3.1 Expression of CC chemokine in patients with skin cutaneous melanoma
Firstly, to investigate the role of the 28 CCL chemokines in tumorigenesis, progression and prognosis, we examined the RNA sequencing data of all 32 cancer types in TCGA for CCL transcript levels in GEPIA. We found that in Breast invasive carcinoma (BRCA), the highly expressed chemokines were CCL5 and CCL11 and the low expressed chemokines were CCL7, CCL14, CCL21, CCL23 and CCL28. In colon adenocarcinoma (COAD), CCL3, CCL4, CCL5, CCL15, CCL18, CCL20 and CCL24 were highly expressed and CCL2, CCL11, CCL13, CCL14, CCL21 and CCL23 were lowly expressed. In Cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC), the highly expressed chemokines were CCL3, CCL4, CCL17, CCL18, CCL20 and CCL22; the low expressed chemokines were CCL14, CCL21, CCL23 and CCL24.In Head and Neck squamous cell carcinoma (HNSC), the highly expressed chemokines were CCL3, CCL5, CCL11, CCL18 and CCL20; the low expressed chemokines were CCL2 and CCL14. In lung squamous cell carcinoma (LUSC), the highly expressed chemokines were CCL20 and CCL26; the low expressed chemokines were CCL2, CCL13, CCL14, CCL15, CCL21 and CCL23. In Liver hepatocellular carcinoma (LIHC), the highly expressed chemokines were CCL15, CCL18, CCL20 and CCL21; the low expressed chemokines were CCL14, CCL19 and CCL23. In Ovarian serous cystadenocarcinoma (OV), the highly expressed chemokines were CCL2, CCL3, CCL4, CCL5, CCL8, CCL11, CCL18, CCL20 and CCL28; the low expressed chemokines were CCL14, CCL21 and CCL26.In Pancreatic adenocarcinoma (PAAD), the highly expressed chemokines were CCL2, CCL3, CCL4, CCL5, CCL11, CCL13, CCL15, CCL17, CCL18, CCL19, CCL20, CCL21, CCL22, CCL22, CCL24, CCL26 and CCL28. Specific results can be found in exhibits Supplement Figure 1-9. In skin cutaneous melanoma we found that CCL3, CCL4, CCL5, CCL8 and CCL18 were highly expressed in tumor tissue, while some CC chemokines CCL14, CCL21, CCL22, CCL23 and CCL27 were found to be expressed at low levels in tumor tissue. The results are presented in Figure 1. Our results suggest that CCL family is differentially expressed in a variety of cancer tissues, including skin cutaneous melanoma. We also compared the relative expression levels of individual CC chemokines in SKCM and found that CCL5 had the highest relative expression of all the CC chemokines we evaluated, as shown in Figure 2.
To identify CC chemokines associated with staging of SKCM, we investigated the correlation between CCL expression levels and staging of differentially expressed CCL. The results revealed that the expression of CCL4 (p=0.00168), CCL5 (p=0.00137), CCL8 (p=0.000343), CCL23 (p=0.0137) and CCL27 (p=0.0156) were significantly correlated with pathological staging (Figure 3).
The expression of CCL4, CCL5, CCL8, CCL23 and CCL27 increased with tumor progression. These data suggest that these CC chemokines play an important role in the development and progression of SKCM.
