Colorectal cancer is driven by the accumulation of driver mutations, but the contributions of specific mutations to different steps in malignant progression are not fully understood. In this study, we generated mouse models harboring different combinations of key colorectal cancer driver mutations () in intestinal epithelial cells to comprehensively investigate their roles in the development of primary tumors and metastases. mutation caused intestinal adenomas and combination with mutation or deletion induced submucosal invasion. The addition of mutation yielded epithelial-mesenchymal transition (EMT)-like morphology and lymph vessel intravasation of the invasive tumors. In contrast, combinations of with and mutation were insufficient for submucosal invasion, but still induced EMT-like histology. Studies using tumor-derived organoids showed that was critical for liver metastasis following splenic transplantation, when this mutation was combined with either plus or deletion, with the highest incidence of metastasis displayed by tumors with a genotype. RNA sequencing analysis of tumor organoids defined distinct gene expression profiles characteristic for the respective combinations of driver mutations, with upregulated genes in tumors found to be similarly upregulated in specimens of human metastatic colorectal cancer. Our results show how activation of Wnt and Kras with suppression of TGFβ signaling in intestinal epithelial cells is sufficient for colorectal cancer metastasis, with possible implications for the development of metastasis prevention strategies. These findings illuminate how key driver mutations in colon cancer cooperate to drive the development of metastatic disease, with potential implications for the development of suitable prevention strategies. .

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 ApcΔ716 Trp53LSL•R270H villin-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 ApcΔ716 Trp53R270H/R270H homozygous tumors like human CRC. Although p53 was distributed to the cytoplasm in ApcΔ716 Trp53+/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 ApcΔ716 Trp53R270H/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.

Missense-type mutant p53 plays a tumor-promoting role through gain-of-function (GOF) mechanism. In addition, the loss of wild-type TP53 through loss of heterozygosity (LOH) is widely found in cancer cells. However, malignant progression induced by cooperation of TP53 GOF mutation and LOH remains poorly understood. Here, we show that mouse intestinal tumors carrying Trp53 GOF mutation with LOH (AKTP M/LOH) are enriched in metastatic lesions when heterozygous Trp53 mutant cells (AKTP +/M) are transplanted. We show that Trp53 LOH is required for dormant cell survival and clonal expansion of cancer cells. Moreover, AKTP M/LOH cells show an increased in vivo tumor-initiating ability compared with AKTP Null and AKTP +/M cells. RNAseq analyses reveal that inflammatory and growth factor/ MAPK pathways are specifically activated in AKTP M/LOH cells, while the stem cell signature is upregulated in both AKTP M/LOH and AKTP Null cells. These results indicate that TP53/Trp53 LOH promotes TP53/Trp53 GOF mutation-driven metastasis through the activation of distinct pathway combination.

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