The goal of this study was to determine type, frequency, and pattern of cMNR frameshift mutations in protein PTP genes in MSI-H colorectal cancer cell lines and primary colon tumors. This gene family was selected because PTPs are expected to exert tumor suppressive function and thus represent potential targets of inactivating mutations in DNA mismatch repair deficient tumors. Among the large number of PTP genes in the human genome we selected 16 PTP genes that contained coding region microsatellites of increased length (≥ 7 repetitive units). Our results provide evidence for frameshift mutations in six of these PTP genes (PTPN21, PTPRS, PTPN5, PTPN23, PTPRA, PTPRE) affecting about 32% of MSI-H colorectal cancers. This frequency differs slightly from a previous study that reported PTP mutations in 26% of unselected colorectal carcinomas . Although most of the coding repeat-harboring PTP genes (13/16 candidates) analyzed by us have also been examined by Wang and co-workers, different sets of mutated PTP genes were identified in both studies. In particular, we observed tumor-specific genetic alterations in PTPN21, PTPN23, PTPN5, PTPRA, PTPRE and PTPRS whereas mutations reported by Wang et al. were confined to PTPRF, PTPRG, PTPRT, PTPN3, PTPN13, and PTPN14. This difference most probably can be explained by the different screening strategies used in these two studies. Wang et al. performed their initial mutational pre-screening on a set of 18 unselected colorectal tumors, This subset would be expected to include only 2 to 3 MSI tumors based on a general MSI frequency of 15% reported for unselected colorectal cancers. Since the type of mutation and the spectrum of affected genes is remarkably different among MSS and MSI-H tumors, the observed differences are not unexpected. However, there were also some consistent findings in both studies. For example, PTPN13 was examined by both groups but no cMNR mutations were observed in a cumulative number of 69 MSI-H CRC samples ( and data presented here). Interestingly, Wang et al. described two nonsense or frameshift mutations at the expected MNR position within PTPN13. Unfortunately, any information about the MSI status of these tumors is missing. So, our investigation represent a reasonable completion of a systematic analysis of human PTP genes in human colorectal cancers.
Recently, we proposed a statistical model that allows to predict positively or negatively selected target genes of MSI tumorigenesis in an organ-specific manner based on cMNR mutation frequencies . According to the actual release of this model (http://www.seltarbase.org, release 200711), cMNRs of 7 or 8 repetitive units – in the absence of any biological selection pressure – are expected to show a mean somatic mutation rate of about 4% and 9% respectively. In order to qualify as positively selected MSI target genes, tumor-specific mutation frequencies for cMNRs of this length have to exceed 22% and 28%, respectively. The three most frequently mutated PTPs identified in the present study (PTPN21 [A8], PTPRS [C7], PTPN5 [C7]) show mutation frequencies of 17%, 12%, and 6%, respectively. The final overall mutation frequency of PTPN21 A8 (14.4%) is even slightly lower, when considering data from the literature . Certainly, we did not extend our mutation search to the entire coding sequence of each PTP gene, and therefore, additional mutations outside the cMNR sequence cannot be excluded. Furthermore, detailed studies of individual tumor types have provided compelling evidence that mutations of different genes but within the same pathway can have similar functional effects, i.e. leading to its disruption and providing a growth advantage to affected cells , as it has been proposed for apoptotic genes like Fas, Apaf-1, and Bcl-10 . In conclusion, although these gene-specific mutation rates are higher than the mean mutation frequencies for this length of repeats they do not reach statistical significance and prediction about positive Selective Target Genes in MSI-H tumorigenesis is not feasible. Therefore, human PTP genes do not seem to play a common role in MSI-H tumorigenesis. However, contribution to individual tumor development cannot be excluded. Furthermore, the observation of 2 mutations within a set of 17 MSI-H adenomas in PTPN21 argues for an early event in malignant transformation.
Despite some structural diversity, the six PTP proteins found to be mutated in the present study share a catalytically active phosphatase that resides in one (PTPN21, PTPN23, PTPN5, PTPRS) or two (PTPRA) C-terminal domains . Notably, all cMNR frameshift mutation sites are located upstream or within the first half of these catalytic domains (see Figure 2) resulting in truncated proteins that are expected to show partial or complete loss of phosphatase activity. However, all PTP frameshift mutations only affected a single allele leaving the cMNR on the remaining allele intact. The presence of contaminating normal inflammatory cells in these tumors may well account for this observation. Alternatively, the remaining wildtype copy may be silenced by epigenetic mechanisms. The absence of biallelic mutations in the analyzed tumors could be due to a dominant negative fashion or affection of gene dosage [46, 47]. Shortened transcripts of murine PTPs lacking catalytic or interaction domains by alternative splicing can act in a dominant negative manner .
From our analysis of preneoplastic lesions we further conclude that PTPN21 frameshift mutations also occur in MSI-H colorectal adenomas albeit at lower frequency (12%) thereby indicating an early step during MSI tumorigenesis. At the protein level, PTPN21 is known to bind to and activate c-Src and Etk protein kinases [49, 50]. Interestingly, activated Etk has been reported to trigger apoptosis in breast cancer cells via Stat1 and p21 . By analogy, we hypothesize that inactivation of PTPN21 by cMNR frameshift mutations in MSI-H colon cancer cells might lead to Etk inactivation and subsequent inhibition of apoptosis.