Detecting activation of ribosomal protein S6 kinase by complementary DNA and tissue microarray analysis

Background: Studies by comparative genomic hybridization (CGH) have shown t hat chromosomal region 17q23 is amplified in up to 20% of primary breast ca ncers. We used microarray analyses to measure the expression levels of gene s in this region and to explore their prognostic importance. Methods: A mic roarray that contained 4209 complementary DNA (cDNA) clones was used to ide ntify genes that are overexpressed in the MCF-7 breast cancer cell line as compared with normal mammary tissue. Fluorescence in situ hybridization was used to analyze the copy number of one overexpressed gene, ribosomal prote in S6 kinase (S6K), and to localize it to the 17q23 region. Northern and we stern blot analyses were used to measure S6K gene and protein expression, a nd an enzymatic assay was used to measure S6K activity. Tumor tissue microa rray analysis was used to study amplification of S6K and the HER-2 oncogene , another 17q-linked gene, and the relationship between amplification and p rognosis was analyzed, The Kaplan-Meier method was used for data analysis, and the log-rank test was used for statistical analysis. All P values are t wo-sided. Results: S6K was amplified and highly overexpressed in MCF-7 cell s relative to normal mammary epithelium, and protein expression and enzyme activity were increased. S6K was amplified in 59 (8.8%) of 668 primary brea st tumors, and a statistically significant association between amplificatio n and poor prognosis (P = .0021) was observed. Amplification of both S6K an d HER-2 implied particularly poor survival (P = .0001). Conclusions: The co mbination of CGH information with cDNA and tissue microarray analysts can b e used to identify amplified and overexpressed genes and to evaluate the cl inical implications of such genes and genomic rearrangements. S6K is likely to be one of the genes at 17q23 that is amplified during oncogenesis and m ay adversely affect the prognosis of patients with this amplification.