CITED2 and NCOR2 in anti-oestrogen resistance and progression of breast cancer

Abstract

Background: Endocrine therapies of breast cancer are effective but ultimately fail because of the development of treatment resistance. We have previously revealed several genes leading to tamoxifen resistance in vitro by retroviral insertion mutagenesis. To understand the manner in which these genes yield tamoxifen resistance, their effects on global gene expression were studied and those genes resulting in a distinct gene expression profile were further investigated for their clinical relevance.

Methods: Gene expression profiles of 69 human breast cancer cell lines that were made tamoxifen resistant through retroviral insertion mutagenesis were obtained using oligonucleotide arrays and analysed with bioinformatic tools. mRNA levels of NCOR2 and CITED2 in oestrogen receptor-positive breast tumours were determined by quantitative RT-PCR. mRNA levels were evaluated for association with metastasis-free survival (MFS) in 620 patients with lymph node-negative primary breast cancer who did not receive systemic adjuvant therapy, and with clinical benefit in 296 patients receiving tamoxifen therapy for recurrent breast cancer.

Results: mRNA expression profiles of most tamoxifen-resistant cell lines were strikingly similar, except for the subgroups of cell lines in which NCOR2 or CITED2 were targeted by the retrovirus. Both NCOR2 and CITED2 mRNA levels were associated with MFS, that is, tumour aggressiveness, independently of traditional prognostic factors. In addition, high CITED2 mRNA levels were predictive for a clinical benefit from first-line tamoxifen treatment in patients with advanced disease.

Conclusions: Most retrovirally targeted genes yielding tamoxifen resistance in our cell lines do not impose a distinctive expression profile, suggesting that their causative role in cell growth may be accomplished by post-transcriptional processes. The associations of NCOR2 and CITED2 with outcome in oestrogen receptor-positive breast cancer patients underscore the clinical relevance of functional genetic screens to better understand disease progression, which may ultimately lead to the development of improved treatment options.

Figure 1

Hierarchical clustering of 251 genes in tamoxifen-resistant breast cancer cell lines. Cell lines (three hybridisations each) were grouped together according to the location of the retrovirus in the cellular genome (columns). The number of different cell lines in a group is indicated below the name. Genes (rows) were selected by class comparison analysis and clustering was performed using the unweighted pair-group method with arithmetic mean in Spotfire. Gene expression above the mean is shown in white, that below the mean is indicated in black. Target genes in the integration loci are marked with arrows (from left to right: BCAR1, BCAR3, CITED2 and NCOR2). The order of the genes, their names and identifiers are provided in Supplementary Table S1.

Figure 2

Metastasis-free-survival of 620 lymph node-negative patients with ERα-positive breast cancer. Kaplan–Meier curves for MFS for subgroups of patients as a function of the NCOR2 (A) or CITED2 (B) mRNA levels of primary tumours. Patients were divided into two groups having primary tumours with high (above median) or low mRNA levels. The y axis shows the percentage of patients without distant metastasis. Patients at risk (i.e., without event and not censored) at 24-month intervals are indicated. N, number of patients; F, number of patients with distant recurrences.