Abstract 4507: Study of CCCTC-binding factor (CTCF) genetic aberrations and dysregulation in head and neck squamous cell carcinoma (HNSCC)

Abstract

Abstract CCCTC-binding Factor (CTCF) is a DNA-binding protein that acts as a global genome organizer in higher eukaryotes. CTCF folds the genome into spatial domains that is crucial for regulation of genes, including oncogenes and tumor suppressor genes. Recently, Bornstein et al. has reported that CTCF truncating mutations are associated with progression of Head and Neck Squamous Cell Carcinoma (HNSCC), suggesting a potential role of CTCF and its aberrations in HNSCC progression. Survival analysis of the US-HNSCC The Cancer Genome Atlas (TCGA) Provisional cohort (N=502) revealed that CTCF mutations are associated with poor overall survival (P = 0.0206, Median survival of15 months vs 56.9 months) and disease-free survival (DFS; P = 0.0078, median DFS of 12.975 months vs 71.22 months). Furthermore, CTCF truncating mutations are associated with poor overall survival (P = 0.0153, Median survival of 14.98 months vs 56.9 months) and disease-free survival (DFS; P = 0.0010, median DFS of 8.97 months vs 71.22 months). Intriguingly, RNA-seq analysis of 43 pairs of normal-tumor tissues from the TCGA reveal a tumor-specific upregulation of CTCF mRNA (P < 0.0001). Subsequent Western blot analyses demonstrated at least 2.5-fold and 1.2-fold CTCF protein upregulation in HNSCC patient primary cultures and HNSCC cancer cell lines relative to normal head and neck squamous cell and normal immortalized head and neck squamous cell line respectively. Based on these preliminary genomic findings, we hypothesized that CTCF upregulation may have pro-tumorigenic effects in HNSCC. Therefore, cancer cell proliferation was monitored following exogenous overexpression by retroviral introduction of the human CTCF gene or specific knockdown (by shRNA) of CTCF in HNSCC cell lines. We found that exogenous overexpression of CTCF promoted HNSCC cell proliferation by about 40% (N=6). However, with multiple trials, specific knockdown of CTCF only resulted in about 10% growth inhibition of HNSCC cells (N=6) with minimal apparent knockdown of CTCF protein. This unexpected challenge of CTCF protein knockdown could likely be due to potentially long CTCF protein half-life in HNSCC. In fact, we found that CTCF protein is highly stable in HNSCC cell line and HNSCC normal immortalized cell line, with protein level reduction by only 20 to 40% after two to three days of cycloheximide inhibition. Our results demonstrated a growth-promoting role of CTCF in HNSCC, and this CTCF protein appears to be highly stable with overexpression in HNSCC, potentially serving as a stable push for tumorigenesis. Citation Format: Sze Man Chan, Hoi-Lam Ngan, Yuchen Liu, Wenying Piao, Chin Wang Lau, Jason Ying Kuen Chan, Yu Xiong Su, Vivian Wai Yan Lui. Study of CCCTC-binding factor (CTCF) genetic aberrations and dysregulation in head and neck squamous cell carcinoma (HNSCC) [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4507.