Skin is the largest organ of human beings. There are about 1,500 melanocytes in human epidermis per square millimeter, equivalent to nearly 3 billion melanocytes in an ordinary human skin [16]. The incidence of melanoma continues to increase every year. Because of its highly aggressive and treatment-resistant, melanoma is not projected to reduce the death rate in the next few years [17]. Melanoma is associated with a huge burden of genetic alterations [18]. Research on genetic characterizations of melanoma has made a great progress [19, 20]. BRAF, NRAS and NF1 are related to initiation and proliferation of tumor cells [21]. CDKN2A and TERT are associated with progression and cell cycle control [19]. While PTEN and KIT play important roles in advanced progression and metabolism [22]. Targeted therapeutic drugs for the above mutations have been rapidly developed and used for the treatment of melanoma with an improvement in overall survival [23]. The classical progression models of melanoma are from nevus to primary melanoma, to invasive melanoma [6]. However, the lack of research on different periods limits people's understanding of the key genes of melanoma in these periods, especially the initial stage and metastasis stage. Most of the existing targeted drugs are aimed at patients with advanced melanoma, which has the extensive genetic heterogeneity and genomic instability [24]. These two reasons may lead to the emergence of drug resistance,as seen in targeted therapeutic approaches for BRAF inhibitors [25]. Cancer molecular prevention suggests that we can interrupt the prime driver at an early stage of tumor [26]. The shortage of intermediate biomarkers has been a barrier for the development of new molecular target drugs. In addition, melanoma is a highly immunogenic cancer. It is of great value to clarify the potential mechanism of immune activation and immunosuppression in tumor microenvironment. Microarray technology enables exploration of immune mechanism and new targets for melanoma.
In the present study, a total of 308 DEGs and 12 hub genes were identified from three microarray datasets. GO analysis indicated that changes in DEGs were significantly enriched in the melanin biosynthetic process, inflammatory response, extracellular region, extracellular space, chemokine activity and transcriptional activator activity. Pathway analysis of KEGG demonstrated that those DEGs played pivotal roles in pathways in cytokine-cytokine receptor interaction, chemokine signaling pathway and pathways in cancer. These results demonstrated a correlation between DEGs and the tumor microenvironment, immune response and melanoma tumorigenesis. Subsequently, we focused on assessing potential value of 12 hub genes. Interestingly, 8 of 12 hub genes (CXCL10, CCL4, CCL5, IL6, CXCL2, PTGER3, GAL, NPY1R) were also found in the predicted drug-gene interaction, as shown in Fig. 8. Furthermore, seven genes (CCL4, CCL5, NMU, GAL, CXCL9, CXCL10, CXCL13) were identified with significant overall survival outcomes. Subsequently, we chose these seven genes for further analysis. GEPIA verified that the expression these genes except NMU and GAL has significant difference (P values < 0.05) between tumor tissues and normal. In addition, five of these seven hub genes were found to be related to clinical stages (P values < 0.05), including CCL5, CXCL13, CCL4, CXCL9, CXCL10. In recent years,a variety of new biomarkers, such as microRNAs (miRNAs) and lncRNA, have become powerful new tools for diagnosis and monitoring in cancer patients [27]. Therefore, we constructed the related miRNA and lncRNA networks.
C–X–C motif ligand 9,10 (CXCL9, CXCL10) belong to CXC family chemokine. They are predominantly expressed by immune cells such as macrophages and T cells. CXCL9, CXCL10 exert their function through binding to C-X-C motif receptor CXCR3 that was highly expressed on the activated T cells [28]. Research shows that CD8 + T cell infiltration is CXCR3 dependent [29]. CD8 + T cell plays important role in tumor microenvironment, which inhibits the proliferation and metastasis of tumor cells. In our study, high expression of CXCL9 and CXCL10 associated with better overall survival in melanoma patients. What’s more, CXCL9 and CXCL10 were found to be highly expressed in stage I melanoma compared to other stages. A recent research found that the CXCL10-CXCR3 axis may relate to melanoma brain metastasis [30]. Additionally, the CXCR3 ligands enhanced the response rates to immune checkpoint blockade (anti-PD-1, anti-CTLA-4) by recruiting T cells [31]. Kim J et al. found that CXCR3-deficient natural killer cells fail to migrate to B16F10 melanoma cells [32]. These results reveal the importance of manipulating this axis, which provide us opportunities to manipulate chemokine expression for tumor therapy and prevention.
C-X-C motif ligand 13 (CXCL13) also belongs to CXC family. There are very few studies on the relationship between CXCL13 and melanoma. One research suggested that CXCL13 is closely interact with NRAS, BARF and MITF in MAPK signaling pathway, which have implications for melanoma genesis and metastatic progression [33]. In our study, CXCL13 showed similar outcome in overall survival and expression with CXCL9, CXCL10. Pathway analysis suggested they were involved in peptide ligand-binding receptors and chemokine receptors bind chemokine. Therefore, CXCL13 may play the same role as CXCL9, CXCL10 in the occurrence and development of melanoma. Our results revealed that CXCL9, CXCL10, CXCL13 are positively associated with immune cell infiltration. Interestingly, CXCL10 and CXCL2 were found in the predicted drug-gene interaction, providing new thoughts for melanoma treatment. Whether to detect the level of chemokine expression in peripheral blood can help us diagnose melanoma and its stage is unknown. Further external experiments are needed to verify this conjecture.
