Identification of metastasis-related genes by DNA microarray analyses on samples generated from a metastatic mouse model of head and neck cancer.

204 To identify genes that mediate metastasis of squamous cell carcinoma of head and neck (SCCHN), a novel SCCHN xenograft mouse model has been established (Zhang et al. Cancer 95:1663, 2002). Using this model we performed in vivo selection from a SCCHN cell line. In the fourth round of in vivo selection, significant incidences of metastases in lymph nodes (7/10) and lungs (6/10) were achieved from a derived SCCHN cell line as compared with its parental cell line (1/5 in lymph nodes and 0/5 in lungs). Highly metastatic SCCHN cell lines were then established from lymph node metastatic tumors. The RNA from 18 metastatic cell lines (M) and 6 parental cell lines (P) isolated from nonmetastatic xenograft tumors were subjected to DNA microarray analysis using an Affymetrix gene chip HG-U133A. GCRMA in Bioconductor (www.bioconductor.org) was used for probe analysis of the microarray data. Statistical analysis (one-way ANOVA), false discovery rate calculation (q-value 1.5) were sequentially performed to select the most significant genes. A total of 5234 genes were identified from 22,123 genes on the HG-U133A gene chip. Ontological classification and k-mean clustering of these genes revealed specific features of the metastatic SCCHN cells that are different from the non-metastatic parental cells, including (1) loss of expression of cell-cell adhesion proteins, such as down-regulation of cellular adhesion molecule E-cadherin [mean signal values (log-base 2): 2.3 (M) vs. 10.4 (P)]; (2) down-regulation of cellular feature proteins, such as a putative tumor suppressor caveolin-1 [2.6 (M) vs. 12 (P)] and a differentiation marker involucrin [2.3 (M) vs. 5.2 (P)], and up-regulation of prostate stem cell antigen [8.9 (M) vs. 3.5 (P)]; (3) up-regulation of signal transducers that induce migration and invasion, such as chemokine (C-X-C) motif receptor 4 [CXCR4, 9.0 (M) vs. 2.1 (P)]; (4) switching expression pattern of small Ca2+ binding proteins, such as enhancement of S100A4 [13.6 (M) vs. 6.3 (P)], but reduction of S100A2 [5.4 (M) vs. 13.9 (P)]; and (5) alterations of expression pattern of several cytokeratins. These genotypic changes support a phenotypic alteration, epithelia-mesenchymal transition, observed between the parental cells and their metastatic derivatives. After experimental validation, these genes as a group can potentially be used as “predictors” for lymph node metastases of SCCHN (Supported by NIH grant R21 DE014767).