3.2 Effect of CC chemokine on the prognosis of patients with skin cutaneous melanoma
We used three methods, UALCAN, GEPIA and Prognoscan, to assess the phototropism of CC chemokine expression and clinical prognosis of SKCM and obtained relatively consistent results. We found that CCL expression significantly affected the prognosis of SKCM. By UALCAN analysis, we found that high expression of CCL3 (p=0.0018), CCL4 (p<0.0001), CCL5 (p<0.0001), CCL8 (p<0.0001), CCL23 (p=0.021) was associated with a better prognosis for patients with SKCM, and the results of the survival curves can be seen in Figure 4. Our study in GEPIA found that SKCM OS had a better prognosis associated with CCL3 (p=0.0018, r=0.65), CCL4 (p=8.2e-08, r=0.48), CCL5 (p=3.2e-06, r=0.53), CCL8 (p=e-06, r=0.52), CCL22 (p=0.0063, r=0.69), CCL23 (p=0.0031, r=0.67) were associated with high expression, and better DFS prognosis was associated with high expression of CCL4 (p=0.0026, r=0.76), CCL5 (p=0.0027, r=0.76). The results are presented in Figure 5. In our analysis using Prognoscan, we found that a better prognosis for OS in SKCM was associated with high expression of CCL15, CCL19, CCL21 and CCL23. In dataset GSE19234 using probe HG-U133_Plus_2, High expression of CCL15 had a good prognostic outcome in SKCM (OS p=0.044292, HR=0.42, 95%CI=0.18-0.98). High expression of CCL19 had a good prognostic outcome (OS p=0.001221, HR=0.62, 95%CI=0.46-0.83). High expression of CCL21 had a good prognostic outcome in SKCM (OS p=0.046, HR=0.74, 95%CI=0.55-1.00). High expression of CCL23 had a good prognostic outcome in SKCM (OS p=0.034096, HR=0.58, 95% CI=0.35-0.96). (figure 6).
3.3 Analysis of genetic alterations, co-expression, neighborhood gene networks and interactions in CCL families of skin cutaneous melanoma patients
To investigate the molecular characterization of differentially expressed CCL, we used cBioPortal on the TCGA dataset to analyze genetic alterations in differentially expressed CCL. As shown in Figure 7a and 7b, genetic alterations were dominated by mutations, profound deletions and multiple alterations. CCL3, CCL4, CCL5, CCL8, CCL18, CCL14, CCL21, CCL22, CCL23 and CCL27 were found in 0.5, 0.5, 1.4, 0.9, 0.9, 0.9, 0.9, 1.4, 0.5 and 0.9% of SKCM samples were altered. We also obtained genes related to CCL family genes in SKCM patients include PKP4, OSGIN1, LYG1, LMAN2L, CPNE2, LINC01242, LOC100506422, TLK1, ZSCAN25 and NCK2. The results are shown in Table 1.
We further analyzed the relationship of differentially expressed genes in skin cutaneous melanoma, their co-expression genes and interactions using the STRING database and GeneMANIA to explore how our CCL family and its associated genes function in concert. In STRING, we obtained 10 nodes and 22 edges in the PPI network. We found that the functions of these differentially expressed CCLs were associated with chemokine signaling pathways, viral protein-cytokine, cytokine receptor interactions, and inflammatory responses, as shown in Figure 8a. GeneMANIA results also showed that the functions of the differentially expressed CCLs and related genes were mainly associated with cytokine activity, chemokine receptor binding, cellular responses to chemokines, targeting of chemokines, lymphocyte migration, neutrophil migration, G protein-coupled receptor binding, granulocyte chemotaxis, monocyte migration and cytokine receptor binding. The specific results can be seen in Figure 8b and Table 2.
3.4 Functional enrichment analysis of CCL family genes in skin cutaneous melanoma patients
We performed a functional enrichment analysis of differentially expressed CCL genes in SKCM using three sites to explore the biological processes involved and the functions acted. The richest features of CC in omicshare were extracellular region, cytosol cytoplasmic part, cytoplasm, intracellular part, intracellular, cell, cell part and cellular component. The richest features in MF in omicshare were CCR chemokine receptor binding, chemokine activity, chemokine receptor binding, cytokine activity, cytokine receptor binding, G protein-coupled receptor binding, receptor ligand activity, receptor regulator activity, CCR1 chemokine receptor binding and signaling receptor binding. In category BP, lymphocyte chemotaxis, lymphocyte migration, monocyte chemotaxis, chemokine-mediated signaling pathway, mononuclear cell, migration, response to chemokine, cellular response to chemokine, neutrophil chemotaxis, leukocyte chemotaxis, neutrophil migration and other functions were enriched. The KEGG pathway analysis revealed that the functions of Signaling molecules and interaction, Immune system, Infectious diseases Signal transduction and Neurodegenerative diseases, etc. are closely related to SKCM. These are shown in Figure 10.