C-C motif ligand CCL4, CCL5 belong to CC family chemokine. CCL4, CCL5, CXCL9, CXCL10 were confirmed to be preferentially expressed in tumors that contained T cells [34]. This suggests that CCL4, 5 and CXCL9,10 may act in the same pathway in T-cell recruitment. The experimental results carried out by Michelle H et al. showed that expression of CXCR3 ligands and CCL5 in chemotherapy tumors did lead to a synergistic increase in T-cell infiltration [35]. In our study, these four genes were mainly enriched in cytokine-cytokine receptor interaction, chemokine signaling pathway and toll-like receptor signaling pathway. The expression of CCL4, CCL5 showed positive correlation with multiple immune cells infiltration (B-cell, CD8 + T cells, CD4 + T cells, macrophages, Neutrophils, Dendritic cells). It is also been reported that CCL5 released by tumor cells acts its function via paracrine signaling to attract NKs to the tumor bed [36]. However, previous studies showed that tumor growth is delayed in CCR5-/- mice, associated with reduced tumor regulatory T cells infiltration [37]. Edwin L et al. found that a selective increase in CXCL10, CCL4, and CCL17 may contribute to aggressive development of brain metastasis [38]. In the present study, high expression of CCL4 and CCL5 associated with better overall survival and were found to be closely related to stage I melanoma patients compared to other stages. Taken together, we assumed that CCL4 and CCL5 may be potent biomarkers and targets for melanoma.
Neuromedin U (NMU), a neuropeptide belonging to the neuromedin family, is involved in stress, obesity, immune, and energy metabolism [39]. A recent study found the YAP1–NMU axis is associated with pancreatic cancer progression and poor prognosis [40]. High expression of NMU was found to be related to reginal metastasis of head and neck squamous cell carcinoma [41]. NMU upregulation in the breast cancer tissues was proposed as prognostic biomarker for poor outcome, but only in HER2-overexpressing tumor [42]. Moreover, patients with NSCLC (non-small-cell lung carcinoma) and NMU-positive tumor showed significantly shorter survival times than patients with NMU-negative tumor [43]. All these findings suggest that NMU is associated with cancer development and NMU may serve as a new biomarker of poor prognosis. Furthermore, NMU has been proved to be an important resistance-enhancing factor. Overexpression of NMU in sensitive cells makes it resistant to the tested drugs, while NMU silencing sensitized resistant cells [42]. Therefore, silencing NMU can be an effective way for cancer treatment. Researchers found the results in proliferation and survival of NMU appeared to be cancer-type dependent [44, 45]. However, research on melanoma and NMU is still lacking. In our study, high expression of NMU result in poor overall survival. NMU receptors are expressed on macrophages and endothelial cells, indicating that NMU is highly related to the tumor microenvironment [46]. Therefore, NMU is a potential tumor growth and progression biomarker in melanoma and silencing NMU may be an effective way for melanoma treatment. More research is needed to reveal its role in melanoma microenvironment.
Galanin and GAMP prepropeptide (GAL) is a neuropeptide, which is found to be expressed in several cancers such as gastrointestinal cancer, head and neck squamous cell carcinoma, brain tumor and salivary duct carcinoma [47]. Galanin acts its function through binding to three receptors (GALR1, GALR2, GALR3). The activation of GALR1 is usually anti-proliferation, while the activation of GALR2 may have the effect of promoting proliferation or anti-proliferation [48]. GALR1 and GALR2 inhibit proliferation of head and neck squamous cell carcinoma (HNSCC) cells. Hypermethylation of GALR1, GALR2 is associated with poor survival and high recurrence in HNSCC [49]. Therefore, galanin and galanin receptors (GALRs) are gradually used as new biomarkers for prognosis in HNSCC. Another research found that the down-regulation of Galanin and GALR1 is related to development of gastric cancer [50]. Besides, Galanin and GAL receptors have been found to be expressed in human skin [51]. Few studies have proved the role of GAL in melanoma. In our results, overall survival of samples with high expression of GAL showed in poor outcome. Four drugs interacted with GAL in the predicted drug-gene interaction. The relationship between GAL, GALRs and melanoma remains unknown, and more research is needed to verify it.
In our study, we used ClueGO to investigate pathway in hub genes. The most important pathways include interleukin-10 signaling, peptide ligand-binding receptors, chemokine receptors bind chemokine, toll-like receptor signaling pathway, cytosolic DNA-sensing pathway. Interleukin-10 signaling mediates immunosuppressive via its immune-regulatory function, while chemokine and its receptors usually attract immune cells such as CD8 + T cell in tumor microenvironment for immune activation [52]. Melanoma is a highly immunogenic cancer. It is valuable to clarify the potential mechanism of immune activation and immunosuppression in tumor microenvironment. Our results revealed a strong positive correlation between gene expression (CCL4, CCL5, CXCL9, CXCL10 and CXCL13) and immune cell infiltration (B-cell, CD8 + T cells, CD4 + T cells, macrophages, Neutrophils, Dendritic cells). Furthermore, activating the immune system and abrogating immune checkpoints (PD1, CTLA-4) are the keys for cancer therapy. Studies conducted by Tsukamoto H et al. indicated that combined blockade of IL6 and PD-1/PD-L1 signaling abrogates mutual regulation of their immunosuppressive effects in the tumor microenvironment [53]. Altogether, these hub genes and pathways may play critical role in formation, prognosis and therapy of melanoma, while further investigation is necessary.