1. Cancer Biology
  2. Genetics and Genomics

Crosstalk with keratinocytes causes GNAQ oncogene specificity in melanoma

  1. Oscar Urtatiz
  2. Amanda Haage
  3. Guy Tanentzapf
  4. Catherine D Van Raamsdonk  Is a corresponding author
  1. Department of Medical Genetics, Life Sciences Institute, University of British Columbia, Canada
  2. Department of Cellular and Physiological Sciences, Life Sciences Institute, University of British Columbia, Canada
https://doi.org/10.7554/eLife.71825
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Cite this article
  1. Oscar Urtatiz
  2. Amanda Haage
  3. Guy Tanentzapf
  4. Catherine D Van Raamsdonk
(2021)
Crosstalk with keratinocytes causes GNAQ oncogene specificity in melanoma
eLife 10:e71825.
https://doi.org/10.7554/eLife.71825
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7 figures, 2 videos, 1 table and 4 additional files

Figures

Figure 1
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Forced GNAQQ209L signaling reduces the number of melanocytes in the interfollicular epidermis (IFE).

(A) Representative example of LacZ-positive cells within scales of WT-LacZ and GNAQ -LacZ tails at 1 or 8 weeks post-tamoxifen (TM) treatment. (B) Quantification of the average number of …

Figure 2
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Fluorescent activated cell sorting (FACS) sorted GNAQQ209L and BRAFV600E interfollicular epidermis (IFE) melanocytes cultured on fibronectin have increased survival compared to wildtype (WT).

(A) Representative examples of FACS dot plots of single-cell suspension from the IFE epidermis of WT and GNAQQ209L mice. (B) Average percentage of tdTomato+ cells sorted from the IFE of WT and GNAQQ2…

Figure 3
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The interfollicular epidermis (IFE) impairs survival and alters pseudopod dynamics in GNAQQ209L melanocytes.

(A) Survival of unsorted wildtype (WT) melanocytes plated with its IFE, compared to sorted IFE WT melanocytes plated onto fibronectin. The presence of IFE increased the survival of WT melanocytes. …

Figure 4
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The interfollicular epidermis (IFE) impairs cell division of GNAQQ209L melanocytes.

(A) Migration plots of wildtype (WT) and GNAQQ209L melanocytes co-cultured with IFE over 20 hr. (B) Quantification of total distance traveled in 20 hr. (Each point represents the measurement from …

Figure 5
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Interfollicular epidermis (IFE) control of melanocytes was maintained in a transwell culture system.

(A) Comparison of survival for wildtype (WT) melanocytes in direct contact with IFE (‘direct co-culture’) versus a transwell system where the two populations are separated by a permeable membrane. …

Figure 6
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Analysis of GNAQQ209L expressing melanocytes in the interfollicular epidermis (IFE) reveals alterations in the actin cytoskeleton and cell death.

(A) Significant terms identified by Gene Ontology analysis for differentially expressed (DE) genes (log2 fold change [LFC] >2 or <−2) in GNAQQ209L melanocytes. All were enriched in the GNAQQ209L …

Figure 7
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Model for microenvironmental control of the Gαq-PLCB4 signaling outcome and how this leads to GNAQ oncogene specificity in melanoma.

G alpha q (GNAQ) and G alpha 11 (GNA11) are classic signaling components that transmit G protein-coupled receptor (GPCR) activation to phospholipase C-beta (PLC-B) inside the cell. Either GNAQ, …

Videos

Video 1
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Wildtype (WT) melanocytes co-cultured with interfollicular epidermis (IFE), 625 min time lapse.

Cell protrusions, cell movements, and cell division events can be seen in the melanocytes in this video. Cell protrusions are shorter in WT cells than in GNAQQ209L. One of the cell division events …

Video 2
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GNAQQ209L melanocytes co-cultured with interfollicular epidermis (IFE), 625 min time lapse.

