Induction of Blimp1 is mediated via Bcl-2. (A) For the left panels, NF639 cells were transiently transfected with expression vectors for either control shRNA (contl shRNA) or Bcl2 shRNA, and WCEs were analyzed for ERα, Bcl-2 (which confirmed effective knockdown), and β-actin protein levels. For the right panels, ZR-75 and MCF-7 cells were transiently transfected with EV or full-length Bcl-2 expression vector. WCEs were analyzed for ERα and β-actin protein levels and for Bcl-2 (which confirmed the expected changes in levels [data not shown]). (B) NF639 cells were transiently transfected with expression vectors for either control shRNA (contl shRNA), Bcl2 shRNA (upper panels), or EV or full-length Bcl-2 (bottom panels). For the left panels, WCEs were analyzed for Blimp1 and β-actin. For the right panels, RNA was subjected to RT-PCR analysis determine the levels of Blimp1 and Gapdh. (C) RNA (top panels) and nuclear extracts (bottom panels) were prepared from ZR-75 and MCF-7 cells transfected with control EV DNA or vector expressing Bcl-2 and subjected to RT-PCR for PRDM1 and GAPDH expression (upper panels) and Blimp1 and lamin B levels (lower panels), respectively. (D) Stable mixed populations of MCF-7 cells expressing Bcl-2 or pBabe (pB) DNA were transiently transfected with EV DNA (−) or vector expressing dnBlimp (+). Cells were subjected, in triplicate, to a migration assay for 24 h, as in Fig. 4A. The increase in migration induced by Bcl-2 is significantly reduced by dnBlimp, P = 3.8e⁻⁷. Western blot analysis confirmed the expression of the dnBlimp (not shown).

Blimp1 induces a migratory phenotype in breast cancer cells via repression of ERα. (A) NF639 cells were transiently transfected with control siRNA (si-con) or siBlimp1 RNA. (Left panel) Cells were subjected, in triplicate, to a migration assay for 4 h. Cells that migrated to the lower side of the filter were quantified by spectrometric determination of the optical density at 410 nm. The average migration from three independent experiments ± the SD is presented relative to si-con (set at 100%) (*, P = 7.0e⁻⁵). (Right panel) WCEs were subjected to immunoblotting for E-cadherin (E-Cadh), ERα, Blimp1, and β-actin. (B) ZR-75 cells were transiently transfected with 6 μg of EV or Blimp1 expression vector. (Left panel) Transfected ZR-75 cells were subjected, in triplicate, to a migration assay for 24 h, as in panel A. *, P = 0.0007. (Right panel) WCEs were analyzed by immunoblotting for Blimp1, E-cadherin, γ-catenin (γ-Caten), and β-actin. (C) ZR-75 cells were transiently transfected with 6 μg of Blimp1 expression vector or EV DNA in the absence or presence of ERα expression vector (1.5 μg) or EV DNA, as indicated. (Left panel) After 24 h, ZR-75 cells were subjected to migration assays for 24 h, as in panel A. The enhanced migration induced by Blimp1 is significantly decreased by ERα. *, P = 0.0001. (Right panel) After 48 h, WCEs were prepared and analyzed by immunoblotting for E-cadherin, γ-catenin, ERα, Blimp1, and β-actin. (D) ZR-75 and MCF-7 cells were transiently transfected with dnBlimp (+) or EV DNA (−). After 24 h, ERα validated Stealth RNAi (+) or Stealth RNAi negative control (−) were introduced in cells using a Lipofectamine RNAiMAX reagent by reverse transfection as previously described (56). After 24 h, WCEs were prepared and analyzed by immunoblotting for E-cadherin, ERα, and β-actin.

