Comprehensive characterization of the DNA amplification at 13q34 in human breast cancer reveals TFDP1 and CUL4A as likely candidate target genes

Abstract

Introduction: Breast cancer subtypes exhibit different genomic aberration patterns with a tendency for high-level amplifications in distinct chromosomal regions. These genomic aberrations may drive carcinogenesis through the upregulation of proto-oncogenes. We have characterized DNA amplification at the human chromosomal region 13q34 in breast cancer.

Methods: A set of 414 familial and sporadic breast cancer cases was studied for amplification at region 13q34 by fluorescence in situ hybridization (FISH) analysis on tissue microarrays. Defining the minimal common region of amplification in those cases with amplification at 13q34 was carried out using an array-based comparative genomic hybridization platform. We performed a quantitative real-time - polymerase chain reaction (qRT-PCR) gene expression analysis of 11 candidate genes located within the minimal common region of amplification. Protein expression levels of two of these genes (TFDP1 and CUL4A) were assessed by immunohistochemical assays on the same tissue microarrays used for FISH studies, and correlated with the expression of a panel of 33 antibodies previously analyzed.

Results: We have found 13q34 amplification in 4.5% of breast cancer samples, but the frequency increased to 8.1% in BRCA1-associated tumors and to 20% in basal-like tumors. Tumors with 13q34 amplification were associated with high grade, estrogen receptor negativity, and expression of EGFR, CCNE, CK5, and P-Cadherin, among other basal cell markers. We have defined a 1.83 megabases minimal common region of genomic amplification and carried out mRNA expression analyses of candidate genes located therein, identifying CUL4A and TFDP1 as the most likely target genes. Moreover, we have confirmed that tumors with 13q34 amplification significantly overexpress CUL4A and TFDP1 proteins. Tumors overexpressing either CUL4A or TFDP1 were associated with tumor proliferation and cell cycle progression markers.

Conclusions: We conclude that 13q34 amplification may be of relevance in tumor progression of basal-like breast cancers by inducing overexpression of CUL4A and TFDP1, which are both important in cell cycle regulation. Alternatively, as these genes were also overexpressed in non-basal-like tumor samples, they could play a wider role in cancer development by inducing tumor proliferation.

Figure 1

Genomic definition of the human 13q34 amplification I. (a) Chromosome 13 genomic profiles by conventional-comparative genomic hybridization (cCGH) of four breast cancer cases analyzed in a previous study by our group [21]. (b) FISH assays, detecting the 13q34 region (red probe) and the 13q12.11 region as a reference (green probe), show amplification at the 13q34 region in nuclei cell from a breast cancer sample (left, seven to eight red and one to two green signals) and in a metaphase of the MDA-MB-157 breast cancer cell line (right, six red and two green signals). (c) Boxplots displaying the range of copy signals for 13q12.11 (green box) and 13q34 (red box) in those cases with Amp13q34 found in our FISH screening.

Figure 2

Genomic definition of the human 13q34 amplification II. (a) Genomic pattern of chromosome 13 by array-based comparative genomic hybridization (aCGH) in seven breast cancer samples (six familial breast tumors from the Spanish National Cancer Research Center, one sporadic breast tumor sample from the University of Pennsylvania, and MDA-MB-157 cell line) from centromere (top, CEN) to telomere (bottom, TEL). An idiogram showing chromosomal bands is depicted on the left. Each row represents a BAC array-clone sorted by their position in the UCSC Genome Browser (Human Feb 2009, assembly (hg 19)). Each clone is colored for each sample according to its level of genomic copies: loss (red), normal (white), gain (light green), amplification (dark green). Grey cells reflect clone data rejected after quality tests for signal intensity and replicate reproducibility. The green and red stars correspond to the location of the BAC probes used for FISH analyses. (b) aCGH genomic pattern of the 13q31-q34 chromosomal region. The BAC clone name and mid-position Mb is displayed. Colors mean the same as in A. The red star shows names and locations of the FISH clones used for the 13q34 probe. The striped dark green box represents the 1.83 Mb minimal common region of DNA amplification within the 13q34 site. A list of genes located within this minimal region is supplied in Table 2.

Figure 3

Gene expression analysis by qRT-PCR of the eleven candidate genes located in the minimal common regions of Amp13q34. Relative expression levels of the candidate genes in two sample cohorts: breast tumors with no genomic aberration at 13q34 (grey), and breast tumors with Amp13q34 (black). The expression of the candidate genes was evaluated by real-time quantitative RT-PCR and normalized with a housekeeping gene (ACTB). P values are shown for those genes with statistically significant differences when comparing the two groups in a U-Mann Whitney Test (P < 0.05).

Figure 4

Immunohistochemical staining for CUL4A (a-c) and TFDP1 (d-f) in tumors with and without Amp13q34. (a) Null CUL4A staining in a non-amplifier tumor sample. (b) Moderate CUL4A staining in a non-amplifier tumor sample. (c) Strong CUL4A staining in a tumor with the Amp13q34. (d) Negative TFDP1 expression in a tumor without Amp13q34. (e) and (f) Strong TFDP1 expression in tumors with Amp13q34, the arrow in (f) points out the absence of expression in a lymphocyte infiltrate compared with the breast tumor cells.