Analysis of the complete sequence of the novel metastasis-associated candidate gene, mtal, differentially expressed in mammary adenocarcinoma and breast cancer cell lines

Abstract

To understand the genes and gene products involved in breast cancer invasion and metastasis, we previously isolated ten differentially expressed genes by differential cDNA library screening techniques, using the 13762NF rat mammary adenocarcinoma metastatic system. In this study, we further analysed a novel candidate metastasis-associated gene, mtal, previously designated clone 10.14. Northern blotting analyses showed that the steady-state mRNA expression level of mtal was fourfold higher in a highly metastatic line (MTLn3) than in a nonmetastatic line (MTC.4). The mtal gene was expressed at low levels in various normal rat organs, except testis, where it was expressed in high amounts. The mRNA expression levels of the human homologue of this gene were also examined in two human breast cancer metastatic systems; the ratios of mRNA were estimated to be MCF-7 (nonmetastatic):MCF7/LCC1 (invasive):MCF7/LCC2 (metastatic)= 1:2:4 and MDA-MB-468 (nonmetastatic):MDA231 (metastatic)= 1:4. Thus, the expression of this gene directly correlated with metastatic potential in two human systems, as well as in the rat metastatic system. Clone 10.14 was used to isolate a full-length cDNA clone for mtal, yielding the clone p10.14-C4.5, which was sequenced and analysed. Clone p10.14-C4.5 was 2756-bp long and contained a single open reading frame that could encode a protein of 703 amino acid (aa) residues. The aa sequence of mtal was found to be novel by database homology search and contained possible phosphorylation sites for tyrosine kinase, protein kinase C and casein kinase II. A Pro-rich stretch was found at the C-terminal end that completely matched the consensus sequence for the SH3-binding motif. Thus, the novel gene mtal may encode an important molecule that is functional in breast cancer invasion and metastasis, and could prove to be a useful probe to assess the malignancy of breast cancers.