Prolactin inhibits BCL6 expression in breast cancer through a Stat5a-dependent mechanism

Abstract

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.

Figure 1

Prolactin suppression of BCL6 protein and mRNA in human breast cancer lines. (A) Immunoblot of representative time-course showing protein levels of BCL6, pY-Stat5, Stat5 and GAPDH in T47D and SKBr3 cells treated with or without prolactin for up to 48h. (B) Corresponding densitometry of BCL6 and pY-Stat5 (three experiments) normalized to GAPDH loading controls. (C) Time-course of BCL6 mRNA levels in T47D and SKBr3 cells (three experiments) in response to prolactin by qRT-PCR. Values are normalized to untreated control cells. Inset: prolactin stimulated CISH mRNA and reduced BCL6 mRNA by agarose gel. (D) BCL6 mRNA levels in ZR75.1 and MCF7 human breast cancer lines 24h post prolactin stimulation.

Figure 2

Prolactin inhibits BCL6 expression via Stat5a but not Stat5b, ERK or AKT pathways. (A) qRT-PCR analyses of BCL6 mRNA in SKBr3 cells treated with or without prolactin with or without ERK (U0126) or AKT (LY294002) inhibitor (top panel). Densitometry (middle panels) and immunoblotting (bottom panel) showing BCL6, pY-Stat5, ERK, p-ERK, AKT, p-AKT protein levels in SKBr3 cells in the absence or presence of prolactin with or without U0126 or LY294002. (B) Stat5a is required for prolactin suppression of BCL6. qRT-PCR assay showing relief of BCL6 mRNA suppression in cells expressing Stat5a shRNA (5a2) but not Stat5b shRNA (5b6) (top panel). Densitometry (three experiments; middle panel) and a representative immunoblot (bottom panel) of BCL6, Stat5a, Stat5b and GAPDH proteins in SKBr3 cells expressing shRNA targeting either Stat5a (5a2 or 5a3) or Stat5b (5b3 or 5b6) or a non-target control shRNA (NT) using lentiviral delivery (* P<0.001 compared to levels in prolactin treated cells exposed to non-target (NT) control shRNA). (C) qRT-PCR analyses of BCL6 or CISH mRNA levels in SKBr3 cells overexpressing Stat5a (top left), Stat5b (top right), Stat5a-Δ713, Stat5a-S710F (bottom) using adenoviral gene delivery and 16h later treated with or without prolactin for 6h.

Figure 3

Stat5 directly interacts with BCL6 regulatory region in T47D breast cancer cells. (A) Graphic representation of BCL6 regulatory Region B containing four GAS sites. (B) Chromatin immunoprecipitation enriched for CISH and BCL6 regulatory regions in prolactin-stimulated cells, coinciding with increased pY-Stat5 levels. (C) Corresponding qPCR analyses of BCL6 and CISH regulatory sequences purified by anti-Stat5 chromatin immunoprecipitation from T47D cells treated with or without prolactin. (D) Luciferase reporter assays showing reconstitution of prolactin suppression of BCL6 regulatory region upon genomic integration of reporter gene into T47D cells.

Figure 4

BCL6 blocks prolactin-induced Stat5 target gene expression in breast cancer. (A) Luciferase assays demonstrating that overexpression of BCL6 abolished prolactin-stimulated β-casein and CIS reporter genes in T47D cells. (B) Corresponding immunoblots of BCL6 and pY-Stat5 proteins. (C) Luciferase assays demonstrating that BCL6 overexpression abolished prolactin induction of β-casein and CIS reporters in MDA-MB-231 cells expressing prolactin receptor and Stat5a.

Figure 5

Prolactin suppression of BCL6 expression in vivo. (A) Representative immunohistochemistry of pY-Stat5 and BCL6 proteins in T47D xenotransplant tumors in mice treated with or without prolactin for 48h. (B) Corresponding qRT-PCR analyses of BCL6 mRNA levels in T47D xenograft tumors. (C) Immunohistochemistry of two surgical human breast cancer tissue explants treated ex vivo with or without prolactin (100 nM) for 1h. Specimen 1 but not Specimen 2 is responsive to prolactin stimulation as measured by pY-Stat5 induction. (D) qRT-PCR quantified CISH and BCL6 mRNA levels in response to prolactin in Specimens 1 and 2.

Figure 6

AQUA immunohistochemical quantification of BCL6 and pY-Stat5 protein levels in human breast tissues. (A) Representative immunofluorescent images of normal human breast tissue or primary breast cancer stained for pY-Stat5 (red) or BCL6 (red) and cytokeratin (green) and DNA (blue). Case 1, Case 2 and normal tissue were selected from a progression series to demonstrate a range of BCL6 and pY-Stat5 levels. (B) AQUA quantification of pY-Stat5 and BCL6 levels in a progression array of normal, DCIS and primary invasive breast cancer (grades 1–3), and metastases (left panel). Correlation analyses of levels of cellular BCL6 protein and nuclear localized pY-Stat5 in the human breast tissues (right panel). (C) Correlation analyses between levels of cellular BCL6 protein and nuclear Stat5a protein (left) or nuclear Stat5b protein (right).