RelB represses ERα synthesis in breast cancer cells. (A) Stable MCF-7 clones expressing either RELB or EV DNA, isolated as described previously (56) and termed RELB(1), RELB(2), EV(1), and EV(2), were transfected with 0.5 μg of ERE-TK luciferase reporter, 0.5 μg of SV40-β-Gal. Normalized ERE activity, set to 100% in the stable EV cells, was decreased in both RelB-expressing lines (mean ± the SD from three separate experiments). (B) ZR-75 cells were transiently transfected with 0.5 μg of ERE-TK luciferase DNA plus 0.5 μg of the indicated NF-κB subunit expression vectors, 0.5 μg of SV40-β-Gal, and EV pcDNA3 DNA to a 3.0 μg of DNA total. Normalized ERE activity, set to 100% in the EV-transfected cells, was reduced by RelB alone or in combination with p50 or p52 (mean ± the SD from three separate experiments). (C) RNA, isolated from EV(1) and RELB(1) stable MCF-7 clones, was subjected to RT-PCR analysis for expression of ERα target genes CATHEPSIN D (CATHD), RARα, pS2, MTA3, and GAPDH, as a loading control. (D) ZR-75 or MCF-7 cells were transiently transfected with 3 μg of each of the vectors expressing RelB and either p50 or p52 or 6.0 μg of EV DNA. Isolated RNA was subjected to RT-PCR analysis for RARα, CATHEPSIN D (CATHD), and GAPDH. (E and F) ZR-75 or MCF-7 cells were transiently transfected with 3.0 μg of vectors expressing RelB, and p50 or p52 or EV DNA. WCEs and RNA were isolated, which were subjected to immunoblot analysis for the expression of ERα and β-actin (E) and to RT-PCR analysis for the expression of ERα and GAPDH (F), respectively. (Samples in panels E and F were run on the same gels, and the lanes were brought into contiguous positions.) (G) ZR-75 and MCF-7 cells were transiently transfected with 0.5 μg of ERα proB luciferase reporter plus 0.25 μg of vectors expressing RelB and p52 or 0.5 μg of EV pcDNA3 DNA, 0.5 μg of SV40-β-Gal, and EV DNA to a 3.0 μg of DNA total. Normalized proB activity values are presented as the means ± the SD from three experiments (control EV DNA set to 1). (H and I). Protein and RNA, isolated from RELB(1), RELB(2), EV(1), and EV(2) stable MCF-7 clones, were subjected to immunoblotting for the expression of ERα, RelB, and β-actin (H), and to RT-PCR analysis for the expression of ERα and GAPDH (I), respectively. (J) RELB(1), RELB(2), EV(1), and EV(2) MCF-7 clones were transiently transfected with 0.5 μg of ERα proB luciferase reporter, 0.5 μg of SV40-β-Gal, and EV DNA to a 3.0 μg of DNA total. Normalized proB activity values are presented as the means ± the SD from three experiments (control EV DNA set to 1).

Induction of Blimp1 is mediated via Bcl-2 and Ras activation. (A) ZR-75 and MCF-7 cells were transiently transfected with EV or vector expressing constitutively active Ras (Ras CA), and nuclear extracts were analyzed for Blimp1 and lamin B. (B) A box plot of data from the Bild_CellLine microarray data set of primary human mammary epithelial cells transfected with the vectors expressing GFP, E2F3, β-catenin (β-cat), c-Myc, c-Src, or H-Ras (reporter number 228964_at) was accessed for PRDM1 mRNA levels by using the Oncomine Cancer Profiling Database and is plotted on a log scale (2). A Student t test, performed directly through the Oncomine 3.0 software, showed that the difference in PRDM1 mRNA levels between the means of the H-Ras versus all others groups combined was significant (*, P = 1.7e⁻¹⁰). (C and D) Stable mixed populations of MCF-7 cells expressing Bcl-2 or pBabe DNA were transiently transfected with 6 μg of vectors expressing Ras WT or Ras C186S, as indicated. (C) After 48 h, WCEs were prepared and analyzed by immunoblotting for E-cadherin (E-Cadh), ERα, Bcl-2, and β-actin. (D) Alternatively, cells were subjected to migration assays. Analysis of variance shows that the difference between MCF-7/pBABE + EV and MCF-7/Bcl-2 + Ras C186S was not statistically significant (P = 0.84), whereas the differences between MCF-7/pBABE + EV and either MCF-7/Bcl-2 +EV or MCF-7/Bcl-2 + Ras WT were significant, as indicated. (E) Summary scheme. Bcl-2, induced by RelB/p52 as shown previously (56), interacts with and activates Ras, apparently in the mitochondrial membrane. In turn, Ras induces expression of Blimp1, which represses ERα.

