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

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

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 3

Cancer cells educate macrophages toward an invasion-promoting phenotype via ADAM17-dependent soluble factors.

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Cancer cells educate macrophages toward an invasion-promoting phenotype ...
(A) Western blot of ADAM17 WT and Adam17–/– (A17–/–) clones 1 and 2 4T1 and CT26 cell lines (representative of 3 repeats). β-Actin served as control. (B) Experimental setup for C, E, and G. (C) Average invaded cells/field of WT 4T1 and CT26 cells alone or with BMDMs educated with WT or Adam17–/– cells (n = 3). (D) ADAM17 Western blot of Adam17–/– 4T1 and CT26 cell lines expressing empty vector (negative control, NC) or mouse ADAM17 (mADAM17) (representative of 3 repeats). β-Actin served as control. (E) Invaded cells/field of WT 4T1 or CT26 cell lines alone or with BMDMs educated with Adam17–/– NC or mADAM17 cancer cells (n = 3). (F) ADAM17 Western blot of MDA-231 and SW480 cells transfected with NC or ADAM17 (A17) siRNA (representative of 3 repeats). β-Actin served as control. (G) Invaded cells/field of WT cell lines with THP-1-derived macrophages (THP-1MΦ) educated by NC or ADAM17 siRNA–transfected MDA-231 and SW480 cells (n = 3). (H) Experimental setup for the zebrafish embryo dissemination assay in I–K. (I) Example of tail foci in embryonic zebrafish injected with SW480 cells alone or with THP-1MΦ educated with NC or A17 siRNA–transfected SW80 cells. Arrows: green: cancer cells, red: macrophages, yellow: both cancer cells and macrophages. Scale bar: 200 μm (top), 100 μm (bottom). Quantification of cancer cell (J) and macrophage (K) foci in tail regions 24 hours after injection with SW480 cells alone (n = 19) or with THP-1MΦ educated with NC siRNA–transfected SW80 cells (n = 53) or A17 siRNA–transfected SW80 cells (n = 57). Mean and standard deviation indicated. Data in C, E, and J were analyzed by 1-way ANOVA with Dunnett’s multiple comparison test, and data in G were analyzed by Kruskal-Wallis with Dunn’s multiple comparison test. Data in K were analyzed using unpaired 2-sided Student’s t test. *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.001.