Human papillomavirus type 38 alters wild-type p53 activity to promote cell proliferation via the downregulation of integrin alpha 1 expression.

Tumor suppressors can exert pro-proliferation functions in specific contexts. In the beta human papillomavirus type 38 (HPV38) experimental model, the viral proteins E6 and E7 promote accumulation of a wild-type (WT) p53 form in human keratinocytes (HKs), promoting cellular proliferation. Inactivation of p53 by different means strongly decreases the proliferation of HPV38 E6/E7 HKs. This p53 form is phosphorylated at S392 by the double-stranded RNA-dependent protein kinase PKR, which is highly activated by HPV38. PKR-mediated S392 p53 phosphorylation promotes the formation of a p53/DNMT1 complex, which inhibits expression of integrin alpha 1 (ITGA1), a repressor of epidermal growth factor receptor (EGFR) signaling. Ectopic expression of ITGA1 in HPV38 E6/E7 HKs promotes EGFR degradation, inhibition of cellular proliferation, and cellular death. Itga1 expression was also inhibited in the skin of HPV38 transgenic mice that have an elevated susceptibility to UV-induced skin carcinogenesis. In summary, these findings reveal the existence of a specific WT p53 form that displays pro-proliferation properties.

An Emerging Issue in Oncogenic Virology: the Role of Beta Human Papillomavirus Types in the Development of Cutaneous Squamous Cell Carcinoma.

Evidence suggests that beta human papillomaviruses (HPVs), together with ultraviolet radiation, contribute to the development of cutaneous squamous cell carcinoma. Beta HPVs appear to be not the main drivers of carcinogenesis but rather facilitators of the accumulation of ultraviolet-induced DNA mutations. Beta HPVs are promoters of skin carcinogenesis, although they are dispensable for the maintenance of the malignant phenotype. Therefore, beta HPV represents a target for skin cancer prevention, especially in high-risk populations.

Beta HPV38 oncoproteins act with a hit-and-run mechanism in ultraviolet radiation-induced skin carcinogenesis in mice.

Cutaneous beta human papillomavirus (HPV) types are suspected to be involved, together with ultraviolet (UV) radiation, in the development of non-melanoma skin cancer (NMSC). Studies in in vitro and in vivo experimental models have highlighted the transforming properties of beta HPV E6 and E7 oncoproteins. However, epidemiological findings indicate that beta HPV types may be required only at an initial stage of carcinogenesis, and may become dispensable after full establishment of NMSC. Here, we further investigate the potential role of beta HPVs in NMSC using a Cre-loxP-based transgenic (Tg) mouse model that expresses beta HPV38 E6 and E7 oncogenes in the basal layer of the skin epidermis and is highly susceptible to UV-induced carcinogenesis. Using whole-exome sequencing, we show that, in contrast to WT animals, when exposed to chronic UV irradiation K14 HPV38 E6/E7 Tg mice accumulate a large number of UV-induced DNA mutations, which increase proportionally with the severity of the skin lesions. The mutation pattern detected in the Tg skin lesions closely resembles that detected in human NMSC, with the highest mutation rate in p53 and Notch genes. Using the Cre-lox recombination system, we observed that deletion of the viral oncogenes after development of UV-induced skin lesions did not affect the tumour growth. Together, these findings support the concept that beta HPV types act only at an initial stage of carcinogenesis, by potentiating the deleterious effects of UV radiation.

Correction: E6 and E7 from Beta Hpv38 Cooperate with Ultraviolet Light in the Development of Actinic Keratosis-Like Lesions and Squamous Cell Carcinoma in Mice.

[This corrects the article DOI: 10.1371/journal.ppat.1002125.].

Human papillomavirus type 38 E6 and E7 act as tumour promoters during chemically induced skin carcinogenesis.

