Dohun Pyeon

Fundamental Differences in Cell Cycle Deregulation in Human Papillomavirus–Positive and Human Papillomavirus–Negative Head/Neck and Cervical Cancers

Human papillomaviruses (HPV) are associated with nearly all cervical cancers, 20% to 30% of head and neck cancers (HNC), and other cancers. Because HNCs also arise in HPV-negative patients, this type of cancer provides unique opportunities to define similarities and differences of HPV-positive versus HPV-negative cancers arising in the same tissue. Here, we describe genome-wide expression profiling of 84 HNCs, cervical cancers, and site-matched normal epithelial samples in which we used laser capture microdissection to enrich samples for tumor-derived versus normal epithelial cells. This analysis revealed that HPV(+) HNCs and cervical cancers differed in their patterns of gene expression yet shared many changes compared with HPV(-) HNCs. Some of these shared changes were predicted, but many others were not. Notably, HPV(+) HNCs and cervical cancers were found to be up-regulated in their expression of a distinct and larger subset of cell cycle genes than that observed in HPV(-) HNC. Moreover, HPV(+) cancers overexpressed testis-specific genes that are normally expressed only in meiotic cells. Many, although not all, of the hallmark differences between HPV(+) HNC and HPV(-) HNC were a direct consequence of HPV and in particular the viral E6 and E7 oncogenes. This included a novel association of HPV oncogenes with testis-specific gene expression. These findings in primary human tumors provide novel biomarkers for early detection of HPV(+) and HPV(-) cancers, and emphasize the potential value of targeting E6 and E7 function, alone or combined with radiation and/or traditional chemotherapy, in the treatment of HPV(+) cancers.

Establishment of Human Papillomavirus Infection Requires Cell Cycle Progression

Human papillomaviruses (HPVs) are DNA viruses associated with major human cancers. As such there is a strong interest in developing new means, such as vaccines and microbicides, to prevent HPV infections. Developing the latter requires a better understanding of the infectious life cycle of HPVs. The HPV infectious life cycle is closely linked to the differentiation state of the stratified epithelium it infects, with progeny virus only made in the terminally differentiating suprabasal compartment. It has long been recognized that HPV must first establish its infection within the basal layer of stratified epithelium, but why this is the case has not been understood. In part this restriction might reflect specificity of expression of entry receptors. However, this hypothesis could not fully explain the differentiation restriction of HPV infection, since many cell types can be infected with HPVs in monolayer cell culture. Here, we used chemical biology approaches to reveal that cell cycle progression through mitosis is critical for HPV infection. Using infectious HPV16 particles containing the intact viral genome, G1-synchronized human keratinocytes as hosts, and early viral gene expression as a readout for infection, we learned that the recipient cell must enter M phase (mitosis) for HPV infection to take place. Late M phase inhibitors had no effect on infection, whereas G1, S, G2, and early M phase cell cycle inhibitors efficiently prevented infection. We conclude that host cells need to pass through early prophase for successful onset of transcription of the HPV encapsidated genes. These findings provide one reason why HPVs initially establish infections in the basal compartment of stratified epithelia. Only this compartment of the epithelium contains cells progressing through the cell cycle, and therefore it is only in these cells that HPVs can establish their infection. By defining a major condition for cell susceptibility to HPV infection, these results also have potentially important implications for HPV control.

APOBEC3A Functions as a Restriction Factor of Human Papillomavirus

ABSTRACT Human papillomaviruses (HPVs) are small DNA viruses causally associated with benign warts and multiple cancers, including cervical and head-and-neck cancers. While the vast majority of people are exposed to HPV, most instances of infection are cleared naturally. However, the intrinsic host defense mechanisms that block the early establishment of HPV infections remain mysterious. Several antiviral cytidine deaminases of the human APOBEC3 (hA3) family have been identified as potent viral DNA mutators. While editing of HPV genomes in benign and premalignant cervical lesions has been demonstrated, it remains unclear whether hA3 proteins can directly inhibit HPV infection. Interestingly, recent studies revealed that HPV-positive cervical and head-and-neck cancers exhibited higher rates of hA3 mutation signatures than most HPV-negative cancers. Here, we report that hA3A and hA3B expression levels are highly upregulated in HPV-positive keratinocytes and cervical tissues in early stages of cancer progression, potentially through a mechanism involving the HPV E7 oncoprotein. HPV16 virions assembled in the presence of hA3A, but not in the presence of hA3B or hA3C, have significantly decreased infectivity compared to HPV virions assembled without hA3A or with a catalytically inactive mutant, hA3A/E72Q. Importantly, hA3A knockdown in human keratinocytes results in a significant increase in HPV infectivity. Collectively, our findings suggest that hA3A acts as a restriction factor against HPV infection, but the induction of this restriction mechanism by HPV may come at a cost to the host by promoting cancer mutagenesis. IMPORTANCE Human papillomaviruses (HPVs) are highly prevalent and potent human pathogens that cause >5% of all human cancers, including cervical and head-and-neck cancers. While the majority of people become infected with HPV, only 10 to 20% of infections are established as persistent infections. This suggests the existence of intrinsic host defense mechanisms that inhibit viral persistence. Using a robust method to produce infectious HPV virions, we demonstrate that hA3A, but not hA3B or hA3C, can significantly inhibit HPV infectivity. Moreover, hA3A and hA3B were coordinately induced in HPV-positive clinical specimens during cancer progression, likely through an HPV E7 oncoprotein-dependent mechanism. Interestingly, HPV-positive cervical and head-and-neck cancer specimens were recently shown to harbor significant amounts of hA3 mutation signatures. Our findings raise the intriguing possibility that the induction of this host restriction mechanism by HPV may also trigger hA3A- and hA3B-induced cancer mutagenesis.

