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

TFAP4/AP4蛋白属于碱性-螺旋-环-螺旋亮氨酸拉链（bHLH-LZ）转录因子。AP4只形成同型二聚体，与E-box基序CAG/CCTG结合，从而抑制或激活靶基因的表达。我们之前发现AP4基因是c-MYC的直接转录靶点。AP4在人类结肠隐窝和结直肠癌（CRC）的祖细胞/瞬时扩增（TA）细胞中表达，表达模式与c-MYC相似。原型癌基因c-MYC是APC (adenomatous polyposis coli)/Wnt （Wingless/Int-1）通路的直接靶点，也是肠道中APC失活诱导肿瘤形成的重要介质。先前的研究表明，AP4可能通过调节参与上皮-间质转化（epithelial-mesenchymal transition， EMT）和增殖的基因参与CRC的进展。在之前的资助期内，我们在ApcMin小鼠（一种遗传性结直肠癌的临床前模型）中删除了Ap4。Ap4缺乏使小鼠平均生存期延长110天，并减少肠腺瘤和肿瘤源性类器官的形成。Ap4缺失的影响可能是由于可适应腺瘤启动突变事件的功能性肠干细胞（ISCs）数量减少。Ap4的缺失也使结肠干细胞数量减少，Paneth细胞数量增加。表达谱分析显示，在ap4缺陷腺瘤和肠道类器官中，ISC特征以及Wnt/β-catenin和Notch信号通路下调。ap4相关特征在小鼠腺瘤和人类结直肠癌样本之间是保守的。我们的研究结果表明Ap4是腺瘤起始的限速介质，也是肠和结肠干细胞和Paneth细胞稳态的调节剂。然而，在我们的研究中，关于AP4的功能和作用的许多问题仍未得到解答，因为ApcMin模型中的肿瘤通常不具有侵袭性，因此不会形成转移。在新的提案中，Ap4在侵袭和转移中的作用将在两种CRC小鼠模型中进行研究，这些模型通过在体内删除Ap4或异位Ap4表达来显示侵袭和局部以及远处转移。此外，我们将评估Ap4在DSS治疗的ApcMin小鼠结肠肿瘤维持中的作用。此外，在Lgr5-CreERT2、Apc fl/fl小鼠中，ISCs中Ap4的特异性缺失对肿瘤起始的影响也将被询问。最后，我们将通过谱系追踪研究Ap4在肠成体干细胞中的作用及其在辐照损伤后再生中的作用。下一代测序将用于鉴定ap4控制表型的关键介质。此外，将分析来自肠上皮的类器官。综上所述，这些研究将有助于阐明AP4在肠道生物学和癌症中的作用。

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.