Defining the Regulatory Function of SOX2 in Squamous Cell Carcinoma

Project Summary/Abstract Squamous cell carcinoma (SCC) is one of the most common cancers worldwide, with over one million deaths annually. While treatments such as chemotherapy and radiation are available, their severe side effects highlight the need for targeted therapies. SCCs from diverse tissues often share the overexpression of the transcription factor SOX2, which has been implicated in tumor initiation and maintenance. Despite its central role in SCC, therapeutic targeting of SOX2 remains challenging, necessitating a deeper understanding of its oncogenic transcriptional program and regulatory mechanisms. Our preliminary work demonstrates that SOX2 acts primarily as a transcriptional activator in SCC, binding promoters and distal enhancers to upregulate cancer- relevant genes via chromatin loops. Interestingly, many SOX2 binding sites are co-occupied by the oncogenic transcription factor TP63, a known master regulator in SCC. TP63 also functions as a pioneer factor, opening chromatin to facilitate binding by other factors including SOX2. Despite their established co-regulatory role, little is known about the mechanisms by which SOX2 and TP63 interact to regulate target gene expression in SCC. We hypothesize that SOX2 and TP63 co-occupy enhancers and upregulate target genes driving oncogenic pathways in SCC. This study aims to elucidate the functional regulatory role of SOX2 in SCC by addressing two key aims. First, characterizing the cancer-specific transcriptional program regulated by SOX2. Using an integrated genomics approach combining CRISPR-Cas9-mediated SOX2 knockout RNA-seq, SOX2 ChIP-seq, and H3K27ac HiChIP, we will identify direct SOX2 target genes. We will then prioritize genes relevant to human SCC using data from the Cancer Genome Atlas. We will evaluate the role of these prioritized target genes in cell proliferation and tumor growth using an in vitro competition assay and in vivo xenograft models. Second, we will explore the interplay between SOX2 and TP63 at shared enhancer binding sites. We hypothesize that TP63 facilitates chromatin accessibility, enabling SOX2 binding, and together, they regulate critical oncogenic pathways. This will be tested using CRISPR Cas9-based enhancer motif disruption, and RNA-seq and ATAC-seq following TP63 knockout to assess changes in chromatin accessibility and gene expression. This work will reveal SOX2 target genes, and how SOX2 along with TP63 drives SCC, providing insights into cancer-specific pathways that could serve as therapeutic targets. Furthermore, the insights gained may extend to other cancers where SOX2 activation is implicated, broadening the impact of this work.