Summary To define the cellular composition and architecture of cutaneous squamous cell carcinoma (cSCC), we combined single-cell RNA sequencing with spatial transcriptomics and multiplexed ion beam imaging from a series of human cSCCs and matched normal skin. cSCC exhibited four tumor subpopulations, three recapitulating normal epidermal states, and a tumor-specific keratinocyte (TSK) population unique to cancer, which localized to a fibrovascular niche. Integration of single-cell and spatial data mapped ligand-receptor networks to specific cell types, revealing TSK cells as a hub for intercellular communication. Multiple features of potential immunosuppression were observed, including T regulatory cell (Treg) co-localization with CD8 T cells in compartmentalized tumor stroma. Finally, single-cell characterization of human tumor xenografts and in vivo CRISPR screens identified essential roles for specific tumor subpopulation-enriched gene networks in tumorigenesis. These data define cSCC tumor and stromal cell subpopulations, the spatial niches where they interact, and the communicating gene networks that they engage in cancer.

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Cutaneous beta-papillomaviruses are associated with non-melanoma skin cancers that arise in patients who suffer from a rare genetic disorder, Epidermodysplasia verruciformis (EV) or after immunosuppression following organ transplantation. Recent studies have shown that the E6 proteins of the cancer associated beta human papillomavirus (HPV) 5 and HPV8 inhibit NOTCH and TGF-β signaling. However, it is unclear whether disruption of these pathways may contribute to cutaneous HPV pathogenesis and carcinogenesis. A recently identified papillomavirus, MmuPV1, infects laboratory mouse strains and causes cutaneous skin warts that can progress to squamous cell carcinoma. To determine whether MmuPV1 may be an appropriate model to mechanistically dissect the molecular contributions of cutaneous HPV infections to skin carcinogenesis, we investigated whether MmuPV1 E6 shares biological and biochemical activities with HPV8 E6. We report that the HPV8 and MmuPV1 E6 proteins share the ability to bind to the MAML1 and SMAD2/SMAD3 transcriptional cofactors of NOTCH and TGF-beta signaling, respectively. Moreover, we demonstrate that these cutaneous papillomavirus E6 proteins inhibit these two tumor suppressor pathways and that this ability is linked to delayed differentiation and sustained proliferation of differentiating keratinocytes. Furthermore, we demonstrate that the ability of MmuPV1 E6 to bind MAML1 is necessary for papilloma formation in experimentally infected mice. Our results, therefore, suggest that experimental MmuPV1 infection in mice will be a robust and useful experimental system to model key aspects of cutaneous HPV infection, pathogenesis and carcinogenesis.

We present the first disinfectant susceptibility data on HPV16 native virions, which show that commonly used clinical disinfectants, including those used as sterilants in medical and dental healthcare facilities, have no effect on HPV16 infectivity. Policy changes concerning disinfectant use are needed. The unusually high resistance of HPV16 to disinfection supports other data suggesting the possibility of fomite or non-sexual transmission of HPV16.

Most DNA viruses associate with, and reorganize, nuclear domain 10 (ND10) bodies upon entry into the host nucleus. In this study, we examine the roles of the ND10 components PML, Sp100, and Daxx in the establishment of human papillomavirus type 18 (HPV18) infection of primary human keratinocytes. HPV18 DNA or HPV18 quasivirus was introduced into primary human keratinocytes depleted of each ND10 protein by small interfering RNA technology, and genome establishment was determined by using a quantitative immortalization assay and measurements of viral transcription and DNA replication. Keratinocyte depletion of Sp100 resulted in a substantial increase in the number of HPV18-immortalized colonies and a corresponding increase in viral transcription and DNA replication. However, Sp100 repressed viral transcription and replication only during the initial stages of viral establishment, suggesting that Sp100 acts as a repressor of incoming HPV DNA.

A small group of human papillomaviruses (HPVs) cause almost all cervical carcinoma and a significant percentage of other anogenital tract and oral carcinoma. Another group of HPVs causes non-melanoma skin cancers in genetically predisposed or immune suppressed patients upon UV exposure. HPV genome replication requires the host cell’s DNA synthesis machinery and HPVs encode proteins that maintain differentiated epithelial cells in a replication competent state. The resulting rewiring of cellular signal transduction circuits triggers several innate cellular tumor suppressor responses that HPVs need to inactivate in order to establish persistent and/or productive infections. This review emphasizes this interplay between virus and the infected host cells and points out biological similarities and differences between different groups of HPVs.

