Detection of novel amplicons in prostate cancer by comprehensive genomic profiling of prostate cancer cell lines using oligonucleotide-based arrayCGH

Abstract

Background: The purpose of this study was to prove the feasibility of a longmer oligonucleotide microarray platform to profile gene copy number alterations in prostate cancer cell lines and to quickly indicate novel candidate genes, which may play a role in carcinogenesis.

Methods/results and findings: Genome-wide screening for regions of genetic gains and losses on nine prostate cancer cell lines (PC3, DU145, LNCaP, CWR22, and derived sublines) was carried out using comparative genomic hybridization on a 35,000 feature oligonucleotide microarray (arrayCGH). Compared to conventional chromosomal CGH, more deletions and small regions of gains, particularly in pericentromeric regions and regions next to the telomeres, were detected. As validation of the high-resolution of arrayCGH we further analyzed a small amplicon of 1.7 MB at 9p13.3, which was found in CWR22 and CWR22-Rv1. Increased copy number was confirmed by fluorescence in situ hybridization using the BAC clone RP11-165H19 from the amplified region comprising the two genes interleukin 11 receptor alpha (IL11-RA) and dynactin 3 (DCTN3). Using quantitative real time PCR (qPCR) we could demonstrate that IL11-RA is the gene with the highest copy number gain in the cell lines compared to DCTN3 suggesting IL11-RA to be the amplification target. Screening of 20 primary prostate carcinomas by qPCR revealed an IL11-RA copy number gain in 75% of the tumors analyzed. Gain of DCTN3 was only found in two cases together with a gain of IL11-RA.

Conclusions/significance: ArrayCGH using longmer oligonucleotide microarrays is feasible for high-resolution analysis of chomosomal imbalances. Characterization of a small gained region at 9p13.3 in prostate cancer cell lines and primary prostate cancer samples by fluorescence in situ hybridization and quantitative PCR has revealed interleukin 11 receptor alpha gene as a candidate target of amplification with an amplification frequency of 75% in prostate carcinomas. Frequent amplification of IL11-RA in prostate cancer is a potential mechanism of IL11-RA overexpression in this tumor type.

Figure 1. Comparison of cCGH (A, C) and arrayCGH (B, D) results from chromosome 9 of CWR22 and chromosome 14 of DU145-MN1.

A small region of gain on 9p close to the centromer (B, arrowhead) and a small deletion on 14q (D, arrowhead) are only detected by arrayCGH.

Figure 2. Whole genome plot of gained (A) and lost (B) chromosomal regions of 11 prostate cancer cell lines, as detected with aCGH.

Numbers above the plot indicate chromosome numbers and vertical lines boundaries between chromosomes.

Figure 3. Validation of amplicons by FISH analysis on cell lines.

Identification of increased copies of signals of the BAC clone RP11-165H19 mapped to 9p13.3 amplicon in cell line CWR22 and CWR22-Rv1 (A and B). C Optimal signal and lack of cross hybridization was verified using normal metaphase spreads showing two signals for each probe. BAC clone RP11-165H19 signals are green; red signals indicate chromosome 9 centromere (D9Z1).

Figure 4. DNA copy number quantification of DCTN3 and IL11-RA genes in prostate cancer cell lines and 20 primary prostate carcinoma samples using quantitative real time PCR.

IL-11RA showed a significant increase in gene copy number above normal in CWR22, CWR22-Rv1, DU145HOM and DU145-MN1 and in 15 out of the 20 (75%) prostate cancer samples. For DCTN3, no copy number gain was detected in any cell line and in only 2 out of the 20 (10%) prostate cancer samples. Values above cut off line being assigned as increased gene copy number compared to normal.