Mastomys coucha: A natural animal model for papillomavirus-induced skin carcinogenesis

Transcription

1 Mastomys coucha: A natural animal model for papillomavirus-induced skin carcinogenesis Kai Schaefer, Julia Nafz, Myriam Ibberson and Frank Rösl Angewandte Tumorvirologie, Deutsches Krebsforschungszentrum, Heidelberg

2 Risk Factors of Non Melanoma Skin Cancer (NMSC) Cumulative UV exposure Outdoor workers (farmers, sailors) Outdoor sport (tennis, golf etc.) Age Individual susceptibility Skin type Hair type Immunosuppression HPV infection

3 Human Papillomaviruses (HPV) more than 100 types known to date Mucosal types low risk: anogenital warts high risk: HPV16/18 cervical cancer Skin types warts, papillomas, keratoacanthomas Non Melanoma Skin Cancer (NMSC)?

4 HPV and Non-melanoma Skin Cancer (NMSC) Association between HPV and NMSC first described in patients with Epidermodysplasia verruciformis (EV) EV-HPV types (HPV 5, 8, 9, 12, 14, 15, 17, 19-25, 38) 30-60% develop multiple cutaneous squamous cell carcinoma (SCC) on UV-exposed sites after years These SCC mostly harbour the oncogenic types HPV 5 and HPV 8

5 HPV and Non-melanoma Skin Cancer Patients under systemic immunosuppression (e.g. organ transplant recipients) multiple warts and NMSC at UV-exposed sites HPV-DNA in up to 80 % of tumors, but here not necessarily every cell is HPV positive kindly provided by Prof. E. Stockfleth, Charité Berlin

6 Causal Role of Viruses in Carcinogenesis as indirect carcinogen 1. Regular presence of the genome or parts of it in every cell is not given. 2. Transfection of the nucleic acid into tissue culture cells should lead to immortalization or tumor induction. Animal models. 3. Prevention of DNA repair and apoptosis 4. Epidemiological case/control studies should identify this virus as an essential co-factor for the respective tumor 5. Vaccination against this agent should provide protection

7 UV-exposure irradiation of the skin Non-infected cells HPV-positive cells p53 induction p53 induction Growth arrest & DNA repair Normal phenotype Bak induction & Apoptosis Elimination of cells with abnormal phenotype p53 mediated E6-mediated induction of Bak degradation & DNp73 XRCC1 inactivation E7-mediated MMP induction Accumulationof cells with an abnormal phenotype

8 Non-melanom skin cancer (NMSC) kindly provided by Prof. E. Stockfleth, Charité Berlin

9 Requirement of a Model

10 Mastomys coucha african multimammate mouse natural occurring animal model immune competent Mastomys population latently infected with Mastomys natalensis Papillomavirus (MnPV) spontaneous development of benign epithelial tumors of the skin and the anogenital tract age-dependent appearance in 40% of the animals (> 8 months) treatment with a tumor promoter: skin cancer

11 Spontaneous appearance of lesions papillomas, keratoacanthomas (skin, eyes, snout, ears and anus)

12 Time course of lesion development Skin Papilloma Papilloma eyelid Condyloma Papilloma ear Tumor incidence (No. of cases) Time [months] 0

13 MnPV genomic organisation URR E6 E7 7 ORFs L E5 is missing like in other 6000 MnPV 7687 bps 2000 E1 EV-HPVs and cutaneous HPV types L E2 one coding DNA strand E4

14 Histological detection of viral DNA in infected tissues DNA-In Situ-Hybridisation Skin Skin tumor 50 µm

15 Is there any oncogenic potential of MnPV?

16 Expression of MnPV E6 in transgenic mice

17 Carcinoma-Bearing Animals (%) Keratoakanthoma-Bearing Animals (%) Papillomas per Animal Papilloma Incidence (%) Time course of tumor formation A B Weeks after DMBA Treatment Weeks after DMBA Treatment C D MnPVE6-transgenic Wildtype Weeks after DMBA Treatment Weeks after DMBA Treatment

18 Detection of H-ras mutations in MnPV E6 transgenic mice by RT-PCR RFLP analysis DMBA induces a A T transversion at codon 61 G G T C A A G A A 268 bp G G T C T A Xba I G A A 155 bp 113 bp

19 tg scc 1 tg scc 2 tg scc 3 tg scc 4 tg scc 5 tg scc 6 tg scc 7 HP22-42 scc cell line tg kerato. 1 tg kerato. 2 tg kerato. 3 tg pap. 1 tg pap. 2 tg pap. 3 Inverse correlation between Ha-ras activation and E6 expression in SCC of MnPV E6 transgenic mice 268 bp H-ras 155 bp H-ras mutant 113 bp 599 bp -actin 269 bp MnPV E6 599 bp -actin 269 bp MnPV E6

20 H-ras mutations in MnPV E6 transgenic animals Papillomas pos. 12 0/34 pos. 13 0/34 pos /34 Keratoacanthomas pos. 12 0/14 pos. 13 0/14 pos. 61 4/14 SCC pos. 12 0/23 pos. 13 0/23 pos. 61 0/23

21 Distribution and propagation of Mastomys Papillomaviruses in different tissues

22 Absence of MnPV in anogenital and tongue lesions MnPV L1 MnPV E6 actin = normal skin 2 = papilloma eye 3 = condyloma anus = normal skin 2 = papilloma eye 3 = condyloma vulva = papilloma eye 2 = condyloma anus 3 = papilloma tongue

23 Mastomys coucha Papillomavirus 2 (McPV2) URR E6 E7 L McPV bps 2000 E L2 E2 Condylomas benign tumors of the anogenital tract conjunctional epithelia