Many findings support a possible involvement of a subgroup of human papillomaviruses (HPVs), called cutaneous beta HPV types, in the development of non-melanoma skin cancer. The skin of transgenic (Tg) mice expressing viral oncoproteins E6 and E7 from different cutaneous beta HPV types, including HPV38, showed an increased susceptibility to UV-induced and/or chemically induced skin carcinogenesis compared with wild-type animals. In this study, we show that beta HPV38 E6 and E7 oncoproteins act as promoter and progression factors in multi-stage skin carcinogenesis, strongly cooperating with the initiator and DNA damage agent 7,12-dimethylbenz[a]anthracene. In contrast, exposure of HPV38 E6/E7 Tg mice to the promoter 12-O-tetradecanoylphorbol-13-acetate did not significantly result in the development of skin lesions. These findings further support the role of beta HPV types in skin carcinogenesis, providing additional insight into their precise contribution to the multi-step process.

E6 and E7 from beta HPV38 cooperate with ultraviolet light in the development of actinic keratosis-like lesions and squamous cell carcinoma in mice.

Cutaneous beta human papillomavirus (HPV) types appear to be involved in the development of non-melanoma skin cancer (NMSC); however, it is not entirely clear whether they play a direct role. We have previously shown that E6 and E7 oncoproteins from the beta HPV type 38 display transforming activities in several experimental models. To evaluate the possible contribution of HPV38 in a proliferative tissue compartment during carcinogenesis, we generated a new transgenic mouse model (Tg) where HPV38 E6 and E7 are expressed in the undifferentiated basal layer of epithelia under the control of the Keratin 14 (K14) promoter. Viral oncogene expression led to increased cellular proliferation in the epidermis of the Tg animals in comparison to the wild-type littermates. Although no spontaneous formation of tumours was observed during the lifespan of the K14 HPV38 E6/E7-Tg mice, they were highly susceptible to 7,12-dimethylbenz(a)anthracene (DMBA)/12-0-tetradecanoylphorbol-13-acetate (TPA) two-stage chemical carcinogenesis. In addition, when animals were exposed to ultraviolet light (UV) irradiation, we observed that accumulation of p21(WAF1) and cell-cycle arrest were significantly alleviated in the skin of Tg mice as compared to wild-type controls. Most importantly, chronic UV irradiation of Tg mice induced the development of actinic keratosis-like lesions, which are considered in humans as precursors of squamous cell carcinomas (SCC), and subsequently of SCC in a significant proportion of the animals. In contrast, wild-type animals subjected to identical treatments did not develop any type of skin lesions. Thus, the oncoproteins E6 and E7 from beta HPV38 significantly contribute to SCC development in the skin rendering keratinocytes more susceptible to UV-induced carcinogenesis.

HPV38 impairs UV-induced transcriptional activation of the IL-18 pro-inflammatory cytokine.

IMPORTANCE: Here, we demonstrate that the direct binding of p53 on the IL-18 promoter region regulates its gene expression. However, the presence of E6 and E7 from human papillomavirus type 38 impairs this mechanism via a new inhibitory complex formed by DNA methyltransferase 1 (DNMT1)/PKR/ΔNp73α, which binds to the region formerly occupied by p53 in primary keratinocytes.

Detection of stage I HPV-driven oropharyngeal cancer in asymptomatic individuals in the Hamburg City Health Study using HPV16 E6 serology - A proof-of-concept study.