Molecular transitions from papillomavirus infection to cervical precancer and cancer: Role of stromal estrogen receptor signaling

Significance Cervical cancer (CxCa) is the second most frequent cancer in women and the third leading cause of cancer death in women worldwide. Our global analysis of gene expression in normal, precancerous, and cancerous cervical tissue shows increased DNA replication/repair and cell proliferation followed by substantial metabolic shifts. We observed a dramatic, progressive decrease in estrogen receptor alpha (ERα) in tumor progression, and ranking specimens by estrogen-responsive gene expression correlated remarkably with histopathology. Whereas ERα expression shuts off in tumor epithelium, stromal fibroblasts in the microenvironment retain ERα, and the data indicate estrogen-related alteration of several candidate stroma–tumor signaling pathways. Our findings strongly support a role of stromal estrogen signaling in CxCa development with implications for CxCa management and control. To study the multistep process of cervical cancer development, we analyzed 128 frozen cervical samples spanning normalcy, increasingly severe cervical intraepithelial neoplasia (CIN1– CIN3), and cervical cancer (CxCa) from multiple perspectives, revealing a cascade of progressive changes. Compared with normal tissue, expression of many DNA replication/repair and cell proliferation genes was increased in CIN1/CIN2 lesions and further sustained in CIN3, consistent with high-risk human papillomavirus (HPV)-induced tumor suppressor inactivation. The CIN3-to-CxCa transition showed metabolic shifts, including decreased expression of mitochondrial electron transport complex components and ribosomal protein genes. Significantly, despite clinical, epidemiological, and animal model results linking estrogen and estrogen receptor alpha (ERα) to CxCa, ERα expression declined >15-fold from normalcy to cancer, showing the strongest inverse correlation of any gene with the increasing expression of p16, a marker for HPV-linked cancers. This drop in ERα in CIN and tumor cells was confirmed at the protein level. However, ERα expression in stromal cells continued throughout CxCa development. Our further studies localized stromal ERα to FSP1+, CD34+, SMA− precursor fibrocytes adjacent to normal and precancerous CIN epithelium, and FSP1−, CD34−, SMA+ activated fibroblasts in CxCas. Moreover, rank correlations with ERα mRNA identified IL-8, CXCL12, CXCL14, their receptors, and other angiogenesis and immune cell infiltration and inflammatory factors as candidates for ERα-induced stroma–tumor signaling pathways. The results indicate that estrogen signaling in cervical cancer has dramatic differences from ERα+ breast cancers, and imply that estrogen signaling increasingly proceeds indirectly through ERα in tumor-associated stromal fibroblasts.

Prostaglandin E2 Increases Bovine Leukemia Virus tax and pol mRNA Levels via Cyclooxygenase 2: Regulation by Interleukin-2, Interleukin-10, and Bovine Leukemia Virus

