Identification of Core Genes and Pathways in Melanoma Metastasis via Bioinformatics Analysis

Metastasis is the leading cause of melanoma-related mortality. Current therapies are rarely curative for metastatic melanoma, revealing the urgent need to identify more effective preventive and therapeutic targets. This study aimed to screen the core genes and molecular mechanisms related to melanoma metastasis. A gene expression profile, GSE8401, including 31 primary melanoma and 52 metastatic melanoma clinical samples, was downloaded from the Gene Expression Omnibus (GEO) database. The differentially expressed genes (DEGs) between melanoma metastases and primary melanoma were screened using GEO2R tool. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genome (KEGG) analyses of DEGs were performed using the Database for Annotation Visualization and Integrated Discovery (DAVID). The Search Tool for the Retrieval of Interacting Genes (STRING) and Cytoscape with Molecular Complex Detection (MCODE) plug-in tools were utilized to detect the protein–protein interaction (PPI) network among DEGs. The top 10 genes with the highest degrees of the PPI network were defined as hub genes. In the results, 425 DEGs, including 60 upregulated genes and 365 downregulated genes, were identified. The upregulated genes were enriched in ECM–receptor interactions and the regulation of actin cytoskeleton, while 365 downregulated genes were enriched in amoebiasis, melanogenesis, and ECM–receptor interactions. The defined hub genes included CDK1, COL17A1, EGFR, DSG1, KRT14, FLG, CDH1, DSP, IVL, and KRT5. In addition, the mRNA and protein levels of the hub genes during melanoma metastasis were verified in the TCGA database and paired post- and premetastatic melanoma cells, respectively. Finally, KRT5-specific siRNAs were utilized to reduce the KRT5 expression in melanoma A375 cells. An MTT assay and a colony formation assay showed that KRT5 knockdown significantly promoted the proliferation of A375 cells. A Transwell assay further suggested that KRT5 knockdown significantly increased the cell migration and cell invasion of A375 cells. This bioinformatics study provided a deeper understanding of the molecular mechanisms of melanoma metastasis. The in vitro experiments showed that KRT5 played the inhibitory effects on melanoma metastasis. Therefore, KRT5 may serve important roles in melanoma metastasis.

Identification of Keratinocyte Differentiation-Involved Genes for Metastatic Melanoma by Gene Expression Profiles

Background. Melanoma is the deadliest type of skin cancer. Until now, its pathological mechanisms, particularly the mechanism of metastasis, remain largely unknown. Our study on the identification of genes in association with metastasis for melanoma provides a novel understanding of melanoma. Methods. From the Gene Expression Omnibus (GEO) database, the gene expression microarray datasets GSE46517, GSE7553, and GSE8401 were downloaded. We made use of R aiming at analyzing the differentially expressed genes (DEGs) between metastatic and nonmetastatic melanoma. R was also used in differentially expressed miRNA (DEM) data mining from GSE18509, GSE19387, GSE24996, GSE34460, GSE35579, GSE36236, and GSE54492 datasets referring to Li’s study. Based on the DEG and DEM data, we performed functional enrichment analysis through the application of the DAVID database. Furthermore, we constructed the protein-protein interaction (PPI) network and established functional modules by making use of the STRING database. Through making use of Cytoscape, the PPI results were visualized. We predicted the targets of the DEMs through applying TargetScan, miRanda, and PITA databases and identified the overlapping genes between DEGs and predicted targets, followed by the construction of DEM-DEG pair network. The expressions of these keratinocyte differentiation-involved genes in Module 1 were identified based on the data from TCGA. Results. 239 DEGs were screened out in all 3 datasets, which were inclusive of 21 positively regulated genes and 218 negatively regulated genes. Based on these 239 DEGs, we finished constructing the PPI network which was formed from 225 nodes and 846 edges. We finished establishing 3 functional modules. And we analyzed 92 overlapping genes and 26 miRNA, including 11 upregulated genes targeted by 11 negatively regulated DEMs and 81 downregulated genes targeted by 15 positively regulated DEMs. As proof of the differential expression of metastasis-associated genes, eleven keratinocyte differentiation-involved genes, including LOR, EVPL, SPRR1A, FLG, SPRR1B, SPRR2B, TGM1, DSP, CSTA, CDSN, and IVL in Module 1, were obviously downregulated in metastatic melanoma tissue in comparison with primary melanoma tissue based on the data from TCGA. Conclusion. 239 melanoma metastasis-associated genes and 26 differentially expressed miRNA were identified in our study. The keratinocyte differentiation-involved genes may take part in melanoma metastasis, providing a latent molecular mechanism for this disease.

