Gene products of chromosome 11q and their association with CCND1 gene amplification and tamoxifen resistance in premenopausal breast cancer

Abstract

Introduction: The amplification event occurring at chromosome locus 11q13, reported in several different cancers, includes a number of potential oncogenes. We have previously reported amplification of one such oncogene, namely CCND1, to be correlated with an adverse effect of tamoxifen in premenopausal breast cancer patients. Over-expression of cyclin D1 protein, however, confers tamoxifen resistance but not a tamoxifen-induced adverse effect. Potentially, co-amplification of an additional 11q13 gene, with a resulting protein over-expression, is required to cause an agonistic effect. Moreover, during 11q13 amplification a deletion of the distal 11q region has been described. In order to assess the potential impact of the deletion we examined a selected marker for this event.

Method: Array comparative genomic hybridization analysis was employed to identify and confirm changes in the gene expression of a number of different genes mapping to the 11q chromosomal region, associated with CCND1 amplification. The subsequent protein expression of these candidate genes was then examined in a clinical material of 500 primary breast cancers from premenopausal patients who were randomly assigned to either tamoxifen or no adjuvant treatment. The protein expression was also compared with gene expression data in a subset of 56 breast cancer samples.

Results: Cortactin and FADD (Fas-associated death domain) over-expression was linked to CCND1 amplification, determined by fluorescence in situ hybridization, but was not associated with a diminished effect of tamoxifen. However, deletion of distal chromosome 11q, defined as downregulation of the marker Chk1 (checkpoint kinase 1), was associated with an impaired tamoxifen response, and interestingly with low proliferative breast cancer of low grade. For Pak1 (p21-activated kinase 1) and cyclin D1 the protein expression corresponded to the gene expression data.

Conclusions: The results indicate that many 11q13 associated gene products are over-expressed in conjunction with cyclin D1 but not linked to an agonistic effect of tamoxifen. Finally, the deletion of distal 11q, linked to 11q13 amplification, might be an important event affecting breast cancer outcome and tamoxifen response.

Figure 1

Genomic profiles of chromosome 11 from three breast tumors demonstrating different amplification patterns. Breast tumor samples were analyzed by array CGH. Bold dashed lines correspond to a log2(ratio) of ± 0.3 and represent gain or loss. The high-level peaks on 11q13 comprising (among others) CCND1, FADD, CTTN (panels a, b, and c) and PAK1 (panels b and c) represent gene amplification. The distal part of chromosome 11q, telomeric to the amplified region, including the CHK1 gene, has been hemizygously deleted, consistently among all three tumors. CGH, comparative genomic hybridization.

Figure 2

Expression of cortactin, FADD, and Chk1 in breast cancer cell lines and primary breast tumors. (a) Five human breast cancer cell lines were examined for cortactin, FADD, and Chk1 protein expression by Western blot and immunocytochemistry. Protein levels were equivalent between the two methods. (b) Tumor staining reveals cytoplasmic staining of cortactin and FADD, whereas Chk1 protein is mainly present in the nuclei. Staining intensity for cortactin and FADD was evaluated as negative (0) or low (1; left panels: cortactin low, FADD negative), intermediate (2; middle panels), and high (3; right panels). Chk1 staining was evaluated according to fraction positive nuclei (0% to 5% left panel, 6% to 50% middle panel, 51% to 100% right panel). In tumors with highly stained nuclei Chk1 was also present in the cytoplasm. Scale bar = 25 μm. Chk1, checkpoint kinase 1; FADD, Fas-associated death domain.

Figure 3

Recurrence-free survival according to protein expression of cortactin, FADD, and Chk1. Kaplan-Meier curves showing the effect of (a) cortactin, (b) FADD, (c,d) and Chk1 expression on recurrence-free survival in the subgroup of untreated control patients. Expression of cortactin or Chk1 did not influence the recurrence rate. However, over-expression of FADD was associated with shorter recurrence-free survival compared with lower expression. Chk1, checkpoint kinase 1; FADD, Fas-associated death domain.

Figure 4

Predicted tamoxifen response in patient subgroups defined by protein expression of the 11q gene products. Hazard ratios (black boxes, with 95% confidence intervals) were calculated for each subgroup in univariate analysis using a Cox proportional hazards regression model. The number of patients in each subgroup receiving no adjuvant treatment versus tamoxifen, and P values are indicated. In patients with ER-α-positive tumors tamoxifen had an antagonistic effect, whereas patients with ER-α-negative tumors did not respond to tamoxifen. The expression of cortactin and FADD did not influence the tamoxifen response significantly, whereas patients with tumors showing deviant Chk1 expression did not respond to tamoxifen, relative to patients with tumors exhibiting normal expression. Tamoxifen response was impaired in patients with tumors exhibiting over-expression of Pak1 (defined as positive nuclei) or cyclin D₁, and the effect was potentially agonistic in patients with CCND1 amplified tumors. Chk1, checkpoint kinase 1; ER, estrogen receptor; FADD, Fas-associated death domain; Pak1, p21-activated kinase 1.

Figure 5

11q protein data in the cohort of premenopausal breast cancer patients. Protein expression of the 11q gene products and the associated response to tamoxifen was examined in primary breast tumors. Chk1, checkpoint kinase 1; FADD, Fas-associated death domain; Pak1, p21-activated kinase 1.