Proteomic and Phosphoproteomic Analysis Reveals that Neurokinin-1 Receptor (NK1R) Blockade with Aprepitant in Human Keratinocytes Activates a Distinct Subdomain of EGFR Signaling: Implications for the Anti-Pruritic Activity of NK1R Antagonists.

Shawn G Kwatra,Madan M Kwatra,Cory Nanni,Madison Krischak,Kyle A Williams,Emily Boozalis,Callie M Roberts,Raveena Khanna,Yevgeniy R Semenov,Amy H Huang,Robert F Burns

doi:10.3390/medicines6040114

Abstract

Background: Epidermal growth factor receptor (EGFR) inhibitors can cause serious cutaneous toxicities, including pruritus and papulopustular acneiform skin eruptions. Increasingly, the neurokinin-1 receptor (NK1R) antagonist aprepitant is being utilized as an anti-pruritic agent in the treatment of EGFR-inhibitor induced pruritus. Aprepitant is believed to reduce itching by blocking NK1R on the surface of dermal mast cells. However, the effects of aprepitant on human keratinocytes remains unexplored. Methods: Herein, we examine the effects of aprepitant on EGFR stimulation in HaCaT cells using a phosphoproteomic approach including reverse phase protein arrays and Ingenuity Pathway Analysis. Changes in EGFR phosphorylation were visualized using Western blotting and the effect of EGF and aprepitant on the growth of HaCaT cells was determined using the WST-1 Cell Proliferation Assay System. Results: We found that aprepitant increased the phosphorylation of EGFR, as well as 10 of the 23 intracellular proteins phosphorylated by EGF. Analysis of phosphoproteomic data using Ingenuity Pathway Analysis software revealed that 5 of the top 10 pathways activated by EGF and aprepitant are shared. Conclusions: We propose that aprepitant produces its antipruritic effects by partially activating EGFR. Activation of EGFR by aprepitant was also seen in primary human keratinocytes. In addition to itch reduction through partial activation of shared EGFR pathways, aprepitant exerts a dose-dependent cytotoxicity to epithelial cells, which may contribute to its antitumor effects.

Highlights

Epidermal growth factor receptor (EGFR) inhibitors such as erlotinib can cause serious cutaneous toxicities, including papulopustular acneiform skin eruptions and severe pruritus [1,2,3]
To better understand the effect of aprepitant on human keratinocytes, we examined the effects of aprepitant on EGFR signaling in HaCaT cells—an immortalized line of human keratinocytes [14]—using reverse phase protein array (RPPA) technology
We examined whether aprepitant-induced EGFR activation seen in HaCaT cells, a cell line derived from human keratinocytes, was seen in primary human keratinocytes (NHEK) cells

Summary

Introduction

Epidermal growth factor receptor (EGFR) inhibitors such as erlotinib can cause serious cutaneous toxicities, including papulopustular acneiform skin eruptions and severe pruritus [1,2,3]. A loss-of-function mutation of EGFR in a human child exhibited skin toxicity resembling that seen in patients taking EGFR inhibitors [5]. Epidermal growth factor receptor (EGFR) inhibitors can cause serious cutaneous toxicities, including pruritus and papulopustular acneiform skin eruptions. The neurokinin-1 receptor (NK1R) antagonist aprepitant is being utilized as an anti-pruritic agent in the treatment of EGFR-inhibitor induced pruritus. Methods: we examine the effects of aprepitant on EGFR stimulation in HaCaT cells using a phosphoproteomic approach including reverse phase protein arrays and Ingenuity Pathway Analysis.

Methods

Results

Conclusion