Abstract 3526: The transcription factor ZEB1 shapes osteosarcoma aggressiveness by affecting tumor cell differentiation and stemness features

Abstract

Abstract Osteosarcoma (OS) is a mesenchymal bone tumor 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 therapies. OS is considered a differentiation disease, because of the incapability of mesenchymal stem cells or osteoblastic progenitors to proceed toward terminal differentiation. We tested the effects on OS cells of targeting ZEB1, a transcription factor known to be critical for the maintenance of mesenchymal and stemness features in cancer. We inhibited ZEB1 expression in a murine OS cell line by lentiviral vectors based on CRISPR-Cas9 technology. We firstly tested the effects of ZEB1 deficiency in vitro, evaluating OS cell stemness potential by sarcosphere forming assay and osteogenic differentiation by alkaline phosphatase (Alp) staining. Gene expression profile (GEP) comparing ZEB1 KO clones and control ones was also performed to investigate the transcriptional changes induced by ZEB1 deletion and potentially identify alternative, more easily druggable, therapeutic targets. We also investigated in vivo the effect of ZEB1 inhibition. ZEB1 KO clones showed decreased stemness potential and increased Alp staining, both features suggestive of a more differentiated phenotype. Preliminary GEP analysis showed 849 down-regulated genes 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. In vivo we observed a reduced tumor growth in ZEB1 KO clones that were also characterized by a more differentiated morphology in comparison to controls. Interestingly, the absence of ZEB1 in tumor cells affected also the immune infiltrate, as tumors derived from ZEB1 KO clones were significantly less infiltrated by pro-tumoral CD206+ M2-like macrophages. Moreover, we observed a reduced metastatic potential in ZEB1 KO clones than controls when we inject the cells intravenously. Taken together, these results support the hypothesis of ZEB1 as a key factor in OS aggressiveness, affecting tumor cell differentiation, stemness ability and in vivo growth capability. Citation Format: Caterina Cascini, Daniele Lecis, Laura Botti, Chiara Ratti, Valeria Cancila, Katia Scotlandi, Claudio Tripodo, Mario Paolo Colombo, Claudia Chiodoni. The transcription factor ZEB1 shapes osteosarcoma aggressiveness by affecting tumor cell differentiation and stemness features. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3526.