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Proteolytic processing of PD-L1 by ADAM proteases in breast cancer cells

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Abstract

Expression of programmed death ligand 1 (PD-L1) on the surface of tumor cells and its interaction with programmed cell death protein 1 (PD-1) on tumor-infiltrating lymphocytes suppress anti-tumor immunity. In breast tumors, PD-L1 expression levels are the highest in estrogen receptor-negative, progesterone receptor-negative, and human epidermal growth factor receptor 2-negative (triple-negative) cancers. In this study, we show that a portion of PD-L1 exogenously expressed in several triple-negative breast cancer cell lines, as well as endogenous PD-L1, is proteolytically cleaved by cell surface metalloproteases. The cleavage generates an ~ 37-kDa N-terminal PD-L1 fragment that is released to the media and a C-terminal PD-L1 fragment that remains associated with cells but is efficiently eliminated by lysosomal degradation. We identify ADAM10 and ADAM17, two closely related members of the ADAM family of cell surface metalloproteases, as enzymes mediating PD-L1 cleavage. Notably, treatment of cells with ionomycin, a calcium ionophore and a known activator of ADAM10, or with phorbol 12-myristate 13-acetate, an activator of ADAM17, dramatically increases the release of soluble PD-L1 to the media. We postulate that ADAM10 and/or ADAM17 may contribute to the regulation of the PD-L1/PD-1 pathway and, ultimately, to anti-tumor immunity in triple-negative breast cancer.

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Abbreviations

ADAM:

A disintegrin and metalloprotease

AEBSF:

4-(2-Aminoethyl)benzenesulfonyl fluoride hydrochloride

BCA:

Bicinchoninic acid

FBS:

Fetal bovine serum

GAPDH:

Glyceraldehyde 3-phosphate dehydrogenase

GEO:

Gene expression omnibus

HOPS:

Homotypic fusion and vacuole protein sorting

HS:

Horse serum

FcγRIII:

Low-affinity IgγFc region receptor III

LAG-3:

Lymphocyte activation gene 3 protein

M/F:

Myc/FLAG tag

MMP:

Matrix metalloprotease

PD-1:

Programmed cell death protein 1

PD-L1:

Programmed death ligand 1

PMA:

Phorbol 12-myristate 13-acetate

siRNA:

Small interfering RNA

sPD-L1:

Soluble programmed death ligand 1

COSMIC:

The catalogue of somatic mutations in cancer database

TAPI-2:

Tumor necrosis factor protease inhibitor 2

TNBC:

Triple-negative breast cancer

VPS18:

Vacuolar protein sorting-associated protein 18

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Funding

This work was supported by the National Institutes of Health (US) Grant R01CA172222 and by funds from Kansas State University Johnson Cancer Research Center to Anna Zolkiewska.

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Contributions

YR and RW performed the experiments and contributed to writing the manuscript. AZ conceived the study, performed the experiments, and wrote the manuscript. All authors read and approved the final manuscript.

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Correspondence to Anna Zolkiewska.

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The authors declare that they have no conflict of interest.

Ethical approval and ethical standards

The studies described in this paper were performed using commercially available cell lines. This article does not contain any experiments involving patients or experimental animals. Therefore, no IACUC approval and no informed consent from the donors were required.

Cell line authentication

MDA-MB-231, BT549, MCF10A, A549, and DU-145 cell lines were from the American Type Culture Collection (Manassas, VA). SUM149 and SUM159 cell lines were obtained from Asterand (Detroit, MI). These cell lines were authenticated by their suppliers using short tandem repeat analysis and have been passaged for fewer than 6 months after culture initiation from an early passage number.

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Romero, Y., Wise, R. & Zolkiewska, A. Proteolytic processing of PD-L1 by ADAM proteases in breast cancer cells. Cancer Immunol Immunother 69, 43–55 (2020). https://doi.org/10.1007/s00262-019-02437-2

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