Chromosome 20q gene signature associated with colorectal cancer progression

Abstract

Amplification of human chromosome 20q has been reported as the most frequently recurring genetic abnormality associated with large scale changes in mRNA and protein levels in sporadic colorectal carcinomas. While some studies have found 20q amplification to be consistent between primary and metastatic samples from the same patient with a role in the development of metastasis and worse patient prognosis, others have reported association with improved overall survival for a subset of these patients with colorectal cancer (CRC). To fine map the Minimal Common Regions (MCRs) of amplification on chromosome 20q and identify the candidate genes playing roles in progression of the disease, microarray comparative genomic hybridization analyses of two in vitro cultured CRC liver metastasis cell line model systems was utilized. Microarray expression analysis led to the identification of a candidate gene signature comprising of four genes, BMP7, DNMT3B, UBE2C and YWHAB, residing in the MCRs that were over expressed in CRC cells. By validating our results in a training set of 23 adenocarcinomas (tumors) and five adenomas (polyps) using reverse transcription‑quantitative PCR, as well as analyses of two larger colorectal cancer test data sets derived from 195 The Cancer Genome Atlas and 182 MD Anderson Cancer Center patients with colorectal adenocarcinoma patients, this gene signature was ascertained to be associated with lymph node spread and/or distant metastasis (P<0.05). Previously reported functional studies of the gene signature indicated their involvement in inflammatory and immune response pathways driving CRC progression.

Keywords: CGH; CRC progression; RT‑qPCR; SKY; chromosome 20q; gene signature.

Figure 1.

High-resolution genomic profile of chromosome 20 in colorectal cancer metastasis cell line models. (A) Heat map representation of copy number along chromosome 20 (the Y-axis measured in Mb from pter to qter) in each of the cell lines (X-axis). Copy number is represented by color, with green representing 0 copies, yellow representing 2 copies (normal) and reds of increasing intensity representing gain of copies. Gain of part (for SW620) or complete 20q is observed in all cell lines. The vertical black line along the right side represents the three minimal common gain intervals, with the boundaries represented by the black horizontal lines that cross the heat map. Black arrows represent the position within the intervals of the BAC clones used as FISH probes in copy number verification experiments. (B, D and F) Line graph represents the 29–34 Mb interval (B), 40–44 Mb interval (D) and 53–56 Mb interval copy number in the five cell lines (KM12C in red, KM12L4A in green, KM12SM in blue, SW480 in light blue and SW620 in pink). The black arrow indicates position of the BAC clones used in FISH probes. (C, E and G) FISH analysis of the indicated cell lines using the probes 1, 2 and 3 shown in B, D and F respectively. Scale bar, 10 µm. FISH, fluorescence in situ hybridization.

Figure 2.

Heat map representation of copy number along the q arm of chromosome 20 (the Y-axis measured in Mb) in tumor and polyp samples along with HCT116 cell line, with different intensities of blue and red representing copy gain and copy loss respectively, white representing no copy change and gray representing no detected signal. The three regions outlined by the black boxes represent the three minimal common regions of amplification respectively.

Figure 3.

Validation of expression array data by RT-qPCR. Fold changes of gene signature were compared in six cell lines, twenty-three tumor and five polyp samples. All assays were run in duplicate and the relative expression of the selective genes in terms of fold change in the cancer cell line, tumor and polyp samples, compared with normal tissue samples was calculated using the comparative CT (2^−ΔΔCq) method, in reference to the endogenous control to normalize the data, where CT (cycle threshold) is defined as the number of cycles required for the FAM signal to cross the threshold in RT-qPCR. ΔCT was calculated by subtracting the CT values of control gene from the CT values of the gene of interest. ΔΔCT was then calculated by subtracting the mean ΔCT of the control samples from the ΔCT of test samples. RT-qPCR, reverse transcription-quantitative PCR.