Alternative TFAP2A isoforms have distinct activities in breast cancer

Abstract

IntroductionAP-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.MethodsExpression 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.ResultsWe 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.ConclusionsOur 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.

Sumoylation Pathway Is Required to Maintain the Basal Breast Cancer Subtype

“…Knockdown of Ubc9 reduced the relative amount of the upper band (Figure 4D), suggesting the potential for sumoylation of TFAP2A. One major site for sumoylation is lysine 10 and the TFAP2A isoform 1A, which was used to generate the chimeric AP-2 proteins, contains the K10 SUMO site (Berlato et al, 2011). To formally demonstrate sumoylation of TFAP2A, SUMO-1, -2 and -3 were expressed in vitro with TFAP2A.…”

Inhibition of neural crest formation by Kctd15 involves regulation of transcription factor AP-2

“…AP-2α occurs in at least three isoforms varying at the N terminus; our standard construct is based on isoform 1b (35). To test whether Kctd15 inhibition is isoform specific, we tested zebrafish isoform 1a in the reporter assay; isoform 1a was active and inhibited by Kctd15 to a similar extent as isoform 1b (Fig.…”

“…To test for functional interactions between Kctd15 and AP-2 we used a reporter construct, AP2-Luc, that contains three copies of the AP-2 consensus binding site controlling the expression of luciferase (24,34,35) (Fig. 2A).…”

Histone Demethylase KDM5B Collaborates with TFAP2C and Myc To Repress the Cell Cycle Inhibitor p21cip (CDKN1A)

“…To date, no clear AP-2 corepressor proteins have been identified, although modification through sumoylation may be required for repressor activity (4,10). TFAP2A has also been reported to interact with other nuclear factors, including Myc, pRB, and p53 (reviewed in reference 9), to regulate the transcription of target genes.…”