Effects of overexpression of eIF4E and erbB2 on gene expression proflies and invasive phenotype in human cancer cells.
Bray, Isabella (2006) Effects of overexpression of eIF4E and erbB2 on gene expression proflies and invasive phenotype in human cancer cells. PhD thesis, Dublin City University.
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Regulation of specific gene expression at the translation level mediated by eukaryotic initiation factor 4E (eIF4E) may play a pivotal role in both tumor formation and metastasis. Non-invasive MCF7 cells, and mildly-invasive DLKP cells were transfected with wild-type eIF4E, eIF4Emut (mutated at serine 209; serine has been replaced with alanine to prevent phosphorylation) and pcDNA (empty plasmid). Up-regulation of eIF4E protein was observed in eIF4E and eIF4E-mutant clones. Increased growth-rate was observed in MCF74E/4Emut and DLKP4E/4Emut compared to the pcDNA clone and parent cell lines. A marked increase in invasion was also observed in DLKP4E and 4Emut clones compared to parent and DLKPpcDNA, but not in the transfected MCF7 clones. MCF74E and MCF74Emut had a greater tendency to grow in suspension, and form colonies in soft agar than parental MCF7. To examine genes related to invasion in a breast cancer cell line, MCF7 cloneH3 (non-invasive) and MCF7H3erbB2 (invasive, erbB2 overexpressing) also were examined.
Whole genome expression microarray experiments and subsequent analysis resulted in gene lists comparing DLKP4E/4Emut to parental DLKP and related to invasion; another specific to MCF7H3erbB2 (compared to non-invasive MCF7, MCF7H3, MCF7pcDNA /4E/4Emut) and related to invasion. A combination of genes with and without previously reported connections to invasion were chosen from each list following application of pathway analysis/literature mining programs to the data. Targets based on analysis of DLKP4E/4Emut had no effect on the rate of invasion of either cell line when individually silenced using siRNA. However, siRNA silencing of EGR1, RPS6KA3, TFPI1 and TNFAIP8, all up-regulated in MCF7erbB2 compared to the parent, caused a decrease in invasion of both invasive DLKP4E and invasive SKBR3 (breast carcinoma) cell lines. THBS1, down-regulated in MCF7H3erbB2 compared to the parent, caused an increase in invasion of mildly-invasive DLKP parent, and non-invasive MCF7 when silenced by siRNA.
This study resulted in the identification of some of the genes involved in development of in vitro invasion, and extended the knowledge of known invasion-related genes. It also provided data on what alterations may occur in cancer cells at the mRNA level if eIF4E levels are altered.
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