Calprotectin controls carcinoma growth and malignant potential

Calprotectin, a heterodimer of S100A8 and S100A9, is differentially regulated in various types of cancer, but is generally down-regulated in head and neck squamous cell carcinoma (HNSCC). When over-expressed in carcinoma cells in vitro and in vivo, calprotectin appears to suppress tumor growth at the G2/M cell cycle checkpoint and inhibit anchorage-independent growth through the C-terminal tail of the S100A9 subunit. Objective: To characterize interactions of calprotectin with specific G2/M regulators and determine whether the loss of calprotectin increases malignant potential and resistance to apoptosis-inducing agents. Methods: Calprotectin-negative KB and calprotectin-positive TR146 carcinoma cells were used for in vitro studies. KB cells were transfected to express S100A8/A9 for gain-of-function, whereas TR146 was transfected with shRNA to knockdown calprotectin expression (S100A8/A9kd) for loss-of-function. KB cells were also transfected to express S100A8/A9 with S100A9 C-terminal deletion (S100A8/A9-Cterm) or S100A9 penultimate threonine (T) deletion (S100A8/A9-T112). Protein-protein interactions were determined by co-immunoprecipitation. Cellular responses to apoptosis-inducing agents, hydrogen peroxide, aspirin and cisplatin, were assessed. Standard soft agar and transwell migration assays were performed to study malignant phenotypes. Results: Protein phosphatase PP2A-Aa co-immunoprecipitated with calprotectin in KB-S100A8/A9 cells. KB-S100A8/A9 and TR146 formed fewer colonies in soft agar than sham-transfected KB, KB-S100A8/A9-Cterm, KB-S100A8/A9-T112 or TR146-S100A8/A9kd cells. Expression of wild-type and mutated calprotectin blocked the ability of the cells to migrate and increased resistance to aspirin treatment. KB-S100A8/A9-Cterm and KB-S100A8/A9-T112 deletion mutants showed loss of viability in response to H2O2. Knock-down of calprotectin in TR146-S100A8/A9kd cells increased resistance to cisplatin treatment. Conclusion: Calprotectin suppresses migration of carcinoma cells and appears to control carcinoma growth through C-terminal domain of the S100A9 subunit and interaction with PP2A-Aa. Knock-down of S100A8/A9 increases carcinoma growth and resistance to anti-neoplastic agent cisplatin, suggesting that calprotectin plays an essential role to prevent malignant transformation in HNSCC. Supported by the NIH/NIDCR grants R01DE11831.