Multi-omics Analysis of Serum Samples Demonstrates Reprogramming of Organ Functions Via Systemic Calcium Mobilization and Platelet Activation in Metastatic Melanoma

Abstract

Pathophysiologies of cancer-associated syndromes such as cachexia are poorly understood and no routine biomarkers have been established, yet. Using shotgun proteomics, known marker molecules including PMEL, CRP, SAA, and CSPG4 were found deregulated in patients with metastatic melanoma. Targeted analysis of 58 selected proteins with multiple reaction monitoring was applied for independent data verification. In three patients, two of which suffered from cachexia, a tissue damage signature was determined, consisting of nine proteins, PLTP, CD14, TIMP1, S10A8, S10A9, GP1BA, PTPRJ, CD44, and C4A, as well as increased levels of glycine and asparagine, and decreased levels of polyunsaturated phosphatidylcholine concentrations, as determined by targeted metabolomics. Remarkably, these molecules are known to be involved in key processes of cancer cachexia. Based on these results, we propose a model how metastatic melanoma may lead to reprogramming of organ functions via formation of platelet activating factors from long-chain polyunsaturated phosphatidylcholines under oxidative conditions and via systemic induction of intracellular calcium mobilization. Calcium mobilization in platelets was demonstrated to alter levels of several of these marker molecules. Additionally, platelets from melanoma patients proved to be in a rather exhausted state, and platelet-derived eicosanoids implicated in tumor growth were found massively increased in blood from three melanoma patients. Platelets were thus identified as important source of serum protein and lipid alterations in late stage melanoma patients. As a result, the proposed model describes the crosstalk between lipolysis of fat tissue and muscle wasting mediated by oxidative stress, resulting in the metabolic deregulations characteristic for cachexia.

Fig. 1.

Regulation of proteins in serum from melanoma patients. Differences of LFQ values (logarithmic scale base two) of proteins determined in serum samples from melanoma patients versus controls (A), as well as from melanoma patients with high tumor load (high TL) or low tumor load (low TL) (B), with corresponding p values (logarithmic scale base 10), are represented as volcano plots. The area above the two black lines encompasses at least 2-fold significantly regulated proteins with a global FDR<0.05 as determined by a permutation-based method. C, Profile plots showing LFQ values (logarithmic scale base two) obtained from each individual measurement. Values for non-regulated proteins (green), proteins upregulated upon escalation of melanoma (dark red), proteins which were downregulated in melanoma versus control serum samples (blue), and one protein (ANXA1) which was upregulated in low TL, but downregulated in high TL (light red) are highlighted. Patients annotated as mel_4a to mel_6b showed increased levels of melanoma marker molecules D, Results from targeted analyses showing that CRP levels were similar in serum samples from patients with high TL or low TL, but elevated in patients with metastatic melanoma in comparison to controls. Progressive FTL increase associated with high TL melanoma was also determined with targeted analysis; TAN, log₂ of normalized total peak area for a selected peptide. E, Calcium levels, as determined in clinics, were higher in serum samples from patients with high TL in comparison to low TL melanoma. Boxplots represent data collected in a four month period of time and consist of 15 samples per group. F, Correlation of quantification data obtained fom MRM and shotgun analyses. TAN, log₂ of the normalized total peak area for proteins obtained by MRM; LFQ intensity, log₂ of the label-free quantification value obtained by shotgun analysis; r, Pearson correlation coefficient.

Fig. 2.

Results of targeted proteomics analyses. The MRM analysis revealed nine proteins which were significantly up- or downregulated in serum samples from patients with with high TL or low TL. For statistical analysis, the MSStats software was used and the significance threshold was set to a fold change higher than 1.5 with a p value lower than 0.05. Sample quantification data from MSStats are represented as log₂ intensities.

Fig. 3.

Metabolomics analysis. A, The concentrations of glycine (Gly), aspartic acid (Asp) and selected phospohatidylcholine acyl alkyl (PC ae) derivatives determined with targeted analysis in samples from healthy donors as well as from melanoma patients are represented. B, The abundance of platelet activating factor (PAF) obtained by shotgun analysis was found increased in high TL melanoma patients; NAUC; normalized area under the curve.

Fig. 4.

Proposed mechanism for cachexia development. A, From long-chain highly unsaturated phosphatidylcholines acyl alkyl metabolites (PC ae) sourcing from adipose tissue platelet activating factor (PAF) and platelet activating factor-receptor (PAF-R) agonists are produced under oxidative conditions. These molecules induce calcium release from ESR in patients with high TL upon binding to PAF-R (two of three with cachexia). To transfer calcium back into the ESR and maintain cytosolic calcium concentrations tolerable, cells may upregulate SERCA pumps, which is an energy consuming process. At increased calcium concentration, S100 proteins may form insoluble complexes which can explain the reduced levels of these proteins. C4A levels are increased as protection mechanism of the cells as response to the elevated levels of calcium concentration. Additionally, membrane protein-receptors are released and calcium dependent signaling pathways may be activated as a consequence of the sustained calcium influx in the cytosol. Disruption of ESR-mitochondria bidirectional communication may also be induced by incorporation of PC ae into membranes. B, The combination of the three processes lipolysis, oxidation and intracellular calcium mobilization may be a critical combination resulting in melanoma damage signature and probably in cachexia establishment. Phosphatidylcholines sourcing from lipolysis of adipose tissues can be converted into PAF and PAF-R agonists upon oxidation which induce intracellular calcium mobilization by activating the PAF-R. Subsequently, systemic intracellular calcium mobilization may eventually result in cachexia.

Fig. 5.

Shotgun analysis of platelets at protein and eicosanoid levels. A, Results for selected proteins included in the mechanistic model of cachexia development, obtained from analysis of untreated and ionomycin-treated platelets isolated from two healthy donors, are shown. Each protein is represented with one or two peptides; TAN, normalized total peak area for a selected peptide. B, Results for selected proteins obtained from the analysis of whole lysates of platelets obtained from healthy donors and melanoma patients. P1 and P2 refer to independent peptides of the same protein. C, The fold change in abundance of proteins and eicosanoids secreted from treated versus untreated platelets are shown. Platelets were isolated from three healthy donors as well as from three melanoma patients. The isolated platelets were treated with ionomycin for 15 min.

Fig. 6.

Eicosanoid analysis in platelets and whole blood samples. A, Results for 12S-HETE and 15S-HETE, obtained from analysis of untreated and ionomycin-treated platelets isolated from two healthy donors, are shown NAUC; normalized area under the curve. B, The shotgun analysis of eicosanoids was applied to 8 ml blood collected from melanoma patients and healthy donors. The results obtained for 12S-HETE and 15S-HETE are represented; AUC, area under the curve of MS1 peak.