Regulation of ERBB2 by oestrogen receptor-PAX2 determines response to tamoxifen

Abstract

Crosstalk between the oestrogen receptor (ER) and ERBB2/HER-2 pathways has long been implicated in breast cancer aetiology and drug response, yet no direct connection at a transcriptional level has been shown. Here we show that oestrogen-ER and tamoxifen-ER complexes directly repress ERBB2 transcription by means of a cis-regulatory element within the ERBB2 gene in human cell lines. We implicate the paired box 2 gene product (PAX2), in a previously unrecognized role, as a crucial mediator of ER repression of ERBB2 by the anti-cancer drug tamoxifen. We show that PAX2 and the ER co-activator AIB-1/SRC-3 compete for binding and regulation of ERBB2 transcription, the outcome of which determines tamoxifen response in breast cancer cells. The repression of ERBB2 by ER-PAX2 links these two breast cancer subtypes and suggests that aggressive ERBB2-positive tumours can originate from ER-positive luminal tumours by circumventing this repressive mechanism. These data provide mechanistic insight into the molecular basis of endocrine resistance in breast cancer.

Figure 1

Estrogen Receptor ChIP-on-chip reveals a binding site within the ERBB2 gene region that is bound by Pax2. a. Schematic representation of the ERBB2 gene locus and the intronic ER binding site, as defined by ER ChIP-on-chip experiments. b. Pax motif enriched within ER binding sites. c. Pax2 ChIP after vehicle, estrogen or tamoxifen treatment. d. Changes in ErbB2 mRNA by real time RT-PCR after vehicle, estrogen or tamoxifen treatment. All experiments are the average of three independent replicates ± Std Dev.

Figure 2

Specific silencing of Pax2 reverses the tamoxifen-mediated repression of ErbB2 and growth arrest. a. siRNA to Pax2 was transfected into hormone-depleted MCF-7 cells and stimulated with vehicle (V), estrogen (E) or tamoxifen (T) and total protein was immunoblotted b. Control siRNA (siLuc) (white bars) or siPax2 (gray bars) was transfected and ErbB2 mRNA was assessed. c. siPax2 was transfected into cells in the presence of control or Herceptin (ErbB2 antibody) after which cells were collected and total viable cell number was determined. The control transfection (siLuc) data is in Supplementary figure 7. d. Control siRNA (siLuc) or siPax2 was performed as described and cells were treated with vehicle, estrogen or tamoxifen. AIB-1 binding to the ERBB2 enhancer was determined by ChIP. All experiments are the average of three independent replicates ± Std Dev.

Figure 3

Pax2 restores tamoxifen sensitivity in tamoxifen resistant breast cancer cells. a. Total protein from wild type MCF-7 or tamoxifen resistant MCF-7 cells (Tam-R) was immunoblotted. b. ChIP of ER, Pax2, AIB-1 and HDAC-1 at the ER binding site in the ERBB2 gene in wild type and Tam-R cells after vehicle or tamoxifen treatment. c. Control or Pax2 expressing plasmids were transfected into Tam-R cells, followed by vehicle (V) or tamoxifen (T) treatment. Total protein was immunoblotted. Following Pax2 expression in Tam-R cells, total viable cell numbers were determined after vehicle or tamoxifen treatment. The immunoblots were cropped and the original figures are in Supplementary data 11. All experiments are the average of three independent replicates ± Std Dev.

Figure 4

Pax2 predicts clinical outcome in breast cancer patients. Kaplan-Meier curve representing percent relapse free survival in tumours based on Pax2 and AIB-1 staining (n = 109). The percentage of ErbB2 over-expressing tumours within each category is provided.