The fatty-acid amide hydrolase inhibitor URB597 inhibits MICA/B shedding

Kazuma Sekiba,Kazuhiko Koike,Yu Miyakawa,Motoyuki Otsuka,Takahiro Seimiya,Eri Tanaka,Kazuyoshi Funato

doi:10.1038/s41598-020-72688-y

Abstract

MICA/B proteins are expressed on the surface of various types of stressed cells, including cancer cells. Cytotoxic lymphocytes expressing natural killer group 2D (NKG2D) receptor recognize MICA/B and eliminate the cells. However, cancer cells evade such immune recognition by inducing proteolytic shedding of MICA/B proteins. Therefore, preventing the shedding of MICA/B proteins could enhance antitumor immunity. Here, by screening a protease inhibitor library, we found that the fatty-acid amide hydrolase (FAAH) inhibitor, URB597, suppresses the shedding of MICA/B. URB597 significantly reduced the soluble MICA level in culture medium and increased the MICA level on the surface of cancer cells. The effect was indirect, being mediated by increased expression of tissue inhibitor of metalloproteinases 3 (TIMP3). Knockdown of TIMP3 expression reversed the effect of URB597, confirming that TIMP3 is required for the MICA shedding inhibition by URB597. In contrast, FAAH overexpression reduced TIMP3 expression and the cell-surface MICA level and increased the soluble MICA level. These results suggest that inhibition of FAAH could prevent human cancer cell evasion of immune-mediated clearance.

Highlights

Abbreviations natural killer (NK) group 2D (NKG2D) Natural killer group 2D fatty-acid amide hydrolase (FAAH) Fatty-acid amide hydrolase tissue inhibitor of metalloproteinases 3 (TIMP3) Tissue inhibitor of metalloproteinases 3 MICA Major histocompatibility complex class I polypeptide-related sequence A NK Natural killer hepatocellular carcinoma (HCC) Hepatocellular carcinoma dimethyl sulfoxide (DMSO) Dimethyl sulfoxide matrix metalloproteinase (MMP) Matrix metalloproteinase a disintegrin and metalloproteinase (ADAM) A disintegrin and metalloproteinase polymerase chain reaction (PCR) Polymerase chain reaction phosphate-buffered saline (PBS) Phosphate-buffered saline qRT-PCR Quantitative reverse-transcription PCR standard deviation (SD) Standard deviation
Because MICA/B polymorphisms and expression are critical in human c ancers[4,5,6,7,8,9,10,11], the immune mechanisms mediated by MICA/B ligands and the NK group 2D (NKG2D) receptor are crucial for antitumor immunity and prevention of carcinogenesis
Using a threshold of a > 30% increase in the cell-surface MICA/B level compared with DMSO in the first screening, we identified eight candidate drugs: actinonin, bestatin, E64-d, epigallocatechin gallate, GM6001, PMSF, URB597, and Z-LeuLeu-CHO (Fig. 1a,b)

Summary

Introduction

Abbreviations NKG2D Natural killer group 2D FAAH Fatty-acid amide hydrolase TIMP3 Tissue inhibitor of metalloproteinases 3 MICA Major histocompatibility complex class I polypeptide-related sequence A NK Natural killer HCC Hepatocellular carcinoma DMSO Dimethyl sulfoxide MMP Matrix metalloproteinase ADAM A disintegrin and metalloproteinase PCR Polymerase chain reaction PBS Phosphate-buffered saline qRT-PCR Quantitative reverse-transcription PCR SD Standard deviation. Major histocompatibility complex class I polypeptide-related sequence A (MICA) and MICB are highly expressed in many infected or transformed human cells. (a) Fold changes in the mean fluorescence intensity (MFI) of surface MICA/B by flow cytometry in HepG2 cells after treatment for 24 h with protease inhibitors. (c) Fold changes in the MFI of surface MICA/B by flow cytometry in Hep3B cells after treatment for 24 h with the eight candidate protease inhibitors. (d) Flow cytometry of surface MICA protein levels in HepG2 cells treated with URB597 (blue line) or DMSO (control, red line). (e) Flow cytometry of surface MICA protein levels in Hep3B cells treated with URB597 (blue line) or DMSO (control, red line). Several methods of inhibiting MICA/B shedding have been reported[3,16,17,18], none are suitable for clinical use

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Results

Conclusion