Blimp1 represses ERα gene expression in breast cancer cells. (A) Nuclear extracts, prepared from ZR-75 cells transfected with Blimp1 expression vector, were used in competition EMSAs with the Blimp1 element from the c-MYC gene as a probe and a 50-fold molar excess of oligonucleotides containing the c-MYC or putative ERα promoter Blimp1 sites 1, 2, or 3 (P1 to P3). (B) Nuclear extracts, prepared from ZR-75 cells transfected with EV (−) or Blimp1 expression vector (+), were used in EMSAs with the putative ERα gene P1, P2, and P3 Blimp1 sites as a probe. (C) Nuclear extracts, prepared from ZR-75 cells transfected with Blimp1 expression vector, were used in supershift EMSAs in the absence (−) or presence (+) of 200 ng of Blimp1 antibody and the putative ERα gene Blimp1 P2 site as a probe. (D) ERα promoter A and B regions with confirmed Blimp1 site indicated with the element sequence given below, where the core is underlined. (E) ChIP analyses were performed on MCF-7 cells transiently transfected with Blimp1 (top panels) or Hs578T cells (bottom panels) using anti-Blimp1 antibody or the corresponding normal IgG as indicated. The DNA, extracted from the immunoprecipitates or the input, was amplified by PCR using pairs of primers specific to the confirmed Blimp1 site of ERα promoter (Blimp1 site) (left panels) or an upstream region as negative control (right panels). (F) ZR-75 and MCF-7 cells were transiently transfected with 5.0 μg of Vxy-puro TBlimp vector expressing the 35-kDa dnBlimp protein or EV DNA, and WCEs were subjected to immunoblotting for ERα, dnBlimp, and β-actin. (G) ZR-75 and MCF-7 cells were transiently transfected with 5.0 μg of Blimp1 expression vector or EV DNA, and WCEs were subjected to immunoblotting for ERα, Blimp1, and β-actin. Densitometry indicated the ERα levels in the ZR-75 and MCF-7 cells were reduced to 28.0 ± 3.4 and 73.9 ± 5.0, respectively, upon ectopic Blimp1 expression compared to EV DNA, set at 100. (H) ZR-75 cells were transiently transfected with 5.0 μg of vector expressing either Blimp1 or dnBlimp protein (left or right panels, respectively) or the corresponding EV DNA, and RNA was subjected to RT-PCR for ERα and GAPDH. (I) MCF-7 cells carrying an inducible C4_bsrR(TO-RelB) RelB construct, plated in the absence or presence of 1 μg of doxycycline/ml, were transiently transfected with 6.0 μg of dnBlimp expression vector. After 48 h, WCEs were analyzed for levels of ERα, RelB, dnBlimp, and β-actin.

RelB induces Blimp1 in breast cancer cells. (A) Nuclear extracts were isolated from the indicated human and mouse breast cancer cell lines and subjected to immunoblotting for Blimp1 and β-actin, which confirmed equal loading. (B) RNA, isolated from the indicated human breast cancer lines, was subjected to RT-PCR for PRDM1 for either 28 or 30 cycles (as indicated) and for GAPDH levels. (C) In the left panel, a box plot of data from the Van de Vijver_Breast carcinoma microarray data set (reporter number NM_001198) (50) was accessed by using the Oncomine Cancer Profiling Database (www.oncomine.org) and is plotted on a log scale. The data set includes 69 ERα-negative and 226 ERα-positive human primary breast carcinoma samples. A Student t test, performed directly through the Oncomine 3.0 software, showed the difference in PRDM1 expression between the two groups was significant (P = 1.3e⁻⁶). In the right panel, a box plot of data from the Bittner study (see Results) showed that the difference in PRDM1 expression between the two groups was significant (P = 7.3e⁻⁷). (D) RNA was isolated from stable Hs578T transfectants expressing either RELB siRNA (Hs578T RELB siRNA) or sense RELB (Hs578T control siRNA) (Con) and analyzed for PRDM1 levels. (E) Nuclear proteins were isolated from Hs578T RELB siRNA and Hs578T control siRNA (Con) cells and analyzed for Blimp1, lamin B, and RelB levels by immunoblotting. (F) ZR-75, MCF-7, and NF639 cells were transiently transfected with 3 μg each of RelB and p52 expression vectors or 6 μg of EV DNA and RNA subjected to RT-PCR for human PRDM1 or mouse Blimp1 RNA levels. (G) ZR-75, MCF-7, and NF639 cells were transiently transfected with 3 μg each of RelB and p52 expression vectors or 6 μg of EV DNA. Nuclear extracts (ZR-75 and MCF-7 [left panels]) and WCEs (NF639 [right panels]) were analyzed by immunoblotting for Blimp1 and either lamin B or β-actin, respectively. (H and I). Nuclear extracts and RNA, isolated from RELB(1), RELB(2), EV(1), and EV(2) stable MCF-7 clones, were subjected to immunoblotting for the expression of Blimp1 and lamin B (H) and to RT-PCR analysis for the expression of PRDM1 and GAPDH (I), respectively. (J) MCF-7 cells were transiently transfected with 0.5 μg of ERα proB luciferase reporter, 0.5 μg of SV40-β-Gal, and EV DNA to a 3.0 μg of DNA total. Normalized proB activity values are presented as the means ± the SD from three experiments (control EV DNA set to 1).

