Identification of Novel Candidate Oncogenes in Chromosome Region 17p11.2-p12 in Human Osteosarcoma

Osteosarcoma is the most common primary malignancy of bone. The tumours are characterized by high genomic instability, including the occurrence of multiple regions of amplifications and deletions. Chromosome region 17p11.2–p12 is amplified in about 25% of cases. In previous studies, COPS3 and PMP22 have been identified as candidate oncogenes in this region. Considering the complexity and variation of the amplification profiles for this segment, the involvement of additional causative oncogenes is to be expected. The aim of the present investigation is to identify novel candidate oncogenes in 17p11.2–p12. We selected 26 of in total 85 osteosarcoma samples (31%) with amplification events in 17p11.2–p12, using quantitative PCR for 8 marker genes. These were subjected to high-resolution SNP array analysis and subsequent GISTIC analysis to identify the most significantly amplified regions. Two major amplification peaks were found in the 17p11.2–p12 region. Overexpression as a consequence of gene amplification is a major mechanism for oncogene activation in tumours. Therefore, to identify the causative oncogenes, we next determined expression levels of all genes within the two segments using expression array data that could be generated for 20 of the selected samples. We identified 11 genes that were overexpressed through amplification in at least 50% of cases. Nine of these, c17orf39, RICH2, c17orf45, TOP3A, COPS3, SHMT1, PRPSAP2, PMP22, and RASD1, demonstrated a significant association between copy number and expression level. We conclude that these genes, including COPS3 and PMP22, are candidate oncogenes in 17p11.2–p12 of importance in osteosarcoma tumourigenesis

Oncogenic Properties of Candidate Oncogenes in Chromosome Region 17p11.2p12 in Human Osteosarcoma

Osteosarcomas are primary tumors of bone that most often develop in adolescents. They are characterized by complex genomic changes including amplifications, deletions, and translocations. The chromosome region 17p11.2p12 is frequently amplified in human high grade osteosarcomas (25% of cases), suggesting the presence of one or more oncogenes. In previous studies, we identified 9 candidate oncogenes in this region (GID4, ARGHAP44, LRRC75A-AS1, TOP3A, COPS3, SHMT1, PRPSAP2, PMP22, and RASD1). The aim of the present study was to determine their oncogenic properties. Therefore, we generated osteosarcoma cell lines overexpressing these genes, except for LRRC75A-AS1 and PRPSAP2, and subjected these to functional oncogenic assays. We found that TOP3A, SHMT1, and RASD1 overexpression provided increased proliferation and that ARGHAP44, COPS3, and PMP22 overexpression had a stimulatory effect on migration and invasion of the cells. COPS3 and PMP22 overexpression additionally improved the ability of the cells to form new colonies. No oncogenic effect could be demonstrated for GID4 overexpression. We conclude that the concerted amplification-mediated overexpression of these genes in 17p11.2p12 may contribute to the oncogenic process in malignant osteosarcom

Focal chromosomal copy number aberrations identify CMTM8 and GPR177 as new candidate driver genes in osteosarcoma

Osteosarcoma is an aggressive bone tumor that preferentially develops in adolescents. The tumor is characterized by an abundance of genomic aberrations, which hampers the identification of the driver genes involved in osteosarcoma tumorigenesis. Our study aims to identify these genes by the investigation of focal copy number aberrations (CNAs, <3 Mb). For this purpose, we subjected 26 primary tumors of osteosarcoma patients to high-resolution single nucleotide polymorphism array analyses and identified 139 somatic focal CNAs. Of these, 72 had at least one gene located within or overlapping the focal CNA, with a total of 94 genes. For 84 of these genes, the expression status in 31 osteosarcoma samples was determined by expression microarray analysis. This enabled us to identify the genes of which the over- or underexpression was in more than 35% of cases in accordance to their copy number status (gain or loss). These candidate genes were subsequently validated in an independent set and furthermore corroborated as driver genes by verifying their role in other tumor types. We identified CMTM8 as a new candidate tumor suppressor gene and GPR177 as a new candidate oncogene in osteosarcoma. In osteosarcoma, CMTM8 has been shown to suppress EGFR signaling. In other tumor types, CMTM8 is known to suppress the activity of the oncogenic protein c-Met and GPR177 is known as an overexpressed upstream regulator of the Wnt-pathway. Further studies are needed to determine whether these proteins also exert the latter functions in osteosarcoma tumorigenesi

Representative examples of copy number (CN) changes along chromosome 17 in osteosarcomas OS2, OS11, and OS20.

<p>SNP-array data were processed using the OverUnder algorithm described by Attiyeh <i>et al </i><a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030907#pone.0030907-Attiyeh1" target="_blank">[<i>22</i>]</a>, and copy numbers were calculated using Illumina BeadStudio.</p

Top 11 genes most frequently overexpressed through amplification in 17p11.2-p12.

<p>Genes were scored for the number of times a feature was found in the tumour (data taken from Supplemental <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0030907#pone.0030907.s001" target="_blank">Figure S1</a>). A: number of tumours with amplification of a gene; O: number of tumours with overexpression of that gene; O/A: number of tumours with overexpression and amplification of that gene; %: percentage of tumours in which the amplified gene is overexpressed.</p

GISTIC amplification analysis.

<p>A. Chromosomal regions showing significant amplification by SNP-array profiling, generated by GISTIC analysis. The significance threshold (q-value 0.25) is indicated by the green line. B. Detailed GISTIC analysis of chromosome arm 17p. Positions of previously suggested candidate oncogenes <i>COPS3</i> in 17p11.2 and <i>PMP22</i> in 17p12 are indicated. The significance threshold (q-value 0.25) is indicated by the green line.</p

Pearson's correlation coefficient <i>R</i> estimating the relationship between gene copy number and expression level for the top 11 genes most frequently overexpressed through amplification in 17p11.2-p12.

<p>Pearson's correlation coefficient <i>R</i> estimating the relationship between gene copy number and expression level for the top 11 genes most frequently overexpressed through amplification in 17p11.2-p12.</p

Genome-wide frequency plot of all copy number aberrations (top) and of focal copy number aberrations (bottom) in 26 osteosarcoma samples.

<p>Frequencies of gains (in red) and losses (in blue) are indicated. Vertical bars in B represent detected focal copy number aberrations (<3 Mb).</p

Genes in focal losses with underexpression (FC<0.75, A) and in focal gains with overexpression (FC>1.5, B) in more than 35% of osteosarcomas.

<p>Green: expression fold change (FC) <0.75 of the gene in the tumor relative to osteoblasts (underexpression). Red: expression fold change (FC) >1.50 of the gene in the tumor relative to osteoblasts (overexpression). For each gene, the number (n) and fraction (%) of osteosarcomas with the indicated fold change (FC) are given.</p