The combined immunodetection of AP-2alpha and YY1 transcription factors is associated with ERBB2 gene overexpression in primary breast tumors

Abstract

Introduction: Overexpression of the ERBB2 oncogene is observed in about 20% of human breast tumors and is the consequence of increased transcription rates frequently associated with gene amplification. Several studies have shown a link between activator protein 2 (AP-2) transcription factors and ERBB2 gene expression in breast cancer cell lines. Moreover, the Yin Yang 1 (YY1) transcription factor has been shown to stimulate AP-2 transcriptional activity on the ERBB2 promoter in vitro. In this report, we examined the relationships between ERBB2, AP-2alpha, and YY1 both in breast cancer tissue specimens and in a mammary cancer cell line.

Methods: ERBB2, AP-2alpha, and YY1 protein levels were analyzed by immunohistochemistry in a panel of 55 primary breast tumors. ERBB2 gene amplification status was determined by fluorescent in situ hybridization. Correlations were evaluated by a chi2 test at a p value of less than 0.05. The functional role of AP-2alpha and YY1 on ERBB2 gene expression was analyzed by small interfering RNA (siRNA) transfection in the BT-474 mammary cancer cell line followed by real-time reverse transcription-polymerase chain reaction and Western blotting.

Results: We observed a statistically significant correlation between ERBB2 and AP-2alpha levels in the tumors (p < 0.01). Moreover, associations were found between ERBB2 protein level and the combined high expression of AP-2alpha and YY1 (p < 0.02) as well as between the expression of AP-2alpha and YY1 (p < 0.001). Furthermore, the levels of both AP-2alpha and YY1 proteins were inversely correlated to ERBB2 gene amplification status in the tumors (p < 0.01). Transfection of siRNAs targeting AP-2alpha and AP-2gamma mRNAs in the BT-474 breast cancer cell line repressed the expression of the endogenous ERBB2 gene at both the mRNA and protein levels. Moreover, the additional transfection of an siRNA directed against the YY1 transcript further reduced the ERBB2 protein level, suggesting that AP-2 and YY1 transcription factors cooperate to stimulate the transcription of the ERBB2 gene.

Conclusion: This study highlights the role of both AP-2alpha and YY1 transcription factors in ERBB2 oncogene overexpression in breast tumors. Our results also suggest that high ERBB2 expression may result either from gene amplification or from increased transcription factor levels.

Figure 1

Detection of activator protein 2 alpha (AP-2α), Yin Yang 1 (YY1), and ERBB2 by immunohistochemistry in breast tumors. (a) Case with low immunoreactivity for AP-2α. (b) Tumor sample expressing high level of AP-2α protein in more than 80% of the nuclei.(c) Tumor with low immunoreactivity for YY1. (d) Case expressing high level of YY1 protein in more than 80% of the nuclei. (e) Case with no ERBB2 membrane staining, scored as IHC 0. (f) Tumor with partial weak membrane staining, scored as IHC 1+. (g) Case with ERBB2 score of 2+. (h) Tumor with thick circumferential ERBB2 membrane staining, scored as IHC 3+.

Figure 2

Suppression of AP-2α, AP-2γ, and YY1 expression downregulates ERBB2 transcript levels in BT-474 cells. (a) Cells were transfected on days 0 and 2 by small interfering RNAs (siRNAs) directed against AP-2α (siAP-2α) and/or AP-2γ (siAP-2γ) or against luciferase mRNA as control. Graphic shows real-time reverse transcription-polymerase chain reaction (RT-PCR) for AP-2α transcripts on total RNA extracted after 2, 3, or 4 days of treatment with indicated siRNAs. Results are presented as percentages of mRNA level as compared with control cells transfected with luciferase siRNA. Data are means ± standard deviation of three experiments. (b) RT-PCR for AP-2γ transcripts on total RNA. Cells were transfected like in (a). (c) Detection by Western blotting of AP-2α and AP-2γ levels at day 3. Ku70 protein served as control. Cells were transfected like in (a).(d) RT-PCR for ERBB2 transcripts on total RNA. Cells were transfected like in (a). (e) Cells were transfected on day 0 with 30 nM siRNA directed against YY1 (siYY1) or 100 nM combined siRNAs against AP-2α and AP-2γ transcripts (siAP-2α+γ) or both as indicated. Control cells were transfected with a commercially available negative control siRNA (control). Proteins extracted after 24 hours of treatment were detected by Western blotting. Ku70 protein served as control for the protein amount charged on the gel. The percentage of ERBB2 protein level compared with transfection of control siRNA is shown in brackets. AP-2, activator protein 2; YY1, Yin Yang 1.