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Amplification and overexpression of peroxisome proliferator-activated receptor binding protein (PBP/PPARBP) gene in breast cancer - PubMed

  • ️Fri Jan 01 1999

Amplification and overexpression of peroxisome proliferator-activated receptor binding protein (PBP/PPARBP) gene in breast cancer

Y Zhu et al. Proc Natl Acad Sci U S A. 1999.

Abstract

Peroxisome proliferator-activated receptor binding protein (PBP), a nuclear receptor coactivator, interacts with estrogen receptor alpha (ERalpha) in the absence of estrogen. This interaction was enhanced in the presence of estrogen but was reduced in the presence of antiestrogen, tamoxifen. Transfection of PBP in CV-1 cells resulted in enhancement of estrogen-dependent transcription, indicating that PBP serves as a coactivator in ER signaling. To examine whether overexpression of PBP plays a role in breast cancer because of its coactivator function in ER signaling, we determined the levels of PBP expression in breast tumors. High levels of PBP expression were detected in approximately 50% of primary breast cancers and breast cancer cell lines by ribonuclease protection analysis, in situ hybridization, and immunoperoxidase staining. Fluorescence in situ hybridization of human chromosomes revealed that the PBP gene is located on chromosome 17q12, a region that is amplified in some breast cancers. We found PBP gene amplification in approximately 24% (6/25) of breast tumors and approximately 30% (2/6) of breast cancer cell lines, implying that PBP gene overexpression can occur independent of gene amplification. This gene comprises 17 exons that, together, span >37 kilobases. The 5'-flanking region of 2.5 kilobase pairs inserted into a luciferase reporter vector revealed that the promoter activity in CV-1 cells increased by deletion of nucleotides from -2,500 to -273. The -273 to +1 region, which exhibited high promoter activity, contains a typical CCAT box and multiple cis-elements such as C/EBPbeta, YY1, c-Ets-1, AP1, AP2, and NFkappaB binding sites. These observations, in particular PBP gene amplification, suggest that PBP, by its ability to function as ERalpha coactivator, might play a role in mammary epithelial differentiation and in breast carcinogenesis.

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Figures

Figure 1
Figure 1

Binding of PBP to ER and potentiation of estrogen dependent transcription from ER. (A) Binding of PBP to ER. [35S] methionine-labeled PBP generated by in vitro translation was incubated with glutathione-Sepharose beads containing GST, or GST-ER, in the presence of estrogen (E) or tamoxifen (T) or absence of the ligand (-). The bound protein was eluted and analyzed by SDS/PAGE and was autoradiographed. PBP binds to ER in the absence of estrogen and in the presence of tamoxifen, but estrogen increases the interaction of ER with PBP. No binding was seen for GST alone, indicating that the interaction is specific for ER. (B) PBP potentiates the transcriptional activity of ER. CV-1 cell were transiently cotransfected with 1.5 μg of reporter construct ERE-TK-luc, 0.25 μg of pCMV-ER, 0.5 μg of pCMX-PBP or pCMX, and 0.5 μg of pCMVβ in the presence of β-estradiol or absence of ligand. PBP increased the transcription of luciferase gene ≈2- fold in the presence of β-estradiol. Results are the mean of four independent transfections and are normalized to the internal control β-galactosidase expression.

Figure 2
Figure 2

Ribonuclease protection assay of PBP transcript levels. Total RNA (10 μg) was hybridized with radiolabeled antisense PBP probe generated by in vitro transcription. Glyceraldehyde-3-phosphate dehydrogenase probe was included in each hybridization reaction as control. Nuclease resistant fragments were resolved on 6% polyacrylamide-urea gels. High levels of expression of PBP mRNA are seen in breast tumor samples A1, A8, B3, and B6 and in MDA-MD-361 breast cancer cell line (A4). Samples B7 and B8 are breast tumors with no appreciable increase in PBP transcript, and sample C1 is normal human mammary gland RNA.

Figure 3
Figure 3

In situ analysis of PBP mRNA expression and immunoperoxidase localization of PBP in breast carcinoma. Shown are examples of high (A) and low (B) levels of PBP mRNA expression in breast carcinoma. The connective tissue stroma surrounding the tumor tissue exhibits low signal (A). Immunohistochemical analysis showed the nuclear staining of PBP (C).

Figure 4
Figure 4

Chromosomal assignment of the human PBP gene. Shown is in situ hybridization of a biotin-labeled 3.4-kb human PBP cDNA probe to human metaphase cells from phytohemagglutinin-stimulated peripheral blood lymphocytes. The chromosome 17 homologues are identified with arrows; specific labeling was observed at 17q12 (arrowheads).

Figure 5
Figure 5

Analysis of DNA from breast tumors and breast cancer cell lines for PBP gene amplification. I represents semiquantitative slot blots of DNA. DNA (2 μg) was loaded into each slot and was denatured and hybridized with [32P]-labeled cDNA probes. Ia represents the blot probed with PBP. This blot then was stripped and reprobed with Cyp4A1cDNA to assess DNA loading (Ib). A4 (MDA-MB-361) and B4 (MDA-MB-134VI) represented breast cancer cell lines. The rest of samples were primary breast tumors. Samples A1, A4, A8, B1, and B6 suggest amplification of PBP gene. II represents the Southern blot of representative samples. Genomic DNA (10 μg) digested with PstI was probed with full-length PBP cDNA (upper panel marked PBP) and was reprobed with β-actin cDNA to serve as a control. PBP gene amplification is clearly evident in samples A1, A4, A8, B1, and B6.

Figure 6
Figure 6

The nucleotide sequence of the 5′-flanking region of the human PBP gene. Nucleotide positions −308 to +58 relative to the transcription initiation site are shown. A major transcription start site is indicated by an asterisk and is designated as +1, and the ATG methionine is shown (boldface type and underlined). The position of intron 1 is shown. Underlined sequences indicate the putative binding sites for transcription factors.

Figure 7
Figure 7

The human PBP promoter activity. The schematic diagram on the left represents each deletion construct of the PBP gene (dark box) fused into the upstream region of the luciferase gene (luc, white box), with variable 5′-ends to the +125 nucleotide relative to the transcription start site. Each construct was transiently transfected into CV-1 cells. The promoter activity is expressed as fold induction relative to the activity of promoterless pGL3-Basic. The data are the mean ± standard error of triplicate determinations. A repressor region is located between bp −2,500 and −518, 5′-of the PBP transcription start site.

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