Abstract
Tumor suppressor TP53 is frequently mutated in colorectal cancer (CRC), and most mutations are missense type. Although gain-of-functions by mutant p53 have been demonstrated experimentally, the precise mechanism for malignant progression in in vivo tumors remains unsolved. We generated ApcΔ716Trp53LSL•R270Hvillin-CreER compound mice, in which mutant p53R270H was expressed in the intestinal epithelia upon tamoxifen treatment, and examined the intestinal tumor phenotypes and tumor-derived organoids. Mutant Trp53R270H, but not Trp53-null mutation accelerated submucosal invasion with generation of desmoplastic microenvironment. The nuclear accumulation of p53 was evident in ApcΔ716Trp53R270H/R270H homozygous tumors like human CRC. Although p53 was distributed to the cytoplasm in ApcΔ716Trp53+/R270H heterozygous tumors, it accumulated in the nuclei at the invasion front, suggesting a regulation mechanism for p53 localization by the microenvironment. Importantly, mutant p53 induced drastic morphological changes in the tumor organoids to complex glandular structures, which was associated with the acquisition of invasiveness. Consistently, the branching scores of human CRC that carry TP53 mutations at codon 273 significantly increased in comparison with those of TP53 wild-type tumors. Moreover, allografted ApcΔ716Trp53R270H/R270H organoid tumors showed a malignant histology with an increased number of myofibroblasts in the stroma. These results indicate that nuclear-accumulated mutant p53R270H induces malignant progression of intestinal tumors through complex tumor gland formation and acquisition of invasiveness. Furthermore, RNA sequencing analyses revealed global gene upregulation by mutant p53R270H, which was associated with the activation of inflammatory and innate immune pathways. Accordingly, it is possible that mutant p53R270H induces CRC progression, not only by a cell intrinsic mechanism, but also by the generation or activation of the microenvironment, which may synergistically contribute to the acceleration of submucosal invasion. Therefore, the present study indicates that nuclear-accumulated mutant p53R270H is a potential therapeutic target for the treatment of advanced CRCs.
Highlights
Molecular genetic studies have revealed that genetic alterations in driver genes induce the development of colorectal cancer (CRC) through an adenoma carcinoma sequence.[1,2,3] It has recently been demonstrated that cumulative mutations in APC, KRAS, SMAD4 and TP53 in human intestinal cell-derived organoids are associated with the development of transplanted tumors in immunodeficient mice.[4,5] despite these findings, the precise functional role of each driver gene mutation in the malignant progression is still not fully understood
Immunohistochemical analysis revealed that the expression of mesothelin, an invading CRC cell marker,[22] and snail[2], an epithelial-mesenchymal transition marker,[23,24] was induced at the submucosal invasive area of ApcΔ716 Trp53+/R270H adenocarcinomas, but it was not detected in the invasive area of ApcΔ716 Trp53+/flox tumors (Supplementary Figure 1a)
It is possible that Trp53R270H but not Trp53-null mutation promotes the metastatic potential of cancer cells
Summary
Molecular genetic studies have revealed that genetic alterations in driver genes induce the development of colorectal cancer (CRC) through an adenoma carcinoma sequence.[1,2,3] It has recently been demonstrated that cumulative mutations in APC, KRAS, SMAD4 and TP53 in human intestinal cell-derived organoids are associated with the development of transplanted tumors in immunodeficient mice.[4,5] despite these findings, the precise functional role of each driver gene mutation in the malignant progression is still not fully understood. Precisely how the respective genetic alterations in tumor cells contribute to the generation of the microenvironment, which is an important factor in cancer progression, remains to be elucidated. In this regard, mouse genetic studies remain a powerful complementary approach that enables detailed in vivo experimentation and observations for delineating the mechanistic basis of tumorigenesis. 74% of p53 mutations are missense mutations, which results in the formation of a mutant protein.[9,10] Mouse genetic studies have indicated that missense mutations in Trp[53] at codon 172 and 270 cause adenocarcinomas in the lung and intestine, which are not developed in Trp53-null mutant mice, indicating that mutant p53 induces tumors in epithelial organs by a gain-of-function mechanism.[11,12] it has been shown that mutant p53 contributes to the invasion and metastasis of rhabdomyosarcoma,[13] pancreatic cancer[14] and sporadic and chemically induced colitis-associated intestinal tumors.[15,16] Mechanistically, mutant p53 promotes tumorigenesis by the activation of hepatocyte growth factor and platelet-derived growth factor receptor β signaling.[17,18] mutant p53 alters gene expression at a global level through
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