The prognostic value of p53 mutations in MSS phenotypes of Stage III CRCs was assessed, and other molecular markers associated with p53 mutant phenotypes were identified. The prevalence of p53 mutations was higher in CRCs with the MSS phenotype (59%) as compared to the MSI-H phenotype (30%). Both univariate and multivariate survival analyses revealed that p53 mutations in Stage III-MSS-CRCs were associated with shorter cancer-specific survival relative to those with wt-p53. After rigorous validation on four platforms, a molecular signature associated with p53 mutant phenotypes in this subset of CRCs was identified.
The differentially expressed genes are involved in remodeling of the extracellular matrix, adhesion, cytoskeleton plasticity, and signal transduction. The p53 mutant signature was characterized by transcriptional repression, which is distinct from the expression associated with wt-p53. Thus, metastasis mediated by p53 mutations is a selective process with a specific molecular signature of Stage III-MSS CRCs.
In about 50% of human cancers, including CRCs, the p53
gene is mutated. The gain of oncogenicity or loss of tumor suppressor function of p53
due to its inactivation through missense mutations contributes to tumor aggressiveness and results in poor patient survival 
mutations have been considered as metastatic signatures in CRCs 
. The mutations that affect structural or functional domains and those in evolutionary conserved regions are associated with aggressive tumors 
and chemo-resistance 
mutations within certain domains [L2 and L3 loops, and the Loop Sheet Helix (LSH) motif] and mutations in evolutionary non-conserved region of the p53 gene 
are associated with aggressive phenotypes 
. These observations, in conjunction with our previous findings 
, suggest a function for the p53 protein in metastasis due to inactivation and indicate that patients with CRCs exhibiting p53
mutations are at risk for aggressive progression and early death. The present study demonstrates an association between p53
mutations and poor survival in Stage III-MSS-CRCs. Genomic instability, common in CRCs 
, correlates with p53
. Thus, for some Stage III-MSS-CRCs, genomic instability associated with p53
mutations is the cause of developing a risk phenotype and for poor survival.
Previous studies have demonstrated that sporadic colon cancers with the MSI phenotype are less aggressive, because these tumors commonly show a lower frequency of p53 mutations 
, as observed in the current study. This relationship is consistent with the concept that most CRCs develop either along the chromosomal instability pathway associated with TP53
mutations and MSS tumors or the aberrant mismatch repair pathway associated with wild-type p53 and MSI tumors 
. This association may also explain why tumors with MSI seem to have less aggressiveness as compared to those with a proficient mismatch repair system. In the current investigation, there was no significant association between p53 status and poor patient survival for Stage III CRCs with the MSI phenotype, suggesting different effects of p53 mutations in Stage III CRCs with MSS and MSI phenotypes.
Microarray analyses, based on the p53
status in Stage III-MSS CRCs, identified 84 genes differentially expressed in tumors with p53
mutant and wt-p53
phenotypes. In tumors of the p53
mutant phenotype, 35 genes were up-regulated and 49 down-regulated, suggesting that transcriptional repression of genes is important for developing risk of aggressive cancer and for poor survival manifested by p53
mutations. The repression affects known tumor or metastasis suppressor genes, such as TRIM 29
, and LPAR6 
, and the sestrins, SESN1
and SESN3 
; however, in our analyses, ACVR1B, LPAR6, and sestrins were not validated by the qNPA method. Thus, the results support the concept that silencing of genes is essential for tumor progression 
. One explanation for this is that the inactivation of p53
by mutation results in the overexpression of transcriptional repressors, which suppress other genes, including those suppressing metastasis.
Gene microarray is an excellent approach to evaluate the expression profiles of thousands of genes; however, it has several limitations related to accuracy and reproducibility 
. For a differentially expressed gene to serve as a biomarker with high accuracy, verification of analysis of gene expression by multiple methods is required 
. Inaccuracies in identifying the ‘true’ expression of potentially useful molecular markers may be the basis for the lack of translation of the findings obtained from studies involving gene microarrays. Thus, to validate results obtained from these microarrays, the genes selected in the present investigation were subjected to the quantitative nuclease protection assay (qNPA) and qRT-PCR. The qNPA assay validated only seven of the differentially expressed genes identified by Affymetrix microarrays. These seven genes were further validated by qRT-PCR and IHC. There are associations between dysregulation of all seven genes and cancer development 
Although little is known about the properties of TRIM29
, a member of the tripartite motif (TRIM) family, three proteins of this family (TRIM19, TRIM24 and TRIM27) are involved in cellular growth or development 
and become oncogenic as a result of chromosomal translocations 
, suggesting their involvement in tumor progression. Increased expression of TRIM29
is associated with tumor differentiation, tumor growth, tumor invasion, and lymph node metastasis 
. In the present study, TRIM29 was over-expressed in p53
mutant phenotypes, supporting its function in p53 dependent-pathways that lead to aggressive behavior of Stage III-MSS CRCs. Further mechanistic studies are needed to clarify the function of TRIM29 in CRC progression.
IQGAP family proteins modulate cytoskeletal architecture and cell adhesion 
, and they may be involved in metastasis of CRCs, lung cancers, and cholangiocarcinomas 
, which encodes a putative 180-kDa protein with RasGAP, IQG1, CH, and COG5022 domains, regulates cell proliferation through the Ras/ERK signaling cascade 
. Our findings show up-regulation of IQGAP3 in MSS and p53
mutant phenotypes and indicate that, for Stage III-MSS CRCs, the underlying molecular mechanism for IQGAP3 is different in p53
mutant and wt-p53
phenotypes. Also, the results indicate that IQGAP3 is involved in metastasis. Although it might be useful for predicting CRC progression, its function and prognostic capacity remain to be investigated.
It is important to compare the gene expression profiles of MSS Stage III CRCs with and without p53 mutations to understand the molecular mechanisms involved in their progression and to identify aggressive subsets. The present results, demonstrating that p53 mutations are associated with a poor prognosis for Stage III microsatellite-stable CRCs, may lead to development of individualized therapies for Stage III-MSS CRCs. Furthermore, robust validations of the expression profiles, including those for TRIM29 and IQGAP3, may allow identification of novel therapeutic targets.