Functional consequence of the p53 codon 72 polymorphism in colorectal cancer

Abstract

Background: The codon 72 polymorphism in p53 has been implicated in colorectal cancer (CRC) risk, prognosis and CRC health disparities. We examined the functional consequence of this polymorphism in CRC.

Experimental design: Plasmids (pCMV6) that express different phenotypes of p53 [p53 wild type (wt) at codon 72 (R72^wt), R72^wt with mutation at codon 273 cysteine (R72^273Cys), p53 mutation at codon 72 (P72^wt) and P72^wt with mutation at codon 273 (P72^273Cys)] were constructed. The CRC cell line Caco2, which does not express p53 for in vitro studies, was used as host. CRC xenografts were established in severe combined immunodeficient (SCID) mice using established cell lines. CRC surgical specimens, corresponding normal colon, and tumor xenografts were sequenced for codon 72 polymorphism of p53. Proteins signaling mechanisms were evaluated to assess the functional consequence of P72 phenotype of p53.

Results: This study demonstrated a significantly increased survival of cells expressing P72^wt, mutant phenotype, versus R72^wt phenotype. WB analyses revealed that P72^wt induced activation of p38 and RAF/MEK/ extracellular signal-regulated kinase (ERK) MAP kinases. Activation of CREB was found to be higher in tumors that exhibit P72 phenotype. Metastatic lesions of CRC expressed more phospho-CREB than non-metastatic lesions. The expression of P72^wt promoted CRC metastasis.

Conclusions: P72 contributes to the aggressiveness of CRC. Because P72 is over-expressed in CRC, specifically in African-American patients, this suggests a role for P72 in cancer health disparities. This work was supported by NIH/NCI Workforce Diversity Grant R21-CA171251 & U54CA118948.

Keywords: CREB; EMT; codon 72 polymorphism; p38MAPK; p53.

Figure 1. P72 phenotype of p53 induces cell survival and endothelial cell tubular formation

Increased cell survival in cells that express P72^wt phenotype or mutant phenotypes compared to cells that express R72^wt phenotype (A and B). Conditioned medium of P72^wt or mutant phenotypes expressing cells induce HUVEC to form networks of multiple tube-like structures, while conditioned medium of R72^wt expressing cells did not support tubule formation (C).

Figure 2. P72 phenotype of p53 is associated with abundance of tumor promoting phenotypes

The expression of p-NF-kB, p-STAT, CXCR4 and p-ERK was found to be higher in the cell model of P72^wt or mutant phenotypes than in the vector-control or R72^wt cell models.

Figure 3

P72 phenotype promotes EMT process (A and B). P72^wt or mutant phenotype expressing cells displayed decreased expression of the epithelial signature E-cadherin and increased expression of the mesenchymal signature vimentin, fibrinectin, CD44 and p-GSK-3β.

Figure 4. P72 phenotype promotes p38MAPK and RAF/MEK/ERK signaling

Molecular analyses revealed that P72^wt or mutant phenotypes effectively induced the activation of p38 MAPK and RAF/MEK/ERK signaling (A and B). Up-regulation of phosphorylated SEK1/MKK4, upstream kinase of p38 MAPK has been found to be associated with P72^wt or mutant phenotypes (A). Activation SEK1/MKK4 was accompanied by up-regulation of p-MAPKAPK-2, p-Hsp27 and p-CREB downstream targets of p38 MAPK (A). Suppression of RAF/MEK/ERK activation was significantly higher in cells that express P72^wt or mutant phenotypes compared to cells that express R72^wt phenotype. This suppression was accompanied by down-regulation of p-Akt (B).

Figure 5. CREB expression in tumor xenografts by IHC

IHC confirmed the presence of CREB expression in CRC (HT29) and (SW480) (A). Compared to primary CRC lesion, the expression of CREB was markedly increased in metastatic CRC lesions (A). IHC confirmed the expression status of CD31, CREB, and p53 in CRC (HT29) and (SW480) (B). Compared to R72 CRC, the expression of CD31 (tumor angiogenesis) or CREB was markedly increased in P72 CRC. Sequencing analysis revealed that LS174T or HT29 tumors exhibit homozygous for Arg/Arg, whereas SW480 tumor exhibits homozygous for Pro/Pro (B). IHC analysis revealed that LS174T tumor was found to be negative for p53 expression (p53 wild type) (B), whereas HT29 or SW480 tumors were found to be positive for p53 expression (p53 mutants) (B).

Figure 6. CREB expression in CRC by IHC

Immunostaining confirmed the presence of CREB expression in CRC (A). Compared to normal, the expression of CREB was markedly increased in primary CRC lesion or metastatic CRC lesions (A). (B) IHC confirmed the expression status of CD31, CREB, and p53 in CRC (B). Compared to R72 CRC, the expression of CD31 (tumor angiogenesis) or CREB was markedly increased in P72 CRC. IHC analysis revealed that tumors were found to be negative for p53 expression (p53 wild type) (B).