BCL6 is a transcriptional repressor that recognizes DNA target sequences similar to those recognized by signal transducer and activator of transcriptions 5 (Stat5). BCL6 disrupts differentiation of breast epithelia, is downregulated during lactation, and is upregulated in poorly differentiated breast cancer. In contrast, Stat5a mediates prolactin-induced differentiation of mammary epithelia, and loss of Stat5 signaling in human breast cancer is associated with undifferentiated histology and poor prognosis. Here, we identify the mammary cell growth factor prolactin as a potent suppressor of BCL6 protein expression in human breast cancer through a mechanism that requires Stat5a, but not prolactin-activated Stat5b, MEK-ERK, or PI3K-AKT pathways. Prolactin rapidly suppressed BCL6 mRNA in T47D, MCF7, ZR75.1, and SKBr3 breast cancer cell lines, followed by prolonged reduction of BCL6 protein levels within 3 hours. Prolactin suppression of BCL6 was enhanced by overexpression of Stat5a but not Stat5b, was mimicked by constitutively active Stat5a, but did not require the transactivation domain of Stat5a. Stat5 chromatin immunoprecipitation demonstrated physical interaction with a BCL6 gene regulatory region, and BCL6 transcript repression required histone deacetylase activity based on sensitivity to trichostatin A. Functionally, BCL6 overexpression disrupted prolactin induction of Stat5 reporter genes. Prolactin suppression of BCL6 was extended to xenotransplant tumors in nude mice in vivo and to freshly isolated human breast cancer explants ex vivo. Quantitative immunohistochemistry revealed elevated BCL6 in high-grade and metastatic breast cancer compared with ductal carcinoma in situ and nonmalignant breast, and cellular BCL6 protein levels correlated negatively with nuclear Stat5a (r = −0.52; P < 0.001) but not with Stat5b. Loss of prolactin-Stat5a signaling and concomitant upregulation of BCL6 may represent a regulatory switch facilitating undifferentiated histology and poor prognosis of breast cancer.
Prolactin (PRL) receptors (PRLRs) have been considered selective activators of Janus tyrosine kinase (Jak)2 but not Jak1, Jak3, or Tyk2. We now report marked PRL-induced tyrosine phosphorylation of Jak1, in addition to Jak2, in a series of human breast cancer cell lines, including T47D, MCF7, and SKBR3. In contrast, PRL did not activate Jak1 in immortalized, noncancerous breast epithelial lines HC11, MCF10A, ME16C, and HBL-100, or in CWR22Rv1 prostate cancer cells or MDA-MB-231 breast cancer cells. However, introduction of exogenous PRLR into MCF10A, ME16C, or MDA-MB-231 cells reconstituted both PRL-Jak1 and PRL-Jak2 signals. In vitro kinase assays verified that PRL stimulated enzymatic activity of Jak1 in T47D cells, and PRL activated Jak1 and Jak2 with indistinguishable time and dose kinetics. Relative Jak2 deficiency did not cause PRLR activation of Jak1, because overexpression of Jak2 did not interfere with PRL activation of Jak1. Instead, PRL activated Jak1 through a Jak2-dependent mechanism, based on disruption of PRL activation of Jak1 after Jak2 suppression by 1) lentiviral delivery of Jak2 short hairpin RNA, 2) adenoviral delivery of dominant-negative Jak2, and 3) AG490 pharmacological inhibition. Finally, suppression of Jak1 by lentiviral delivery of Jak1 short hairpin RNA blocked PRL activation of ERK and signal transducer and activator of transcription (Stat)3 and suppressed PRL activation of Jak2, Stat5a, Stat5b, and Akt, as well as tyrosine phosphorylation of PRLR. The data suggest that PRL activation of Jak1 represents a novel, Jak2-dependent mechanism that may serve as a regulatory switch leading to PRL activation of ERK and Stat3 pathways, while also serving to enhance PRL-induced Stat5a/b and Akt signaling.
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