CTEN/TNS4 is an oncogene in colorectal cancer (CRC) which enhances cell motility although the mechanism of Cten regulation is unknown. We found an association between high Cten expression and KRAS/BRAF mutation in a series of CRC cell lines (p = 0.03) and hypothesised that Kras may regulate Cten. To test this, Kras was knocked-down (using small interfering (si)RNA) in CRC cell lines SW620 and DLD1 (high Cten expressors and mutant for KRAS). In each cell line, Kras knockdown was mirrored by down-regulation of Cten Since Kras signals through Braf, we tested the effect of Kras knockdown in CRC cell line Colo205 (which shows high Cten expression and is mutant for BRAF but wild type for KRAS). Cten levels were unaffected by Kras knockdown whilst Braf knockdown resulted in reduced Cten expression suggesting that Kras signals via Braf to regulate Cten. Quantification of Cten mRNA and protein analysis following proteasome inhibition suggested that regulation was of Cten transcription. Kras knockdown inhibited cell motility. To test whether this could be mediated through Cten, SW620 cells were co-transfected with Kras specific siRNAs and a Cten expression vector. Restoring Cten expression was able to restore cell motility despite Kras knockdown (transwell migration and wounding assay, p<0.001 for both). Since KRAS is mutated in many cancers, we investigated whether this relationship could be demonstrated in other tumour models. The experiments were repeated in the pancreatic cancer cell lines Colo357 & PSN-1(both high Cten expressors and mutant for KRAS). In both cell lines, Kras was shown to regulate Cten and forced expression of Cten was able to rescue loss of cell motility following Kras knockdown in PSN-1 (transwell migration assay, p<0.001). We conclude that, in the colon and pancreas, Cten is a downstream target of Kras and may be a mechanism through which Kras regulates of cell motility.

Disrupted Ras/Raf signalling is common in CRC. Homozygous KRAS mutations and concomitant KRAS/BRAF mutations may be indicative of a gene dosage effect. The significance of BRAF splice variants is uncertain but may represent another layer of complexity. Finally, if KRAS mutation is to be used for predictive testing, then the whole gene may need to be screened as mutations occur outside codons 12/13.

QMC-PCR with HRM is a simple, robust and inexpensive technique which had greater sensitivity than Sanger sequencing. It allows multiple mutation hotspots to be rapidly screened and is thus highly suited to mutation detection in DNA derived from FFPE tissues.

Mutation detection is important in cancer management. Several methods are available of which high resolution melting (HRM) analysis and pyrosequencing are the most versatile. We undertook a comparative analysis of these techniques. The methods are: To compare the limit of detection (LOD), mutations in KRAS (codon 12/13 hotspot) and BRAF (V600E hotspot) were tested. DNA mixtures containing mutant alleles at a frequency of around 25%/12.5%/6%/3%/ 1.5%/0.8% were analysed. To compare frequency of mutation detection, 22 DNA samples (nine high quality samples from cell lines, 13 low quality samples from formalin-fixed paraffin-embedded tissue) were tested for three hotspots in KRAS (codons 12/13, 61 and 146) and two hotspots in BRAF (V600E and exon 11). HRM analysis of KRAS (codon12/13) and BRAF (V600E) showed that 3% and 1.5% mutant alleles respectively could be reliably detected whilst pyrosequencing reliably detected 6% mutant alleles in each case. Of 110 tests performed on 22 DNA samples, in 109 cases HRM and pyrosequencing gave identical results. Two of the samples tested had previously been called as wild type for KRAS by direct Sanger sequencing but were found to be mutant by both HRM and pyrosequencing. Both HRM and pyrosequencing can detect small numbers of mutant alleles although HRM has a lower limit of detection. Both are suitable for use in mutation detection and are both more sensitive than Sanger sequencing.