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Melanoblast transcriptome analysis reveals pathways promoting melanoma metastasis.

ABSTRACT: Cutaneous malignant melanoma is an aggressive cancer of melanocytes with a strong propensity to metastasize. We posit that melanoma cells acquire metastatic capability by adopting an embryonic-like phenotype, and that a lineage approach would uncover metastatic melanoma biology. Using a genetically engineered mouse model to generate a rich melanoblast transcriptome dataset, we identify melanoblast-specific genes whose expression contribute to metastatic competence and derive a 43-gene signature that predicts patient survival. We identify a melanoblast gene, KDELR3, whose loss impairs experimental metastasis. In contrast, KDELR1 deficiency enhances metastasis, providing the first example of different disease etiologies within the KDELR-family of retrograde transporters. We show that KDELR3 regulates the metastasis suppressor, KAI1, and report an interaction with the E3 ubiquitin-protein ligase gp78, a regulator of KAI1 degradation. Our work demonstrates that the melanoblast transcriptome can be mined to uncover targetable pathways for melanoma therapy.

SUBMITTER: Marie KL

PROVIDER: S-EPMC6965108 | biostudies-literature | 2020 Jan

REPOSITORIES: biostudies-literature

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Melanoblast transcriptome analysis reveals pathways promoting melanoma metastasis.

Marie Kerrie L KL Sassano Antonella A Yang Howard H HH Michalowski Aleksandra M AM Michael Helen T HT Guo Theresa T Tsai Yien Che YC Weissman Allan M AM Lee Maxwell P MP Jenkins Lisa M LM Zaidi M Raza MR Pérez-Guijarro Eva E Day Chi-Ping CP Ylaya Kris K Hewitt Stephen M SM Patel Nimit L NL Arnheiter Heinz H Davis Sean S Meltzer Paul S PS Merlino Glenn G Mishra Pravin J PJ

Nature communications 20200116 1

Cutaneous malignant melanoma is an aggressive cancer of melanocytes with a strong propensity to metastasize. We posit that melanoma cells acquire metastatic capability by adopting an embryonic-like phenotype, and that a lineage approach would uncover metastatic melanoma biology. Using a genetically engineered mouse model to generate a rich melanoblast transcriptome dataset, we identify melanoblast-specific genes whose expression contribute to metastatic competence and derive a 43-gene signature ...[more]

