Regulatory roles of calprotectin in head and neck squamous cell carcinogenesis.

Abstract

Malignant transformation in squamous cell carcinomas (SCC) such as those of the head and neck (HNSCC) remains an enigmatic process that results in abnormal cellular differentiation, loss of growth and cell cycle regulation, gain of replicative immortality and resistance to apoptosis, activation of cellular migration and invasion, increase in energy metabolism, and evasion of immune destruction. These abnormalities in cell functions emerged as the hallmarks of cancer. Calprotectin, a heterodimeric protein complex of calcium regulating S100A8 and S100A9 encoded by genes mapped to the chromosomal locus 1q21.3 of the epidermal differentiation complex (EDC), may play essential roles in the regulation of cell differentiation, cell cycle progression, cellular survival and cell migration that are part of the cancer hallmarks. While highly upregulated in a variety of cancers, calprotectin is down-regulated in squamous cell carcinomas of the cervix, esophagus and the head and neck. Using microarray analysis for gene expression we found that the S100A9 subunit of calprotectin was significantly down-regulated along with other EDC genes in human primary HNSCC cases, suggesting a loss of functional S100A8/A9 protein complex and differentiation during carcinogenesis. Expression of S100A9 correlated strongly with a set of HNSCC downregulated genes putatively involved in loss of cytodifferentiation and control of cell cycle. To probe its role in carcinogenesis, S100A8/A9 was stably expressed in a calprotectin-negative human carcinoma cell line (KB cells). Expression of S100A8/A9 in KB cells up-regulated differentiation and cell-cell contact growth inhibition signaling pathways, re-established epithelial actin microfilament cytoskeletal structures and v cellular adhesion to the extracellular matrix, down-regulated anti-apoptosis gene networks, and suppressed anchorage-independent survival and stress-induced (by serum starvation) cell migration. Calprotectin appeared to induce growth suppression by signaling a reactivation of G2/M cell cycle checkpoint regulators, Chk1 and PP2A, and inactivation of mitotic activators Cdc25C and Cdc2. As a result, we saw a marked increase in Cdc2-PThr14/Tyr15 phosphorylation and down-regulation of cyclin B1, suggesting an inactivation of the mitotic entry promoting Cdc2/cyclin B1 complex, resulting in cell cycle and mitotic arrest at the G2/M checkpoint. When inoculated into nude mice, KB cells producing calprotectin showed reduced tumor growth when compared to sham-transfected control KB cells. Using shRNA, silencing of S100A8/A9 expression in the TR146 human HNSCC cell line increased carcinoma growth and survival and reduced Cdc2 phosphorylation at Thr14/Tyr15. Calprotectin expression in KB and TR146 cells also down-regulated expression of putative HNSCC marker genes, INHBA, PTGS2 (Cox-2) and SULF1, found to be expressed only in HNSCC samples. Calprotectin-mediated control of cellular differentiation and G2/M cell cycle checkpoint is, therefore, a likely suppressive mechanism in human squamous cell carcinomas and may point to new molecular targets for therapy.