ABSTRACT Prostaglandin E2 (PGE2), produced by macrophages, has important immune regulatory functions, suppressing a type 1 immune response and stimulating a type 2 immune response. Type 1 cytokines (interleukin-2 [IL-2], IL-12, and gamma interferon) increase in freshly isolated peripheral blood mononuclear cells (PBMCs) of animals with an early disease stage of bovine leukemia virus (BLV) infection, while IL-10 increases in animals with a late disease stage. Although IL-10 has an immunosuppressive role in the host immune system, IL-10 also inhibits BLV tax and polmRNA levels in vitro. In contrast, IL-2 stimulates BLVtax and pol mRNA and p24 protein expression in cultured PBMCs. The inhibitory effect of IL-10 on BLV expression depends on soluble factors secreted by macrophages. Thus, we hypothesized that PGE2, a cyclooxygenase 2 (COX-2) product of macrophages, may regulate BLV expression. Here, we show that the level of COX-2 mRNA was decreased in PBMCs treated with IL-10, while IL-2 enhanced the level of COX-2 mRNA. Addition of PGE2 stimulated BLV tax and polmRNA levels and reversed the IL-10 inhibition of BLV mRNA. In addition, the specific COX-2 inhibitor, NS-398, inhibited the amount of BLV mRNA detected. Addition of PGE2 increased BLV tax mRNA regardless of NS-398 addition. PGE2 inhibited antigen-specific PBMC stimulation, suggesting that stimulation of BLVtax and pol mRNA levels by PGE2 is independent of cell proliferation. These findings suggest that macrophage-derived COX-2 products, such as PGE2, regulate virus expression and disease progression in BLV infection.

Human papillomavirus infection is inhibited by host autophagy in primary human keratinocytes

Human papillomavirus (HPV) infection is severely limited in its natural host, primary human keratinocytes. Our data show HPV infectivity in primary keratinocytes is over 100- and 1,000-fold lower than in established keratinocyte cell lines NIKS and HaCaT, respectively. Here, we show that the basal level of autophagy in primary human foreskin keratinocytes (HFKs) is higher than in immortalized keratinocytes, and that HPV16 virions significantly induce autophagy in HFKs. Interestingly, HPV16 infectivity is dramatically enhanced by knockdown of essential autophagy genes as well as biochemical inhibition of autophagy. The increase in HPV16 infectivity by autophagy inhibition is most significant in HFKs, showing an inverse correlation with basal HPV16 infectivity in HFK, NIKS, HaCaT, and 293FT cells. Further, inhibition of autophagy delays degradation of HPV16 capsid proteins during virus trafficking, indicating that host autophagy induced by HPV16 virions inhibits infection of primary keratinocytes through rapid degradation of viral capsid proteins.

The antiviral restriction factors IFITM1, 2 and 3 do not inhibit infection of human papillomavirus, cytomegalovirus and adenovirus.

Type I interferons (IFN-α and β) induce dynamic host defense mechanisms to inhibit viral infections. It has been recently recognized that the interferon-inducible transmembrane proteins (IFITM) 1, 2 and 3 can block entry of a broad spectrum of RNA viruses. However, no study to date has focused on the role of IFITM proteins in DNA virus restriction. Here, we demonstrate that IFN-α or -β treatment of keratinocytes substantially decreases human papillomavirus 16 (HPV16) infection while robustly inducing IFITM1, 2 and 3 expression. However, IFITM1, 2 and 3 overexpression did not inhibit HPV16 infection; rather, IFITM1 and IFITM3 modestly enhanced HPV16 infection in various cell types including primary keratinocytes. Moreover, IFITM1, 2 and 3 did not inhibit infection by two other DNA viruses, human cytomegalovirus (HCMV) and adenovirus type 5 (Ad5). Taken together, we reveal that the entry of several DNA viruses, including HPV, HCMV, and Ad5 is not affected by IFITM1, 2 and 3 expression. These results imply that HPV, and other DNA viruses, may bypass IFITM restriction during intracellular trafficking.

Inhibition of nuclear factor-kappa B activation decreases survival of Mycobacterium tuberculosis in human macrophages.

Nuclear factor-kappa B (NFκB) is a ubiquitous transcription factor that mediates pro-inflammatory responses required for host control of many microbial pathogens; on the other hand, NFκB has been implicated in the pathogenesis of other inflammatory and infectious diseases. Mice with genetic disruption of the p50 subunit of NFκB are more likely to succumb to Mycobacterium tuberculosis (MTB). However, the role of NFκB in host defense in humans is not fully understood. We sought to examine the role of NFκB activation in the immune response of human macrophages to MTB. Targeted pharmacologic inhibition of NFκB activation using BAY 11-7082 (BAY, an inhibitor of IκBα kinase) or an adenovirus construct with a dominant-negative IκBα significantly decreased the number of viable intracellular mycobacteria recovered from THP-1 macrophages four and eight days after infection. The results with BAY were confirmed in primary human monocyte-derived macrophages and alveolar macrophages. NFκB inhibition was associated with increased macrophage apoptosis and autophagy, which are well-established killing mechanisms of intracellular MTB. Inhibition of the executioner protease caspase-3 or of the autophagic pathway significantly abrogated the effects of BAY. We conclude that NFκB inhibition decreases viability of intracellular MTB in human macrophages via induction of apoptosis and autophagy.

