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Tumor Microenvironment and Immunobiology

T-Cell Deletion of MyD88 Connects IL17 and IκBζ to RAS Oncogenesis

Christophe Cataisson, Rosalba Salcedo, Aleksandra M. Michalowski, Mary Klosterman, Shruti Naik, Luowei Li, Michelle J. Pan, Amalia Sweet, Jin-Qiu Chen, Laurie G. Kostecka, Megan Karwan, Loretta Smith, Ren-Ming Dai, C. Andrew Stewart, Lyudmila Lyakh, Wang-Ting Hsieh, Asra Khan, Howard Yang, Maxwell Lee, Giorgio Trinchieri and Stuart H. Yuspa
Christophe Cataisson
1Laboratory of Cancer Biology and Genetics, NCI, Bethesda, Maryland.
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Rosalba Salcedo
2Cancer and Inflammation Program (CIP), NCI, Bethesda Maryland.
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Aleksandra M. Michalowski
1Laboratory of Cancer Biology and Genetics, NCI, Bethesda, Maryland.
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Mary Klosterman
1Laboratory of Cancer Biology and Genetics, NCI, Bethesda, Maryland.
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Shruti Naik
3Department of Pathology and Ronald O. Perelman Department of Dermatology, NYU School of Medicine, New York, New York.
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Luowei Li
1Laboratory of Cancer Biology and Genetics, NCI, Bethesda, Maryland.
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Michelle J. Pan
1Laboratory of Cancer Biology and Genetics, NCI, Bethesda, Maryland.
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Amalia Sweet
1Laboratory of Cancer Biology and Genetics, NCI, Bethesda, Maryland.
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Jin-Qiu Chen
4Collaborative Protein Technology Resource, Center for Cancer Research, NCI, Bethesda, Maryland.
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Laurie G. Kostecka
1Laboratory of Cancer Biology and Genetics, NCI, Bethesda, Maryland.
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  • ORCID record for Laurie G. Kostecka
Megan Karwan
5Leidos Biomedical Research, Inc., Frederick, Maryland.
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Loretta Smith
2Cancer and Inflammation Program (CIP), NCI, Bethesda Maryland.
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Ren-Ming Dai
5Leidos Biomedical Research, Inc., Frederick, Maryland.
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C. Andrew Stewart
2Cancer and Inflammation Program (CIP), NCI, Bethesda Maryland.
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Lyudmila Lyakh
2Cancer and Inflammation Program (CIP), NCI, Bethesda Maryland.
6Division of Allergy, Immunology & Transplantation, National Institute of Allergy and Infectious Diseases, NIH, Bethesda Maryland.
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Wang-Ting Hsieh
5Leidos Biomedical Research, Inc., Frederick, Maryland.
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Asra Khan
2Cancer and Inflammation Program (CIP), NCI, Bethesda Maryland.
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Howard Yang
1Laboratory of Cancer Biology and Genetics, NCI, Bethesda, Maryland.
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Maxwell Lee
1Laboratory of Cancer Biology and Genetics, NCI, Bethesda, Maryland.
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Giorgio Trinchieri
2Cancer and Inflammation Program (CIP), NCI, Bethesda Maryland.
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  • For correspondence: yuspas@mail.nih.gov trinchig@mail.nih.gov
Stuart H. Yuspa
1Laboratory of Cancer Biology and Genetics, NCI, Bethesda, Maryland.
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  • For correspondence: yuspas@mail.nih.gov trinchig@mail.nih.gov
DOI: 10.1158/1541-7786.MCR-19-0227 Published August 2019
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    Figure 1.

    Systemic or CD4+ T-cell–targeted deletion of MyD88 inhibits tumor development in chemically induced skin carcinogenesis and reduces the content of IL17A producing cells in TPA-treated skins. A–D, Panels represent the mean number of skin tumors per mouse (mean ± SEM). Mice were treated with DMBA/0.2 mL acetone at time 0 then with 10 nmol TPA/0.2 mL acetone twice a week for up to 20 weeks. MyD88fl/fl (n = 6) MyD88CD4-Cre (n = 8; A); MyD88fl/fl (n = 9) and MyD88CD19-Cre (n = 10; B); MyD88fl/fl (n = 8) and MyD88Lyz2-Cre (n = 9; C); and MyD88fl/fl (n = 5) and MyD88CD11c-Cre (n = 14; D). Significant differences in the number of papillomas that develop were calculated by Student t test (**, P < 0.001). E, Graphs represent the quantification of CD4+ T cells on papilloma sections from MyD88fl/fl and MyD88CD4-Cre. **, P < 0.01 versus MyD88fl/fl. F, qPCR analysis of differentially expressed genes in CD3+-sorted cell population isolated from MyD88fl/fl and MyD88CD4-Cre TPA-treated skins. G, Plots summarizing flow cytometric data of Foxp3, IL17A, IFNγ expression by live CD45+ TCRβ+ infiltrating the skin of MyD88fl/fl and MyD88−/− mice after 9 TPA applications. H, The same analysis was performed comparing MyD88fl/fl and MyD88CD4-Cre mice but IL17A+ TCRγ+ cells were also analyzed. Graphs show means ± SEM of 3 to 8 mice. F and G, **, P < 0.01 MyD88−/− and MyD88CD4-Cre, respectively, versus control group. I, Gene expression changes in skin biopsies collected 6 hours after the last TPA or acetone application (8 total) in WT and MyD88−/− mice. Expression levels were determined by semiquantitative PCR. *, P < 0.05 MyD88−/− versus control group. J, Immunoblot of total skin lysates from WT and MyD88−/− mice collected as described for I. p-, phosphorylated. Each lane represents a skin biopsy from an individual mouse. Data shown in A–I are representative of three independent experiments, while data shown in E and J are representative of two independent experiments. Data in A–J are means ± SEM.

