Interferon alpha-inducible protein 6 regulates NRASQ61K-induced melanomagenesis and growth
- Yale University School of Medicine, United States
- Agency for Science Technology and Research (A*STAR), Brenner Center for Molecular Medicine, Singapore
Figures
Figure 1 with 6 supplements
IFI6 is transcriptionally upregulated by NRASQ61K via MAPK pathway.
(A) Relative IFI6 mRNA expression in MEL-ST/NRASQ61K cells compared to empty vector-expressing MEL-ST cells. (B) Box plots for IFI6 mRNA expression in indicated melanoma gene expression datasets …
Figure 1—figure supplement 1
NRASQ61K transcriptionally upregulates interferon-stimulated genes.
Heatmap shows altered gene expression in MEL-ST cells expressing either an empty vector or NRASQ61K. Upregulated genes are shown in red, and downregulated genes are shown in green. The top five …
Figure 1—figure supplement 2
Regulation of MX1 and IFI6 by NRASQ61K.
Relative mRNA expression of MX1 and IFI27 in MEL-ST cells expressing NRASQ61K or empty vector was analyzed by RT-qPCR. Data are presented as mean ± SEM; ***p<0.0005.
Figure 1—figure supplement 3
Monitoring regulation of IFI6 by BRAFV600E and NF1 loss.
(Top left) Relative expression of IFI6 mRNA in MEL-ST cells expressing BRAFV600E or empty vector was analyzed by RT-qPCR. (Top right) Relative expression of the indicated proteins in BRAFV600E- and …
Figure 1—figure supplement 4
STAT1 is not necessary for IFI6 expression in MEL-ST/NRASQ61K cells.
(Left) IFI6 promoter analysis using rVista 2.0 reveals DNA binding sites for STAT1. (Right) MEL-ST/NRASQ61K cells were analyzed for STAT1 enrichment using the ChIP assay.% STAT1 enrichment (%) …
Figure 1—figure supplement 5
Oncogenic NRASQ61K transcriptionally upregulates IFI6 via transcription factor NF-κB.
(Top Left) mRNA expression of NF-κB was analyzed in NRASQ61K-transformed MEL-ST cells expressing either NF-κB or non-specific (NS) shRNA. Relative expression of NF-κB mRNA in cells expressing NF-κB …
Figure 1—figure supplement 6
IKKβ is necessary for NF-κB activation and NRASQ61K-induced IFI6 upregulation.
(Top left) IKKβ mRNA levels in MEL-ST/NRASQ61K cells expressing IKKβ shRNA or non-specific (NS) shRNA was analyzed by RT-qPCR. Relative mRNA expression of IKKβ in IKKβ shRNA expressing cells in …
Figure 2 with 2 supplements
IFI6 is necessary for NRASQ61K-induced melanocyte transformation.
(A) MEL-ST/NRASQ61K cells expressing IFI6 or non-specific (NS) shRNA were analyzed for colony-forming potential using a soft agar assay. Representative images are shown. (B) Relative colony size for …
Figure 2—figure supplement 1
Monitoring expression of IFI6 and E2F2 proteins following IFI6 knockdown.
Indicated cell lines expressing non-specific (NS) or IFI6 shRNA were analyzed for IFI6, E2F2, and ACTINB protein expression by immunoblotting.
Figure 2—figure supplement 2
Ectopic expression of shRNA-resistant IFI6 ORF rescues tumor growth in MEL-ST/NRASQ61K cells expressing IFI6 shRNA.
(Top panel) Anchorage-independent growth in soft agar was evaluated using MEL-ST/NRASQ61K cells expressing non-specific (NS) shRNA or IFI6 shRNA with or without the shRNA-resistant IFI6 open reading …
Figure 3 with 2 supplements
IFI6 is necessary for NRAS-mutant melanoma tumor growth.
(A) NRAS-mutant melanoma cell lines expressing either non-specific (NS) or IFI6 shRNA were analyzed for anchorage-independent growth using the soft agar assay. Representative images for the …
Figure 3—figure supplement 1
IFI6 is necessary for NRAS-mutant melanoma cell growth.
Representative soft agar well images of NRAS-mutant melanoma cell lines expressing non-specific (NS) or IFI6 shRNAs.
Figure 3—figure supplement 2
Ectopic expression of shRNA-resistant IFI6 ORF rescues tumor growth in YUGASP cells expressing IFI6 shRNA.
(Top panel) Anchorage-independent growth in soft agar was evaluated using YUGASP cells expressing non-specific (NS) shRNA or IFI6 shRNA with or without the shRNA-resistant IFI6 open reading frame …
Figure 4 with 4 supplements
IFI6 loss results in E2F2-mediated dysregulation of DNA replication and induction of cellular senescence in melanoma cells.
(A) YUGASP cells expressing either IFI6 or non-specific (NS) shRNA were analyzed by RT-qPCR. Expression of each gene was calculated in IFI6 knockdown cells relative to NS shRNA-expressing cells. (B) …
Figure 4—figure supplement 1
IFI6 knockdown results in upregulation of E2F2 and its target genes.
(Top) mRNA levels of the indicated genes in SKMEL-103 cells expressing non-specific (NS) or IFI6 shRNA were analyzed by RT-qPCR. Relative mRNA expression for indicated genes in IFI6 shRNA in …
Figure 4—figure supplement 2
Increased enrichment of E2F2 on MCM10 promoter following IFI6 knockdown.