Evasion of host immune defenses by human papillomavirus

A majority of human papillomavirus (HPV) infections are asymptomatic and self-resolving in the absence of medical interventions. Various innate and adaptive immune responses, as well as physical barriers, have been implicated in controlling early HPV infections. However, if HPV overcomes these host immune defenses and establishes persistence in basal keratinocytes, it becomes very difficult for the host to eliminate the infection. The HPV oncoproteins E5, E6, and E7 are important in regulating host immune responses. These oncoproteins dysregulate gene expression, protein-protein interactions, posttranslational modifications, and cellular trafficking of critical host immune modulators. In addition to the HPV oncoproteins, sequence variation and dinucleotide depletion in papillomavirus genomes has been suggested as an alternative strategy for evasion of host immune defenses. Since anti-HPV host immune responses are also considered to be important for antitumor immunity, immune dysregulation by HPV during virus persistence may contribute to immune suppression essential for HPV-associated cancer progression. Here, we discuss cellular pathways dysregulated by HPV that allow the virus to evade various host immune defenses.

Suppression of Antitumor Immune Responses by Human Papillomavirus through Epigenetic Downregulation of CXCL14

ABSTRACT High-risk human papillomaviruses (HPVs) are causally associated with multiple human cancers. Previous studies have shown that the HPV oncoprotein E7 induces immune suppression; however, the underlying mechanisms remain unknown. To understand the mechanisms by which HPV deregulates host immune responses in the tumor microenvironment, we analyzed gene expression changes of all known chemokines and their receptors using our global gene expression data sets from human HPV-positive and -negative head/neck cancer and cervical tissue specimens in different disease stages. We report that, while many proinflammatory chemokines increase expression throughout cancer progression, CXCL14 is dramatically downregulated in HPV-positive cancers. HPV suppression of CXCL14 is dependent on E7 and associated with DNA hypermethylation in the CXCL14 promoter. Using in vivo mouse models, we revealed that restoration of Cxcl14 expression in HPV-positive mouse oropharyngeal carcinoma cells clears tumors in immunocompetent syngeneic mice, but not in Rag1-deficient mice. Further, Cxcl14 reexpression significantly increases natural killer (NK), CD4+ T, and CD8+ T cell infiltration into the tumor-draining lymph nodes in vivo. In vitro transwell migration assays show that Cxcl14 reexpression induces chemotaxis of NK, CD4+ T, and CD8+ T cells. These results suggest that CXCL14 downregulation by HPV plays an important role in suppression of antitumor immune responses. Our findings provide a new mechanistic understanding of virus-induced immune evasion that contributes to cancer progression. IMPORTANCE Human papillomaviruses (HPVs) are causally associated with more than 5% of all human cancers. During decades of cancer progression, HPV persists, evading host surveillance. However, little is known about the immune evasion mechanisms driven by HPV. Here we report that the chemokine CXCL14 is significantly downregulated in HPV-positive head/neck and cervical cancers. Using patient tissue specimens and cultured keratinocytes, we found that CXCL14 downregulation is linked to CXCL14 promoter hypermethylation induced by the HPV oncoprotein E7. Restoration of Cxcl14 expression in HPV-positive cancer cells clears tumors in immunocompetent syngeneic mice, but not in immunodeficient mice. Mice with Cxcl14 reexpression show dramatically increased natural killer and T cells in the tumor-draining lymph nodes. These results suggest that epigenetic downregulation of CXCL14 by HPV plays an important role in suppressing antitumor immune responses. Our findings may offer novel insights to develop preventive and therapeutic tools for restoring antitumor immune responses in HPV-infected individuals. Human papillomaviruses (HPVs) are causally associated with more than 5% of all human cancers. During decades of cancer progression, HPV persists, evading host surveillance. However, little is known about the immune evasion mechanisms driven by HPV. Here we report that the chemokine CXCL14 is significantly downregulated in HPV-positive head/neck and cervical cancers. Using patient tissue specimens and cultured keratinocytes, we found that CXCL14 downregulation is linked to CXCL14 promoter hypermethylation induced by the HPV oncoprotein E7. Restoration of Cxcl14 expression in HPV-positive cancer cells clears tumors in immunocompetent syngeneic mice, but not in immunodeficient mice. Mice with Cxcl14 reexpression show dramatically increased natural killer and T cells in the tumor-draining lymph nodes. These results suggest that epigenetic downregulation of CXCL14 by HPV plays an important role in suppressing antitumor immune responses. Our findings may offer novel insights to develop preventive and therapeutic tools for restoring antitumor immune responses in HPV-infected individuals.