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ADAM17-mediated EGFR ligand shedding directs macrophage-promoted cancer cell invasion
Sebastian P. Gnosa, … , Pauliina Kronqvist, Marie Kveiborg
Sebastian P. Gnosa, … , Pauliina Kronqvist, Marie Kveiborg
Published August 23, 2022
Citation Information: JCI Insight. 2022;7(18):e155296. https://doi.org/10.1172/jci.insight.155296.
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Research Article Oncology

ADAM17-mediated EGFR ligand shedding directs macrophage-promoted cancer cell invasion

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Abstract

Macrophages in the tumor microenvironment have a substantial impact on tumor progression. Depending on the signaling environment in the tumor, macrophages can either support or constrain tumor progression. It is therefore of therapeutic interest to identify the tumor-derived factors that control macrophage education. With this aim, we correlated the expression of A Disintegrin and Metalloproteinase (ADAM) proteases, which are key mediators of cell-cell signaling, to the expression of protumorigenic macrophage markers in human cancer cohorts. We identified ADAM17, a sheddase upregulated in many cancer types, as a protein of interest. Depletion of ADAM17 in cancer cell lines reduced the expression of several protumorigenic markers in neighboring macrophages in vitro as well as in mouse models. Moreover, ADAM17–/– educated macrophages demonstrated a reduced ability to induce cancer cell invasion. Using mass spectrometry–based proteomics and ELISA, we identified heparin-binding EGF (HB-EGF) and amphiregulin, shed by ADAM17 in the cancer cells, as the implicated molecular mediators of macrophage education. Additionally, RNA-Seq and ELISA experiments revealed that ADAM17-dependent HB-EGF ligand release induced the expression and secretion of CXCL chemokines in macrophages, which in turn stimulated cancer cell invasion. In conclusion, we provide evidence that ADAM17 mediates a paracrine EGFR-ligand-chemokine feedback loop, whereby cancer cells hijack macrophages to promote tumor progression.

Authors

Sebastian P. Gnosa, Laia Puig Blasco, Krzysztof B. Piotrowski, Marie L. Freiberg, Simonas Savickas, Daniel H. Madsen, Ulrich auf dem Keller, Pauliina Kronqvist, Marie Kveiborg

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Figure 4

Soluble EGFR ligands are decreased in Adam17–/– cocultures.

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Soluble EGFR ligands are decreased in Adam17–/– cocultures.
(A) Experime...
(A) Experimental setup for secretome analyses by TMT-MS/MS and ELISA of BMDMs cocultured with cancer cells (used in B–D). (B) Volcano plot of proteins identified in secretomes of WT or Adam17–/– (A17–/–) 4T1 cancer cell-BMDM cocultures by TMT-MS/MS. Significantly altered proteins are shown in red. SPP-1, secreted phosphoprotein-1. (C) Relative HB-EGF secretion in WT, Adam17–/–, and Adam17–/– expressing empty vector (NC) or ADAM17 4T1 cells, determined by parallel reaction monitoring–targeted (PRM-targeted) MS. (D) Secretion of HB-EGF, AREG, and TGF-α in WT or Adam17–/– 4T1 (top) and E0771 (bottom) cancer cell-BMDM cocultures, determined by ELISA (n = 3). (E) Experimental setup for HB-EGF and AREG ELISA of BMDM-cancer cell coculture media. (F) HB-EGF and AREG ELISA of WT or Adam17–/– 4T1 and E0771 cell lines and BMDM media, 16 hours after coculture (n = 3). (G) Experimental setup for Western blot of BMDMs upon cancer cell coculture. (H) Western blot of phosphorylated EGFR (p-EGFR) (Tyr1068) and total EGFR in BMDMs cocultured with either WT or Adam17–/– 4T1 cells, quantified as p-EGFR/total EGFR (n = 3). Mean and standard deviation indicated. Data in C were analyzed using Welch ANOVA with correction for multiple comparisons by controlling FDR using Benjamini-Hochberg method, data in D were analyzed using 2-way ANOVA with Holm-Šidák correction for multiple analysis, data in F were analyzed by 1-way ANOVA with Dunnett’s multiple comparison test, and data in H were analyzed using 2-sided, unpaired Student’s t test, *P ≤ 0.05, ***P ≤ 0.001.

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