systemic chemotherapy is the major mode of treatment of metastatic diseases. However, this treatment is usually palliative, but not curative in part due to the poor response of metastatic tumors to chemotherapy. 3,4 Thus, identification of prognostic markers and effective treatment regimens for metastasis are urgently needed. Over the past decade, research effort on metastatic disease has been focused on the biological processes that influence establishment of metastases. It has been well established that tumor metastasis is a complex, multistep process that requires migration, invasion and angiogenes...

Abstract. DNA amplifications activate oncogenes and are hallmarks of nearly all advanced cancers including head and neck squamous cell carcinoma (HNSCC). Some oncogenes show both DNA copy number gain and mRNA overexpression. Chromosomal comparative genomic hybridization and oligonucleotide microarrays were used to examine 8 HNSCC cell lines and a plot of gene expression levels relative to their position on the chromosome was produced. Three highly up-regulated genes, NT5C3, ANLN and INHBA, were identified on chromosome 7p14. These genes were subjected to quantitative real-time RT-PCR on cDNA and genomic DNA derived from 8 HNSCC cell lines. ANLN and INHBA showed a strong positive correlation between mRNA expression and genomic DNA levels and a similar relationship was shown for the known oncogene, EGFR, at 7p11.2. In clinical samples, ANLN and INHBA showed a significantly higher expression in tumors than in normal tissues. Patients with high expression levels of INHBA had a shorter diseasefree survival rate. Therefore, INHBA may be a promising prognostic marker of HNSCC.

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