Re-expression of SPR1 in breast cancer cells by phorbol 12-myristate 13-acetate (PMA) or UV irradiation is mediated by the AP-1 binding site in the SPR1 promoter

Background: Invasive tumor cells are characterized by multiple phenotypic c hanges as a result of the large number of cDNAs being differentially expres sed in tumor cells compared to normal progenitors. Expression genetics focu ses on changes at the RNA level with the aim of identifying functionally im portant genes whose aberrant expression in cancer cells is regulated at the level of transcription. These genes were named class II genes and are dist inguished from class I genes, which are characterized by genomic mutations, deletions, or other alterations. Reversal of the tumor cell phenotype acco mpanying normalization of the expression of such genes may be exploited the rapeutically if gene expression can be specifically modulated by drugs or o ther treatments. Considering that genes are coordinately regulated in compl ex networks, it is Likely that the expression of multiple genes can be simu ltaneously modulated in tumor cells by drugs acting on the signal transduct ion pathway that regulates their expression. The SPR1 gene is associated wi th differentiation and its expression is down-regulated or inactivated in m alignant cells. Analysis of the SPR1 promoter showed that down-regulation o f SPR1 expression in breast tumor cells occurs at the level of transcriptio n. SPR1 presents an example of class II genes, since its expression was up- regulated in tumor cells by phorbol 12-myristate 13-acetate (PMA) or by ult raviolet (UV) irradiation. Materials and Methods: The SPR1 gene was identified by differential display on the basis of its reduced or absent expression in human breast tumor cel l lines compared to normal mammary epithelial cell strains. Differential ex pression was confirmed by Northern blot analysis employing multiple normal and tumor cell lines. The promoter region -619 to +15 of the SPR1 gene was sequenced and analyzed by CAT assays, deletion analysis, and mutagenesis. U p-regulation of SPR1 expression by PMA and UV irradiation was monitored by Northern analysis and analyzed by CAT assays. Results: The mechanism of down-regulation of SPR1 expression in breast tumo r cells was investigated. It was found that the -619 to +15 upstream promot er region is sufficient for SPR1 expression in normal breast cells, but it is transcriptionally silent in most breast tumor cell lines. By deletion an alysis and mutagenesis, two upstream cis-acting promoter elements were iden tified. Our data indicate that the AP-1 element located between -139 and -1 33 acts as a major enhancer of SPR1 transcription only in normal mammary ep ithelial cells but not in corresponding tumor cells, whereas the sequences flanking the AP-1 site do not affect its promoter enhancing activity. In ad dition, a transcriptional repressor was identified that binds unknown facto r(s) and is active in both normal and tumor breast cells. Inhibitor functio n was mapped to a 35-bp element located from -178 to -139 upstream of the h uman SPR1 mRNA start site. The expression of SPR1 could be induced in the 2 1MT-2 metastatic breast tumor cell line by PMA treatment or by short UV irr adiation via a transcriptional mechanism. AP-1 is the cis element mediating the transcriptional activation of SPR1 by PMA, which induces the expressio n of AP-1 factors in 21MT-2 cells. Mutation of the AP-1 site abolishes the induction of SPR1 expression by PMA. Conclusions: Our results demonstrate that loss of SPR1 expression in breast tumor cells results from impaired transactivation through the AP-1 site in the SPR1 promoter, as well as from the presence of a negative regulatory e lement active in both normal and tumor cells. Furthermore, our results prov ide a basis for therapeutic manipulation of down-regulated genes, such as S PR1, in human cancers.