Skin cancer incidence has rapidly increased over the years and is generally divided into malignant melanoma and non-melanoma skin cancer (NMSC), the latter being subdivided into cutaneous squamous cell carcinoma (SCC) and basal cell carcinoma [1]. To date, more than 200 human papillomavirus (HPV) genotypes have been discovered; these viruses can infect the skin and mucosa of several animal species [2]. HPVs are ubiquitous DNA viruses, and HPV infection, especially with high-risk subtypes, can mediate skin tumorigenesis [3]. HPV8, belonging to beta-genus HPV, is suspected to be carcinogenic in some skin cancer types, but the underlying pathological mechanisms of these cancers remain poorly understood [4].

HPV8 E6 and E7 reportedly promote the autophagic degradation of cellular checkpoint kinase-1, which may contribute to the oncogenic potential of the virus [5]. Furthermore, HPV8 E7 expression in the murine epidermis under the control of keratin-14 promoter showed that the E7 protein acts carcinogenically in mice by inducing the invasion of basal keratinocytes, which are regulated by the extracellular protein fibronectin [6]. However, the potential molecular mechanisms and the downstream transcriptional modulation effects of HPV8 E7-infected keratinocytes remain unclear [7, 8].

Changes in the epigenetic modifications, immune response, and metabolic status are associated with tumor occurrence due to HPV infection [9]. For example, clear CpG island methylation occurs in HPV-induced cutaneous warts [10]. Furthermore, recent studies have suggested that the HPV oncogenes E5, E6, and E7 play critical roles in the development of chronic inflammation through various mechanisms and can affect the redox homeostasis of host cells, inducing oxidative stress, which may promote viral integration and cancer development [11, 12]. Moreover, the oncoproteins HPV16 E6 and E7 regulate the cell metabolism to satisfy the energy demands necessary for persistent infection and development of cervical cancer [13]. However, the gene expression profiles of the oncogene HPV8 E7 in human epidermal keratinocytes remain unclear.

Currently, RNA-sequencing technology is widely used to detect transcriptome profiling in cancer research and therapy such as biomarker discovery and cancer heterogeneity, drug resistance, cancer microenvironment, and immunotherapy [14]. However, the transcriptome signatures in HPV8 E7-infected epithelial cells remain unclear. In our study, we took advantage of RNA-sequencing technology and the enrichment analysis of gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) and discovered numerous significantly altered gene transcriptional landscapes that could be clustered into epigenetic modifications, immune regulations, and metabolic reprogramming. This study will elucidate the understanding of the tumorigenic mechanisms of HPV8 E7 expression.

HPV infection, especially with high-risk subtypes, is associated with SCCs that arise in several different organs but share many common biological and immunological properties [18]. In cervical and anal cancers, HPV is the etiology for nearly 90% of malignancies, whereas only 30% of oropharyngeal, vaginal, vulvar, and penile cancers are driven by carcinogen exposure or other causes [19]. Mechanistically, HPV oncogenes not only directly inhibit the tumor suppressors p53 and Rb, but also suppress apoptosis and telomere shortening and promote genomic instability, angiogenesis, invasion, and metastasis to facilitate malignant transformation [20–22]. These properties render HPV infection, induced by factors other than sunlight exposure/UV light, or chemical irritants, an ideal model for the study of the potential mechanisms of HPV-associated cancers.

The functional evaluation of HPVs is far from complete, but currently, the two viral oncoproteins E6 and E7 have been the focus [23]. The HPV8 E6 oncoprotein mainly impairs the DNA damage repair pathway to facilitate carcinogenesis [24]. Moreover, carcinogenic effects are observed in epidermis HPV8 E7 oncoprotein-overexpressing transgenic mice [6]. In vitro, HPV8 E6 counters the cellular response to DNA damage and mitotic errors by destabilizing the functions of P300, a histone acetyltransferase [25]. Furthermore, a recent study finds that HPV8 E7-expressing human primary keratinocytes increase GADD34 and GDF15 expression to facilitate skin tumorigenesis by activating the Src kinase family members Fyn and Lyn [26]. Unfortunately, although the HPV8 E7 oncogene has been identified in more than 90% of SCCs and has been implicated in NMSCs, its exact role in human skin carcinogenesis has not been fully elucidated [3, 27, 28]. In this study, we took advantage of RNA-sequencing technology and the enrichment analysis of GO and KEGG pathways and found that 831 genes were significantly altered upon HPV8 E7 overexpression. Most of the DEGs could be clustered into nucleosomes, nucleosome assembly, viral carcinogenesis pathway, transcriptional misregulation in cancer, and necroptosis pathways. These are the first in vitro mechanistic data on the tumorigenicity of HPV8 E7 and may shed new light on the tumorigenic effects of HPV8 E7 expression.