We obtained these results in DAVID 6.8: the most abundant functions in the BP category were monocyte chemotaxis, cellular response to interferon-gamma, chemokine-mediated signaling pathway, cellular response to interleukin-1 response to interleukin-1, cellular response to tumor necrosis factor, neutrophil chemotaxis, lymphocyte chemotaxis, positive regulation of ERK1 and ERK2 cascade, immune response and cell-cell signaling. The most abundant functions in the CC category were extracellular space, extracellular region, cell and intracellular. The richest features in MF were chemokine activity, CCR chemokine receptor binding, CCR1 chemokine receptor binding, phospholipase activator activity, CCR5 chemokine receptor binding, chemokine receptor binding, chemoattractant activity and protein kinase activity. The first 8 KEGG pathways were Chemokine signaling pathways, Cytokine-cytokine receptor interaction, Toll-like receptor signaling pathway and Cytosolic DNA-sensing pathway. The results are shown in Figure 10. The results of Metascape were similar to the first two analyses. The CCL family plays an important role in the development and progression of SKCM. The results are shown in Figure 11 and Table 3.
3.5 CCL family gene transcription factor targets, kinase targets in skin cutaneous melanoma patients
TRRUST analysis of differentially expressed genes in SKCM identified two transcription factors associated with SKCM, v-rel reticuloendotheliosis viral oncogene homolog A (avian) (RELA) and nuclear factor of kappa light polypeptide gene enhancer in B-cell 1 (NFKB1). RELA is the target of transcription factors for CCL4, CCL22, CCL3 and CCL5 and NFKB1 is the transcription factor for CCL5, CCL3, CCL4 and CCL22. The results are shown in Table 4.
In LinkedOmics, we identified the top three kinase targets associated with CC chemokine. SYK, BCL2, and CASP7 are the three most important targets in the CCL3 kinase-target network. The composition of the CCL4 kinase target network is mainly associated with CASP7, LCK and SYK. CASP7, LCK and ANXA7 are considered to be the CCL5. CASP7, SYK, and CDH1 are mainly associated with CCL8. BCL2L11, SETD2 and MAP2K1, SERPINE1, 53BP1 and MAPK14 are the three most important targets in the CCL14 and CCL18 kinase-target network, respectively. The composition of the CCL 21and CCL22 kinase-target network is mainly associated with SRC, RPS6KB1 and MYH11 as well as MAPK14, BAP1 and YWHAZ. The kinase target network of CCL23 was mainly composed of NDRG1, MAP2K1 and YBX1. CCL27 was associated with PRKAA1, TSC2 and RBM15. The results can be found in Table 5.
3.6 Immune infiltration of the CCL family gene in skin cutaneous melanoma patients
Using the TIMER database, we performed an analysis of the correlation between differentially expressed genes of SKCM and the abundance of immune infiltrates in the CCL family. Tumor infiltrating lymphocytes were an independent predictor of tumor sentinel lymph node status and survival. The results showed that CCL3, CCL4, CCL5, CCL8, CCL11, CCL14, CCL18, CCL21 and CCL23 expression was negatively correlated with tumor purity.
CCL3 expression and B Cell, CD8+ T Cell, CD4+ T Cell, Macrophage, Neutrophil, Dendritic Cell infiltration level were positively correlated. CCL4 expression and B Cell, CD8+ T Cell, CD4+ T Cell, Macrophage, Neutrophil, Dendritic Cell infiltration level were positively correlated. CCL5 expression was positively correlated with B Cell, CD8+ T Cell, CD4+ T Cell, Macrophage, Neutrophil and Dendritic Cell infiltration level. CCL8 expression was positively correlated with B Cell, CD8+ T Cell, CD4+ T Cell, Macrophage, Neutrophil and Dendritic Cell infiltration level. CCL14 expression was positively correlated with CD4+ T Cell and Macrophage infiltration. CCL18 expression was negatively correlated with CD8+ T Cell and Dendritic Cell infiltration level. CCL21 expression was positively correlated with B Cell, CD8+ T Cell, CD4+ T Cell, Macrophage, Neutrophil, Dendritic Cell infiltration level were positively correlated. The results of CCL22 and CCL23 were consistent with those of CCL21. Specific results can be found in Table 6 and Figures 12.