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    Figure 2.

    IL17 stimulates proliferation, upregulates cytokine expression, and inhibits differentiation of normal keratinocytes. Tritiated thymidine incorporation was measured in WT and MyD88−/− (A) or WT and EGFR−/− (B) keratinocyte cultures treated with IL17 or IL22 for 24 hours, bars represent the mean ± SEM value of four replicates; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001. C, Immunoblotting of total cell extracts from primary keratinocytes treated with IL17 for the indicated period. HSP90, heatshock protein 90; p-, phosphorylated. D, Heatmap representation of gene expression analysis performed by real-time PCR quantification from keratinocytes treated for 1, 3, 6, and 12 hours with IL17. E, Total SDS cell extracts from PBS, IL17- or IL22-treated keratinocytes were immunoblotted with specific antibodies recognizing early (K1 and K10) and late (filaggrin) markers of differentiation from cultures maintained under differentiating conditions (0.12 mmol/L Ca++) for 24 hours. F, Real-time PCR quantification of K1 and K10 mRNAs in keratinocytes infected with GFP (control) or degradation-resistant IκBα (IκBsr Ad) adenovirus to block NF-κB activity and treated with IL17 under differentiating conditions (**, P < 0.01; ***, P < 0.001).

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    Figure 3.

    IL17 enhances transcriptional activity and cytokine release but not proliferation of RAS keratinocytes. A, Tritiated thymidine incorporation was measured in control or RAS-transduced WT and Myd88−/− keratinocyte cultures treated with IL17 for 24 hours. Bars represent the mean ± SEM value of four replicates. B, Real-time PCR quantification of mRNAs from control and RAS-transduced keratinocytes stimulated with IL17 for 6 or 24 hours. Bars represent the mean ± SEM value of four replicates. C, Real-time PCR analysis of mRNA expression in control or RAS-transduced keratinocytes treated with PBS or IL1R antagonist (IL1ra, anakinra). D, Cytokine and chemokine concentrations in culture supernatants from control and RAS-transduced keratinocytes cultures collected 24 hours after TPA or IL17 treatments and assayed by multiplex ELISA. For A–E, **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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    Figure 4.

    IL17 enhances transcription of downstream effectors in mouse keratinocytes with endogenous mutant Ras activation and human keratinocytes transformed by human mutant RAS. A, Real-time PCR quantification of mRNAs from GFP and Cre adenovirus-transduced LSL-HrasG12D mouse keratinocytes stimulated with IL17. B, Real-time PCR quantification of mRNAs from GFP, mutant HRASG12V, and KRASG12V lentivirus-transduced human keratinocytes. C, Real-time PCR quantification of mRNAs from GFP and mutant HRASG12V lentivirus-transduced human keratinocytes treated with human IL17A. For A–C, *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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    Figure 5.

    TPA, IL17, and the RAS oncogene are dependent on IkBz signaling for induction of tumor-promoting transcripts and tumor formation in vivo. Tritiated thymidine incorporation was measured in WT or Nfkbiz−/− normal keratinocytes treated with IL17 for 24 hours (A) or RAS-transduced keratinocytes (B). Bars represent the mean ± SEM value of four replicates. C, Total SDS cell extracts from Nfkbiz+/− and Nfkbiz−/− keratinocytes were immunoblotted with specific antibodies recognizing K1 and K10 from cultures under basal (0.05 mmol/L Ca++) conditions or treated with IL17 under differentiating conditions (0.12 mmol/L Ca++). Real-time PCR quantification of mRNAs from Nfkbiz+/− and Nfkbiz−/− keratinocytes treated for 1, 3, 6, and 12 hours with IL17 (D) or from WT or Nfkbiz−/− normal keratinocyte treated with TPA (E) or from control and RAS-transduced WT and Nfkbiz−/− keratinocytes (F). Symbols represent the mean ± SEM value of three replicates. G, Immunoblotting of nuclear extracts from control and RAS-transduced WT and Nfkbiz−/− keratinocytes. Arrow head denotes specific band. H, Primary keratinocytes Nfkbiz+/− or Nfkbiz−/− were transduced with a retrovirus expressing oncogenic RAS and grafted together with WT dermal fibroblasts onto the backs of athymic mice. Each dot represents mean volume of individual tumors at day 20 postgrafting. Data shown represent two independent experiments and are reported as mean ± SEM. For all panels: *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

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    Figure 6.

