We have investigated the relationship between MSI, TS and p53 status using standard genotyping and immunohistochemical methods in early stage CRC. Although only loosely correlating with TP53 mutation, p53 nuclear over-expression detected by IHC has been found to be a marker of worse prognosis in many previously published analyses of CRC datasets [21, 30, 31]. Our results indicate a highly significant association between p53 status and TS expression, with CRCs expressing high TS levels more likely to over-express p53, regardless of MSI status.
In normal cells, regulation of both TS and p53 are independently tightly controlled. In addition to it's role in enzyme catalysis, TS also functions as a RNA binding protein [32, 33], regulating it's own expression by a negative autoregulatory mechanism [33, 34], as well as binding to it's own RNA, to form TS-ribonucleoprotein complexes with several RNA species including c-myc and TP53 . Although in vitro data indicates that p53 and TS have the ability to regulate each-other in non-malignant cells [32, 33, 36], evidence for a relationship in CRC has been conflicting, with some studies reporting that TS negatively regulates p53 expression , whilst others have shown no such relationship [38, 39]. In addition, the potential for wild-type p53 to regulate TS expression has also been demonstrated using a luciferase-based system, which showed that p53 expression can inhibit TYMS promoter activity . These results are, however, based on in vitro analysis, and no studies have characterised the role of mutant p53 or whether aberrant mismatch repair impacts on the relationship. Our results suggest a relationship between TP53 status and TS expression implying that the poor prognosis and chemoresistance observed in studies of CRC patients with either high TS expression or TP53 mutation/p53 over-expression, may have been impacted on by either co-variate.
A number of potential mechanisms may account for our findings. TP53 mutation is associated with loss of transcriptional activity control resulting in up- or down-regulation of downstream p53 effectors. Thus, inactive p53 might disinhibit an inhibitory role of p53 on TS expression. Alternatively, according to the gain-of-function paradigm , mutant p53 might acquire novel activities that promote cell growth and survival , perhaps through enhanced TS expression. An example of the latter case is the 273 Arg-His mutation, which has strong transactivating activity . Although the role of this specific mutation in regulating TS expression is unknown, it is feasible that specific p53 mutations retain transcriptional regulatory activity, which may be partially responsible for control of transcriptional activity of proteins such as TS. Supporting this, Lenz et al. investigated the relationship between TP53 mutation and TS expression in a series of 36 CRCs and demonstrated that CRCs with p53 mutations affecting the zinc-binding domains had higher TS expression than those with mutation outside these domains . Zinc domains are involved in direct DNA contact, protein stabilization, and structural activity, indicating that these mutations may have a severe impact on the transcriptional activity of p53.
Nine other studies have investigated the relationship between p53 and TS expression, based on immunohistochemistry, using p53 over-expression as a surrogate of TP53 mutation [8, 45–52]. However, most have been based on small sample sizes (median 66, range 22–691). Our results are consistent with four [44–46, 50] of these studies, which also demonstrated a relationship between p53 and TS expression. Moreover, our study is the only one to assess this relationship stratified by MSI status. The relationship between p53 and TS was observed both in tumours with MSI and those with MSS, implying that aberrant mismatch repair is unlikely to impact on any mechanism relating p53 to TS expression. However, given the small number of samples with MSI, this cannot be entirely excluded.
As expected a highly significant inverse relationship between p53 status and MSI was observed. This relationship was independent of TS status, observed in both tumours expressing low and high TS levels (p ≤ 0.03), and is consistent with the concept that most CRCs develop either along the chromosomal instability pathway associated with TP53 mutation and MSS tumours or the aberrant mismatch repair pathway associated with wild-type p53 and MSI tumours [53–56]. This association may also explain why tumours with MSI seem to have an improved prognosis compared to those with intact mismatch repair. However, we demonstrated no relationship between MSI and TS status. This finding was independent of p53 status. Whilst the precise mechanisms by which cells with MSI seem resistant to 5FU in vitro has been poorly defined, Ricciardiello et al.  have suggested that this may, in part, be due to TS over-expression TS. In their analysis of 192 CRCs the authors demonstrated an association between CRCs with MSI and high TS expression . This observation was, however, contrary to an earlier study based on an analysis of 53 CRCs, which also observed no relationship between TS expression and the MSI phenotype . Moreover, the study by Ricciardiello et al.  was based on only 24 CRCs with MSI and the rate of high TS expression in their study was at the lower end of that previously reported (21%)  and may have biased findings. Our data, based on a sample size over two times larger, precludes at least a 16% difference between MMR status and high TS expression at the 5% threshold, with 90% power.
Our results provide little evidence that TS expression plays a major role in defining chemoresistance in microsatellite unstable CRCs, but gives support to previous reports of an inverse relationship between MSI and p53 status. In addition, we demonstrated that TS expression is related to p53 status, and this relationship may in part account for the poorer prognosis and relative chemoresistance seen in these tumours.