Characterization of the Recurrent 8p11-12 Amplicon Identifies PPAPDC1B, a Phosphatase Protein, as a New Therapeutic Target in Breast Cancer (original) (raw)
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Multiple Interacting Oncogenes on the 8p11-p12 Amplicon in Human Breast Cancer
Cancer Research, 2006
The 8p11-p12 genomic region is amplified in 15% of breast cancers and harbors several candidate oncogenes. However, functional evidence for a transforming role for these genes is lacking. We identified 21 genes from this region as potential oncogenes based on statistical association between copy number and expression. We further showed that three of these genes (LSM1, BAG4, and C8orf4) induce transformed phenotypes when overexpressed in MCF-10A cells, and overexpression of these genes in combination influences the growth factor independence phenotype and the ability of the cells to grow under anchorage-independent conditions. Thus, LSM1, BAG4, and C8orf4 are breast cancer oncogenes that can work in combination to influence the transformed phenotype in human mammary epithelial cells.
Research Article Multiple Interacting Oncogenes on the 8p11-p12 Amplicon in Human Breast Cancer
2013
The 8p11-p12 genomic region is amplified in 15% of breast cancers and harbors several candidate oncogenes. However, functional evidence for a transforming role for these genes is lacking. We identified 21 genes from this region as potential oncogenes based on statistical association between copy number and expression. We further showed that three of these genes (LSM1, BAG4, and C8orf4) induce transformed phenotypes when overexpressed in MCF-10A cells, and overexpression of these genes in combination influences the growth factor independence phenotype and the ability of the cells to grow under anchorage-independent conditions. Thus, LSM1, BAG4, and C8orf4 are breast cancer oncogenes that can work in combination to influence the transformed phenotype in human mammary epithelial cells.
Genomic and expression analysis of the 8p1112 amplicon in human breast cancer cell lines
Cancer research, 2004
Gene amplification is an important mechanism of oncogene activation in breast and other cancers. Characterization of amplified regions of the genome in breast cancer has led to the identification of important oncogenes including erbB-2/HER-2, C-MYC, and fibroblast growth factor receptor (FGFR) 2. Chromosome 8p11-p12 is amplified in 10-15% of human breast cancers. The putative oncogene FGFR1 localizes to this region; however, we show evidence that FGFR inhibition fails to slow growth of three breast cancer cell lines with 8p11-p12 amplification. We present a detailed analysis of this amplicon in three human breast cancer cell lines using comparative genomic hybridization, traditional Southern and Northern analysis, and chromosome 8 cDNA microarray expression profiling. This study has identified new candidate oncogenes within the 8p11-p12 region, supporting the hypothesis that genes other than FGFR1 may contribute to oncogenesis in breast cancers with proximal 8p amplification. MATERIALS AND METHODS Bacterial Artificial Chromosome (BAC) Array CGH. Array CGH was carried out using arrays of 2464 BAC clones each printed in triplicate (Hum-Array1.14) according to published protocols (16, 17). Briefly, cell line and normal male reference DNA (300 ng each) were labeled by random priming in separate 50-l reactions to incorporate Cy3 and Cy5, respectively. The labeled DNAs were combined with 100 g human Cot-1 DNA and hybridized to the BAC arrays for ϳ48 h at 37°C. After posthybridization washes, the arrays were mounted in a solution containing 90% glycerol, 10% PBS, and 1 M 4Ј,6-diamidino-2-phenylindole, and sealed with a coverslip. A custom built CCD camera system was used to acquire 16 bit 1024 ϫ 1024 pixel 4Ј,6diamidino-2-phenylindole, Cy3 and Cy5 images (18). Image analysis was carried out using University of California San Francisco SPOT software (19). The log 2 ratio of the total integrated Cy3 and Cy5 intensities for each spot after background subtraction was calculated, normalized to the median log 2 ratio of all of the