P2Y2 receptors mediate nucleotide-induced EGFR phosphorylation and stimulate proliferation and tumorigenesis of head and neck squamous cell carcinoma cell lines

Lucas T Woods; Kimberly J Jasmer; Kevin Muñoz Forti; Vinit C Shanbhag; Jean M Camden; Laurie Erb; Michael J Petris; Gary A Weisman

doi:10.1016/j.oraloncology.2020.104808

P2Y₂ receptors mediate nucleotide-induced EGFR phosphorylation and stimulate proliferation and tumorigenesis of head and neck squamous cell carcinoma cell lines

Oral Oncol. 2020 Jun 12:109:104808. doi: 10.1016/j.oraloncology.2020.104808. Online ahead of print.

Authors

Lucas T Woods¹, Kimberly J Jasmer¹, Kevin Muñoz Forti¹, Vinit C Shanbhag¹, Jean M Camden¹, Laurie Erb¹, Michael J Petris², Gary A Weisman³

Affiliations

¹ Division of Biochemistry, University of Missouri, Columbia, MO 65211-7310 USA; Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211-7310 USA.
² Division of Biochemistry, University of Missouri, Columbia, MO 65211-7310 USA; Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211-7310 USA; Department of Ophthalmology, University of Missouri School of Medicine, Columbia, MO 65211-7310 USA.
³ Division of Biochemistry, University of Missouri, Columbia, MO 65211-7310 USA; Christopher S. Bond Life Sciences Center, University of Missouri, Columbia, MO 65211-7310 USA. Electronic address: weismang@missouri.edu.

Abstract

Objectives: To assess functional expression of the P2Y₂ nucleotide receptor (P2Y₂R) 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, P2Y₂R contributions to extracellular nucleotide-induced changes in intracellular free Ca²⁺ concentration and EGFR and extracellular signal-regulated kinase (ERK1/2) phosphorylation were determined using the ratiometric Ca²⁺ indicator fura-2 and immunoblot analysis, respectively. Genetic knockout of P2Y₂Rs using CRISPR technology or pharmacological inhibition with P2Y₂R-selective antagonist AR-C118925 defined P2Y₂R contributions to in vivo tumor growth.

Results: P2Y₂R agonists UTP and ATP increased intracellular Ca²⁺ levels and ERK1/2 and EGFR phosphorylation in CAL27 and FaDu cells, responses that were inhibited by AR-C118925 or P2Y₂R knockout. P2Y₂R-mediated EGFR phosphorylation was also attenuated by inhibition of the adamalysin family of metalloproteases or Src family kinases. P2Y₂R 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: P2Y₂Rs mediate HNSCC cell responses to extracellular nucleotides and genetic or pharmacological blockade of P2Y₂R signaling attenuates tumor cell proliferation and tumorigenesis, suggesting that the P2Y₂R represents a novel therapeutic target in HNSCC.

Keywords: Adenosine 5′-Triphosphate; Cetuximab; Epidermal Growth Factor Receptor; Head and Neck Squamous Cell Carcinoma; Purinergic P2Y(2) Receptor Antagonist; Purinergic P2Y(2) Receptors; Tumor Microenvironment.

Abstract

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