The Inflammasome Adaptor ASC Delays UV-Induced Skin Tumorigenesis in Beta HPV38 E6 and E7 Transgenic Mice

Non-melanoma skin cancer is the most common in fair-skinned adults and comprises basal cell carcinoma squamous (SCC). Its incidence reaches 1 of 100 person-year Australia (Lomas et al., 2012Lomas A. Leonardi-Bee J. Bath-Hextall F. A systematic review worldwide nonmelanoma cancer.Br J Dermatol. 2012; 166: 1069-1080Crossref PubMed Scopus (1126) Google Scholar) still on rise (Madan 2010Madan V. Lear J.T. Szeimies R.M. cancer.Lancet. 2010; 375: 673-685Abstract Full Text PDF (587) Scholar). The development non-melanoma depends genetic environmental factors. Particularly, there clear evidence that cumulative sun exposure contributes to (Ananthaswamy 1997Ananthaswamy H.N. Loughlin S.M. Cox P. Evans R.L. Ullrich S.E. Kripke M.L. Sunlight cancer: inhibition p53 mutations UV-irradiated mouse by sunscreens.Nat Med. 1997; 3: 510-514Crossref (193) Scholar, Madan In addition, fact immunosuppression strongly increases susceptibility supports role infectious agents this pathologic condition. Indeed, a subgroup cutaneous human papillomavirus (HPV) types belonging genus Beta HPV phylogenetic tree has been implicated as etiological factor SCC (Chahoud 2016Chahoud Semaan Chen Y. Cao M. Rieber A.G. Rady al.Association between β-Genus immunocompetent individuals-a meta-analysis.JAMA 2016; 152: 1354-1364Crossref (82) Rollison 2019Rollison D.E. Viarisio D. Amorrortu R.P. Gheit T. Tommasino An emerging issue oncogenic virology: β carcinoma.J Virol. 2019; 93: e01003-e01018Crossref (55) Inflammation double-edged sword promoting tumorigenesis participating antitumoral immune responses. Inflammasomes are pillars inflammation response UV (Fenini 2018Fenini G. Grossi S. Contassot E. Biedermann Reichmann French L.E. al.Genome editing primary keratinocytes CRISPR/Cas9 reveals an essential NLRP1 inflammasome UVB sensing.J Invest 2018; 138: 2644-2652Abstract (40) infections. multiprotein complexes leading activation inflammatory CASP1, CASP4, CASP5 humans (CASP1 CASP11 mice), resulting release proinflammatory cytokines IL-1β IL-18. through cleavage GasderminD, leads death termed pyroptosis. (KCs), major sensor acts with ASC, adaptor, promote UVB-induced cytokine inflammasomes controversial and, our knowledge, studied only chemical-induced carcinogenesis (Drexler 2012Drexler S.K. Bonsignore L. Masin Tardivel Jackstadt R. Hermeking H. al.Tissue-specific opposing functions adaptor ASC regulation epithelial carcinogenesis.Proc Natl Acad Sci USA. 109: 18384-18389Crossref (99) Gasparoto 2014Gasparoto T.H. de Oliveira C.E. Freitas L.T. Pinheiro C.R. Hori J.I. Garlet G.P. al.Inflammasome critical protective during chemically induced carcinoma.PLoS One. 2014; 9e107170Crossref (14) molecules CASP1 remains unknown. To investigate issue, we used transgenic model expresses beta HPV38 E6 E7 under control K14 promoter active layer epidermis (K14-HPV38 E6/E7Tg). two oncoproteins block action several ways (Accardi 2006Accardi Dong W. Smet Cui Hautefeuille Gabet A.S. al.Skin type 38 alters accumulation deltaNp73.EMBO Rep. 2006; 7: 334-340Crossref (96) Wallace 2014Wallace N.A. Robinson K. Galloway D.A. expression inhibits stabilization tolerance genomic instability.J 88: 6112-6127Crossref (36) K14-HPV38 E6/E7Tg mice highly susceptible developing UV-mediated (Viarisio 2011Viarisio Mueller-Decker Kloz U. Aengeneyndt B. Kopp-Schneider Gröne H.J. al.E6 from cooperate ultraviolet light actinic keratosis-like lesions mice.PLoS Pathog. 2011; 7e1002125Crossref (115) were crossed PycardKO (Pycard encodes ASC). Initially, C57BL/6 background, first rendered albino (e.g., tyrosinase-mutated) crosses B6(Cg)-Tyrc-2J/J populations. referred AscKO-alb. Littermates AscWT-alb controls. Similarly, generated Casp1/11DKO background assess influence CASP tumorigenesis. All experiments approved ethics committee (Comité d'Evaluation Commun au Centre Léon Bérard, à l'Animalerie transit l'ENS, PBES laboratoire P4) University Lyon, France (#7960-201611 0313531963) performed strict accordance European laws. Mice irradiated escalating doses for 30 weeks (Figure 1a). As previously reported Scholar), absence transgene, wild-type did not develop any tumor following irradiation 1b d). no was observed AscKO-alb or Casp1/11DKO-alb d), indicating deficiency sufficient chronic irradiation. Long-term transgene synergistically promoted (littermate controls) thus recapitulating previous findings Interestingly, developed