Tumor heterogeneity for ATF2 and its impact for invasive behavior of colorectal cancer

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Colorectal cancer (CRC) is one of the most commonly diagnosed cancers worldwide. The majority of CRC-related deaths are associated with metastasis. Peritoneal metastasis occurs in appr. 25% of metastatic CRC and is associated with worse prognosis. The molecular mechanisms behind this type of metastatic spread are unknown. Tumor heterogeneity is considered to be the major reason for tumor relapse, metastasis, and therapy resistance. Although molecular CRC consensus subtypes based on gene expression pattern have been already described in 2015, the problem of intratumoral heterogeneity was not solved so far. Only transcriptomic analyses on single-cell resolution have the potential to identify invasive „driver cells“ in genetically heterogeneous tumors. Interestingly, we could demonstrate at CRC tumor invasion front a remarkable heterogeneity for expression of the transcription factor ATF2. This might have so far prevented causal analyses for ATF2-dependent functions and have masked its role for tumor invasion. We showed that ATF2 acts as tumor suppressor, especially as inhibitor of peritoneal metastasis. Using CRISPR/Cas9 generated ATF2 knockout (KO) cell lines we observed an altered invasion pattern in spheroids, mouse, and chicken xenografts. The ATF2 heterogeneous tumor was surrounded by a marginal group of cells with ATF2 loss and strong E-Cadherin expression suggesting the phenotype of so-called leader cells. From our pre-work, we hypothesize a novel function for ATF2 in inhibition of de-adhesion, the first step in the metastatic cascade. With our project, we will continue our studies and analyze in detail the role of ATF2 loss for the invasive potential of CRC. We will simulate tumor heterogeneity for ATF2 in up-to-date genetically modified 3D tumor models such as human and mouse organoids. We will trace the ATF2 loss with stably transduced fluorescence markers. We have available a novel conditional Atf2 KO-mouse for mechanistic studies of the ATF2 loss-regulation network. Single-cell RNA analysis will help to identify new therapeutic targets of the ATF2 signaling pathway and for the first time inhibitors of TROP2, one of the identified ATF2 targets, will be tested in chicken, organoids, and mouse models for their anti-invasive effects in CRC.