    The RAS transcriptome is dependent on IκBz. A, Heatmap visualization of relative expression of the top 82 genes whose expression is altered by at least 2-fold when comparing RAS-keratinocytes Nfkbiz+/+ with RAS keratinocytes Nfkbiz−/−. Genes are ordered by fold change. B, GSEA enrichment plots are shown for top-enriched or top-depleted gene sets in RAS-transduced Nfkbiz−/− keratinocytes. The black line is the running enrichment score calculated along the ranked gene list; the vertical light blue bars in the plot indicate the position of the genes from the respective gene set. Supplementary Table S4 lists all GSEA estimates for these gene sets, including “leading edge” genes. C, Network visualization of top-enriched functional gene sets from MSigDB collections (GSEA FDR < 5.0%) in RAS-transduced Nfkbiz−/− keratinocytes. Network nodes represent subsets of the enriched gene sets including GSEA core enriched genes (“leading edge”) and are shown in blue and red for gene sets underexpressed and overexpressed in the RAS-transduced Nfkbiz−/− keratinocytes, respectively. Edges in the network represent mutual overlap of genes between the gene sets and the thickness of an edge is proportional to the combined similarity coefficient, ranging between 0.375 to 1.0. Supplementary Table S4 contains the underlying network data.

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    Figure 7.

    IkBz-dependent RAS gene signature predicts disease free survival in COAD, PAAD, and LUAD. A, A 16-gene sum index from the most overexpressed genes in RAS keratinocytes depleted of Nfkbiz was used to divide patients with COAD with high- and low-index groups to plot Kaplan–Meier curves. The patients with high index had worse prognosis with HR = 3.6 (1.5–8.65) and log-rank P = 0.003. A 19-gene weighted sum index from the same profile was used to divide the patients with PAAD (B), LUAD (C), and LUSC (D) with high- and low-index groups. The 16-gene set is a subset of the 19 genes and the weights used to compute the index were log2-fold changes derived from Nfkbiz-dependent gene in mouse RAS keratinocytes (Supplementary Table S3). The patients with PAAD with high index had worse prognosis with HR = 1.76 (1.13–2.74) and the log-rank P = 0.006. The patients with LUAD with high index had worse prognosis with HR = 1.67 (1.24–2.27) and the log-rank P = 0.002. But for the patients with LUSC, the high- and low-index groups had no significant difference in survival.

Additional Files

  • Figures
  • Supplementary Data

    • Table S1 - Supplemental table 1: RNA profiling of differentially expressed genes in CD3+ sorted cell population isolated from MyD88fl/fl and MyD88CD4-Cre chronically TPA-treated skins. (A) List of genes from RNA profiling that are significantly affected (by at least 1.3-fold change and FDR &lt;5%) by MyD88 deficiency in T cells. (B) Ingenuity Pathways Analysis (IPA) of top significant genes.
    • Table S2 - Supplemental table 2: List of genes from RNA profiling, the expression of which is significantly affected (by at least 1.5-fold change and FDR &lt;10%) by IL-17 in RAS-keratinocytes.
    • Table S3 - Supplemental table 3: List of genes from RNA profiling, the expression of which is significantly affected (by at least 1.5-fold change and FDR &lt;5%) by IkBz deficiency in RAS-keratinocytes.
    • Table S4 - Supplemental table 4: GSEA estimates underlying data presented in figure 6B and 6C.
    • Table S5 - Primers used for mouse colonies genotyping.
    • Supplemental Figures 1-3 - Supplemental Figures 1-3
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Molecular Cancer Research: 17 (8)
August 2019
Volume 17, Issue 8
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T-Cell Deletion of MyD88 Connects IL17 and IκBζ to RAS Oncogenesis
Christophe Cataisson, Rosalba Salcedo, Aleksandra M. Michalowski, Mary Klosterman, Shruti Naik, Luowei Li, Michelle J. Pan, Amalia Sweet, Jin-Qiu Chen, Laurie G. Kostecka, Megan Karwan, Loretta Smith, Ren-Ming Dai, C. Andrew Stewart, Lyudmila Lyakh, Wang-Ting Hsieh, Asra Khan, Howard Yang, Maxwell Lee, Giorgio Trinchieri and Stuart H. Yuspa
Mol Cancer Res August 1 2019 (17) (8) 1759-1773; DOI: 10.1158/1541-7786.MCR-19-0227

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T-Cell Deletion of MyD88 Connects IL17 and IκBζ to RAS Oncogenesis
Christophe Cataisson, Rosalba Salcedo, Aleksandra M. Michalowski, Mary Klosterman, Shruti Naik, Luowei Li, Michelle J. Pan, Amalia Sweet, Jin-Qiu Chen, Laurie G. Kostecka, Megan Karwan, Loretta Smith, Ren-Ming Dai, C. Andrew Stewart, Lyudmila Lyakh, Wang-Ting Hsieh, Asra Khan, Howard Yang, Maxwell Lee, Giorgio Trinchieri and Stuart H. Yuspa
Mol Cancer Res August 1 2019 (17) (8) 1759-1773; DOI: 10.1158/1541-7786.MCR-19-0227
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