BCAR4 induces antioestrogen resistance but sensitises breast cancer to lapatinib

Abstract

Background: High BCAR4 and ERBB2 mRNA levels in primary breast cancer associate with tamoxifen resistance and poor patient outcome. We determined whether BCAR4 expression sensitises breast cancer cells to lapatinib, and identifies a subgroup of patients who possibly may benefit from ERBB2-targeted therapies despite having tumours with low ERBB2 expression.

Methods: Proliferation assays were applied to determine the effect of BCAR4 expression on lapatinib treatment. Changes in cell signalling were quantified with reverse-phase protein microarrays. Quantitative reverse-transcriptase polymerase chain reaction (RT-PCR) of ERBB2 and BCAR4 was performed in 1418 primary breast cancers. Combined BCAR4 and ERBB2 mRNA levels were evaluated for association with progression-free survival (PFS) in 293 oestrogen receptor-α (ER)-positive patients receiving tamoxifen as first-line monotherapy for recurrent disease.

Results: BCAR4 expression strongly sensitised ZR-75-1 and MCF7 breast cancer cells to the combination of lapatinib and antioestrogens. Lapatinib interfered with phosphorylation of ERBB2 and its downstream mediators AKT, FAK, SHC, STAT5, and STAT6. Reverse transcriptase-PCR analysis showed that 27.6% of the breast cancers were positive for BCAR4 and 22% expressed also low levels of ERBB2. The clinical significance of combining BCAR4 and ERBB2 mRNA status was underscored by the finding that the group of patients having BCAR4-positive/ERBB2-low-expressing cancers had a shorter PFS on tamoxifen treatment than the BCAR4-negative group.

Conclusion: This study shows that BCAR4 expression identifies a subgroup of ER-positive breast cancer patients without overexpression of ERBB2 who have a poor outcome and might benefit from combined ERBB2-targeted and antioestrogen therapy.

Figure 1

Knockdown of ERBB receptors reduces proliferation of antioestrogen-resistant MCF7/BCAR4 cells. MCF7/BCAR4 (A) and MCF7/vector (B) cells were cultured in the absence (open bars) or presence (closed bars) of the antioestrogen ICI182,780. The inhibition of ERBB receptors by specific siRNAs was measured with a proliferation assay. Average of five replicates and SDs are shown. Significance was determined by the Mann–Whitney U-test. *P<0.05, compared with cells cultured without siRNAs. Abbreviation: AU=arbitrary units.

Figure 2

BCAR4 sensitises ZR-75-1 cells to lapatinib. ZR/vector control (▴), ZR/BCAR4 (○), ZR/BCAR1 (◊), ZR/BCAR3 (Δ) or ZR/EGFR cells (□) were plated in oestradiol-containing medium (A), or 4-hydroxytamoxifen-containing medium (B) with increasing doses of lapatinib as indicated. Concentrations of lapatinib (X axis) are presented on a logarithmic scale. Results are expressed as a percentage of maximal growth as measured with a WST-1 proliferation assay. Average of five replicates and SDs are presented.

Figure 3

Lapatinib treatment inhibits ERBB2 and ERBB3 signalling in ZR/BCAR4 cells. (A) Molecular network analysis of ZR-75-1-derived antioestrogen-resistant cell lines (horizontal axis) treated with lapatinib. Lysates were analysed with reverse-phase protein microarrays. The heatmap presents the different total and phosphorylated proteins (n=31; vertical axis) that showed at least two-fold difference with the vector control cultured in the presence of oestradiol (colour version in Supplementary Figure 1 and protein data in Supplementary Table 1). Higher relative levels are represented in white; lower levels in black. Cells were cultured with oestradiol (E) or 4-hydroxytamoxifen (T), or T and EGF (T+) and treated for 17 h, without, or with 0.01 or 0.1 μℳ lapatinib (triangles represent increasing lapatinib concentrations, from left to right). (B–H) Effects of lapatinib treatment on ERBB2 and ERBB3 signalling. Phosphorylation intensity (Y axis) of ERBB2 (B), ERBB3 (C), AKT (D), FAK (E), SHC (F), STAT5 (G), and STAT6 (H) in the different cell lines (horizontal axis) is presented. Results of two independently derived pools of transduced cells were averaged. Phosphorylation intensity values for antibody staining were negative control subtracted and normalised for total protein concentration. An average of three measurements is presented. SD values <2% across the replicates, the three bars for each cell line represent the three different conditions (no or 0.01 or 0.1 μℳ lapatinib).

Figure 4

Lapatinib-treated cells utilise ER signalling for survival. ZR/BCAR4 (A) and ZR/vector (B) cells were plated in the absence (•), 0.01 μℳ lapatinib (○), 0.1 (▪), or 1 μℳ (□) of lapatinib and different concentrations of oestradiol, as indicated. Results are expressed as a percentage of maximal growth (cultures with 1 nℳ oestradiol but without lapatinib), as measured with a WST-1 proliferation assay. Average of three replicates and SD values are presented.

Figure 5

Clinical relevance of combined BCAR4 and ERBB2 status. Expression of BCAR4 and ERBB2 mRNA levels was measured in a cohort of primary breast carcinomas. Patients were stratified according to the combined BCAR4 and ERBB2 status as indicated. BCAR4 was divided in negative (no call), and positive low and high by the mean. There is no relation observed between BCAR4 and ERBB2 expression. (A) Prevalence of breast tumours expressing BCAR4 and low levels of ERBB2. Number of tumours and percentages are shown for each group. (B) Progression-free survival of patients with ER-positive breast cancer who received first-line tamoxifen monotherapy for recurrent breast cancer. Patients with ERBB2-high tumours were grouped irrespective of their BCAR4 status. The patients at-risk at 12-month intervals are indicated. Abbreviations: neg=negative, pos=positive.