DIFFERENTIAL EXPRESSION ANALYSIS OF MICROSATELLITE UNSTABLE COLORECTAL CANCERS IDENTIFIES NOVEL GENE TARGETS

Abstract

Molecular profiling of sporadic colorectal cancers has led to the demarcation of three distinct groups: those with a high level of microsatellite instability (MSI‐H), low level of instability (MSI‐L) and microsatellite stable tumours (MSS) (no instability). Each subclass can be characterised by a number of distinguishing genetic features which effect the phenotype and drug responsiveness of the tumour and may indicate patient prognosis. Tumours arise as a result of altered protein function and this is associated with concomitant alteration in gene expression levels. Based on this premise, suppressive subtractive hybridisation (SSH) was utilised to identify genes down‐regulated in MSH‐H tumour cells when compared with the corresponding normal mucosa. In addition to amplifying differentially expressed genes, this technique suppresses the amplification of high copy number genes and therefore selects for expression differences in rare transcripts. Two subtraction experiments were performed in parallel with varying starting material. SSH1 utilised paired normal and tumour tissue from an MSI‐H cancer. SSH2 used pure MSI‐H tumour cells after passage in a nude mouse paired with colonic crypts isolated from the corresponding normal mucosa. The subtracted libraries were cloned and 400 randomly picked colonies from each SSH were subjected to a secondary screen to confirm differential expression. All remaining clones were sequenced and compared with known genes through the ANGIS nonredundant nucleic acid databases to identify clones which had been isolated in duplicate or from different regions of the same gene. Contiguous sequences were constructed using the Sequencher assembly program. This resulted in a total of 146 unique clones of which 31 matched known sequences. Four clones were isolated from both SSH experiments. The differentially expressed genes included those associated with transcription, checkpoint control, DNA mismatch repair, ion transport, mucin synthesis, metabolism, apoptosis and cell adhesion. Novel cDNA clones were further characterised bioinformatically to identify putative open reading frames, functional domains and chromosomal location. SAGE and virtual northern analysis confirmed many clones to be of colonic origin, often isolated from normal mucosa and down‐regulated in tumours. Clones were prioritised for further study based on known functional relevance, presence of coding region repeat tract or 5′ CpG island which may allow epigenetic gene silencing by methylation. Detailed functional significance of known genes and confirmatory expression analysis of selected candidates will be discussed. This technique has been proven sensitive for the detection of functionally relevant and novel genes in the progression of MSI‐H tumours, which may provide novel points of therapeutic intervention.