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.