Alternative TFAP2A isoforms have distinct activities in breast cancer

Abstract

Introduction: AP-2α is a transcription factor implicated in the regulation of differentiation and proliferation in certain tissues, including the mammary gland. In breast tumours, continued expression of AP-2α has been correlated with a better prognosis, but this is hard to reconcile with a reported role in the upregulation of the ERBB2 oncogene. The existence of TFAP2A isoforms, deriving from alternative first exons and differing in their N-terminal sequence, has been described in some mammals, but their relative abundance and activity has not been investigated in the human breast.

Methods: Expression levels of four TFAP2A isoforms were assayed at the level of RNA and protein (via the generation of isoform-specific antibodies) in a panel of breast tumour cell lines and in tissue from normal breast and primary tumour samples. Expression constructs for each isoform were used in reporter assays with synthetic and natural promoters (cyclin D3 and ERBB2) to compare the activation and repression activity of the isoforms.

Results: We demonstrate that the two isoforms AP-2α 1b and AP-2α 1c, in addition to the originally cloned, AP-2α 1a, are conserved throughout evolution in vertebrates. Moreover, we show that isoform 1c in particular is expressed at levels at least on a par with the 1a isoform in breast epithelial lines and tissues and may be more highly expressed in tamoxifen resistant tumours. The isoforms share a similar transactivation mechanism involving the recruitment of the adaptors CITED2 or 4 and the transactivators p300 or CBP. However, isoform 1b and 1c are stronger transactivators of the ERBB2 promoter than isoform 1a. In contrast, AP-2α 1a is the only isoform able to act as a repressor, an activity that requires an intact sumoylation motif present within the N-terminus of the protein, and which the other two isoforms lack.

Conclusions: Our findings suggest that TFAP2A isoforms may be differentially regulated during breast tumourigenesis and this, coupled with differences in their transcriptional activity, may impact on tumour responses to tamoxifen therapy. These data also have implications for the interpretation of tumour studies that seek to correlate outcomes with TFAP2A expression level.

Figure 1

TFAP2A gene contains four alternative first exons which encode conserved protein sequences. (a) Schematic representation of TFAP2A 5' gene structure (exons 1 to 3). Exons are shown as rectangles, introns as horizontal lines, drawn in proportion to their actual length. Closed rectangles represent translated regions, open rectangles represent untranslated regions. Isoforms 1b and 1c are orthologous to the murine isoform 3 [16] and ovine variant 6 [17], respectively. (b) TFAP2A protein sequences encoded by the four alternative first exons. Possible starting methionine residues are underlined. AP-2α isoform/variant 4 (as described in [16] and [17], respectively), generated by initiation of transcription upstream of exon 2, does not have a human paralog due to the presence of an in-frame stop codon upstream of exon 2. (c) Alignment of TFAP2A isoform 1c protein sequences in H. sapiens, D. rerio and X. tropicalis generated by ClustalW. Sequences for rhesus, mouse, dog and elephant are identical to the human sequence. * indicates an identical amino acid; : and . indicate conserved and semi-conserved substitutions, respectively. A conserved TATA-box is present 237 bp upsteam of the ATG. (d) Alignment of TFAP2A isoform 1b protein sequence to TFAP2B isoform 1b (EST: BM727695), TFAP2D (NM_172238.3) and TFAP2E (NM_178548.3) generated by ClustalW. Human sequences are shown.

Figure 2

AP-2α isoforms 1a, 1b, and 1c are expressed in breast cell lines and tissues. (a) cDNA was generated from the indicated breast cell lines (left) and normal breast samples (right) and the relative amount of the different AP-2α isoforms was determined by real-time PCR as described in the Materials and methods. (b) HepG2 cells were transfected with pcDNA3 expression vectors encoding each isoform and harvested after 48 h. Two different amounts of cell lysate (5 and 2.5 μg) were loaded for each isoform. Western blot analysis was performed with a pan-AP-2α antibody, 3B5, which recognises an epitope in the DNA-binding domain of the protein common to all isoforms. (c) Protein lysates (30 μg) derived from the indicated cell lines were analysed by western blot with isoform-specific antibodies. As a loading control, two amounts of lysate from HepG2 cells overexpressing each isoform from (b) were loaded on each gel and the blots were also reprobed for actin (lower panels).

