Genetic analysis of AP4 function in stem cells, tumor maintenance and metastasis formation

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The TFAP4/AP4 protein belongs to the class of basic-helix-loop-helix leucine zipper (bHLH-LZ) transcription factors. AP4 exclusively forms homodimers, which bind to the E-box motif CAG/CCTG and thereby either repress or activate the expression of target genes. We previously identified the AP4 gene as a direct transcriptional target of c-MYC. AP4 is expressed in progenitor/transient amplifying (TA) cells in human colonic crypts, and in colorectal cancer (CRC) in a pattern similar to c-MYC. The prototypic oncogene c-MYC is a direct target of the APC (adenomatous polyposis coli)/Wnt (Wingless/Int-1) pathway and an essential mediator of tumor formation induced by inactivation of Apc in the intestine. Previous studies suggested that AP4 may contribute to the progression of CRC by regulating genes involved in epithelial–mesenchymal transition (EMT) and proliferation. In the previous funding period we deleted Ap4 in ApcMin mice, a preclinical model of inherited colorectal cancer. Ap4 deficiency extended their average survival by 110 days and decreased the formation of intestinal adenomas and tumor-derived organoids. The effects of Ap4 deletion were presumably due to the reduced number of functional intestinal stem cells (ISCs) amenable to adenoma-initiating mutational events. Deletion of Ap4 also decreased the number of colonic stem cells and increased the number of Paneth cells. Expression profiling revealed that ISC signatures, as well as the Wnt/β-catenin and Notch signaling pathways were downregulated in Ap4-deficient adenomas and intestinal organoids. AP4-associated signatures were conserved between murine adenomas and human colorectal cancer samples. Our results establish Ap4 as a rate-limiting mediator of adenoma initiation, as well as a regulator of intestinal and colonic stem cell and Paneth cell homeostasis. However, many questions regarding the function and role of AP4 remained unanswered in our study since tumors in the ApcMin model are generally not invasive and therefore do not form metastases. Within the new proposal the role of Ap4 in invasion and metastasis will be studied in two CRC mouse models that displays invasion and local as well as distant metastases by deleting Ap4 or ectopic Ap4 expression in vivo. Furthermore, the role of Ap4 in colonic tumor maintenance in DSS treated ApcMin mice will be evaluated. Also, the effect of specific deletion of Ap4 in ISCs on tumor initiation in Lgr5-CreERT2, Apc fl/fl mice will be interrogated. Finally, the effect of deleting Ap4 in adult stem cells of the intestine and its role in regeneration after injury by irradiation will be studied by lineage tracing. Next-generation sequencing will be used to identify critical mediators of Ap4-controlled phenotypes. In addition, organoids derived from intestinal epithelia will be analyzed. Taken together, these studies will critically contribute to the illumination of the role of AP4 in intestinal biology and cancer.