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1.
Mol Oncol ; 16(17): 3146-3166, 2022 09.
Article in English | MEDLINE | ID: mdl-35451191

ABSTRACT

Survival of ovarian carcinoma is associated with the abundance of immunosuppressed CD163high CD206high tumor-associated macrophages (TAMs) and high levels of arachidonic acid (AA) in the tumor microenvironment. Here, we show that both associations are functionally linked. Transcriptional profiling revealed that high CD163 and CD206/MRC1 expression in TAMs is strongly associated with an inhibition of cytokine-triggered signaling, mirrored by an impaired transcriptional response to interferons and IL-6 in monocyte-derived macrophages by AA. This inhibition of pro-inflammatory signaling is caused by dysfunctions of the cognate receptors, indicated by the inhibition of JAK1, JAK2, STAT1, and STAT3 phosphorylation, and by the displacement of the interferon receptor IFNAR1, STAT1 and other immune-regulatory proteins from lipid rafts. AA exposure led to a dramatic accumulation of free AA in lipid rafts, which appears to be mechanistically crucial, as the inhibition of its incorporation into phospholipids did not affect the AA-mediated interference with STAT1 phosphorylation. Inhibition of interferon-triggered STAT1 phosphorylation by AA was reversed by water-soluble cholesterol, known to prevent the perturbation of lipid raft structure by AA. These findings suggest that the pharmacologic restoration of lipid raft functions in TAMs may contribute to the development new therapeutic approaches.


Subject(s)
Neoplasms , Tumor Microenvironment , Arachidonic Acid/metabolism , Humans , Macrophages/metabolism , Membrane Microdomains/metabolism , Neoplasms/metabolism , STAT1 Transcription Factor/metabolism , Signal Transduction
2.
Theranostics ; 11(3): 1377-1395, 2021.
Article in English | MEDLINE | ID: mdl-33391540

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.


Subject(s)
Arachidonic Acid/pharmacology , Macrophages/drug effects , Ovarian Neoplasms/drug therapy , Phosphorylation/drug effects , Signal Transduction/drug effects , Calcium/metabolism , Extracellular Vesicles/drug effects , Extracellular Vesicles/metabolism , Female , Group II Phospholipases A2/metabolism , Humans , Neoplasm Recurrence, Local/drug therapy , Neoplasm Recurrence, Local/metabolism , Ovarian Neoplasms/metabolism , Reactive Oxygen Species/metabolism , Transcription, Genetic/drug effects , Tumor Microenvironment/drug effects
3.
Sci Rep ; 8(1): 15278, 2018 10 15.
Article in English | MEDLINE | ID: mdl-30323297

ABSTRACT

Monitoring the drug efficacy or resistance in vitro is usually carried out by measuring the response of single few proteins. However, observation of single proteins instead of an integral cell response may lead to results that are not consistent with patient's response to a drug. We present a Raman spectroscopic method that detects the integral cell response to drugs such as tyrosine kinase inhibitors (TKIs). Non-small cell lung cancer (NSCLC) patients with EGFR mutations develop acquired resistance to first (erlotinib)- and third (osimertinib)-generation TKIs. Large erlotinib-induced differences were detected by Raman micro-spectroscopy in NSCLC cells without T790M EGFR mutation but not in cells with this mutation. Additionally, Raman difference spectra detected the response of NSCLC cells with T790M EGFR mutation to second- (neratinib) and third-generation (osimertinib) TKIs, and the resistance of cells with T790M/C797S EGFR mutation to osimertinib. Thus, the in vitro Raman results indicated that NSCLC cells with T790M and T790M/C797S EGFR mutations are resistant to erlotinib- and osimertinib, respectively, consistent with the observed responses of patients. This study shows the potential of Raman micro-spectroscopy to monitor drug resistance and opens a new door to in vitro companion diagnostics for screening personalized therapies.


Subject(s)
Carcinoma, Non-Small-Cell Lung/drug therapy , Drug Monitoring/methods , Drug Resistance, Neoplasm , Lung Neoplasms/drug therapy , Protein Kinase Inhibitors/therapeutic use , Spectrum Analysis, Raman , Amino Acid Substitution , Carcinoma, Non-Small-Cell Lung/diagnosis , Carcinoma, Non-Small-Cell Lung/genetics , Drug Resistance, Neoplasm/genetics , Drug Screening Assays, Antitumor , ErbB Receptors/genetics , Humans , Lung Neoplasms/diagnosis , Lung Neoplasms/genetics , Molecular Targeted Therapy , Precision Medicine , Spectrum Analysis, Raman/methods , Treatment Outcome , Tumor Cells, Cultured
4.
Angew Chem Int Ed Engl ; 57(24): 7250-7254, 2018 06 11.
Article in English | MEDLINE | ID: mdl-29645336

ABSTRACT

Tyrosine kinase receptors are one of the main targets in cancer therapy. They play an essential role in the modulation of growth factor signaling and thereby inducing cell proliferation and growth. Tyrosine kinase inhibitors such as neratinib bind to EGFR and HER2 receptors and exhibit antitumor activity. However, little is known about their detailed cellular uptake and metabolism. Here, we report for the first time the intracellular spatial distribution and metabolism of neratinib in different cancer cells using label-free Raman imaging. Two new neratinib metabolites were detected and fluorescence imaging of the same cells indicate that neratinib accumulates in lysosomes. The results also suggest that both EGFR and HER2 follow the classical endosome lysosomal pathway for degradation. A combination of Raman microscopy, DFT calculations, and LC-MS was used to identify the chemical structure of neratinib metabolites. These results show the potential of Raman microscopy to study drug pharmacokinetics.


Subject(s)
Lysosomes/metabolism , Neoplasms/metabolism , Protein Kinase Inhibitors/metabolism , Quinolines/metabolism , Cell Line, Tumor , ErbB Receptors/metabolism , Humans , Receptor, ErbB-2/metabolism , Spectrum Analysis, Raman
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