Phosphoproteomics identify arachidonic-acid-regulated signal transduction pathways modulating macrophage functions with implications for ovarian cancer

Raimund Dietze^1#, Mohamad K. Hammoud^1#, María Gómez-Serrano¹, Annika Unger¹, Tim Bieringer^1,6, Florian Finkernagel¹, Anna M. Sokol^2,3, Andrea Nist⁴, Thorsten Stiewe⁴, Silke Reinartz¹, Viviane Ponath⁵, Christian Preußer⁵, Elke Pogge von Strandmann⁵, Sabine Müller-Brüsselbach¹, Johannes Graumann^2,3, Rolf Müller^1✉

1. Tranlational Oncology Group, Center for Tumor Biology and Immunology, Philipps University, Marburg, Germany.
2. Biomolecular Mass Spectrometry, Max-Planck-Institute for Heart and Lung Research, Bad Nauheim, Germany.
3. The German Centre for Cardiovascular Research (DZHK), Partner Site Rhine-Main, Max Planck Institute for Heart and Lung Research, Bad Nauheim, Germany.
4. Genomics Core Facility, Philipps University, Marburg, Germany.
5. Institute for Tumor Immunology, Philipps University, Marburg, Germany.
6. Present address: Hochschule Landshut, 84036 Landshut, Germany.
#These authors contributed equally to this work.

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Citation:
Dietze R, Hammoud MK, Gómez-Serrano M, Unger A, Bieringer T, Finkernagel F, Sokol AM, Nist A, Stiewe T, Reinartz S, Ponath V, Preußer C, von Strandmann EP, Müller-Brüsselbach S, Graumann J, Müller R. Phosphoproteomics identify arachidonic-acid-regulated signal transduction pathways modulating macrophage functions with implications for ovarian cancer. Theranostics 2021; 11(3):1377-1395. doi:10.7150/thno.52442. Available from https://www.thno.org/v11p1377.htm

Abstract

Arachidonic acid (AA) is a polyunsaturated fatty acid present at high concentrations in the ovarian cancer (OC) microenvironment and associated with a poor clinical outcome. In the present study, we have unraveled a potential link between AA and macrophage functions.

Methods: AA-triggered signal transduction was studied in primary monocyte-derived macrophages (MDMs) by phosphoproteomics, transcriptional profiling, measurement of intracellular Ca²⁺ accumulation and reactive oxygen species production in conjunction with bioinformatic analyses. Functional effects were investigated by actin filament staining, quantification of macropinocytosis and analysis of extracellular vesicle release.

Results: We identified the ASK1 - p38δ/α (MAPK13/14) axis as a central constituent of signal transduction pathways triggered by non-metabolized AA. This pathway was induced by the Ca²⁺-triggered activation of calmodulin kinase II, and to a minor extent by ROS generation in a subset of donors. Activated p38 in turn was linked to a transcriptional stress response associated with a poor relapse-free survival. Consistent with the phosphorylation of the p38 substrate HSP27 and the (de)phosphorylation of multiple regulators of Rho family GTPases, AA impaired actin filament organization and inhibited actin-driven macropinocytosis. AA also affected the phosphorylation of proteins regulating vesicle biogenesis, and consistently, AA enhanced the release of tetraspanin-containing exosome-like vesicles. Finally, we identified phospholipase A₂ group 2A (PLA2G2A) as the clinically most relevant enzyme producing extracellular AA, providing further potentially theranostic options.

Conclusion: Our results suggest that AA contributes to an unfavorable clinical outcome of OC by impacting the phenotype of tumor-associated macrophages. Besides critical AA-regulated signal transduction proteins identified in the present study, PLA2G2A might represent a potential prognostic tool and therapeutic target to interfere with OC progression.

Keywords: arachidonic acid, phosphoproteomics, macropinocytosis, ovarian cancer