The GO resource (http://geneontology.org) is a structured, computable database on gene function [29]. In the present study, most HPV8 E7 overexpression-induced genes could be clustered into protein heterodimerization activity and DNA binding (molecular function), nucleosomes (cellular component), and nucleosome assembly (biological process). Our results support the hypothesis that viral proteins interact and manipulate not only cytoplasmic components but also nuclear factors for their survival and propagation [30, 31]. The KEGG pathway database is a reference knowledge base that helps reconstruct molecular network systems [32]. In this study, the KEGG pathway enrichment analysis revealed that HPV8 E7 overexpression mainly affected gene expression in the viral carcinogenesis pathway, transcriptional misregulation in cancer, and necroptosis pathways, which were either important for HPV infection-driven carcinogenesis or could be an important marker for diagnosis and prognosis in HPV-related cancer [33, 34].

Metabolic reprogramming is an important hallmark in cancer [13]. However, the mechanisms of how HPV viral oncoproteins alter cell metabolism to satisfy their own energy demands and how this may contribute to tumorigenesis remain unknown [35]. Interestingly, the spare mitochondrial respiratory capacity of HPV8 E7-expressing cells is markedly increased, and this effect may be due to the direct interaction between HPV8 E7 and the mitochondrial ATP-synthase, which would dramatically result in decreased glycolytic activity [35]. Furthermore, the function of cell metabolism biomolecules, such as glucose, amino acids, and lipids, has been altered to promote cell proliferation, inhibit cell death, and affect genomic instability after HPV infection [9]. Indeed, in the current study, we not only detected DEG profiles in glycolysis pathways in HPV8 E7-expressing NHEKs, but also clustered DEGs into glycerophospholipid metabolism and inositol phosphate metabolism signaling pathways. Glycerophospholipids, the major components of cell membranes, play an important role in viral infection and are crucial for the morphogenesis of progeny virus and viral pathogenicity [36, 37]. Moreover, the G/G genotype and G allele of the inositol 1,4,5-trisphosphate 3-kinase C rs28493229 polymorphism may raise the risk of development of cervical SCC, and could be a potential marker for genetic susceptibility to CSCC [38].

It has been reported viral proteins can regulate epigenetic modifications to affect biological functions, such as cell proliferation and apoptosis [39]. Epigenetic mechanisms related to HPV infection include methylation changes to host and viral DNA and chromatin modification in host species [40]. In this study, we detected 10 epigenetic-associated genes significantly upregulated upon HPV8 E7 overexpression in epidermal keratinocytes, and these DEGs were clustered into DNA and RNA methylation, histone acetylation and deacetylation, and histone methylation and demethylation. HPV E7 promotes the expression of SUV39H1, which is a chromatin repressor and can inhibit the mRNA levels of RIG-I, cGAS, and STING genes by epigenetic silencing, and thereby blocks the interferon secretion in HPV-transformed cells [41]. Furthermore, changed methylated CpGs and regions reportedly regulate gene expression and affect prognosis in HPV-positive head and neck SCC [42]. Further, the epigenetic regulators histone deacetylases (1 and 2) are overexpressed in oncoprotein E6/E7-expressing cells, and histone deacetylase inhibitors could be a therapeutic intervention for skin cancer induced by HPV [43, 44]. An epigenome-wide analysis of HPV-driven common warts reveals that H3F3A, CDKN1A, and MAPK13 are the most differentially methylated genes [45]. Furthermore, HPV viral proteins can directly target histone methyltransferases for activity inhibition and modulate specific gene expression [39].

While the association between global immune compromise and HPV-related malignancies is well established, our understanding of how HPV perpetuates a state of immune suppression in the microenvironment remains limited [18]. Viral oncoproteins can control various cellular circuit programs, such as innate immunity to promote the proliferation of HPV-transformed cells [41]. However, the mechanisms of how persistent HPV8 infection affects the immune response to promote the carcinogenic process remain unknown. HPV E7 promotes the expression of H3K9-specific methyltransferase, which can regulate the host innate immune response to facilitate immune evasion [32]. However, in this study, we found many acute immune response genes, such as TNFAIP3, and those encoding interleukins and chemokines expression in HPV8 E7-expressing NHEKs, which were associated with the clearance of HPV infection.

However, the present study only provided the transcriptional regulation landscape of HPV8 E7-expressing NHEKs; our study limitation is that data interpretation is based only on an in vitro experiment and that it provides only theoretical evidence. Further, these experiments were conducted on a single keratinocyte, and whether the same results can be obtained from normal keratinocytes of other species remains uncertain; therefore, the exact tumorigenic mechanisms of HPV8 E7 need to be further corroborated.

In conclusion, our results demonstrated the pro-carcinogenic effect of HPV8 expression in epithelial cells which could contribute to the regulatory effect of oncogene E7 on gene expression associated with nucleosomes, nucleosome assembly, viral carcinogenesis pathway, transcriptional misregulation in cancer, necroptosis pathways, epigenetic modifications, immune response, and metabolic status. Although our data are based on an in vitro experiment, it provides more effective evidence that the development of squamous cell carcinoma can be caused by HPV.