D108-25 A Non-canonical Role of Smad4 in Regulating 3d Genome Architecture to Inhibit Lung Squamous Cell Carcinoma Development

Rationale

Lung squamous cell carcinoma (LUSC) lacks clearly defined key drivers and effective targeted therapies, reflecting an incomplete understanding of its molecular pathogenesis. While the role of three-dimensional (3D) genome architecture in lung adenocarcinoma has been established, its contribution to the development of LUSC remains elusive. We undertook this study to identify SMAD family member 4 (SMAD4) as a critical regulator of 3D genome organization in LUSC and uncover a mechanistic link between tumor suppressor loss and oncogenic transcriptional activation.

Methods

Smad4 loss was evaluated in CCSPiCreLkb1f/f spontaneous LUSC development mice. The mRNA and protein levels of Smad4 and Sox2 were assessed by quantitative reverse transcription polymerase chain reaction (RT-qPCR) and Western blot. Chromatin interactions were analyzed by High-throughput Chromosome Conformation Capture (Hi-C) in SMAD4 knockout cell line to map Sox2-associated loops, and the frequency of this loop was validated by chromosome conformation capture qPCR (3C-qPCR). The enhancer region within the identified loop were knockout using gRNA. Peaks in the Sox2-associated loop region were identified from publicly available ChIP-Seq data (ChIP-Atlas database) and confirmed by chromatin immunoprecipitation qPCR (ChIP-qPCR). Protein interactions between EP300 and SMAD4 were examined by co-immunoprecipitation in cells, using EP300-specific antibody and IgG control, followed by Western blot detection.

Results

By integrating clinical datasets, genetically engineered mouse models, human and murine LUSC cell lines, and multi-omics analyses, we demonstrated that SMAD4 deficiency promotes LUSC progression by unleashing EP300-mediated enhancer-promoter looping at the SOX2 locus. Mechanistically, SMAD 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 tumor cell proliferation.

Conclusions

Collectively, by linking SMAD4 deficiency to EP300-driven enhancer-promoter looping and SOX2 activation, our work provides new mechanistic insight into LUSC development and highlight 3D genome regulation as a promising therapeutic vulnerability in squamous cell lung cancer.

This abstract is funded by: This abstract is funded 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), 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).