HPV8-induced STAT3 activation led keratinocyte stem cell expansion in human actinic keratoses

Despite epidermal turnover, the skin is host to a complex array of microbes, including viruses, such as HPV, which must infect and manipulate skin keratinocyte stem cells (KSCs) to survive. This crosstalk between the virome and KSC populations remains largely unknown. Here, we investigated the effect of HPV8 on KSCs using various mouse models. We observed that the HPV8 early region gene E6 specifically caused Lrig1+ hair follicle junctional zone KSC proliferation and expansion, which would facilitate viral transmission. Within Lrig1+ KSCs specifically, HPV8 E6 bound intracellular p300 to phosphorylate the STAT3 transcriptional regulatory node. This induced ΔNp63 expression, resulting in KSC expansion into the overlying epidermis. HPV8 was associated with 70% of human actinic keratoses. Together, these results define the “hit-and-run” mechanism for HPV8 in human actinic keratosis as an expansion of KSCs, which lack melanosome protection and are thus susceptible to sun light–induced malignant transformation.


Deposited data
Sequence data (Figure 1 and 2; Supplemental Figure 1)

This paper GSE248056
Sequence data (Figure 4; Supplemental

Cell lines
HaCaT and PM1 cells represent spontaneously immortalized human keratinocyte cell lines.

Immunofluorescence staining and microscopy of OCT sections
For paraffin embedded samples, slides were heated at 60°C for 30 minutes, then deparaffinized with xylene (2 x 10 minutes) and decreasing concentrations of ethanol (100-75%) for 5 minutes each, followed by a 5 min wash in PBS.For antigen retrieval, samples were submerged in citrate buffer (pH6.0) and heated in a microwave for 10 minutes in a pressure cooker and allowed to cool to room temperature gradually.Samples were washed for 5 minutes in PBS, then a hydrophobic barrier pen was used to draw around the tissue sample.Sections were then incubated in blocking buffer (10% goat serum in PBS) for 1 hour at room temperature.Primary antibodies (Supplemental Table 5) were diluted in 5% goat serum and incubated overnight at 4°C.Samples were washed 4 x 5 minutes in PBS Tween-20 (0.05%) before incubating with fluorescence-conjugated secondary antibodies in PBS for 1 hour in the dark at room temperature.Samples were again washed 4 x 5 minutes in PBS Tween-20 (0.05%) before mounting with Vectashield mounting solution (Vector Labs).For frozen OCT samples, sections were fixed in 4% paraformaldehyde for 10 minutes at room temperature, then washed 3 x 5 minutes in PBS before being permeabilized with 0.25% PBS/Tween-20 for 10 minutes at room temperature.Blocking, primary and secondary antibody steps were carried out as described above.Finally, sections were mounted with Vectashield and scanned on a Olympus Slideview VS2000 slide scanner.
Sections were washed in PBS-Tween (0.05%) then incubated for 1 hour at room temperature with biotinylated species-specific secondary antibodies diluted in 5% serum.
Secondary antibodies used were: Goat anti-Mouse IgG Antibody (Vector Laboratories, BP-9200) and Goat anti-Rabbit IgG Antibody (Vector Laboratories, BP-9100).Expression was detected using ABC-HRP for 30 minutes at room temperature and then DAB stained until optimal staining was observed.Sections were then counterstained with haematoxylin (Atom Scientific) and dehydrated using a series of ethanol and xylene rinses and mounted with DPX mountant (Merck).Images were acquired using an Olympus Slideview VS2000 slide scanner.

Fluorescence-activated cell sorting (FACS) or analysis
Samples were gated on the basis of forward-and side-scatter.Doublets and dead cells were excluded.eBioscience™ Fixable Viability Dye eFluor™ 780 (Thermo Fisher Scientific #65-0865-14) was used to gate out dead cells when sorting Confetti (GFP, YFP, RFP and CFP) mice.Single stained samples were used as compensation controls and use of an isotype control was used to determine background fluorescence.Data was processed using FlowJo analysis software (FlowJo, LLC).