Inducible T-Cell Costimulator Ligand Plays a Dual Role in Melanoma Metastasis upon Binding to Osteopontin or Inducible T-Cell Costimulator

Recently, we demonstrated that inducible T-cell costimulator (ICOS) shares its unique ligand (ICOSL) with osteopontin (OPN), and OPN/ICOSL binding promotes tumor metastasis and angiogenesis in the 4T1 breast cancer model. Literature showed that OPN promotes melanoma metastasis by suppressing T-cell activation and recruiting myeloid suppressor cells (MDSC). On the opposite, ICOS/ICOSL interaction usually sustains an antitumor response. Here, we engineered murine B16F10 melanoma cells, by transfecting or silencing ICOSL. In vitro data showed that loss of ICOSL favors anchorage-independent growth and induces more metastases in vivo, compared to ICOSL expressing cells. To dissect individual roles of the three molecules, we compared data from C57BL/6 with those from OPN-KO, ICOS-KO, and ICOSL-KO mice, missing one partner at a time. We found that OPN produced by the tumor microenvironment (TME) favors the metastasis by interacting with stromal ICOSL. This activity is dominantly inhibited by ICOS expressed on TME by promoting Treg expansion. Importantly, we also show that OPN and ICOSL highly interact in human melanoma metastases compared to primary tumors. Interfering with this binding may be explored in immunotherapy either for nonresponding or patients resistant to conventional therapies.

Low-Dose Albendazole Inhibits Epithelial-Mesenchymal Transition of Melanoma Cells by Enhancing Phosphorylated GSK-3β/Tyr216 Accumulation

Albendazole (ABZ) is an effective broad-spectrum anthelmintic agent that has been widely used for humans and animals. Previous studies have reported that ABZ exhibits antitumor effects against melanoma and other different cancer types; however, it is unknown whether ABZ exerts the inhibitory effect against melanoma metastasis. In this study, we aimed to investigate the inhibitory effect of ABZ on melanoma cells. Through in vitro studies, we discovered that low-dose ABZ treatment significantly inhibited the migration and invasion, but not the proliferation, of A375 and B16-F10 cells in a dose-dependent manner. Further analysis revealed that ABZ treatment reduced the expression level of snail family transcriptional repressor 1 (Snail) in the cytoplasm and nucleus by decreasing the levels of phosphorylated AKT (pAKT) Ser473/GSK-3β (pGSK-3β) Ser9 and increasing pGSK-3β/Tyr216, resulting in a significant upregulation of E-cadherin and downregulation of N-cadherin and ultimately reversing the epithelial-mesenchymal transition (EMT) process of melanoma cells. In contrast, the continuous activation of AKT via transfected plasmids elevated the protein levels of pAKT Ser473/pGSK-3β Ser9 and Snail and antagonized the inhibitory action of ABZ. We also confirmed that ABZ treatment effectively inhibited the lung metastasis of melanoma in nude mice in vivo. Subsequent immunohistochemical analysis verified the decreased pAKT Ser473/pGSK-3β Ser9 and increased pGSK-3β/Tyr216 levels in ABZ-treated subcutaneous tumors. Therefore, our findings demonstrate that ABZ treatment can suppress the EMT progress of melanoma by increasing the pGSK-3β/Tyr216-mediated degradation of Snail, which may be used as a potential treatment strategy for metastatic melanoma.

Exploration and Validation of Metastasis-associated Genes for Skin Cutaneous Melanoma

Background: Skin cutaneous melanoma is a malignant and highly metastatic skin tumor, and its morbidity and mortality are still rising worldwide. However, the molecular mechanisms that promote melanoma metastasis are unclear. Methods: Two datasets (GSE15605 and GSE46517) were retrieved to identify the differentially expressed genes (DEGs), including 23 normal skin tissues (N), 77 primary melanoma tissues (T) and 85 metastatic melanoma tissues (M). Gene Ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment were performed to explore the functions of the DEGs. The protein–protein interaction (PPI) network was constructed using the STRING tool and Cytoscape software. We used the cytoHubba plugin of Cytoscape to identify the most significant hub genes by five topological analyses (Degree, Bottleneck, MCC, MNC, and EPC). Hub gene expression was validated using the UALCAN website. Clinical relevance was investigated using The Cancer Genome Atlas (TCGA) resources. Finally, we explored the association between metastasis-associated genes and immune infiltrates through the Tumor Immune Estimation Resource (TIMER) database and performed drug-gene interaction analysis using the Drug-Gene Interaction database.Results: A total of 294 specific genes were related to melanoma metastasis and were mainly involved in the positive regulation of locomotion, mitotic cell cycle process, and epithelial cell differentiation. Four hub genes (CDK1, FOXM1, KIF11, and RFC4) were identified from the cytoHubba plugin of Cytoscape. CDK1 was significantly upregulated in metastatic melanoma compared with primary melanoma, and high expression of CDK1 was positively correlated with poor prognosis. We found that CDK1 expression correlated positively with the infiltration levels of macrophage cells (Rho = -0.164, P = 2.02e-03) and neutrophil cells (Rho = 0.269, P = 2.72e-07) in SKCM metastasis. In addition, we identified that CDK1 had a close interaction with 10 antitumor drugs. Conclusions: CDK1 was identified as a hub gene involved in the progression of melanoma metastasis and may be regarded as a therapeutic target for melanoma patients to improve prognosis and prevent metastasis in the future.

