Advancing Education, Research, and Quality of Care for the Head and Neck oncology patient.
Program Number: AHNS20
Session Name: Scientific Session 4 - Cancer Biology
Session Date: Thursday, May 15, 2025
Session Time: 9:00 AM - 9:45 AM
Epigenomic features and vulnerabilities of head and neck squamous cell carcinoma metastasis
Wesley L Cai, MD, PhD1; Baris Sergi, BS2; Chareeni E Kurukulasuriya3; Ceyda Acilan, PhD2; Umamaheswar Duvvuri, MD, PhD3; 1University of Pittsburgh Medical Center; 2Koc University; 3New York University Langone HealthIntroduction: Regional metastasis to lymph nodes is a critical prognostic factor in head and neck squamous cell carcinoma (HNSCC). Metastasis is a complex biological process involving tumor cells undergoing phenotypic changes triggered by genetic, epigenetic, and transcriptional alterations. The Assay for Transposase-Accessible Chromatin (ATAC) and RNA-sequencing are powerful tools that provide insights into the epigenome and transcriptome of tumors, respectively. Although lymph node and distant metastasis are linked to poor outcomes, the mechanisms driving metastasis in HNSCC remain unclear. This study aims to use matched human tissue samples to identify bona fide transcriptional drivers of metastasis and identify novel therapeutic targets.
Methods: This single-institution study included n=18 patient cases of HPV-negative pT3-4pN1+ HNSCC from 2019-2022 without neoadjuvant treatment. Fresh primary tumor, adjacent normal, and metastatic lymph node tissue were collected from surgical cases and archived in liquid N2 immediately after excision. RNA-seq and ATAC-seq were performed on all samples (n=54 samples) in the cohort. After alignment and processing of sequencing data, differential gene expression and chromatin accessibility were obtained. Motif analysis was performed on differentially accessible chromatin to predict enriched transcription factors proteins (TF). To test the functional significance of predicted TFs, the human cell line CAL-27 was subjected to CRISPR knockout of TF targets. Control and target cell lines were used in scratch assay to assess changes in migration capacity.
Results: 15 out of 18 cases passed our quality control cut-off and 45 unique tissue samples were included in the final analysis. We found that promoter regions with increased chromatin accessibility are associated with increased RNA expression. Gene pathways activated in metastatic tumors included “TNF-alpha signaling” and “unfolded protein response”. To uncover enriched transcription factor motifs, matched tissue samples from the same patient were compared. Comparing primary tumors to matched adjacent normal tissue, the NFY-A transcription factor motif was enriched in 12/15 (80%) of patients. For comparisons between metastasis and primary tumors, motifs for CREB1, EGR1, and SP-family transcription factors were significantly enriched in 13/15 (87%) of patient cases. The top 10 transcription factor targets were selected and 2 sgRNAs were designed against each of these genes. CAL27 cells were transduced with targeting and control CRISPR constructs, and cell migration was assessed with scratch assay. Remarkably, we found that CREB1 and EGR1 knock-out significantly impaired cell migration compared to non-targeting control.
Conclusions: Using a cohort of matched primary tumor, adjacent normal, and lymph node metastatic tissue obtained from the operating room, we characterized epigenomic changes in HNSCC metastasis, and uncovered gene pathways and transcription factor motifs that are enriched in metastatic tumors. Through in vitro experimentation we show that CREB1 and EGR1 are important for tumor cell migration and promising novel targets for metastatic HNSCC.