Protein extraction and quantification
Cells in culture were collected and counted.Adherent cells in culture were detached using Versene or TrypLE (ThermoFisher Scientific).Cells were centrifuged at 250xg for 5 minutes and pellet was washed with PBS, and again pelleted.PBS was removed, and pellet resuspended in 100 µL per 1x10 6 cells of RIPA buffer containing protease inhibitor cocktail (Cell Signalling).Samples were homogenized by pipetting and incubated for 30 minutes on ice.Following incubation, samples were centrifuged at 10,000xg for 10 minutes at 4°C and the supernatant was collected.Nuclear protein lysates were extracted as outlined in manufacturers guidelines (ThermoFisher Scientific).The BCA assay kit (Pierce, ThermoFisher Scientific) was used to determine protein concentrations.
densitometry, using ImageLab software (BioRad).The pixel density over the selected areas was quantified and compared.

Co-Immunoprecipitation
Briefly, columns were prepared by incubating 50µL of coupling resin to 50 µg of YAP (Cell Signalling, 14074) and STAT3 (Cell Signalling, 4904) antibodies.1x10 6 cells were lysed in 100µL of lysis buffer to yield ~300µg of protein.Control agarose resin slurry was used to pre-clear the protein lysate, before being added to the antibody-coupled resin and incubated with gentle mixing overnight at 4°C.Resins were then washed and coimmunoprecipitated proteins YAP and STAT3 were identified by Western blot with the antibodies anti-YAP (1:500, Cell Signalling, 12395) and anti-STAT3 (1:1000, Cell Signalling, 9139) respectively.To ensure Co-IP specificity, all pull downs were performed with the following controls: 1) a non-activated control resin to ensure no non-specific binding of the antibodies and lysate to the resin, 2) a quenched antibody coupling resin which is a resin that has been processed in the exact same way as the test resin but with the antibody present and 3) a non-relevant antibody which was unrelated to the study for all pulldowns shown.

Chromatin Immunoprecipitation (ChIP)-qPCR
The input amount of cells for each reaction was 25mg.Briefly, cells were cross-linked using 1% formaldehyde in cell culture media for 10 minutes at room temperature, and then quenched with 1.25 M Glycine on ice.Cells were collected by centrifugation at 250xg for 5 minutes, and pellet washed with ice cold PBS.Cells were pelleted again, resuspended in working lysis buffer and incubated on ice for 10 minutes, before being vortexed and centrifuged at 3000 rpm for 5 minutes.Supernatant was carefully removed and pellet

Cell proliferation assay
HaCaT/PM1-PLXSN, -E6 and -E7 cells were plated (5,000 cells per well) in a white-walled 96 well plate and left overnight to adhere.Cell proliferation was determined using the BrdU cell proliferation ELISA kit (Abcam, ab126556) as per manufacturers guidelines.BrdU was allowed to incorporate with cells for 24 hours before ELISA assay was conducted.
Luminescence was recorded using a CLARIOstar plate reader with wells containing only media serving as a blank.Blank corrected values were used to calculate the amount of proliferation when compared to vector control cells.

Migration assay (established cell lines)
HaCaT/PM1-PLXSN, -E6 and -E7 cells were seeded into ibidi culture inserts (2 well, Ibidi, #80209) at a density of 100,000 cells in 70 µL media (for each half of the insert) in a 24 well plate and allowed to adhere for 8 hours in the incubator.Cells were then checked for confluence and culture inserts were removed gently using a forceps.Cells were washed to remove any debris using PBS, and fresh media was placed in each well, before being placed in a Leica DM6000 timelapse microscope overnight for image acquisition every hour for 16 hours.Data were quantified by plotting the cell covered area against the time to show the rate of gap closure.The linear phase of gap closure was used to calculate the percentage of gap closure over a defined period of time relative to vector control cells.