Indicated cells expressing a non-specific (NS) or IFI6 shRNA were analyzed for E2F2 enrichment on MCM10 promoter or as control on ACTINB promoter by ChIP assay.% enrichment of E2F2 relative to input …
Figure 4—figure supplement 3
IFI6 knockdown affects expression of E2F2 but not the expression of other E2F family genes.
mRNA levels of the indicated E2F genes in the indicated cells expressing non-specific (NS) or IFI6 shRNA were analyzed by RT-qPCR. Relative mRNA expression for the indicated E2F genes in the indicate…
Figure 4—figure supplement 4
Simultaneous knockdown of IFI6 and E2F2 restores DNA replication defect.
(Top) YUGASP cells expressing non-specific (NS), IFI6, or E2F2 shRNA (alone or in combination) were analyzed by FACS. Histograms for each condition are shown. (Bottom) DNA replication of YUGASP …
Figure 5
E2F2 mediates the loss of IFI6-induced tumor suppression.
(A) YUGASP cells expressing either non-specific (NS) or IFI6 shRNA, alone or in combination with E2F2 shRNA, were analyzed for E2F2 mRNA expression by RT-qPCR. A relative E2F2 mRNA expression under …
Figure 6 with 1 supplement
E2F2 mediates the loss of IFI6-induced dysregulated DNA replication.
(A) YUGASP cells expressing either non-specific (NS) or IFI6 shRNA, alone or in combination with E2F2 shRNA were analyzed by FACS. The percentages of cells in each cell cycle stage are shown. (B) …
Figure 6—figure supplement 1
E2F2 shRNA does not affect the mRNA expression of other E2F family genes.
mRNA levels of E2F family genes in cells expressing non-specific (NS) or E2F2 shRNA were analyzed by RT-qPCR. Relative mRNA expression for the indicated E2F genes in indicated cells expressing E2F2 …
Figure 7 with 1 supplement
IFI6 loss results in DNA replication stress and senescence induction in primary human melanocytes.
(A) Primary human melanocytes expressing NRASQ61K or an empty vector were analyzed for the indicated genes by qRT-PCR. Relative gene expression is shown. (B) Primary human melanocytes expressing …
Figure 7—figure supplement 1
DNA fiber assay results of primary human melanocytes expressing IFI6 shRNA.
DNA replication in primary human melanocytes expressing non-specific (NS) or IFI6 shRNA was analyzed by DNA fiber assay. Representative images are shown.
Figure 8 with 2 supplements
IFI6 loss results in DNA replication stress and apoptosis induction in MEL-ST/NRASQ61K cells.
(A) MEL-ST cells expressing empty vector or NRASQ61K were analyzed by RT-qPCR. The expression of each gene in MEL-ST/NRASQ61K cells is shown relative to expression in empty vector control. (B) …
Figure 8—figure supplement 1
DNA fiber assay results in MEL-ST/NRASQ61K cells.
DNA replication in NRASQ61K-transformed MEL-ST cells expressing either non-specific (NS) or IFI6 shRNA was analyzed by DNA combing assay. Representative images are shown.
Figure 8—figure supplement 2
IFI6 knockdown in MEL-ST/NRASQ61K does not induce senescence.
(Left panel) MEL-ST/NRASQ61K cells expressing non-specific (NS) or IFI6 shRNA were analyzed for senescence induction by using the SA-β-gal assay. Representative images are shown. (Right panel) MEL-ST…
Figure 9 with 1 supplement
Targeting the DNA replication stress resistance pathway to treat NRAS-mutant melanoma.
(A) Indicated melanoma cell lines were treated with indicated drugs and analyzed by MTT assay after 48 hr of drug treatment. Survival was determined relative to vehicle-treated cells. (B) Indicated …
Figure 9—figure supplement 1
IFI6 knockdown in non-NRAS mutant melanoma cells does not induce expression of E2F2 or its target genes.
mRNA levels of the indicated genes in melanoma cell lines expressing non-specific (NS) shRNA or IFI6 shRNA were analyzed by RT-qPCR. Relative mRNA expression for IFI6 shRNAs expressing melanoma …
Figure 10
Proposed model for the role of IFI6 in melanoma tumor growth.
The model shows the mechanism by which IFI6 contributes to NRASQ61K-induced transformation and the maintenance of oncogenic NRAS-mutant melanoma tumor growth.
Additional files
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Supplementary file 1
Files related to microarray analysis and tables for reagents.
(A) List of genes that are significantly upregulated (p<0.05, Fold-change = 2 fold or more) in NRASQ61K expressing MEL-ST cells. (B) Fold change (FC) for significantly altered genes (p-value<0.05) in YUGASP cells expressing IFI6 shRNAs. (C) Biological pathway enrichment analysis report. (D) Primer sequences for RT-qPCR analysis; clone ID and catalog numbers for shRNAs (Open Biosystems); antibodies used; source and concentration of chemical inhibitors used.
- https://doi.org/10.7554/eLife.16432.031
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Supplementary file 2
Analysis of MAP kinase regulated and BRAF-signature genes for correlation with BRAF/NRAS/NF1 mutation status using melanoma TCGA dataset.
- https://doi.org/10.7554/eLife.16432.032