Background: The lack of detectable precancerous lesions poses challenges to the early detection of human papillomavirus-driven oropharyngeal cancer (HPV-OPC). Antibodies against HPV16 early proteins, especially E6, are uniquely sensitive and specific biomarkers detectable years prior to HPV-OPC diagnosis. Thus, HPV16 early protein serology warrants clinical investigation for HPV-OPC screening. Methods: Using multiplex serology, we analyzed HPV16 serum antibodies of the first 5000 participants (n=4,424 sera, recruited 2016-2017) of the Hamburg City Health Study, a population-based prospective cohort (45-74 years). Participants seropositive for HPV16 E6 and at least one additional early protein (E1, E2, E7) were considered at high risk for HPV-OPC development and invited to six-monthly non-invasive head and neck follow-up (FU) examinations (visual inspection, endoscopy, ultrasonography, performed 2019-2020). Participants with suspicious lesions were examined by magnetic resonance imaging and panendoscopy with biopsy. Histologically confirmed OPC cases were treated according to standard of care. Findings: In total, 35 out of 4,424 study participants (0·8%, 95% confidence interval (CI) 0·6-1·1%) were HPV16 E6 seropositive. Among these, eleven (0·3%, 95%CI 0·1-0·5%) were considered at high risk for HPV-OPC of which nine were successfully re-contacted and invited to regular clinical FU examinations. Two males and one female were diagnosed with stage I HPV-OPC within 1·3 years of clinical FU (3-4 years after initial blood draw), representing one diagnosis of prevalent advanced disease, one incident diagnosis of advanced disease, and one incident diagnosis of early disease. The remaining participants showed no detectable signs of cancer, and undergo regular examinations (median clinical FU: 1·0 years, median total FU from blood draw to last clinical FU visit: 4·7 years). Interpretation: HPV16 early antibodies allowed identifying three asymptomatic stage I HPV-OPC patients, out of eleven participants considered at high risk. However, two of the three cases already showed signs of advanced disease at diagnosis. Targeting multiple early proteins may considerably improve the positive predictive value of HPV16 serology and may have clinical utility for HPV-OPC screening. Funding: This work was funded by DKFZ and UKE intramural funding.

Transforming Properties of Beta-3 Human Papillomavirus E6 and E7 Proteins.

The beta human papillomaviruses (HPVs) are subdivided into 5 species (beta-1 to beta-5), and they were first identified in the skin. However, the beta-3 species appears to be more highly represented in the mucosal epithelia than in the skin. Functional studies have also highlighted that beta-3 HPV49 shares some functional similarities with mucosal high-risk (HR) HPV16. Here, we describe the characterization of the in vitro transforming properties of the entire beta-3 species, which includes three additional HPV types: HPV75, HPV76, and HPV115. HPV49, HPV75, and HPV76 E6 and E7 (E6/E7), but not HPV115 E6 and E7, efficiently inactivate the p53 and pRb pathways and immortalize or extend the life span of human foreskin keratinocytes (HFKs). As observed for HR HPV16, cell cycle deregulation mediated by beta-3 HPV E6/E7 expression leads to p16INK4a accumulation, whereas no p16INK4a was detected in beta-2 HPV38 E6/E7 HFKs. As shown for HPV49 E6, HPV75 and HPV76 E6s degrade p53 by an E6AP/proteasome-mediated mechanism. Comparative analysis of cellular gene expression patterns of HFKs containing E6 and E7 from HR HPV16, beta-3 HPV types, and beta-2 HPV38 further highlights the functional similarities of HR HPV16 and beta-3 HPV49, HPV75, and HPV76. The expression profiles of these four HPV HFKs show some similarities and diverge substantially from those of beta-3 HPV115 E6/E7 and beta-2 HPV38 E6/E7 HFKs. In summary, our data show that beta-3 HPV types share some mechanisms with HR HPV types and pave the way for additional studies aiming to evaluate their potential role in human pathologies.IMPORTANCE Human papillomaviruses are currently classified in different genera. Mucosal HPVs belonging to the alpha genus have been clearly associated with carcinogenesis of the mucosal epithelium at different sites. Beta HPV types have been classified as cutaneous. Although findings indicate that some beta HPVs from species 1 and 2 play a role, together with UV irradiation, in skin cancer, very little is known about the transforming properties of most of the beta HPVs. This report shows the transforming activity of E6 and E7 from beta-3 HPV types. Moreover, it highlights that beta-3 HPVs share some biological properties more extensively with mucosal high-risk HPV16 than with beta-2 HPV38. This report provides new paradigms for a better understanding of the biology of the different HPV types and their possible association with lesions at mucosal and/or cutaneous epithelia.

Cancer susceptibility of beta HPV49 E6 and E7 transgenic mice to 4-nitroquinoline 1-oxide treatment correlates with mutational signatures of tobacco exposure.