Cutaneous ␤-human papillomavirus (␤-HPV) E6 proteins inhibit NOTCH signaling by associating with the transcriptional coactivator MAML1. NOTCH has tumor suppressor activities in epithelial cells and is activated during keratinocyte differentiation. Here we report that HPV type 8 (HPV8) E6 subverts NOTCH activation during keratinocyte differentiation by inhibiting RBPJ/MAML1 transcriptional activator complexes at NOTCH target DNA. NOTCH inhibition impairs epithelial differentiation and may thus contribute to ␤-HPV replication and viral oncogenesis. Papillomaviridae are a large family of epitheliotropic viruses with double-stranded circular ϳ8-kb DNA genomes. More than 150 human papillomaviruses (HPVs) have been isolated (1). Some HPVs infect mucosal epithelia, whereas other HPVs infect cutaneous epithelial cells. HPV infections can be nonsymptomatic or cause hyperplastic skin lesions, commonly referred to as warts. Mucosal ␣-HPVs have been studied in great detail since some of these viruses, the "high-risk" HPVs, are the causative agents of cervical carcinoma as well as other anogenital cancers and oropharyngeal carcinomas. Cutaneous ␤-HPVs are less studied. While ␤-HPVs appear to be part of the normal "skin flora" and infections are often asymptomatic, they can cause cutaneous warts, which undergo malignant progression in patients suffering from the rare genetic disease epidermodysplasia verruciformis (EV) or in systemically immune-suppressed organ transplant patients, particularly in sun-exposed areas of the body (2-5). ␤-HPVs may also contribute to nonmelanoma skin cancers (NMSCs) in patients with a normal immune system, but this has not been formally proven.Cutaneous ␤-HPV E6 proteins have intrinsic transforming activities (6, 7) but do not associate with known cellular target proteins of high-risk mucosal ␣-HPV E6 proteins, including p53, UBE3A, and cellular PDZ proteins (8). Several groups have recently reported that the E6 proteins from HPV5, HPV8, and other ␤-HPVs can bind the NOTCH coactivator mastermind-like 1 (MAML1), thereby inhibiting NOTCH transcriptional programs (9-11). This is particularly interesting since NOTCH functions as a tumor suppressor in epithelial cells and mice with expression of a dominant negative MAML in basal epithelial cells develop squamous cell carcinoma (12). Since NOTCH signaling is important for cellular differentiation, we wanted to determine whether HPV8 E6 inhibits NOTCH signaling during keratinocyte differentiation. To experimentally address this issue, we generated populations of human keratinocytes immortalized with human telomerase reverse transcriptase (hTERT) (cl398) (13) and stably expressing amino-terminally hemagglutinin (HA)/FLAG epitopetagged HPV8 E6 (8E6-iHFKs) by lentiviral transduction. Vector-infected cells were generated as controls (C-iHFKs). To confirm the interaction of HPV8 E6 with components of the NOTCH transcriptional complex in these cells, we prepared lysates of 8E6-iHFKs and C-iHFKs in mammalian cell lysis buffer (MCLB; 50 mM Tris [pH 7.5], 150 mM...

Infections with cutaneous papillomaviruses have been linked to cutaneous squamous cell carcinomas that arise in patients who suffer from a rare genetic disorder, epidermodysplasia verruciformis, or those who have experienced long-term, systemic immunosuppression following organ transplantation. The E6 proteins of the prototypical cutaneous human papillomavirus (HPV) 5 and HPV8 inhibit TGF-β and NOTCH signaling. The Mus musculus papillomavirus 1, MmuPV1, infects laboratory mouse strains and causes cutaneous skin warts that can progress to squamous cell carcinomas. MmuPV1 E6 shares biological and biochemical activities with HPV8 E6 including the ability to inhibit TGF-β and NOTCH signaling by binding the SMAD2/SMAD3 and MAML1 transcription factors, respectively. Inhibition of TGF-β and NOTCH signaling is linked to delayed differentiation and sustained proliferation of differentiating keratinocytes. Furthermore, the ability of MmuPV1 E6 to bind MAML1 is necessary for wart and cancer formation in experimentally infected mice. Hence, experimental MmuPV1 infection in mice will be a robust and valuable experimental system to dissect key aspects of cutaneous HPV infection, pathogenesis, and carcinogenesis.