Cancer-Associated Fibroblasts Regulate Kinase Activity in Mesothelioma Cell Lines via Paracrine Signaling and Thereby Dictate Cell Faith and Behavior

Abstract

Background: Malignant pleural mesothelioma (MPM) has an infaust prognosis due to resistance to systemic treatment with platin-analoga. MPM cells modulate the immune response to their benefit. They release proinflammatory cytokines, such as TGF-ß, awakening resting fibrocytes that switch their phenotype into activated fibroblasts. Signaling interactions between cancer cells and cancer-associated fibroblasts (CAFs) play an integral part in tumor progression. This study aimed to investigate the role CAFs play in MPM progression, analyzing the impact this complex, symbiotic interaction has on kinase-related cell signaling in vitro.

Methods: We simulated paracrine signaling in vitro by treating MPM cell lines with conditioned medium (CM) from fibroblasts (FB) and vice versa. NCI-H2052, MSTO-211H, and NCI-H2452 cell lines representing the three mayor MPM subtypes, while embryonal myofibroblast cell lines, IMR-90 and MRC-5, provide a CAFs-like phenotype. Subsequently, differences in proliferation rates, migratory behavior, apoptosis, necrosis, and viability were used as covariates for data analysis. Kinase activity of treated samples and corresponding controls were then analyzed using the PamStation12 platform (PamGene); Results: Treatment with myofibroblast-derived CM revealed significant changes in phosphorylation patterns in MPM cell lines. The observed effect differs strongly between the analyzed MPM cell lines and depends on the origin of CM. Overall, a much stronger effect was observed using CM derived from IMR-90 than MRC-5. The phosphorylation changes mainly affected the MAPK signaling pathway.; Conclusions: The factors secreted by myofibroblasts in fibroblasts CM significantly influence the phosphorylation of kinases, mainly affecting the MAPK signaling cascade in tested MPM cell lines. Our in vitro results indicate promising therapeutic effects by the use of MEK or ERK inhibitors and might have synergistic effects in combination with cisplatin-based treatment, improving clinical outcomes for MPM patients.

Keywords: MAPK signaling; MEK and ERK inhibitors; cancer-associated fibroblasts; kinases; microenvironment; phosphorylation; pleural mesothelioma.

Figure 1

Unsupervised clustering of treated cell lines and controls. MPM cell lines (NCI−H2052, NCI−H2452, and MSTO−211H) were treated with conditioned medium of fibroblast cell lines IMR-90 or MRC-5. In controls, conditioned medium of the respective MPM cell line itself was used.

Figure 2

Volcano plot of phosphotsites associated with changes in apoptosis of FB CM-treated MPM cell lines.

Figure 3

Gene set enrichment analysis in MPM cells being treated with FB CM. Among others, particularly the MAPK-RAP1 and the Fc receptor signalling pathway showed significant associations. These pathways were activated in treated MPM cell lines.

Figure 4

Gene set enrichment analysis in relation to apoptosis in MPM cells being treated with FB CM. Particularly, the MAPK-RAP1 and the Fc receptor signaling pathway showed significant associations. These pathways were activated in treated MPM cell lines.

Figure 5

Gene set enrichment analysis in relation to viability of MPM cells being treated with FB CM. Particularly, the C-type lectin receptor signaling pathway showed significant associations (p = 0.01). This pathway was activated in treated MPM cell lines.