IADR Abstract Archives
Calprotectin (S100A8/A9) Promotes Caspase-Mediated Apoptotic DNA Fragmentation in HNSCC
Objectives: Calprotectin (S100A8/A9), a member of the S100 EF-hand family of calcium-binding proteins, appears to restore the G2/M cell cycle checkpoint, inhibits proliferation, migration and invasion of carcinoma cells in vitro, and suppresses tumorigenesis in vivo. In head and neck squamous cell carcinoma (HNSCC), poor differentiation, EGFR upregulation, resistance to chemotherapy and decreased overall survival rates are associated with loss of S100A8/A9. Calprotectin also participates in post-radiation DNA damage responses through upregulation of DNA repair regulatory proteins 53BP1 and γH2AX. Since calprotectin increases sensitivity to cisplatin, we hypothesized that S100A8/A9 exerts a tumor-suppressive function by inducing cell death in HNSCC.
Methods: S100A8/A9-expressing, shRNA knockdown, and shRNA-control TR146 HNSCC cells were analyzed in vitro and compared to S100A8/A9-negative KB carcinoma cells, KB cells co-transfected to express S100A8/A9 and a sham-control transfectant, KB-EGFP. Double-strand breaks were induced by X-radiation. To assess nuclear fragmentation, comet assays were performed at 10min, 2 and 24h post-radiation. Differential expression of cell death and survival–associated genes between S100A8/A9–high and –low HNSCCs was determined by interrogation of TCGA.
Results: Following X-radiation, calprotectin-overexpressing KB cells showed greater DNA fragmentation and comet tail formation than calprotectin-negative KB and KB-EGFP cells, which also appeared to recover faster after 24h. Silencing calprotectin in TR146 cells conferred resistance to X-radiation, decreased the length and frequency of comet tails and facilitated cell recovery. Interestingly, more than 363 apoptosis-related genes were significantly upregulated by S100A8/A9–high HNSCCs compared to S100A8/A9–low neoplasms. For example, CASP1, -3, -4, -5, -7, -8, -9, -10 and -14 expression was higher (Fold-Change 1.50 to 9.54) in S100A8/A9–high tumors suggesting activation of caspase signaling.
Conclusions: When expressed in HNSCC, intracellular calprotectin appears to promote caspase-mediated DNA fragmentation following radio- and chemotherapy, contributing to function as a tumor-suppressor. S100A8/A9-dependent apoptotic death of carcinomatous cells may contribute to survival of patients with S100A8/A9-high HNSCCs.
Methods: S100A8/A9-expressing, shRNA knockdown, and shRNA-control TR146 HNSCC cells were analyzed in vitro and compared to S100A8/A9-negative KB carcinoma cells, KB cells co-transfected to express S100A8/A9 and a sham-control transfectant, KB-EGFP. Double-strand breaks were induced by X-radiation. To assess nuclear fragmentation, comet assays were performed at 10min, 2 and 24h post-radiation. Differential expression of cell death and survival–associated genes between S100A8/A9–high and –low HNSCCs was determined by interrogation of TCGA.
Results: Following X-radiation, calprotectin-overexpressing KB cells showed greater DNA fragmentation and comet tail formation than calprotectin-negative KB and KB-EGFP cells, which also appeared to recover faster after 24h. Silencing calprotectin in TR146 cells conferred resistance to X-radiation, decreased the length and frequency of comet tails and facilitated cell recovery. Interestingly, more than 363 apoptosis-related genes were significantly upregulated by S100A8/A9–high HNSCCs compared to S100A8/A9–low neoplasms. For example, CASP1, -3, -4, -5, -7, -8, -9, -10 and -14 expression was higher (Fold-Change 1.50 to 9.54) in S100A8/A9–high tumors suggesting activation of caspase signaling.
Conclusions: When expressed in HNSCC, intracellular calprotectin appears to promote caspase-mediated DNA fragmentation following radio- and chemotherapy, contributing to function as a tumor-suppressor. S100A8/A9-dependent apoptotic death of carcinomatous cells may contribute to survival of patients with S100A8/A9-high HNSCCs.