Bcl-2 interaction with Ras induces Blimp1. (A) WCEs (500 μg), prepared from ZR-75 cells transiently transfected with Bcl-2 and Ras expression vectors (left panels) or from untransfected NF639 cells (right panels), were immunoprecipitated with rabbit anti-Bcl-2 or mouse anti-Ras antibodies or the corresponding normal IgG as indicated. Immunoprecipitated proteins were analyzed by immunoblotting for Bcl-2 and Ras. WCEs (5 μg) were used as input. (B) ZR-75 cells, grown on coverslips in six-well dishes, were transfected with 1 μg of Ras-GFP expression plasmid. After staining with MitoTracker, the cells were fixed and subjected to immunohistochemistry for Bcl-2 using an Alexa 647-labeled secondary antibody (purple). Mitochondria are labeled in red with MitoTracker, Ras-GFP is green, and nuclei are labeled with DAPI stain (blue). A composite image was generated from the z-sections and an Orthoganol slice analysis was performed on the composite where XZ is depicted on the horizontal and YZ is depicted on the vertical axis outside the dimension of the composite. Colocalization of Ras and Bcl-2 to the mitochondria is demonstrated by the overlap of signals yielding white spots. Three-dimensional projection images were made from the composites. The Inset shows a 45° rotation of the image of the cell, with arrows indicating areas of colocalization of Ras and Bcl-2 in the mitochondria. (C) ZR-75 and MCF-7 cells were transiently transfected with EV or a vector expressing full-length Bcl-2. After 48 h, cytosol and membrane fractions were prepared as described in Materials and Methods and subjected to immunoblotting for Ras and β-actin. Immunoblotting for VDAC, a mitochondrial ion channel membrane protein, confirmed effective separation of the membrane from the cytoplasmic compartment (data not shown). (D) ZR-75 cells were transiently transfected with EV (−) or vectors expressing Ras WT or Ras C186S mutant protein. After 48 h, WCEs and nuclear extracts were analyzed for levels of ERα and β-actin (upper panels) and Blimp1 and lamin B (lower panels). (E) ZR-75 cells were transiently transfected with EV or vector expressing full-length Bcl-2 in the absence (−) or presence (+) of a vector expressing Ras C186S mutant protein. After 48 h, WCEs were analyzed for ERα protein levels. (F) ZR-75 cells were transiently transfected in triplicate, with 0.5 μg of the 7-kb human PRDM1 promoter-Luc vector, 0.5 μg of Bcl-2 with 0.5 μg of Ras WT (Ras) or 0.5 μg of Ras C186S (C186S) expression vector, 0.5 μg of SV40-β-Gal, and pcDNA3 (EV) to make a total of 3.0 μg of DNA. The luciferase and β-Gal activities were determined, and normalized PRDM1 promoter activity values are presented as the means ± the SD (control EV DNA set to 1). (G) ZR-75 cells were transiently transfected in triplicate with 0.5 μg of the 7-kb human PRDM1 promoter-Luc vector; 0.5 μg of Ras expression vector with 0.5 μg of either EV DNA, WT Bcl-2, Bcl-acta (mitochondrion-localized Bcl-2 mutant), or Bcl-nt (cytoplasm-localized Bcl-2 mutant) expression vectors (64); 0.5 μg of SV40-β-Gal; and pRc/CMV (EV) to make a total of 3.0 μg of DNA. At 48 h after transfection, luciferase and β-Gal activities were determined, and normalized PRDM1 promoter activity values are presented as the means ± the SD from three separate experiments (control EV DNA set to 1). Using a Mann-Whitney test, we determined a P value of <0.05 between each condition, except for Ras plus WT Bcl-2 and Ras plus Bcl-acta.