24 A1d A1c B1a B1b B1c A1a F2a F1a D1c A1b A1a A2a E2a E1a E3a B2a C1c D1a C1a C1b D1b A1b CPV PCPV HPV6b RHPV1 HPV16 HPV54 HPV2 PsPV TtPv2 TmPV MnPV EcPV FDPV PlPV1 COPV ROPVb CRPV ROPV HPV63 HPV1a HPV5 HPV9 HPV92 BPV3 BPV6 BPV4 McPV2 HaOPV HPV101 HPV103 CaPV2 HPV4 HPV60 HPV48 HPV41 EdPV BPV5 EEPV RPV DPV OPV2 OPV1 BPV2 BPV1 FCPV PEPV Phylogenetic tree: difference between MnPV and McPV2

25 Distribution of MnPV and McPV2 DNA: PCR analysis Vulva Skin Anus MnPV-L1 McPV2-L1 localization of tumor Tongue

26 MnPV and McPV2 infection of the tongue EM PCR MnPV-L1 McPV2-L1 localization of tumor anus ear tongue tongue Actin

27 Summary I MnPV can be predominantly detected in skin tumors but also in apparently healthy skin. In transgenic animals, MnPV E6 can substitute or circumwent DMBA induced Ha-ras mutations; 100% formation of SCCs There is no indication for a trans-placental/germ-line transmission route of the virus. MnPV DNA is also present in other organs (cell specificity, viral spread?) McPV2 is responsible for the less frequently occurring tumors of the anogenital tract and other conjunctional epithelia like mouth and eye.

28 Immunological events controlling the virus infection

29 Investigation of humoral immune responses Case-control study: 120 animals analyzed collecting tissue samples and serum PCR GST-Capture-ELISA Detection of viral DNA Detection of antibodies

30 0,0 0,0 2,2 3,6 4,8 7,6 9,2 9,9 10,6 11,3 12,4 12,6 13,3 13,9 14,9 16,0 16,5 16,8 17,8 20,4 6,6 7,8 8,6 9,2 9,3 9,4 9,9 10,3 10,5 10,8 11,7 11,8 12,2 12,5 12,5 13,2 14,0 15,4 16,1 18,0 19,2 Seroreactivity against MnPV-L1 animals without papillomas animals with papillomas 2,5 MnPV-L1 ELISA 2,0 OD 450 1,5 1,0 0,5 0,0 age in months high antibody responses to L1 correlate with the presence of papillomas low antibody level in young animals delayed immune response or latency?

31 Seroreactivity against early proteins MnPV *** ** A 450 Tumor L1 + L1 - E6 + E6 - E7 + E7 - E2 + E2 -

32 Seroreactivity against early proteins McPV2 ** A 450 Tumor L1 + L1 - E6 + E6 - E7 + E7 - E2 + E2 - only low E2 responses compared to MnPV

33 MnPV: early E2-responses E2 seroreactivity: marker for early infection and latency, measurable by ELISA earliest 4 weeks after birth, preceding L1 maternal transmission of E2 antibodies, but no L1 antibodies L1 seroreactivity: productive infection and the onset of tumors

34 How we want to use this animal model?

35 Mastomys as model for a vaccine against skin PV Prophylactic vaccination against cutaneous HPV for OTR before transplantation VLP-vaccination against MnPV-induced skin tumors immunization of young animals induction of neutralizing antibodies at early stages Prevention of primary infections or viral dissemination in older animals

36 Summary MnPV and McPV2 are the etiological agents for a variety of epithelial tumors in M. coucha. The antibody reactivity against L1 is significantly increased in tumorbearing animals. High IgG-titers produced as response to PV-reactivation and the massive viral multiplication in older animals are not sufficient to prevent viral spread and tumor development. MnPV-E2 is highly immunogenic and seroreactivity precedes the L1 response, marking early and latent stages of infection. Unique read-out model for vaccination studies: VLPs will be applied as a prophylactic vaccine to prevent skin tumor development; Testing the efficacy of VLPs vaccines under immunosuppressed conditions

37

38 Maternal transmission of antibodies Mother Offspring (5) (10) 3 (6) 4 (3) 5 (7) 8 7 (3) 6 (7) (12)

39 MnPV: early E2-responses Follow-up study: 84 animals 0,5 0,4 M np V M cp V2 4 weeks after birth: only very weak L1 responses 0,3 0,2 0,1 0, ,8 0,7 E2 L1 4 weeks after birth: E2 responses 0,6 0,5 0,4 0,3 0,2 0,1 0, W E2 responses coincide and precede L1 responses E2 seroreactivity: early infection and latency L1 seroreactivity: productive infection and the onset of tumors

40 Additional perspectives Mastomys coucha Stomach cancer (gastric carcinoids and adenocarcinoma): H2 receptor blocker Loxtidine or by Helicobacter pylori co-infection with PV? (Model of chronic inflammation caused by bacteria and virus?) Transfer of MnPV and McPV2 to nude mice: effect of UV Genetic susceptibility? (CGH, gene profiling)

41

42

43 differentiation Detection of virus particles in skin sections mature viruses are released in stratum corneum Stratum corneum Stratum granulosum Stratum spinosum Stratum basale basal membrane

44 Prophylactic PV vaccine consists of a single protein production in insect cells or yeast 360 copies L1 protein spontaneous assembly virus-like particle (VLP)

45 ISH of McPV2 in lesions of the vulva

46 MnPV-DNA in hair follicle cells: a possible virus reservoir? E hf hf hf hf E = Epidermis hf = hair follicle