Intracellular calprotectin (S100A8/A9) facilitates DNA damage responses and promotes apoptosis in head and neck squamous cell carcinoma.

OBJECTIVES: In head and neck squamous cell carcinoma (HNSCC), poor prognosis and low survival rates are associated with downregulated calprotectin. Calprotectin (S100A8/A9) inhibits cancer cell migration and invasion and facilitates G2/M cell cycle arrest. We investigated whether S100A8/A9 regulates DNA damage responses (DDR) and apoptosis in HNSCC after chemoradiation. MATERIALS AND METHODS: Human HNSCC cases in TCGA were analyzed for relationships between S100A8/A9 and expression of apoptosis-related genes. Next, S100A8/A9-expressing and non-expressing carcinoma lines (two different lineages) were exposed to genotoxic agents and assessed for 53BP1 and γH2AX expression and percent of viable/dead cells. Finally, S100A8/A9-wild-type and S100A8/A9null C57BL/6j mice were treated with 4-NQO to induce oral dysplastic and carcinomatous lesions, which were compared for levels of 53BP1. RESULTS: In S100A8/A9-high HNSCC tumors, apoptosis-related caspase family member genes were upregulated, whereas genes limiting apoptosis were significantly downregulated based on TCGA analyses. After X-irradiation or camptothecin treatment, S100A8/A9-expressing carcinoma cells (i.e., TR146 and KB-S100A8/A9) showed significantly higher 53BP1 and γH2AX expression, DNA fragmentation, proportions of dead cells, and greater sensitivity to cisplatin than wild-type KB or TR146-S100A8/A9-KD cells. Interestingly, KB-S100A8/A9Δ113-114 cells showed similar 53BP1 and γH2AX levels to S100A8/A9-negative KB and KB-EGFP cells. After 4-NQO treatment, 53BP1 expression in oral lesions was significantly greater in calprotectin+/+ than S100A8/A9null mice. CONCLUSIONS: In HNSCC cells, intracellular calprotectin is strongly suggested to potentiate DDR and promote apoptosis in response to genotoxic agents. Hence, patients with S100A8/A9-high HNSCC may encounter more favorable outcomes because more tumor cells enter apoptosis with increased sensitivity to chemoradiation therapy.

Intracellular calprotectin (S100A8/A9) controls epithelial differentiation and caspase-mediated cleavage of EGFR in head and neck squamous cell carcinoma.

OBJECTIVES: Calprotectin (S100A8/A9) appears to function as a tumor suppressor in head and neck squamous cell carcinoma (HNSCC) and expression in the carcinoma cells and patient survival rates are directly related. We seek to characterize the suppressive role of calprotectin in HNSCC. AIMS: (1) Investigate changes in S100A8/A9 expression as oral carcinogenesis progresses and (2) determine whether intracellular calprotectin can regulate epidermal growth factor receptor (EGFR), a negative prognostic factor, in HNSCC. MATERIALS AND METHODS: Using immunohistochemistry (IHC), S100A8/A9 was analyzed in HNSCC specimens (N = 46), including well-differentiated (WD, N = 19), moderately-differentiated (MD, N = 14), poorly-differentiated (PD, N = 5) and non-keratinizing/basaloid (NK/BAS, N = 8), and premalignant epithelial dysplasias (PED, N = 16). Similarly, EGFR was analyzed in HNSCCs (N = 21). To determine whether calprotectin and EGFR expression are mechanistically linked, TR146 HNSCC cells that are S100A8/A9-expressing or silenced (shRNA) were compared for EGFR levels and caspase-3/7 activity using western blotting and immunofluorescence microscopy. RESULTS: In normal oral mucosal epithelium, S100A8/A9 stained strongly in the cytoplasm and nucleus of suprabasal cells; basal cells were consistently S100A8/A9 negative. In PED and HNSCC, S100A8/A9 expression was lower than in adjacent normal epithelial tissues (NAT) and declined progressively in WD, MD, PD and NK/BAS HNSCCs. S100A8/A9 and EGFR levels appeared inversely related, which was simulated in vitro when S100A8/A9 was silenced in TR146 cells. Silencing S100A8/A9 significantly reduced caspase-3/7 activity, whereas EGFR levels increased. CONCLUSIONS: In HNSCC, S100A8/A9 is directly associated with cellular differentiation and appears to promote caspase-3/7-mediated cleavage of EGFR, which could explain why patients with S100A8/A9-high tumors survive longer.

Benign and malignant odontogenic neoplasms of the jaws show a concordant nondiscriminatory p63/p40 positive immunophenotype.

OBJECTIVES: Antibody p40, which recognizes exclusively ΔNp63 but not TAp63, has shown diagnostic utility in salivary gland and sinonasal tract malignancies. Although p63 immunophenotypic characterization of odontogenic lesions has been reported, p40 expression has not been previously studied. We aimed to study p40 immunoreactivity in odontogenic tumors (OTs) and odontogenic cysts (OCs) and to investigate possible discriminatory properties of the combined p63/p40 immunoprofile in OTs and OCs. STUDY DESIGN: Fourteen ameloblastomas, 7 adenomatoid odontogenic tumors, 6 calcifying epithelial odontogenic tumors, 1 squamous odontogenic tumor, 4 primary intraosseous odontogenic carcinomas, 5 calcifying odontogenic cysts, 4 glandular odontogenic cysts, 3 odontogenic keratocysts, 3 dentigerous cysts, and 1 each radicular and orthokeratinized cysts were stained for p63 (4A4) and p40 (BC28) antibodies. RESULTS: Ameloblastoma, adenomatoid odontogenic tumor, calcifying epithelial odontogenic tumor, squamous odontogenic tumor, and primary intraosseous odontogenic carcinoma demonstrated concordant p63+/p40+ immunophenotype. P40, similar to p63, highlighted almost all lesional cells of OTs and, overall, the full thickness of the epithelial lining of the cystic areas of OCs and ameloblastoma. The keratin layer of OKC and the adluminal ductal and mucous cells of GOC were p63-/p40-. CONCLUSIONS: Both ΔNp63 and TAp63 isoforms are present in neoplastic and developmental odontogenic lesions; and p63/p40 immunophenotype is nondiscriminatory pertaining to benign and malignant OTs and OCs.