Small Bowel Metastatic Melanoma: An Emblematic “Coal-Black” Appearance at Videocapsule Endoscopy

A 80-year-old woman underwent vulvar melanoma resection and segmental lung resection for pulmonary metastasis. Immunotherapy with Nivolumab was performed. One year later, the patient was admitted for gastrointestinal (GI) recurrent bleeding and severe anemia. Esophagoastroduodenoscopy and colonoscopy did not show any abnormality, while videocapsule endoscopy (VCE) revealed an irregular and exophytic whitish area with a “coal-black” central depression. Small bowel resection was performed and histological examination revealed S100 protein strongly positive melanoma metastasis. The patient died six months later from disease progression. A “coal-black” appearance of intestinal metastatic melanoma has been described only twice before this report. In one case the patient had been treated by immunotherapy with interferon A and dendritic cell-based vaccination. In our patient, it is presumable that the picture we observed was a consequence of Nivolumab treatment inducing the disappearance of melanocytes in the area surrounding the metastasis with the onset of the central coal-black lesion encircled by whitish tissue. This picture should be emblematic of intestinal metastatic melanoma in subjects treated with immunotherapy showing occult/obscure bleeding.

Quantitative Detection of Disseminated Melanoma Cells by Trp-1 Transcript Analysis Reveals Stochastic Distribution of Pulmonary Metastases

A better understanding of the process of melanoma metastasis is required to underpin the development of novel therapies that will improve patient outcomes. The use of appropriate animal models is indispensable for investigating the mechanisms of melanoma metastasis. However, reliable and practicable quantification of metastases in experimental mice remains a challenge, particularly if the metastatic burden is low. Here, we describe a qRT-PCR-based protocol that employs the melanocytic marker Trp-1 for the sensitive quantification of melanoma metastases in the murine lung. Using this protocol, we were able to detect the presence of as few as 100 disseminated melanoma cells in lung tissue. This allowed us to quantify metastatic burden in a spontaneous syngeneic B16-F10 metastasis model, even in the absence of visible metastases, as well as in the autochthonous Tg(Grm1)/Cyld−/− melanoma model. Importantly, we also observed an uneven distribution of disseminated melanoma cells amongst the five lobes of the murine lung, which varied considerably from animal to animal. Together, our findings demonstrate that the qRT-PCR-based detection of Trp-1 allows the quantification of low pulmonary metastatic burden in both transplantable and autochthonous murine melanoma models, and show that the analysis of lung metastasis in such models needs to take into account the stochastic distribution of metastatic lesions amongst the lung lobes.

Remodeling of the tumor microenvironment via disrupting Blimp1+ effector Treg activity augments response to anti-PD-1 blockade

Background Accumulation of Foxp3+ regulatory T (Treg) cells in the tumor often represents an important mechanism for cancer immune evasion and a critical barrier to anti-tumor immunity and immunotherapy. Many tumor-infiltrating Treg cells display an activated phenotype and express the transcription factor Blimp1. However, the specific impact of these Blimp1+ Treg cells and their follicular regulatory T (TFR) cell subset on tumor and the underlying mechanisms of action are not yet well-explored. Methods Various transplantable tumor models were established in immunocompetent wild-type mice and mice with a Foxp3-specific ablation of Blimp1. Tumor specimens from patients with metastatic melanoma and TCGA datasets were analyzed to support the potential role of Treg and TFR cells in tumor immunity. In vitro culture assays and in vivo adoptive transfer assays were used to understand how Treg, TFR cells and antibody responses influence tumor control. RNA sequencing and NanoString analysis were performed to reveal the transcriptome of tumor-infiltrating Treg cells and tumor cells, respectively. Finally, the therapeutic effects of anti-PD-1 treatment combined with the disruption of Blimp1+ Treg activity were evaluated. Results Blimp1+ Treg and TFR cells were enriched in the tumors, and higher tumoral TFR signatures indicated increased risk of melanoma metastasis. Deletion of Blimp1 in Treg cells resulted in impaired suppressive activity and a reprogramming into effector T-cells, which were largely restricted to the tumor-infiltrating Treg population. This destabilization combined with increased anti-tumor effector cellular responses, follicular helper T-cell expansion, enhanced tumoral IgE deposition and activation of macrophages secondary to dysregulated TFR cells, remodeled the tumor microenvironment and delayed tumor growth. The increased tumor immunogenicity with MHC upregulation improved response to anti-PD-1 blockade. Mechanistically, Blimp1 enforced intratumoral Treg cells with a unique transcriptional program dependent on Eomesodermin (Eomes) expression; deletion of Eomes in Blimp1-deficient Treg cells restored tumor growth and attenuated anti-tumor immunity. Conclusions These findings revealed Blimp1 as a new critical regulator of tumor-infiltrating Treg cells and a potential target for modulating Treg activity to treat cancer. Our study has also revealed two FCERIA-containing immune signatures as promising diagnostic or prognostic markers for melanoma patients.