Molecular profiling of transformed and metastatic murine squamous carcinoma cells by differential display and cDNA microarray reveals altered expression of multiple genes related to growth, apoptosis, angiogenesis, and the NF-kappa B signal pathway

To identify changes in gene expression with transformation and metastasis, we investigated differential gene expression in a squamous carcinoma model established in syngeneic mice, We used mRNA differential display (DD) to de tect global differences and cDNA arrays enriched for cancer-associated gene s using mRNA from primary keratinocytes, transformed Pam 212 squamous carci noma cells, and metastases of Pam 212, After DD, 72 candidate cDNAs express ed primarily in transformed and metastatic cells were selected and cloned, Fifty-seven were detected, and 32 were confirmed to be differentially expre ssed by Northern blot analysis. mRNA expression profiles were also generate d using a mouse cDNA array composed of 4000 elements representing known gen es and expressed sequence tags plus the 57 DD candidate cDNAs detected by N orthern analysis to facilitate data validation. cDNA array detected 76.9% o f the differentially expressed mRNAs selected from DD and confirmed by Nort hern blot, whereas low-abundance mRNAs did not reach the threshold for dete ction by the lower-sensitivity array method. Clustering analysis of Do and array results from transformed and metastatic cells identified genes that e xhibited decreased or increased expression with transformation and metastas is, Alterations in the expression of several genes detected during tumor pr ogression were consistent with their functional activities involving growth (p21, p27, and cyclin DI), resistance and apoptosis (glutathione-S-transfe rase, cIAP-1, PEA-15, and Fas ligand), inflammation and angiogenesis [chemo kine growth-regulated oncogene 1 (also called KC)], and signal transduction (c-Met, yes-associated protein, and syk). Strikingly, 10 of 22 genes in th e cluster expressed in metastases have been associated with activation of t he nuclear factor (NF)-kappaB signal pathway. The NF-kappaB-inducible cytok ine Gro-1 was recently shown to promote tumor growth, metastasis, and angio genesis of squamous cell carcinomas in vivo (Loukinova et at, Oncogene, 19: 3477-3486, 2000), The results demonstrate that early response genes relate d to NF-kappaB contribute to metastatic tumor progression. Comparison of ce ll lines and tumor tissue revealed a concordance of similar to 50% by array , and 70% for Northern-confirmed, metastasis-related genes. Functional geno mic approaches comparing expression among cell lines and tumor tissue may p romote a better understanding of the genes expressed by malignant and host cells during tumor progression and metastasis.