Cell protrusions and cell movements can be seen in the melanocytes in this video. Protrusions are longer in GNAQQ209L melanocytes than in wildtype (WT) and some cells take on usual shapes. The …

Tables

Key resources table
Reagent type (species) or resourceDesignationSource or referenceIdentifiersAdditional
information
Strain background
(Mus musculus domesticus,
males and
females)		C3HeB/FeJJackson LaboratoriesJackson Labs:
strain #000658
Gene(Homo
sapiens)		GNAQGenBankGenBank Gene ID: 2776
Genetic reagent(Mus musculus domesticus
males and
females)		Tg(Mitf-cre)
7114Gsb		Alizadeh et al., 2008PMID:18353144RRID:MGI:5702900
Genetic reagent(Mus musculus domesticus males and females)Tg(Tyr-cre/
ERT2)13Bos/J		Jackson LaboratoriesJackson Labs:
strain #012328
Genetic reagent(Mus musculus domesticus
males and females)		Gt(ROSA)
26Sortm1(GNAQ*)Cvrk		Huang et al., 2015PMID:26113083RRID:MGI:5702877
Genetic reagent(Mus musculus domesticus
males and
females)		Gt(ROSA)
26Sortm14(CAG-tdTomato)Hze		Jackson LaboratoriesJackson Labs:
strain #007914
Genetic reagent(Mus musculus domesticus
males and females)		Gt(Rosa)
26Sortm1Sor/J		Jackson LaboratoriesJackson Labs:
strain #003474
Genetic reagent(Mus musculus domesticus
males and females)		Braftm1MmcmJackson LaboratoriesJackson Labs:
strain #017837
Commercial
assay or kit		Annexin-V-
FLUOS
Staining Kit		Millipore
Sigma		MilliporeSigma SKU:11858777001
Commercial
assay or kit		DNeasy
Blood and
Tissue kit		QiagenQiagen ID: 69504
Commercial
assay or kit		HotStar Taq
DNA polymerase		QiagenQiagen ID: 203207
Chemical
compound,
drug		TamoxifenMillipore SigmaMilliporeSigma catalog:T5648
Chemical
compound,
drug		4-Hydroxyt
amoxifen		Millipore SigmaMillipore Sigma catalog:H6278
Software,
algorithm		Custom
Matlab scripts		Haage et al., 2018PMID:30485809https://github.com/Tanentzapf-Lab/ActinOrganization_CellMorphology_Haage
OthereBioscience
Fixable Viability
Dye eFluor 450		ThermoFisherThermoFisher catalog : 501128817
OtherRiboLock RNase inhibitorThermoFisherThermoFisher catalog :EO0381
OtherFibronectinMillipore SigmaMilliporeSigma catalog:F0895Liquid, 0.1%
solution

Additional files

Supplementary file 1

Excel file providing more details of the differentially expressed genes in GNAQQ209L melanocytes sorted from the IFE.

(a) Genes with FPKM >0.1 in wildtype (WT) and/or GNAQQ209L melanocytes. (b) Differentially expressed genes down-regulated in GNAQQ209L interfollicular epidermis (IFE) melanocytes, sorted by Z_score. (c) Differentially expressed genes up-regulated in GNAQQ209L IFE melanocytes, sorted by Z_score. (d) Genes supporting pathway analysis terms related to cell adhesion, focal adhesion, and the extracellular matrix. (e) Genes supporting pathway analysis terms related to axon guidance, nervous system development, and axon cellular component.

https://cdn.elifesciences.org/articles/71825/elife-71825-supp1-v2.xlsx
Supplementary file 2

Word file providing more details on gene expression in mouse tail IFE melanocytes, frequency of oncogenic mutations in GNAQ and GNA11 in human cutaneous melanomas, and statistical tests used in the studies.

(a) Top 20 most highly expressed genes in mouse wildtype (WT) interfollicular epidermis (IFE) melanocytes. (b) Identification of GNAQ hotspot mutations among human malignant melanomas potentially arising in the epidermis. (c) Identification of GNA11 hotspot mutations among human malignant melanomas potentially arising in the epidermis. (d) Information on statistical tests in Figures 1—6.

https://cdn.elifesciences.org/articles/71825/elife-71825-supp2-v2.doc
Supplementary file 3

Prism file with source data used to create graphs and calculate statistics in Figures 1—6.

https://cdn.elifesciences.org/articles/71825/elife-71825-supp3-v2.zip
Transparent reporting form
https://cdn.elifesciences.org/articles/71825/elife-71825-transrepform1-v2.pdf

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