Combined Inactivation of TP53 and MIR34A Promotes Colorectal Cancer Development and Progression in Mice Via Increasing Levels of IL6R and PAI1

BACKGROUND & AIMS: Combined inactivation of the microRNA 34a gene (MIR34A, by methylation) and the TP53 gene (by mutation or deletion) is observed in 50% of colorectal tumors that progress to distant metastases. We studied mice with intestinal disruption of Mir34a and Tp53 to investigate mechanisms of colorectal carcinogenesis and identify strategies to block these processes. METHODS: Mice with disruption of Mir34a and/or Tp53 specifically in intestinal epithelial cells (IECs) (Mir34a(Delta IEC) mice, Tp53(Delta IEC) mice, and Mir34a (Delta IEC)/Tp53(Delta IEC) mice) and controls (Mir34a(Fl/Fl)/Tp53(Fl/Fl)) were given azoxymethane to induce colorectal carcinogenesis. Some mice were given intraperitoneal injections of an antibody against mouse interleukin 6 receptor (IL6R), or received an inhibitor of PAI1 (tiplaxtinin) in their chow. Intestinal tissues were collected and analyzed by immunohistochemistry; gene expression profiles were analyzed by RNA sequencing. We determined the expression and localization of PAI1 in 61 human primary colon cancers and compared them to MIR34A methylation and inactivating mutations in TP53. Data on mRNA levels, methylation, and clinical features of 628 colon and rectal adenocarcinomas were obtained from The Cancer Genome Atlas portal. RESULTS: Mir34a(Delta IEC)/Tp53(Delta IEC) mice developed larger and more colorectal tumors, with increased invasion of surrounding tissue and metastasis to lymph nodes, than control mice or mice with disruption of either gene alone. Cells in tumors from the Mir34a(Delta IEC)/Tp53(Delta IEC) mice had decreased apoptosis and increased proliferation compared to tumor cells from control mice, and expressed higher levels of genes, that regulate inflammation (including Il6r and Stat3) and epithelial-mesenchymal transition. The gene expression pattern of the tumors from Mir34a(Delta IEC)/Tp53(Delta IEC) mice was similar to that of human colorectal tumor consensus molecular subtype 4 (mesenchymal, invasive). We identified the Pai1 messenger RNA as a target of Mir34a; levels of PAI1 protein were increased in primary colon cancer samples, that displayed methylation of MIR34A and mutational inactivation of TP53. Administration of tiplaxtinin or anti-IL6R antibody to Mir34a(Delta IEC)/Tp53(Delta IEC) mice decreased proliferation of cancer cells, and reduced colorectal tumor invasion and metastasis. CONCLUSIONS: In mice, we demonstrated that combined inactivation of Mir34a and Tp53 promotes azoxymethane-induced colorectal carcinogenesis and tumor progression and metastasis by increasing levels of IL6R and PAI1. Strategies to inhibit these processes might be developed to slow progression of colorectal cancer.