title: The transcription factor ZEB1 shapes osteosarcoma aggressiveness by affecting tumour cell differentiation and stemness features
Creators: name: Cascini, Caterina
Publisher: name: The Open University
Year: 2024

description: Osteosarcoma (OS) is a mesenchymal bone tumour affecting mainly children and young adults, characterized by a particularly aggressive behavior, with 20% of patients showing lung micro metastasis already at diagnosis. To date, treatment options for OS are still based on multi-drug chemotherapy and prognosis for metastatic patients is dismal, making urgent the identification of new, more effective, therapies. OS is considered a differentiation disease, because of the incapability of mesenchymal stem cells or osteoblastic progenitors to proceed toward terminal differentiation.<br> Taking advantage of cellular models, we tested the impact on OS of targeting ZEB1, a transcription factor known to be critical for the maintenance of mesenchymal and stemness features, also in cancer. We inhibited ZEB1 expression in murine OS cell lines by lentiviral vectors based on CRISPR-Cas9 technology for knock-out (KO) or shRNA for knock-down (KD). We firstly tested the effects of ZEB1 deficiency <i>in vitro</i>, evaluating OS cell stemness potential by sarcosphere forming assay and osteogenic differentiation by alkaline phosphatase and alizarin red staining. We found a decreased stemness potential in ZEB1 KO clones, paralleled by an increased osteogenic differentiation. ZEB1 deficiency (or down-modulation) in tumour cells reduced <i>in vivo</i> tumour growth and ZEB1 KO-derived tumours showed a more differentiated morphology in comparison to controls. Interestingly, the absence of ZEB1 in tumour cells affected also the immune infiltrate, as tumours derived from ZEB1 KO clones were significantly less infiltrated by pro-tumoural CD206+ M2-like macrophages. Moreover, we observed a reduced metastatic potential in ZEB1 KO clones when cells were injected intravenously.<br>
Gene expression profile (GEP) comparing ZEB1 KO clones and control ones was performed to investigate the transcriptional changes induced by ZEB1 deletion and potentially identify alternative, more easily druggable, therapeutic targets. GEP analysis showed 849 down-regulated and 1093 up-regulated genes in ZEB1 KO clones versus ZEB1-competent controls. Gene set enrichment analysis indicated a down- modulation of pathways related to cellular proliferation and survival, such as MTORC1 signaling, MYC targets, GM2 checkpoint, E2F targets and oxidative phosphorylation in ZEB1 KO clones. The analysis of differentially expressed genes between ZEB1 KO clones and controls showed an up-regulation of the tumour suppressor <i>Sfrp1</i>, an antagonist of WNT/Beta-catenin pathway. New investigations are ongoing to better define the link between ZEB1 and SFRP1.<br>
Overall, these results support the hypothesis of ZEB1 being a key factor in shaping OS aggressiveness, affecting tumour cell intrinsic features as well as microenvironment- related traits.<br>
In light of the latter aspect, and of the general view of OS as a cold, poorly infiltrated, tumour, we also tested the <i>in vivo</i> therapeutic activity of a TLR-9 agonist (SD101), an immunomodulatory agent able to activate antigen presenting cells. We found that the local administration of SD101 was able not only to reduce tumour growth of a directly-treated lesion, but also of a contralateral, untreated tumour mass.<br> Interestingly, SD101 administration induced a complete reprogramming of the tumour microenvironment, by decreasing the number of M2-like pro-tumoural macrophages and increasing dendritic cells and CD8 T cell infltration, both in treated and untreated lesions.<br>