DNA extraction, precipitation and b-HPV genotyping PCR
Briefly, an equal volume of 5M ammonium acetate was added to each sample and thoroughly mixed.Two volumes of 100% ethanol was added to each sample, thoroughly mixed and placed at -80°C for 30 minutes.Samples were removed from -80°C and centrifuged for 30 minutes at 14,000 x g at 4°C.After carefully removing the supernatant, 1 mL of 70% ethanol was added, thoroughly mixed, and centrifuged at 14,000 x g for 10 minutes at 4°C.The supernatant was again carefully discarded, and samples were placed in a hot block for 10-15 minutes at 37°C until the residual ethanol evaporated.Each sample was then resuspended in 50 µL nuclease free water and DNA concentration determined using a NanoDrop 2000 (Thermo Fisher Scientific).
PCR was performed on each sample to amplify the relatively conserved β-HPV E1 region and PCR-amplified samples were run through a genotype hybridization protocol as per manufacturer's instructions.The positive PCR control was used from the kit, and Milli-Q water without DNA was used as a negative control.The master mix was first added to each PCR tube and 10 µL of sample was added and mixed by pipetting.PCR tubes were briefly spun and placed in a thermocycler as per the PCR protocol in the manual.Amplified PCR products were then hybridized for detection.10 µL of the PCR-amplified sample, 10 µL Denaturation solution and 10 µL of 3B solution were added to their respective trough and mixed by pipetting.Following a 5-minute incubation at room temperature, 2 mL pre-warmed Hybridization solution was added to each trough and mixed by gently shaking the troughs.
Test strips were then immediately placed into their respective troughs and troughs were placed into a pre-heated 50°C shaking water bath for 60 minutes at approximately 80 rpm.
Following incubation, test troughs were removed from the shaking water bath and the troughs were carefully aspirated using a vacuum aspirator.Test strips were washed twice in 2 mL pre-warmed Stringent Wash Solution for 10 seconds by gently shaking the troughs.A final washing step was then performed via a 30-minute shaking water bath incubation with 2 mL Stringent Wash Solution.All wash steps and incubations were next performed on a rocker at room temperature using the highest possible speed and avoiding spillages.
Samples were washed twice in diluted Rinse Solution for 1 minute.A 30-minute incubation was performed using 2 mL of Conjugate Solution.A further two wash steps were performed with 2mL Diluted Rinse Solution, and a third wash step using 2 mL Substrate Diluent.
Colour development was performed through incubation of each test strip with 2 mL Substrate Solution for 30 minutes and covering the troughs with foil.To stop colour development, strips were washed twice for 3 minutes in Milli-Q water.Strips were then removed from the troughs and placed on absorbent paper until dry.Each strip was then cello taped to the results page and results were recorded.

Bioinformatic analysis
For the Lrig1 vs CD34 experiment, cDNA libraries were generated and NEBNext sequencing adaptors were added along with sample barcodes.Libraries were then sequenced on a Illumina HiSeq4000 at a depth of 20M reads per sample.Sequenced reads were then run through FastQC and subsequently mapped to the murine GRCM38 reference genome using STAR.FeatureCounts were then used to quantify reads and DEG analysis was performed via the standard DESeq2 pipeline.For the confetti-positive cells (with and without Lrig1 cell surface expression) experiment, libraries were generated using the Illumina TruSeq RNA Library Prep Kit (v2) and were subsequently sequenced on a NovaSeq 6000 PE150 to generate a total of 20-million paired-end reads per sample.Raw sequencing data was processed by NovoGenes in-house pipelines to remove bad quality reads, map to the mouse GRCm39 reference genome using Hisat2 (v2.0.5) and generate normalized reads using featureCounts (v1.5.0).Normalized reads were used to generate PCA plots in order assess sample clustering.DEGs were then generated using edgeR (v3.22.5) using a false discovery rate (FDR) of 0.1 and then filtered for significance (adjusted p value < 0.05).DEGs were then returned to us from Novogene, where we performed pathway analysis with Gene Set Enrichment Analysis (GSEA) GSEAPreRanked tool and Ingenuity Pathway Analysis (Qiagen) software where causal analysis was run with default parameters.

Supplemental Figure 2 .
Figure 5. (A) QPCR of RNA from vector control and HPV8 E7 transduced HaCaT keratinocytes for STAT3regulated genes, with b-actin as an internal control (n=3 per cell line).(B) In vitro proliferation assessed by 24 hours BrdU incorporation of HPV8 E6 and E7 transduced PM1 keratinocytes compared to vector control (n=3 per cell line).Data are presented as mean ± SEM.Dotted line is the comparator.NS, non-significant.(C) CFE of 500 vector control and HPV8 E6 and E7 transduced PM1 keratinocytes per well (n=7 per cell line).**p < 0.01 by one-way ANOVA.Data are presented as mean ± SEM. (D) Cell migration assay of HPV8 E6 and E7 transduced PM1 keratinocytes assessed by percentage of closure over 24 hours compared to vector control cells (n=3 per cell line).*p < 0.05 by one-way ANOVA.Data are presented as mean ± SEM. (E) Immunoblot of pSTAT3 Y705, total STAT3 and GAPDH in HPV8 E6 and HPV8 E6 (K136N) transduced N/TERT