clones on the array, and the average of the triplicates calculated using a second custom program, SPROC. Automatic data filtering to reject data points based on low 4Ј,6-diamidino-2-phenylindole intensity, low correlation between Cy3 and Cy5 within each segmented spot, and low reference/4Ј,6diamidino-2-phenylindole signal intensity was also carried out using SPROC. Data files were subsequently manually edited by rejecting clones for which only one spot of the triplicate survived after SPROC analysis and for which the SD of the log 2 ratio of the triplicate was Ͼ0.2. For each tumor, the data are plotted as the mean log 2 ratio of the triplicate spots for each clone normalized to the genome median log 2 ratio. The clones are ordered by position in the genome according to the University of California-Santa Cruz Biotechnology (UCSC) Human Genome Working Draft. 6 Cell Lines and FGFR Inhibitor Growth Experiments. The isolation and culture of the SUM-44, SUM-52, and SUM-225 HBC cell lines have been described in detail previously (14, 20, 21). SUM-44 cells were grown under serum-free conditions using a base medium of Ham's F-12 supplemented with 0.1% BSA, insulin (5 g/ml), hydrocortisone (1 g/ml), 5 mM ethanolamine, 10 mM HEPES, transferrin (5 g/ml), triiodothyronine (10 g/ml), 50 M sodium selenite, gentamicin (5 g/ml), and fungizone (0.5 g/ml). SUM-52 and SUM-225 cells were routinely cultured in Ham's F-12 supplemented with 5% fetal bovine serum, insulin (5 g/ml), hydrocortisone (1 g/ml), gentamicin (5 g/ml), and fungizone (0.5 g/ml). Detailed information regarding the SUM series of HBC cell lines is accessible online. 7 The MCF10A cell line, a spontaneously immortalized normal mammary epithelial cell line (22), was cultured in the same serum-free medium supplemented with epidermal growth factor (10 ng/ml).
A 1 Mb minimal amplicon at 8p11–12 in breast cancer identifies new candidate oncogenes
Oncogene, 2005
Amplification of 8p11-12 is a well-known alteration in human breast cancers but the driving oncogene has not been identified. We have developed a high-resolution comparative genomic hybridization array covering 8p11-12 and analysed 33 primary breast tumors, 20 primary ovarian tumors and 27 breast cancer cell lines. Expression analysis of the genes in the region was carried out by using real-time quantitative PCR and/or oligo-microarray profiling. In all, 24% (8/33) of the breast tumors, 5% (1/20) of the ovary tumors and 15% (4/27) of the cell lines showed 8p11-12 amplification. We identified a 1 Mb segment of common amplification that excludes previously proposed candidate genes. Some of the amplified genes did not show overexpression, whereas for others, overexpression was not specifically attributable to amplification. The genes FLJ14299, C8orf2, BRF2 and RAB11FIP, map within the 8p11-12 minimal amplicon, two have a putative function consistent with an oncogenic role, these four genes showed a strong correlation between amplification and overexpression and are therefore the best candidate driver oncogenes at 8p12.
Comprehensive Profiling of 8p11-12 Amplification in Breast Cancer
Molecular Cancer Research, 2005
In human carcinomas, especially breast cancer, chromosome arm 8p is frequently involved in complex chromosomal rearrangements that combine amplification at 8p11-12, break in the 8p12-21 region, and loss of 8p21-ter. Several studies have identified putative oncogenes in the 8p11-12 amplicon. However, discrepancies and the lack of knowledge on the structure of this amplification lead us to think that the actual identity of the oncogenes is not definitively established. We present here a comprehensive study combining genomic, expression, and chromosome break analyses of the 8p11-12 region in breast cell lines and primary breast tumors. We show the existence of four amplicons at 8p11-12 using array comparative genomic hybridization. Gene expression analysis of 123 samples using DNA microarrays identified 14 genes significantly overexpressed in relation to amplification. Using fluorescence in situ hybridization analysis on tissue microarrays, we show the existence of a cluster of breakpo...
