A global view of the oncogenic landscape in nasopharyngeal carcinoma: an integrated analysis at the genetic and expression levels

Abstract

Previous studies have reported that the tumour cells of nasopharyngeal carcinoma (NPC) exhibit recurrent chromosome abnormalities. These genetic changes are broadly assumed to lead to changes in gene expression which are important for the pathogenesis of this tumour. However, this assumption has yet to be formally tested at a global level. Therefore a genome wide analysis of chromosome copy number and gene expression was performed in tumour cells micro-dissected from the same NPC biopsies. Cellular tumour suppressor and tumour-promoting genes (TSG, TPG) and Epstein-Barr Virus (EBV)-encoded oncogenes were examined. The EBV-encoded genome maintenance protein EBNA1, along with the putative oncogenes LMP1, LMP2 and BARF1 were expressed in the majority of NPCs that were analysed. Significant downregulation of expression in an average of 76 cellular TSGs per tumour was found, whilst a per-tumour average of 88 significantly upregulated, TPGs occurred. The expression of around 60% of putative TPGs and TSGs was both up-and down-regulated in different types of cancer, suggesting that the simplistic classification of genes as TSGs or TPGs may not be entirely appropriate and that the concept of context-dependent onco-suppressors may be more extensive than previously recognised. No significant enrichment of TPGs within regions of frequent genomic gain was seen but TSGs were significantly enriched within regions of frequent genomic loss. It is suggested that loss of the FHIT gene may be a driver of NPC tumourigenesis. Notwithstanding the association of TSGs with regions of genomic loss, on a gene by gene basis and excepting homozygous deletions and high-level amplification, there is very little correlation between chromosomal copy number aberrations and expression levels of TSGs and TPGs in NPC.

Figure 1. Immunohistochemical validation of differential regulation.

Panels A–F show normal epithelium on the left and tumour tissue on the right. Panel A uses frozen sections from the same samples that were used in the array analysis (MHAU; normal epithelium: XY23; NPC), Panels B–F are paired specimens from the NPC tissue array. A–C: the upregulated genes EZH2, SKIL and CD44. D–F: the downregulated genes ANXA1, LCN2 and MSH3. Panel G summarises all the tissue array staining. The Y axis shows the log₂ value of the ratio of the paired tumour:normal IHC scores. Some IHC scores were zero resulting in log₂ ratio values of plus or minus infinity. For convenience, these are represented as 4 or−4 on the figure. Except for JAK and CD44, p values were less then 0.05. Individual p values are listed in Tables S1 and S2.

Figure 2. Chromosomal copy number changes.

Panel A shows the major regions of copy number gain (red) or loss (blue) across the genome. The Y axis shows the number of cases (out of 16) at which a region was changed. Chromosomes are ordered from left to right as indicated. Panels B–D show traces of the log2 ratio of the copy number of DNA from the tumour samples compared to the normal controls. B: the homozygous deletion at the FHIT locus in C666-1; C: a hemizygous deletion in C666-1 and a homozygous deletion in tumour XY5, both at 6q22.33; D: homozygous deletions encompassing the CDKN2B locus in tumours MMAH, XY5, XY8 and HKC1. Tumour MMAH also shows a 600 Kb homozygous deletion at 9p24.1 containing the NFIB gene. The sizes of the discrete aberrations are indicated.

Figure 3. The 8p11.21 amplified region of tumour HKD1.

A. Trace of the log2 ratio of the copy number of DNA from the tumour sample compared to the normal controls. The 2.5 Mb amplification is indicated. B. Heat map of the relative expression levels of the genes found within the amplified region. The samples appear in columns and the individual genes within the amplified region form the rows. High level expression is represented by the intensity of red and low level by blue. The brackets at the bottom indicate tumour samples with genome copy numbers of 2 or 3 within this region.