P2Y receptors mediate nucleotide-induced EGFR phosphorylation and stimulate proliferation and tumorigenesis of head and neck squamous cell carcinoma cell lines.

OBJECTIVES

To assess functional expression of the P2Y nucleotide receptor (P2YR) in head and neck squamous cell carcinoma (HNSCC) cell lines and define its role in nucleotide-induced epidermal growth factor receptor (EGFR) transactivation. The use of anti-EGFR therapeutics to treat HNSCC is hindered by intrinsic and acquired drug resistance. Defining novel pathways that modulate EGFR signaling could identify additional targets to treat HNSCC.

MATERIALS AND METHODS

In human HNSCC cell lines CAL27 and FaDu and the mouse oral cancer cell line MOC2, P2YR contributions to extracellular nucleotide-induced changes in intracellular free Ca concentration and EGFR and extracellular signal-regulated kinase (ERK1/2) phosphorylation were determined using the ratiometric Ca indicator fura-2 and immunoblot analysis, respectively. Genetic knockout of P2YRs using CRISPR technology or pharmacological inhibition with P2YR-selective antagonist AR-C118925 defined P2YR contributions to in vivo tumor growth.

RESULTS

P2YR agonists UTP and ATP increased intracellular Ca levels and ERK1/2 and EGFR phosphorylation in CAL27 and FaDu cells, responses that were inhibited by AR-C118925 or P2YR knockout. P2YR-mediated EGFR phosphorylation was also attenuated by inhibition of the adamalysin family of metalloproteases or Src family kinases. P2YR knockout reduced UTP-induced CAL27 cell proliferation in vitro and significantly reduced CAL27 and FaDu tumor xenograft volume in vivo. In a syngeneic mouse model of oral cancer, AR-C118925 administration reduced MOC2 tumor volume.

CONCLUSION

P2YRs mediate HNSCC cell responses to extracellular nucleotides and genetic or pharmacological blockade of P2YR signaling attenuates tumor cell proliferation and tumorigenesis, suggesting that the P2YR represents a novel therapeutic target in HNSCC.