Co-amplification of 8p12 and 11q13 in breast cancers is not the result of a single genomic event
Genes, Chromosomes and Cancer, 2007
Epithelial cancers frequently have multiple amplifications, and particular amplicons tend to occur together. These co-amplifications have been suggested to result from amplification of pre-existing junctions between two chromosomes, that is, translocation junctions. We investigated this hypothesis for two amplifications frequent in breast cancer, at 8p12 and 11q13, which had been reported to be associated in Southern blot studies. We confirmed that both genomic amplification and expression of genes was correlated between the frequently-amplified regions of 8p and 11q, in array CGH and microarray expression data, supporting the importance of co-amplification. We examined by FISH the physical structure of co-amplifications that we had identified by array CGH, in five breast cancer cell lines (HCC1500, MDA-MB-134, MDA-MB-175, SUM44, and ZR-75-1), four breast tumors, and a pancreatic cancer cell line (SUIT2). We found a variety of arrangements: amplification of translocation junctions; entirely independent amplification of the two regions on separate chromosomes; and separate amplification of 8p and 11q sequences in distinct sites on the same rearranged chromosome. In this last arrangement, interphase nuclei often showed intermingling of FISH signals from 8p12 and 11q13, giving a false impression that the sequences were interdigitated. We conclude that co-amplification of the main 8p and 11q amplicons in breast tumors is not usually the result of a preceding translocation event but most likely reflects selection of clones that have amplified both loci. This article contains supplementary material available at
Genome-wide multi-omics profiling of the 8p11-p12 amplicon in breast carcinoma
Oncotarget, 2018
Genomic instability contributes to the neoplastic phenotype by deregulating key cancer-related genes, which in turn can have a detrimental effect on patient outcome. DNA amplification of the 8p11-p12 genomic region has clinical and biological implications in multiple malignancies, including breast carcinoma where the amplicon has been associated with tumor progression and poor prognosis. However, oncogenes driving increased cancer-related death and recurrent genetic features associated with the 8p11-p12 amplicon remain to be identified. In this study, DNA copy number and transcriptome profiling data for 229 primary invasive breast carcinomas (corresponding to 185 patients) were evaluated in conjunction with clinicopathological features to identify putative oncogenes in 8p11-p12 amplified samples. Illumina paired-end whole transcriptome sequencing and whole-genome SNP genotyping were subsequently performed on 23 samples showing high-level regional 8p11-p12 amplification to characteri...
Cancer Research, 2007
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. [Cancer Res 2007;67(9):4104-12]
Transforming function of the LSM1 oncogene in human breast cancers with the 8p11–12 amplicon
Oncogene, 2007
Amplification of the 8p11−12 region occurs in 15−20% of breast cancers, but the driving oncogene at this locus has yet to be definitively identified. We mapped the 8p11−12 amplicon in breast cancer cell lines and primary human breast cancers and identified the candidate oncogene LSM1 based on increases in copy number and expression level relative to human mammary epithelial cells. To examine the oncogenic role of LSM1, we overexpressed this gene in MCF10A mammary epithelial cells and inhibited its production in the SUM44 breast cancer cell line, which has a natural amplification and overexpression of LSM1. Our data confirmed that LSM1 is an oncogene from the 8p11−12 amplicon by showing that human Sm-like protein (hLsm1) overexpression induced growth factor-independent proliferation and soft agar colony formation in MCF10A cells, and hLsm1 inhibition in SUM44 cells dramatically reduced soft agar growth. Little is known about hLsm1 function other than its involvement in mRNA degradation; therefore, we used expression microarray analysis to investigate how hLsm1 affects cell transformation in MCF10A and SUM44 cells. We identified numerous genes altered following hLsm1 overexpression common to SUM44 breast cancer cells that play important roles in cell cycle regulation, cell proliferation and other cancer-promoting processes. Future work will continue to characterize these important changes in the hope of achieving a more complete understanding of the mechanism of hLsm1's effect on cancer progression.
Carcinogenesis, 2014
RNA interference has boosted the field of functional genomics, by making it possible to carry out 'loss-of-function' screens in cultured cells. Here, we performed a small interfering RNA screening, in three breast cancer cell lines, for 101 candidate driver genes overexpressed in amplified breast tumors and belonging to eight amplicons on chromosomes 8q and 17q, investigating their role in cell survival/proliferation. This screening identified eight driver genes that were amplified, overexpressed and critical for breast tumor cell proliferation or survival. They included the well-described oncogenic driver genes for the 17q12 amplicon, ERBB2 and GRB7. Four of six other candidate driver genes-RAD21 and EIF3H, both on chromosome 8q23, CHRAC1 on chromosome 8q24.3 and TANC2 on chromosome 17q23-were confirmed to be driver genes regulating the proliferation/survival of clonogenic breast cancer cells presenting an amplification of the corresponding region. Indeed, knockdown of the expression of these genes decreased cell viability, through both cell cycle arrest and apoptosis induction, and inhibited the formation of colonies in anchorage-independent conditions, in soft agar. Strategies for inhibiting the expression of these genes or the function of the proteins they encode are therefore of potential value for the treatment of breast cancers presenting amplifications of the corresponding genomic region.