Identification of novel amplification gene targets in mouse and human breast cancer at a syntenic cluster mapping to mouse ch8A1 and human ch13q34

Abstract

Serial analysis of gene expression from aggressive mammary tumors derived from transplantable p53 null mouse mammary outgrowth lines revealed significant up-regulation of Tfdp1 (transcription factor Dp1), Lamp1 (lysosomal membrane glycoprotein 1) and Gas6 (growth arrest specific 6) transcripts. All of these genes belong to the same linkage cluster, mapping to mouse chromosome band 8A1. BAC-array comparative genomic hybridization and fluorescence in situ hybridization analyses revealed genomic amplification at mouse region ch8A1.1. The minimal region of amplification contained genes Cul4a, Lamp1, Tfdp1, and Gas6, highly overexpressed in the p53 null mammary outgrowth lines at preneoplastic stages, and in all its derived tumors. The same amplification was also observed in spontaneous p53 null mammary tumors. Interestingly, this region is homologous to human chromosome 13q34, and some of the same genes were previously observed amplified in human carcinomas. Thus, we further investigated the occurrence and frequency of gene amplification affecting genes mapping to ch13q34 in human breast cancer. TFDP1 showed the highest frequency of amplification affecting 31% of 74 breast carcinomas analyzed. Statistically significant positive correlation was observed for the amplification of CUL4A, LAMP1, TFDP1, and GAS6 genes (P < 0.001). Meta-analysis of publicly available gene expression data sets showed a strong association between the high expression of TFDP1 and decreased overall survival (P = 0.00004), relapse-free survival (P = 0.0119), and metastasis-free interval (P = 0.0064). In conclusion, our findings suggest that CUL4A, LAMP1, TFDP1, and GAS6 are targets for overexpression and amplification in breast cancers. Therefore, overexpression of these genes and, in particular, TFDP1 might be of relevance in the development and/or progression in a significant subset of human breast carcinomas.

Figure 1

A, Northern blot analysis of deregulated genes in two p53 null mammary epithelial outgrowth lines: PN1B, mildly aggressive low tumorigenic; PN1A, highly aggressive and tumorigenic, and five mammary tumors derived from the PN1A outgrowth line (Tg5, Tg6, Tg7, 5155, and 2860). Lane wt, p53 wild-type normal mammary epithelium. B, Northern blot analysis of Cul4a, Lamp1, and Tfdp1 in spontaneously generated p53 null mammary tumors (lanes 2–11) compared with p53 wild-type normal mammary epithelium (lane 1).

Figure 2

A, whole-genome mouse BAC microarray CGH of two p53 null mammary tumors. Tumor 2860R (blue) was derived from the outgrowth cell line PN1A, and tumor 8813R (red) was spontaneously generated. Note the increased in hybridization intensity ratios for the DNA in the area close to the centromere. The amplified region corresponding to band 8A1 is shown on the chromosomal ideogram (red line). B, detail of the amplified region on the proximal portion of mouse chromosome 8, band A1. Cul4a, Lamp1, Tfdp1, and Gas6 map to the indicated RP23 BAC clones with high-intensity hybridization ratios, indicative of a gene amplification phenomenon. X-axis, relative chromosomal position in kilobases.

Figure 3

FISH analyses of p53 null mouse mammary model. A, relative position of the BAC clones used as probes in the FISH analyses. TEL, telomere. The loci for Lamp1, Tfdp1, and Gas6 are indicated. B, FISH on touch imprint preparations from a p53 null mouse mammary tumor. Biotin-labeled BAC clones were used as probes. The label was detected with FITC-avidin (green), and the nuclei were counterstained with DAPI (blue). Note that the genomic regions recognized by RP23 BAC probes 270K21 and 470N10 are not amplified and define the boundaries of the amplicon. Yellow, name of genes contained within specific BACs. C, cytogenetic and FISH analysis of the PN1A mammary outgrowth line. At least three distinctive chromosomal markers (identified as M1, M2, and M3) displaying homogenously staining regions (HSR) were identified on these p53 null mouse mammary premalignant cells. BACs identifying the amplified ch8A1 sequences are shown. Yellow, name of genes contained within specific BACs.

Figure 4

A, heat map visualization of amplified human breast carcinomas based on quantitative DNA real-time PCR data. Results of 74 primary breast carcinoma analyzed for amplification of CUL4A, LAMP1, TFDP1, and GAS6 genes. Experiments were done in triplicate for each data point. Results are expressed as log 2 transformation of the mean difference between tumor versus normal control (P < 0.01). Red intensity is a representative of mean difference for each breast tumor. Note that these values do not represent specific copy number increases, but are directly proportional to the level of amplification. Note that eight breast carcinomas showed simultaneous amplification for CUL4A, LAMP1, TFDP1, and GAS6 (black arrows). B, PCA of 74 breast carcinomas analyzed showing a strong association between the amplification of CUL4A, LAMP1, TFDP1, and GAS6 genes (P < 0.001). C, component matrix showing correlation (Kendal's τ -b) and statistical significances (P values) of PCA analysis.

Figure 5

TFDP1 expression profile of human breast carcinoma using publicly available DNA microarray data sets. A, oncomine data output and meta-analysis showing TFDP1 expression patterns positively associated with ERα(–) breast carcinoma (left). Oncomine data output of TFDP1 expression patterns correlated with high-grade breast carcinoma (right). TFDP1 transcripts are represented in normalized expression units (log 2 data transformed, array media set to 0 and array SD to 1). B, Kaplan-Meier curves of overall survival, relapse-free survival, and metastasis-free interval based on high or low expression levels of TFDP1 gene. Kaplan-Meier analysis was assessed using the Van de Vijver et al. (10) data set by means of ITTACA online resources.