HPV8 Field Cancerization in a Transgenic Mouse Model Is due to Lrig1+ Keratinocyte Stem Cell Expansion

Abstract

β-Human papillomaviruses (HPVs) cause near ubiquitous latent skin infection within long-lived hair follicle (HF) keratinocyte stem cells. In patients with epidermodysplasia verruciformis, β-HPV viral replication is associated with skin keratosis and cutaneous squamous cell carcinoma. To determine the role of HF keratinocyte stem cells in β-HPV-induced skin carcinogenesis, we utilized a transgenic mouse model in which the keratin 14 promoter drives expression of the entire HPV8 early region (HPV8tg). HPV8tg mice developed thicker skin in comparison with wild-type littermates consistent with a hyperproliferative epidermis. HF keratinocyte proliferation was evident within the Lrig1+ keratinocyte stem cell population (69 vs. 55%, P < 0.01, n = 7), and not in the CD34+, LGR5+, and LGR6+ keratinocyte stem cell populations. This was associated with a 2.8-fold expansion in Lrig1+ keratinocytes and 3.8-fold increased colony-forming efficiency. Consistent with this, we observed nuclear p63 expression throughout this population and the HF infundibulum and adjoining interfollicular epidermis, associated with a switch from p63 transcriptional activation isoforms to ΔNp63 isoforms in HPV8tg skin. Epidermodysplasia verruciformis keratosis and in some cases actinic keratoses demonstrated similar histology associated with β-HPV reactivation and nuclear p63 expression within the HF infundibulum and perifollicular epidermis. These findings would suggest that β-HPV field cancerization arises from the HF junctional zone and predispose to squamous cell carcinoma.

Figure 1

Phenotypic and histological characterization of HPV8tg mice. (a) Schematic representation of the HPV8 transgenes, showing the human cytokeratin-14 gene promoter upstream of the open reading frames of the HPV8 early region genes. (b) Hematoxylin and eosin staining of paraffin-embedded skin sections from WT (left panel) and HPV8tg (right panel) mouse skin. (c) Toluidine blue staining of mouse whole mount skin including the hair follicle and overlying epidermis from WT (left panel) and HPV8tg (right panel) from the tail. (d) Ear thickness (upper graph) and tail width (bottom graph) measured using calipers on age-matched WT and HPV8tg littermates (n = 8), with mean ± SD (*P < 0.05; unpaired t test). All the images were processed using ImageJ software (National Institutes of Health, Bethesda, MD). All scale bars = 100 μm. HPV, human papillomavirus; SD, standard deviation; WT, wild type.

Figure 2

HPV8 transgenes induce hair follicle changes in HPV8tg mice. (a, b) Adult mice whole mount skin was photographed and analyzed for the area of HF regions and length, WT and HPV8tg were compared, with mean ± SD, using an unpaired t test (n = 20, **P < 0.01). (c) FACS analysis WT and HPV8tg mice skin keratinocyte isolates (n = 6), labeled with Lrig1-FITC and CD34-647 antibodies, with DAPI to select live cells. (d) The number of Lrig1 positive cells determined by FACS (n = 6). (**P < 0.01; unpaired t test), with mean ± SD. (e) Whole mount immunofluorescence of adult WT and HPV8tg tail skin for Ki67 (red) and HF-KSC markers (green). All the images were processed using ImageJ software (National Institutes of Health, Bethesda, MD). All scale bars = 100 μm. HF, hair follicle; HPV, human papillomavirus; KSC, keratinocyte stem cell; SD, standard deviation; WT, wild type.

Figure 3

Lrig1 keratinocyte stem cell proliferation in HPV8tg. (a) Whole mount adult WT and HPV8tg (n = 6) skin sections labeled with Ki67 (red), Lrig1 (green) antibodies, and DAPI (blue). (b) Number of proliferating cells within KSC populations (Lrig1, LGR6, CD34, and LGR5) was enumerated, with mean ± SD, in WT and HPV8tg tissue sections (n = 7); **P < 0.01. (c) Keratinocyte colony-forming assays from flow sorted Lrig1 cells from WT and HPV8tg (n = 6) skin dissociates, with mean ± SD. (d) A representative image. (e) p63 labeled frozen sections from adult WT and HPV8tg mice (n = 3); the broken line indicates the basal layer. Immuno-labeled tissue sections were visualized and photographed by a fluorescent microscope with ×20 magnification and then processed using ImageJ software (National Institutes of Health, Bethesda, MD). All scale bars = 100 μm. HF, hair follicle; HPV, human papillomavirus; KSC, keratinocyte stem cell; SD, standard deviation; WT, wild type.

Figure 4

HPV8 transgenes are uniformly expressed in the epidermis of HPV8tg mice. (a) qRT-PCR of early region genes of WT and HPV8tg mouse skin isolated KSC populations, with mean ± SD. (b) qRT-PCR of murine homologs of EVER1 (TMC6) and EVER2 (TMC8) genes of WT and HPV8tg mouse skin isolated Lrig1+ KSC populations, with mean ± SD. HPV, human papillomavirus; KSC, keratinocyte stem cell; qRT-PCR, real-time quantitative reverse transcription analysis; SD, standard deviation; WT, wild type.

Figure 5

β-HPV-induced keratinocyte stem cell expansion results in keratosis that are predisposed to SCC. (a, b) Reverse transcription-PCR with mean ± SD (**P < 0.01) and (c) western blot gels of p63 isoforms from RNA and protein isolates respectively from WT, HPV8tg skin, papilloma (pap), and SCC (n = 6). (d) Images of tissue sections of HPV8tg mouse skin, human EV keratosis, and AK labeled with p63 (green), β-HPV E4 (red) specific antibodies, and DAPI (blue). (e) Images of tissue sections of HPV8tg mouse and human SCCs labeled with K14 (red), p63 (green) specific antibodies, and DAPI (blue). Immuno-labeled tissue sections were visualized and photographed by fluorescent microscope with ×20 magnification and then processed using ImageJ software (National Institutes of Health, Bethesda, MD). All scale bars = 100 μm. EV, epidermodysplasia verruciformis; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; HPV, human papillomavirus; SCC, cutaneous squamous cell carcinoma; SD, standard deviation; WT, wild type.

Figure 6

β-HPV field cancerization model. The mouse HF (left image) is composed of at least four different KSC populations. In the WT HF, the LGR5 is expressed in the hair bulb, CD34+ and K15+ KSC are located in the bulge, and the Lrig1 and LGR6 KSC are located in the upper and lower junctional zone, respectively. In HPV8tg, the Lrig1 KSC population expands beyond the junctional zone niche and no longer expresses Lrig1, but it maintains KSC function. Similarly, in human β-HPV reactivation (right image), the expanded population occupies the hair follicle infundibulum and adjoining interfollicular epidermis, as shown by the yellow arrow. These changes culminate in the histological phenotype known as Freudenthal’s funnel, the pathognomonic finding in actinic keratosis and skin field cancerization. HF, hair follicle; HPV, human papillomavirus; KSC, keratinocyte stem cell; WT, wild type.