HPV38 E6 and E7 expression is not required for the viability of cancer cells in K14 HPV38 E6/E7 Tg mice.

<p>(A) Electroporated lesions were kept under control and the diameter was recorded weekly. On the day of injection, the lesion diameter varied between 1.2 mm and 2.5 mm for the lesions injected with the Luc plasmid, and between 1.3 mm and 2.6 mm for the lesions injected with the Cre-Luc plasmid. To standardize the measurement, each lesion diameter was set to an arbitrary value of 1 on the day of injection, and the following measurements were adjusted accordingly. The difference in tumour growth between the lesions injected with the Luc plasmid and the lesions injected with the Cre-Luc plasmid was not significant according to an unpaired two-sample Student’s <i>t</i>-test (<i>p</i> = 0.3108, <i>t</i> = 1.052; <i>df</i> = 14). The test was run on data from the fourth week, because afterwards the number of living animals was substantially reduced. (B) Representative images of SCC sections from two different HPV38 E6/E7 Tg mice. Sections were taken from tumours initially electroporated with pS/MARt-Luc plasmid (Luc) or with pS/MARt-Luc-P2A-Cre plasmid (Cre-Luc). The morphological analysis revealed no substantial differences between the specimens; the tumours were all classified as invasive cSCC, with deep penetration into the dermis or into the muscular fibres, and clear and diffuse atypia. The loss of the viral mRNA in the tumours injected with the Cre-Luc plasmid was confirmed by <i>in situ</i> RNA hybridization using a complementary (antisense) riboprobe, while the staining with a sense probe confirmed the specificity of the signal.</p

Cancer-related genes recurrently mutated in cSCCs of K14 HPV38 E6/E7 Tg mice.

<p>(A) Circos presentation of mutations occurring in the same genes between the different mice. From the centre to the outside, the skin samples (white), the lesion samples (yellow), and the SCC samples (grey) are displayed for <i>n</i> = 3 mice each. Each track (three per colour) corresponds to one animal. Red dots represent C:G > T:A mutations, and black dots represent the other types of mutations. For Circos A, only the mutations that occur in genes present in the Cancer Gene Census list from the COSMIC database are displayed, with the number of recurrent mutations in these genes in parentheses. (B) For the epigenetic drivers/modifiers, only the mutations that occur in the epi-driver or the epi-modifier gene lists are displayed. Blue gene names correspond to genes that are only involved in epigenetic processes, and purple gene names correspond to genes that are involved in epigenetic processes and that are present in the Cancer Gene Census list. The total number of recurrent mutations occurring in each of these genes is also displayed in parentheses.</p

Schematic representation of well-known and hypothetical models of virus-associated carcinogenesis.

<p>Schematic representation of well-known and hypothetical models of virus-associated carcinogenesis.</p

ΔNp73α mRNA levels are high in the skin of HPV38 E6/E7 Tg mice, but are decreased in the UV-induced skin lesions harbouring p53 mutations.

<p>Total RNA was extracted from the skin of WT (<i>n</i> = 4) or K14 HPV38 E6/E7 Tg animals (<i>n</i> = 5) as well as histologically confirmed pre-malignant (pre-m) and SCC from three independent mice and harbouring mutated p53. ΔN73α levels were measured by quantitative RT-PCR. The data shown are the mean of two independent experiments. The differences in ΔN73α mRNA levels between WT and K14 HPV38 E6/E7 Tg animals were statistically significant: * <0.05.</p

Luciferase expression vectors can be efficiently electroporated into skin lesions of K14 HPV38 E6/E7 Tg mice.

<p>(A) Schematic diagram of the electroporation procedure of skin lesions of K14 HPV38 E6/E7 Tg mice. (B) Luciferase activity is detected in lesions electroporated with the control vector (Luc) as well as in lesions electroporated with the plasmid coding for the Cre recombinase and luciferase genes (CRE-Luc). Mice and tumour growth are closely monitored at regular intervals.</p

Several genes mutated in human skin lesions are also mutated in the UV-induced skin lesions of cSCCs of K14 HPV38 E6/E7 Tg mice.

<p>(A) Heatmap of significantly mutated genes, corresponding to genes recurrently mutated in at least two mouse SCC samples and reported in the Cancer Gene Census list from the COSMIC database (left panel) or having an impact on epigenetic regulation processes (right panel). The types of mutation represented by colours are chosen according to the most prevalent mutation type in each sample. The data for the human samples displayed in the first column are derived from a previous publication on cutaneous SCC. (B) Heatmap of mutations in genes in normal skin, pre-malignant lesions, and cSCC from different mice (M1–3) reported as significantly mutated in human cSCC.</p

HPV38 E6 and E7 induce an increased steady-state level of UV-induced mutations in mouse skin keratinocytes.