resuspended in 50
µL of ChIP buffer and incubated on ice for 10 minutes.For ChIP-qPCR roughly 100 µg of sheared chromatin and 5 µg of antibody were used.Chromatin was sheared to a fragment size of 200-600bp in a microTUBE-50 AFA Fiber Screw-Cap tube (Covaris) in a waterbath sonicator (Covaris).Sheared chromatin was centrifuged at 12000 rpm at 4°C for 10 minutes and supernatant transferred to a new tube.Input material was reserved.The remainder was included in a ChIP reaction (0.2 mL) PCR tube containing pre-incubated (bound) ChIP grade anti-STAT3 antibody (Cell Signalling, 4904), along with anti-RNA Polymerase II and non-Immune IgG which were used as positive and negative ChIP controls, respectively.Reactions were incubated at room temperature for two hours on an orbital shaker.Wells were then washed with wash buffer and DNA extracted using DNA Release Buffer.Cross-links were reversed by incubating with RNase A solution at 42°C for 30 minutes, then adding Proteinase K and incubating for a further 45 minutes at 60°C.DNA solution was transferred to a fresh PCR tube and incubated at 95°C for 15 minutes.DNA was purified by spin columns provided with the kit and eluted in DNA elution buffer.After purification, qPCR was used to analyze immunoprecipitated DNA with the following primers: ΔNp63-719, 5′-CATAGATGCATCACGTGCA-3′ (sense); ΔNp63-464, 5′-GCAATTACAAAATAAGCTACCTG-3′ (antisense) using SYBR Green qPCR Master mix (ThermoFisher Scientific).(1)Since RNA polymerase II is enriched in the GAPDH gene promoter, DNA immunoprecipitated by the RNA polymerase II antibody was used with GAPDH primers as a positive control.The amount of immunoprecipitated DNA was determined as the fraction of the input (amplification efficiency (Ct Input−Ct ChIP) ) and normalized to IgG control.HPV8-E6 data were then plotted as fold change relative vector control.

3 '
(reverse).The outer set flanked 545bp fragment 828 to 1372 and the nested set flanked 440bp fragment 830 to 1269 (GenBank accession no.M12737.1).DNA amplification was performed using GoTaq® G2 Hot Start Taq (Promega, Madison, WI).DNA extracted (2 µL) from each FFPE sample was used for the first PCR round with the outer primers, then 10 µL from each reaction was aliquoted and diluted 10-fold with Milli-Q water and 2 µL was then used in the reaction with nested primers.The total reaction volume in both rounds was 25 µL including 2 µL of templates, 200 hM of primers, 50 µM of each of the four dNTPs and 0.4 U of Taq polymerase (Promega, Madison, WI).Both rounds of PCR were performed as follows: 94°C, 5 minutes as the hot start step and then 30 seconds at 94°C, 1 minute 30 seconds at 60°C, and 1 minute 30 seconds at 72°C, 35 cycles of amplification.Products from both the first and the second round of PCR were electrophoresed on a 2% agarose gel and visualized under UV light after staining with SafeView (NBS Biologicals, Cambridgeshire, UK).A sample was considered positive if 545bp and 440bp fragments were detected in the first and second round respectively.Negative and positive controls were run in each reaction.The DNA quality was monitored by running a b-globin PCR using DNA extracted from each FFPE sample.b-globin primers were as follows: 5'-GAAGAGCCAAGGACAGGTAC-3' (GH20), 5'-CAACTTCATCCACGTTCACC-3' (PC04), 5'-GCTCACTCAGTGTGGCAAAG-3' (RS42), 5'-GGTTGGCCAATCTACTCCCAGG-3' (KM29), 5'-ATTTTCCCACCCTTAGGCTG-3' (RS40), 5'-TGGTAGCTGGATTGTAGCTG-3' (RS80).The PCR conditions for b-globin PCR were identical to the HPV8 PCR.