Abstract 1289: The role of mutant p53-mediated mechanisms in modulating the tumor microenvironment and promoting lung metastasis
Authors: Efe Gizem , Cunningham Katherine M. , Tang Qiaosi , Sugiura Kensuke , Dunbar Karen , Suzuki Kensuke , Resnick-Silverman Lois , Taylor Alison M. , Manfredi James J. , Prives Carol L. , Rustgi Anil K. 
Published: April 2023

Abstract
INTRODUCTION/METHODS: The multistep process of the tumor invasion-metastasis cascade is a major barrier to effective therapy. Mutations of the tumor suppressor p53 are frequently detected in esophageal squamous cell carcinoma (ESCC) cases, which in turn correlate with high metastatic rates. To understand the mutant p53-mediated mechanisms in promoting ESCC metastasis, we conducted RNA-Seq, ChIP-Seq and cytokine array on isogenic primary and metastatic tumor cells harvested from our mouse model of esophageal cancer, L2-Cre; LSL-Trp53R172H; Rosa26LSL-YFP, in which we have identified macrophage colony stimulating factor 1 (CSF1) to be a direct gene target of p53-R172H. We have targeted CSF1/CSF1R signaling to assess its pro-tumorigenic roles. In addition to studying tumor intrinsic mechanisms, we have established multiplex immunofluorescence and flow cytometry approaches to characterize the changes in tumor microenvironment (TME). Overall, this study aims to investigate the role and mediators of CSF1 signaling through its cognate receptor CSF1R by which missense p53 mutations can promote lung metastasis.
RESULTS/DISCUSSION: We demonstrate that metastatic ESCC has increased Csf1 expression compared to primary tumors and this is dependent upon p53 mutation status, which is reinforced by TCGA data and patient-derived tissue microarrays (TMAs). The TMA analysis also suggested that CSF1 expression is higher in tumors that are poorly differentiated. Furthermore, through the proximity ligation assay (PLA), we have identified that bromodomain and extra-terminal (BET) protein BRD4, which binds to acetylated histones to regulate transcription interacts with p53-R172H at higher levels compared to wild-type p53 to induce Csf1 expression. We show that the BRD4/CSF1/CSF1R signaling axis fosters tumor invasion, subcutaneous tumor growth and metastatic burden in lungs in a mutant p53 background. In accordance, targeting CSF1/CSF1R signaling reduces phosphorylation of STAT3 in the metastatic tumors, which suggests a mechanism for increased invasiveness and metastasis. In parallel, upon inhibiting this axis, we have identified decreased infiltration of F4/80+CD163+ and F4/80+CD206+ M2-polarized macrophages, as well as CD31+ endothelial cells, and increased number of CD3+CD8+ cytotoxic T-cells at the metastatic tumor sites, indicating that the CSF1/CSF1R pathway plays a critical role in shaping the pro-metastatic and immunosuppressive TME. Finally, analysis of squamous cell carcinoma (SCC) datasets reveals that specific p53 mutations are associated with differential survival rates and CSF1 expression.
CONCLUSION: We have demonstrated novel roles and mechanisms of mutant p53-dependent CSF1-CSF1R signaling pathway in fostering ESCC tumor invasion and lung metastasis that may be applicable to other SCCs. We believe this can open up new avenues for therapeutic applications.
Citation Format: Gizem Efe, Katherine M. Cunningham, Qiaosi Tang, Kensuke Sugiura, Karen Dunbar, Kensuke Suzuki, Lois Resnick-Silverman, Alison M. Taylor, James J. Manfredi, Carol L. Prives, Anil K. Rustgi. The role of mutant p53-mediated mechanisms in modulating the tumor microenvironment and promoting lung metastasis [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 1289.