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P-Rex1 is required for efficient melanoblast migration and melanoma metastasis.

ABSTRACT: Metastases are the major cause of death from melanoma, a skin cancer that has the fastest rising incidence of any malignancy in the Western world. Molecular pathways that drive melanoblast migration in development are believed to underpin the movement and ultimately the metastasis of melanoma. Here we show that mice lacking P-Rex1, a Rac-specific Rho GTPase guanine nucleotide exchange factor, have a melanoblast migration defect during development evidenced by a white belly. Moreover, these P-Rex1(-/-) mice are resistant to metastasis when crossed to a murine model of melanoma. Mechanistically, this is associated with P-Rex1 driving invasion in a Rac-dependent manner. P-Rex1 is elevated in the majority of human melanoma cell lines and tumour tissue. We conclude that P-Rex1 has an important role in melanoblast migration and cancer progression to metastasis in mice and humans.

SUBMITTER: Lindsay CR

PROVIDER: S-EPMC3400057 | biostudies-literature | 2011 Nov

REPOSITORIES: biostudies-literature

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P-Rex1 is required for efficient melanoblast migration and melanoma metastasis.

Lindsay Colin R CR Lawn Samuel S Campbell Andrew D AD Faller William J WJ Rambow Florian F Mort Richard L RL Timpson Paul P Li Ang A Cammareri Patrizia P Ridgway Rachel A RA Morton Jennifer P JP Doyle Brendan B Hegarty Shauna S Rafferty Mairin M Murphy Ian G IG McDermott Enda W EW Sheahan Kieran K Pedone Katherine K Finn Alexander J AJ Groben Pamela A PA Thomas Nancy E NE Hao Honglin H Carson Craig C Norman Jim C JC Machesky Laura M LM Gallagher William M WM Jackson Ian J IJ Van Kempen Leon L Beermann Friedrich F Der Channing C Larue Lionel L Welch Heidi C HC Ozanne Brad W BW Sansom Owen J OJ

Nature communications 20111122

Metastases are the major cause of death from melanoma, a skin cancer that has the fastest rising incidence of any malignancy in the Western world. Molecular pathways that drive melanoblast migration in development are believed to underpin the movement and ultimately the metastasis of melanoma. Here we show that mice lacking P-Rex1, a Rac-specific Rho GTPase guanine nucleotide exchange factor, have a melanoblast migra ...[more]

PMID: 22109529

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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:ADAMTS20 (Adisintegrin-like and metalloprotease domain with thrombospondin type-1 motifs) is a member of a family of secreted metalloproteases that can process a variety of extracellular matrix (ECM) components and secreted molecules. Adamts20 mutations in belted (bt) mice cause white spotting of the dorsal and ventral torso, indicative of defective neural crest (NC)-derived melanoblast development. The expression pattern of Adamts20 in dermal mesenchymal cells adjacent to migrating melanoblasts led us to initially propose that Adamts20 regulated melanoblast migration. However, using a Dct-LacZ transgene to track melanoblast development, we determined that melanoblasts were distributed normally in whole mount E12.5 bt/bt embryos, but were specifically reduced in the trunk of E13.5 bt/bt embryos due to a seven-fold higher rate of apoptosis. The melanoblast defect was exacerbated in newborn skin and embryos from bt/bt animals that were also haploinsufficient for Adamts9, a close homolog of Adamts20, indicating that these metalloproteases functionally overlap in melanoblast development. We identified two potential mechanisms by which Adamts20 may regulate melanoblast survival. First, skin explant cultures demonstrated that Adamts20 was required for melanoblasts to respond to soluble Kit ligand (sKitl). In support of this requirement, bt/bt;Kit(tm1Alf)/+ and bt/bt;Kitl(Sl)/+ mice exhibited synergistically increased spotting. Second, ADAMTS20 cleaved the aggregating proteoglycan versican in vitro and was necessary for versican processing in vivo, raising the possibility that versican can participate in melanoblast development. These findings reveal previously unrecognized roles for Adamts proteases in cell survival and in mediating Kit signaling during melanoblast colonization of the skin. Our results have implications not only for understanding mechanisms of NC-derived melanoblast development but also provide insights on novel biological functions of secreted metalloproteases.

Dataset Information

P-Rex1 is required for efficient melanoblast migration and melanoma metastasis.

Publications

P-Rex1 is required for efficient melanoblast migration and melanoma metastasis.

Similar Datasets

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