Array CGH identifies distinct DNA copy number profiles of oncogenes and tumor suppressor genes in chromosomal- and microsatellite-unstable sporadic colorectal carcinomas

Abstract

DNA copy number changes represent molecular fingerprints of solid tumors and are as such relevant for better understanding of tumor development and progression. In this study, we applied genome-wide array comparative genomic hybridization (aCGH) to identify gene-specific DNA copy number changes in chromosomal (CIN)- and microsatellite (MIN)-unstable sporadic colorectal cancers (sCRC). Genomic DNA was extracted from microdissected, matching normal colorectal epithelium and invasive tumor cells of formalin-fixed and paraffin-embedded tissues of 22 cases with colorectal cancer (CIN = 11, MIN = 11). DNA copy number changes were determined by aCGH for 287 target sequences in tumor cell DNAs, using pooled normal DNAs as reference. aCGH data of tumor cell DNAs was confirmed by fluorescence in situ hybridization (FISH) for three genes on serial tissues as those used for aCGH. aCGH revealed DNA copy number changes previously described by metaphase CGH (gains 7, 8q, 13q, and 20q; losses 8p, 15q, 18q, and 17p). However, chromosomal regions 20q, 13q, 7, and 17p were preferentially altered in CIN-type tumors and included DNA amplifications of eight genes on chromosome 20q (TOP1, AIB1, MYBL2, CAS, PTPN1, STK15, ZNF217, and CYP24), two genes on chromosome 13q (BRCA2 and D13S25), and three genes on chromosome 7 (IL6, CYLN2, and MET) as well as DNA deletions of two genes on chromosome 17p (HIC1 and LLGL1). Finally, additional CIN-tumor-associated DNA amplifications were identified for EXT1 (8q24.11) and MYC (8q24.12) as well as DNA deletions for MAP2K5 (15q23) and LAMA3 (18q11.2). In contrast, distinct MIN-tumor-associated DNA amplifications were detected for E2F5 (8p22-q21.3), GARP (11q13.5-q14), ATM (11q22.3), KAL (Xp22.3), and XIST (Xq13.2) as well as DNA deletions for RAF1 (3p25), DCC (18q21.3), and KEN (21q tel). aCGH revealed distinct DNA copy number changes of oncogenes and tumor suppressor genes in CIN- and MIN-type sporadic colorectal carcinomas. The identified candidate genes are likely to have distinct functional roles in the carcinogenesis and progression of CIN- and MIN-type sporadic CRCs and may be involved in the differential response of CIN- and MIN-type tumor cells to (adjuvant) therapy, such as 5-fluorouracil.

Control of tumourigenesis by the Scribble/Dlg/Lgl polarity module

“…Consistent with this, genomic array analysis has identified the loss of Lgl1 as an unfavourable copy number aberration associated with reduced survival in glioblastoma (Korshunov et al, 2006). Lgl1 deletion has been observed in a subset of colorectal carcinomas, with particular relevance to chromosomally unstable sporadic cases (Lassmann et al, 2007). Moreover, the loss of Lgl1 transcript and/or protein have been associated with other solid tumours, notably melanoma, prostate, breast and lung (Grifoni et al, 2004;Schimanski et al, 2005;Kuphal et al, 2006).…”

Aurora A is differentially expressed and regulated in chromosomal and microsatellite instable sporadic colorectal cancers

“…Appropriate tissues samples were selected after re-classification 51,52 of hematoxylin and eosin-stained sections by an experienced pathologist (MD), as described before for 41 of the cases. 45,46 The study had been approved by the local Ethics Committees (#251/04 Ethik-Kommission, Albert-Ludwigs-Universität, Freiburg, Germany; and the University Medicine and Pharmacy, Department of Pathology, Iasi, Romania).…”

“…Aurora A gene copy numbers 46,53 and tumour cell aneuploidy were assessed as follows: Serial tissue microarray sections of 5 mm thickness were subjected to deparaffination, pre-treatment in pH6 citrate buffer in a microwave oven (180 W) for The table summarizes the major clinicopathological parameters of the investigated cases and the tissue materials used for all cases as well as for chromosomal and microsatellite instable cases separately. Note: *For three cases, no MSI-status could be obtained; **no neoadjuvant radio/chemotherapy.…”

“…Subsequently, sections were denatured in 2 Â SSC/50% Formamide (room temperature), and 2 Â SSC/70% Formamide (751C, 15 min), immersed in ice cold ethanols (70, 95 and 100%, 5 min each) and dried at 371C. In the meantime, the fluorescence in situ hybridization (FISH) probes had been denatured (751C, 5 min) and were hybridised to the dried sections (16 h, 371C) as follows: Section (i) probe specific for the aurora A gene and centromere 20 (chromosome enumeration probe, CEP20; Chrombios, Raubling, Germany) 46,53 and section (ii) to (v) probes specific for CEP7, CEP8, CEP11 (Abbott Vysis) and CEP13 (QBiogen). After hybridization, sections were washed in 2 Â SSC (room temperature) and in 2 Â SSC (731C, for 2 min), cover-glassed with DAPI-containing mounting medium (Vector) and stored at À201C until analysis.…”

“…45 In addition, our array-based comparative genomic hybridisation analysis suggested that chromosomal instable carcinomas exhibit preferential amplification of the aurora A gene locus at 20q13. 46 Similarly, Nishida et al 41 found that microsatellite instable colorectal cancers rarely have aurora A gene amplification, as measured by PCR. By mRNA expression profiling of microdissected invasive cancer cells from formalinfixed and paraffin-embedded colorectal cancers, we recently also detected aurora A as one prominently expressed gene in chromosomal instable tumours.…”