A Non-Canonical Role of SMAD4 in Regulating 3D Genome Architecture to Inhibit Lung Squamous Cell Carcinoma Development

Abstract Lung squamous cell carcinoma (LUSC) lacks clearly defined key drivers and effective targeted therapies, reflecting an incomplete understanding of its molecular pathogenesis. Here, we identify SMAD4 as a critical regulator of three-dimensional (3D) genome organization in LUSC and uncover a mechanistic link between tumor suppressor loss and oncogenic transcriptional activation. By integrating clinical datasets, genetically engineered mouse models, human and murine LUSC cell lines, and multi-omics analyses, we demonstrate that SMAD4 deficiency promotes LUSC progression by unleashing EP300-mediated enhancer-promoter looping at the SOX2 locus. Mechanistically, SMAD4 does not directly bind SOX2 regulatory elements but instead constrains chromatin looping by sequestering EP300 away from loop anchor regions. Loss of SMAD4 leads to enhanced H3K27ac deposition, aberrant SOX2 activation, and increased LUSC tumor cell proliferation. Together, these findings reveal a non-canonical role for a transcription factor (e.g., SMAD4) in regulating dysregulated 3D genome architecture to inhibit tumor development. Competing Interest Statement The authors have declared no competing interest. Funder Information Declared This work was supported by grants to Dr. Jian Liu from the Natural Science Foundation (NSF) of China (General Grant: 82172899 and 82472637), the NSF of Zhejiang Province (Continuation Grant of Distinguished Young Scholars: LRG26H160001), Noncommunicable Chronic Diseases-National Science and Technology Major Project (2023ZD0502900/2023ZD0502902 and 2023ZD0507500/2023ZD0507501), ZJE Career Development Programme, Dr. Li Dak Sum & Yip Yio Chin Development Fund for Regenerative Medicine, Zhejiang University, the Open Fund of Zhejiang Provincial Key Laboratory of Pulmonology (KF202302), ZJE seed funding, ZJE 2024 International Campus Talent Special Funding Program, ZJE-UoE Joint Research Project, and Startup Funding of Tenure-track Assistant Professor of Zhejiang University. This work was also supported by the Zhejiang Province Pioneer Research and Development Project (No.2025C02091). This work was also supported by Zhejiang University-University of Edinburgh Institute (ZJE) and the Department of Respiratory and Critical Care Medicine, The Second Affiliated Hospital, Zhejiang University School of Medicine, Zhejiang University.Haining People’s Hospital Tertiary-A Public Hospital Establishment Project Municipal Finance Support Fund; the Joint University-Local Cooperation Fund between ZJU-UoE Institute and Haining People’s Hospital. Supported by Sanming Project of Medicine in Shenzhen (No. SZSM202403006). This study was partially supported by the Guangxi Key Research and Development Program (GuiKe-AB25069017), the Joint Project on Regional High-Incidence Diseases Research of Guangxi Natural Science Foundation (2024GXNSFAA010101. 2024GXNSFBA010032).