PMID: 31949145

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Project description:Purpose: We hypothesized that embryonic melanocytic pathways would be reinitiated in melanoma metatastasis. To identify nove melanoblast signaling pathways with which to explore our hypothesis we used the inducible Dct-GFP mouse model, in which GFP is expressed in both immature melanoblasts and mature melanocytes. Mouse melanoblasts/melanoytes were extracted using Fluorescence Activated Cell Sorting and transcriptomes were analyzed using RNA-sequencing. Abstract: Cutaneous malignant melanoma is an aggressive cancer of melanocytes with a strong propensity to metastasize. We posit that melanoma cells acquire metastatic capability by adopting an embryonic-like phenotype, and that a lineage approach would uncover novel metastatic melanoma biology. Using a genetically engineered mouse model to generate a rich melanoblast transcriptome dataset, we identify melanoblast-specific genes whose expression contribute to metastatic competence and derive a 43-gene signature that predicts patient survival. We identify a melanoblast gene, KDELR3, whose loss impairs experimental metastasis. In contrast, KDELR1 deficiency enhances metastasis, providing the first example of different disease etiologies within the KDELR-family of retrograde transporters. We show that KDELR3 regulates the metastasis suppressor, KAI1, and report an interaction with the E3 ubiquitin-protein ligase gp78, a regulator of KAI1 degradation. Our work demonstrates that the melanoblast transcriptome can be mined to uncover targetable pathways for melanoma therapy. Method: FVB/N iDct-GFP mice were used and doxycycline was administered to activate GFP expression. Multiple litters were used for each developmental stage, and embryos/pups from each stage were pooled to ensure adequate numbers of GFP+ cells. At E17.5, P1 and P7 stages, most melanocytes have reached the dermis, thus only the skin was collected from these developmental stages. Back skin was immersed in a shallow layer of 1X PBS and subcutaneous fat was scraped off until skin appeared translucent. E15.5 was the youngest age assessed due to the necessity to capture sufficient cells for RNA-sequencing. Embryos of the same developmental age that were heterozygous for the TRE-H2B-GFP gene but lacked the Dct-rtTA gene were used as negative controls. Cell doublets were excluded from the analysis. Cells were sorted based on GFP expression and SSC-A. Based on these reference sorts, gates were set so that background cells represented less than 10% of sorted cells. Cells were lysed in 10-fold TRIzol reagent (w/v), phases were separated by addition of 0.2X volume of chloroform, the aqueous phase was combined with an equal volume of 70% ethanol and applied to a RNeasy Micro column (Qiagen) and processed as per the manufacturer’s instructions. Paired-end sequencing libraries were prepared using 1 μg of purified RNA following the mRNA-Seq Sample Prep Kit according to the manufacturer’s instructions (Illumina). RNA-Seq libraries were sequenced on two lanes each of an Illumina GAIIx Genome Analyser to a minimum depth of 49 million reads. Sequence reads were aligned to the mm9 genome using the TopHat software (https://ccb.jhu.edu/software/tophat/index.shtml). Quantified Fragments Per Kilobase of transcript per Million mapped reads (FPKM) values were generated using the Cufflinks software (http://cole-trapnell-lab.github.io/cufflinks/). The UCSC KnownGenes gene models were used for guided alignment and quantification. Results: DESeq2 analysis identified melanoblast-specific gene expression, which was shown by further analyses using patient data and wet lab techniques to likewise be upregulated in metastatic melanoma and facilitate metastasis.

Project description:BackgroundMetastasis is the major cause of death in breast cancer patients. Although the strategies targeting metastasis have promoted survival, the underlying mechanisms still remain unclear. In this study, we used microarray data of primary breast tumor, tumor derived from bone and liver, and skin metastatic tissue, to identify the key genes and pathways that are involved in metastasis in breast cancer.MethodsWe first calculated the differentially expressed genes (DEGs) between three metastatic tissues and primary tumor tissue, and then used it to perform Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Further, we analyzed the correlation of genes enriched in GO terms and KEGG pathways with survival of breast cancer patients. To identify the key genes and pathways associated with metastasis, we overlapped the DEGs and KEGG pathways. In our in vitro experiments, we knocked down the key gene, ERLIN2, and detected the PI3K expression in tumor cells to evaluate their effect on tumor metastasis.ResultsWe identified six genes (ALOX15, COL4A6, LMB13, MTAP, PLA2G4A, TAT) that correlated with survival. Seven key genes (SNRPN, ARNT2, HDGFRP3, ERO1LB, ERLIN2, YBX2, EBF4) and seven signaling pathways (metabolic pathways, phagosome pathway, PI3K-AKT signaling pathway, focal adhesion, ECM-receptor interaction, pancreatic secretion, human papillomavirus infection) associated with metastasis were also identified. Our in vitro experiments revealed that ERLIN2 was highly expressed in MDA-MB231 cells compared to MCF-7 cells. Moreover, knockdown of ERLIN2 increased apoptosis, while inhibiting the proliferation, invasion, and migration ability of breast cancer cells. The PI3K/AKT signaling pathway was also found to be highly expressed in MDA-MB231 cells.ConclusionOur results reveal the key genes and signaling pathways that contribute to metastasis, and highlight that strategic targeting of ENLIN2 and PI3K/AKT signaling pathways could inhibit metastasis of breast cancer.

Project description: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.

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Melanoblast transcriptome analysis reveals pathways promoting melanoma metastasis.

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Melanoblast transcriptome analysis reveals pathways promoting melanoma metastasis.

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