tumors significantly faster (median tumor-free time = 22 weeks, n 15, P 0.021 Mantel‒Cox Log-rank test) than 26 15). Appearance preceded reddish patches, which characterized precancerous keratosis 1e, bottom left panel) also appeared earlier 1c). These results demonstrate mimicking synergy infection individuals, provides advantage against development. Histology at end surviving obvious differences different genotypes. Various ranging papilloma top right invasive observed. suggest affects kinetics but process per se. Surprisingly, although effector downstream significant littermate controls terms 1d; 0.66). Phenotypic AscKO have might be because redundancy ASC. can recruit activate CASP8 trigger IL-18 processing (Gringhuis 2012Gringhuis S.I. Kaptein T.M. Wevers B.A. Theelen van der Vlist Boekhout al.Dectin-1 extracellular pathogen induction via noncanonical caspase-8 inflammasome.Nat Immunol. 13: 246-254Crossref (376) Pierini 2013Pierini Perret Djebali Juruj C. Michallet M.C. Förster I. al.ASC IFN-γ levels IL-18-dependent manner caspase-1-deficient infected Francisella novicida.J 2013; 191: 3847-3857Crossref (30) contrast KCs, murine KCs express very low undetectable components (Sand 2018Sand Haertel al.Expression proteins occurs human, keratinocytes.Cell Death Dis. 9: 24Crossref (54) (Supplementary Figure S1a). consequence macrophages, they do produce lipopolysaccharide various NLRP3 stimuli S1b c). Thus, assessing KC-intrinsic possible model. described study likely reflects crosstalk UVB-exposed E6- and/or E7-expressing inflammasome-expressing myeloid cells, although, stage, cannot rule out level allows KC-specific inflammasome-independent Importantly, originally known target methylation silencing 1, downregulated numerous cancers, including SSC (Meier 2016Meier Drexler Eberle F.C. Lefort Yazdi Silencing 11e0164742Crossref (20) studying impact its loss relevant. Together, highlights triggered long-term presence E7. individual effectors, IL-1β, IL-18, protection their therapeutic potentials SCC. No datasets analyzed study. Flora Magnotti: http://orcid.org/0000-0002-3803-9934 Séverine Valsesia: http://orcid.org/0000-0002-4503-5202 Purnima Gupta: http://orcid.org/0000-0003-4389-3024 Christa Flechtenmacher: http://orcid.org/0000-0003-2040-6246 Pierre Contard: http://orcid.org/0000-0001-5556-0206 Daniele Viarisio: http://orcid.org/0000-0002-3123-688X Assunta Venuti: http://orcid.org/0000-0001-5438-1724 Mélanie Wencker: http://orcid.org/0000-0003-4200-0079 Massimo Tommasino: http://orcid.org/0000-0002-0890-8596 Jacqueline Marvel: http://orcid.org/0000-0001-6241-459X Thomas Henry: http://orcid.org/0000-0002-0687-8565 authors state conflict interest We acknowledge contribution SFR Biosciences facilities (AniRA-PBES, AniRA cytometry, Platinum) funding Plan Cancer (C16014CS). work here undertaken tenure postdoctoral fellowship International Agency PG. Where identified personnel Research World Health Organization, alone responsible views expressed article necessarily represent decisions, policy, Organization. Conceptualization: MT, JM, TH; Formal Analysis: FM, SV, Funding Acquisition: Investigation: PG, CF, PC, MW; Project Administration: MW, Resources: DV; Supervision: DV, AV, Visualization: Writing - Original Draft Preparation: Writing-Review Editing: TH bred (Lyon animal facility, ENS France) used. Genotypes selected, littermates isoflurane anesthesia three times week starting 120 mJ/cm2 presented 1a using BS-02 chamber (Opsytec, Mannheim, Germany). monitored appearance macroscopic tumors. euthanized if one reached diameter cm. Primary cells obtained framework protocols validated local ethical (ENS_2017_024 ENS_2012_061). Bone marrow‒derived macrophages after 6 days culture bone marrow DMEM, 10% fetal calf serum, supplemented 3T3–macrophage colony-stimulating supernatant. isolated tail 3–9 old mice. Briefly, incubated 18 hours 4 °C dispase II (4 mg/ml; Roche, Basel Switzerland). epidermal sheet then separated dermis treated minutes room temperature 0.1% Trypsin EDTA solution (Gibco, ThermoFisher Scientific, Waltham, MA). Keratinocytes gently scraped plated 105 cells/cm2 keratinocyte growth medium 2 (Promocell, Heidelberg, Cells ng/ml ultrapure (Invivogen, San Diego, CA) before addition Nigericin (5 μM, Invivogen) adenosine triphosphate Sigma, St. Louis, MI). Supernatant collected 3 later dosage (DuoSet, R&D Systems, Minneapolis, MN). For irradiation, primed indicated dose (50–150 mJ/cm2) Vilber Lourmat irradiator set 312 nm. later.