Figure 3

AP-2α isoforms exert similar transactivation activity when cotransfected with CITED2 or 4 and p300 or CBP. HepG2 cells were transfected with 0.05 μg pcDNA3-AP-2α, 0.25 μg 3xAP2-Bluc, 0.25 μg phRG-renilla, 0.25 μg pcDNA3-CITED2/4, 1 μg pCI-p300 or pRc/CMV CBP as indicated. Relative firefly luciferase activity normalised to renilla luciferase activity is shown. Average and standard error from four independent experiments is reported.

Figure 4

AP-2α isoforms differ in their transcriptional repression activity. HepG2 cells were transfected with 0.25 μg/well pGL4.74 (Renilla), 0.4 μg/well cyclin D3 reporter construct, and the indicated ratios of pcDNA3-AP-2α (corresponding to 0.2 and 0.4 μg/well). Results are reported as relative firefly luciferase activity normalised to renilla luciferase activity. Average and standard error from three independent experiments is shown.

Figure 5

AP-2α isoform 1a can be sumoylated leading to decreased transactivation activity. (a) HepG2 cells were transfected with 0.3 μg/well of the different pcDNA3-AP-2α constructs, without and with 0.1 μg/well pSG5 Ubc9 and 0.6 μg/well pSG5 SUMO1 in a six-well format. 48 h after transfection, lysates were harvested in RIPA buffer containing IAA and NEM, and analysed by western blot with a pan AP-2α antibody (3B5). One experiment representative of three is shown. A second, weaker sumoylation site (IKKG) was predicted (using "SUMOplot") within the C-terminal half of the protein which could explain the low levels of sumoylation observed for AP-2α K10R and the other isoforms using longer exposures. (b) HepG2 cells were transfected with 0.05 μg/well of the different pcDNA3-AP-2α constructs, 0.25 μg/well 3xAP2-Bluc, 0.25 μg/well phRG-renilla, 0.25 μg/well pcDNA3-CITED2, 0.75 μg/well pCI-p300, and 0.25 or 0.5 μg/well pSG-SUMO1/2 as indicated. Relative firefly luciferase activity normalised to renilla luciferase activity is shown. The average and standard error of three experiments is reported. (c) HepG2 cells were transfected with 0.2 μg/well pGL4.74 (Renilla), 0.3 μg/well cyclin D3 reporter, 0.15 μg/well pcDNA3-AP-2α isoform 1a, 0.3 μg/well pSG-Ubc9 and 0.15 μg/well pSG-SUMO1. Average and standard error from three independent experiments is represented.

Figure 6

AP-2α isoforms exert different levels of transactivation activity at the ERBB2 promoter. HepG2 cells were transfected with 0.12 μg/well p500 ErbB2-luc, 0.02 μg/well renilla hRG, 0.06 μg/well pCI-p300, 0.06 μg/well pcDNA3-CITED2, and indicated ratios (corresponding to a total of 0.06, 0.12, 0.24 and 0.48 μg/well) of the different pcDNA3-AP-2α constructs. The average and standard error of three independent experiments is represented.

Figure 7

AP-2α isoform expression is differentially regulated in tamoxifen resistant cell lines and breast tumour samples. (a, b): Levels of AP-2α isoform 1a and 1c were determined by Western blot in a series of wild-type ER+ breast tumour lines and in their tamoxifen-resistant counterparts. The signal from a number of different non-saturated exposures was quantified with ImageJ and is graphically represented in (b). (c, d): Normal breast (4) and tumour samples (10) were analysed by real-time PCR for AP-2α isoform expression levels. The relative levels of AP-2α 1c normalised using GAPDH levels (c) and the ratio between AP-2α 1c and 1a (d) are represented. The differences remain statistically significant even when the outliers are eliminated from the analysis.