<p>(A) UV-induced cSCCs in K14 HPV38 E6/E7 Tg mice have a vast number of somatic mutations. SCCs display a very high mutational load, with each Tg animal (Tg1–3) harbouring almost 3 times the number of variants compared with pre-malignant lesions (Pre-m). All differences in number of DNA mutations among the tree types of specimens were statistically significant: * ≤0.05; ** ≤0.01; **** ≤0.0001. (B) cSCCs of K14 HPV38 E6/E7 Tg mice display the classic UV-induced mutation signature with a very high number of C:G > T:A mutations. This type of mutation represents the majority of the SNV type in SCC samples of the three Tg animals. (C) Mutation spectrum of pooled SCC samples from the three mice. This spectrum displays the high prevalence of C:G > T:A mutations, especially in the 5′-T_N-3′ and 5′-C_N-3′ context. The <i>y</i> axis represents the percentage of mutations, and the <i>x</i> axis the trinucleotide sequence context.</p

Oligo sequences.

<p>Oligo sequences.</p

Loss of p53 inhibition by 16E6 restores IRF6 activity.

<p>(A) Table defining the 16E6 mutations that alter p53, E6AP or PDZ binding sites. (B) NIKs were co-transfected with IL-1β promoter luciferase construct along with the HPV16E6 WT and mutated constructs at the indicated concentration. n = 5. (C) Human primary keratinocytes were transfected with cas9/sgRNA for p53 or control and 36h later cells were examined for mRNA levels for p53, IL-1β or IRF6. n = 4. (D) siRNAE6AP (+) or siRNA scramble control (-) was transfected into PLXSN or 16E6 transduced human keratinocytes for 48 h, cells were harvested for protein and RNA. Western blot analysis for p53 and β-actin. Left top, RT-qPCR for IL-1β and, left below IRF6. n = 3, (E) NIKs were co-transfected with the IL-1β promoter and pLXSN, E6, p53 or E6 with p53. Luciferase activity was measured 48 h post infection. n = 4. (F) 16E6 transduced primary keratinocytes were transfected with vector control (-), p53 or IRF6 expression vectors. Twenty-four hours later cells were harvested and pro-IL-1β, p53 or IRF6 levels were examined by immunoblotting. n = 4. Data are representative of n independent experiments performed in triplicate. Shown are the mean ± SEM with ***, P < 0.0001, based on an one or two way (applicable to > 2 conditions) ANOVA test. For immunoblotting data, 1 out of 4 experiments is shown.</p

IRF6 and not IRF8 is recruited to the IL-1β promoter which is blocked by HPV16E6.

<p>(A) HEK293 cells were co-transfected with IL-1β promoter luciferase construct along with the empty vector pUNO, IRF8 or IRF6 plasmid at the indicated concentration. Post 48 h cells were lysed and luciferase activity measured. n = 4. (B) Oligo pulldown assay for WT or the mutated ISRE site using protein lysates from HEK293 cells transfected with IRF6 or IRF8. Bound proteins were assessed by immunoblotting for IRF8 or IRF6. Input controls (10%). n = 4. (C) Immunoblot analysis of IRF6 protein levels in in pLXSN and 16E6 transduced human primary keratinocytes. n = 4. (D) IRF6 relative levels were measured in pLXSN, 16E6 and 16E7 transduced human primary keratinocytes by RT-qPCR. n = 4. (E) Immunofluorescent staining of IRF6 in human keratinocytes transduced with pLXSN or HPV16E6. Left, semi-quantative analysis of IRF6 was examined by calculating immunofluorescent intensity. The mean and S.E.M of five fields were plotted. n = 4. (F) Immunoblot analysis of IRF6 protein levels in C33A and NIKs. n = 4. (G) IRF6 mRNA levels detected by RT-qPCR in NIKs and CaSki cells. n = 4. (H) NIKs and CaSki cells were co-transfected with IL-1β promoter luciferase construct ± siRNA for 16E6. Post 48 h cells were lysed and luciferase activity measured. (I) C33A cells were treated with control PsV or 16QsV at different v.g.e per cell for 24 h and IRF6 protein levels were examined by immunoblot. n = 3. (J) C33A cells were treated with control PsV or HPV16 at different v.g.e for 24h and IRF6 mRNA levels were examined by RT-qPCR and (below) viral DNA expression of E7 vs β2-microgloubulin. n = 3. (K), ChIP using IgG, IRF6 or IRF8 antibodies was performed for the ISRE site on C33A cells infected with HPV16 or PsV for 24 h. n = 3. Data are representative of n independent experiments performed in triplicate. Panels A, E, J and K are shown as the mean ± SEM with ***, P < 0.0001, * P, < 0,01, based on a two way ANOVA test. Panels D and G are shown as the mean ± SEM with ***, P < 0.0001 based on an unpaired T test. For immunoblotting data, 1 out of 4 experiments is shown. For immunoblotting data, 1 out of 4 experiments is shown.</p