Regulation of BRCA1/p21 Axis by Prolactin

Tumor formation/progression is determined by a wide array of factors including downregulation of tumor suppressors and upregulation of oncogenes. In breast cancers, mutation of the tumor suppressor, breast cancer 1 (BRCA1), accounts for the majority of inherited cases, which themselves account for about 10% of the total. Given the importance of BRCA1 in the development of breast cancer and the fact that the majority of breast cancers have wild type BRCA1, the question posed in the current study was what happens to BRCA1 tumor suppressor activity during the development of most breast cancers. To examine this issue, we employed two forms of prolactin that work through the same receptors, differ from one another only by a single amino acid, and have antagonistic activity in terms of cell proliferation and survival. Also, since BRCA1 mutations have recently been shown to be important in ovarian and prostate cancer development, the study additionally included cell lines representative of these cancers. Results show that wild type BRCA1 could be functionally silenced by prolactin in terms of its ability to induce transcription of the cell cycle inhibitor, p21, whereas the prolactin antagonist increased expression of p21 through BRCA1. The functional silencing in response to prolactin occurred as a result of an interaction with the signaling molecule, Stat5 and could be blocked by a dominant negative Stat5. In addition, we showed that prolactin also contributes to tumor progression by regulating p21 at post-transcriptional and post-translational stages. The post-transcriptional regulation was mediated by miRNA. One candidate for this activity is miR-106, a miRNA induced by PRL, likely through an estrogen receptor α (ERα) and c-myc pathway. Prolactin also induced phosphorylation of p21 on threonine 145, causing it to be retained in the cytosol under which circumstances it inhibits apoptosis. At present, the kinase responsible for prolactin-stimulated phosphorylation of p21 is unclear, but preliminary data suggest a possible role for pim-1. The prolactin antagonist employed in these studies is a molecular mimic of naturally phosphorylated prolactin. Given its ability to increase expression of p21 and antagonize the effects of unmodified prolactin, one would predict that the ratio of unmodified prolactin to phosphorylated prolactin may be important to the development/progression of cancer. To test this in a clinical setting, a serum-based assay capable of quantifying phosphorylated prolactin as a separate entity from unmodified prolactin is required. To develop such an assay, regions of the p21 promoter were deleted to determine a minimal promoter responsive to phosphorylated prolactin. A 143bp region containing only the BRCA1 response element and TATA box was determined to be sufficient and specific to the phosphorylated form of the hormone. Establishment of stable cell lines expressing this minimal promoter linked to luciferase has shown the resultant bioassay to be sensitive and consistent