We have previously showed that a transgenic (Tg) mouse model with cytokeratin 14 promoter (K14)-driven expression of E6 and E7 from beta-3 HPV49 in the basal layer of the epidermis and of the mucosal epithelia of the digestive tract (K14 HPV49 E6/E7 Tg mice) are highly susceptible to upper digestive tract carcinogenesis upon exposure to 4-nitroquinoline 1-oxide (4NQO). Using whole-exome sequencing, we show that in K14 HPV49 E6/E7 Tg mice, development of 4NQO-induced cancers tightly correlates with the accumulation of somatic mutations in cancer-related genes. The mutational signature in 4NQO-treated mice was similar to the signature observed in humans exposed to tobacco smoking and tobacco chewing. Similar results were obtained with K14 Tg animals expressing mucosal high-risk HPV16 E6 and E7 oncogenes. Thus, beta-3 HPV49 share some functional similarities with HPV16 in Tg animals.

Nitric oxide reverts the resistance to doxorubicin in human colon cancer cells by inhibiting the drug efflux.

Multidrug resistance (MDR) is a phenomenon by which cancer cells evade the cytotoxic effects of chemotherapeutic agents. It may occur through different mechanisms, but it often correlates with the overexpression of integral membrane transporters, such as P-glycoprotein (Pgp) and MDR-associated proteins (MRPs), with resulting decrease of drug accumulation and cellular death. Doxorubicin is a substrate of Pgp; it has been suggested that its ability to induce synthesis of nitric oxide (NO) could explain, at least in part, its cytotoxic effects. Culturing the human epithelial colon cell line HT29 in the presence of doxorubicin, we obtained a doxorubicin-resistant (HT29-dx) cell population: these cells accumulated less intracellular doxorubicin, were less sensitive to the cytotoxic effects of doxorubicin and cisplatin, overexpressed Pgp and MRP3, and exhibited a lower NO production (both under basal conditions and after doxorubicin stimulation). The resistance to doxorubicin could be reversed when HT29-dx cells were incubated with inducers of NO synthesis (cytokines mix, atorvastatin). Some NO donors increased the drug accumulation in HT29-dx cells in a guarosine-3':5'-cyclic monophosphate-independent way; this effect was associated with a marked reduction of doxorubicin efflux rate in HT29 and HT29-dx cells, and tyrosine nitration in the MRP3 protein. Our results suggest that onset of MDR and impairment of NO synthesis are related; this finding could point to a new strategy to reverse doxorubicin resistance in human cancer.

Human papillomaviruses and carcinogenesis: well-established and novel models.

Human papillomaviruses (HPVs) infect the cutaneous or mucosal epithelia and are classified phylogenetically as genera and species. Persistent infections by the mucosal high-risk (HR) HPV types from genus alpha are associated with cancer development of the genital and upper respiratory tracts. The products of two early genes, E6 and E7, are the major HR HPV oncoproteins, being essential in all steps of the carcinogenic process. Cutaneous beta HPV types are proposed, together with ultraviolet (UV) radiation, to promote non-melanoma skin cancer development. However, in contrast to the HR HPV types, beta HPV types appear to be required only at an early stage of carcinogenesis, facilitating the accumulation of UV-induced DNA mutations. Although findings in experimental models also suggest that beta HPV types and other carcinogens may synergize in the induction of malignancies, these possibilities need to be confirmed in human studies.

Novel ß-HPV49 Transgenic Mouse Model of Upper Digestive Tract Cancer.

The beta genus of human papillomaviruses (ß-HPV) includes approximately 50 different viral types that are subdivided into five species (ß-1 through ß-5). Nonmelanoma cancers may involve some ß-1 and ß-2 HPV types, but the biology of most ß-HPV types and their possible connections to human disease are still little characterized. In this study, we studied the effects of ß-3 type HPV49 in a novel transgenic (Tg) mouse model, using a cytokeratin K14 promoter to drive expression of the E6 and E7 genes from this virus in the basal skin epidermis and the mucosal epithelia of the digestive tract (K14 HPV49 E6/E7-Tg mice). Viral oncogene expression only marginally increased cellular proliferation in the epidermis of Tg animals, compared with wild-type littermates, and we observed no spontaneous tumor formation during their entire lifespan. However, we found that K14 HPV49 E6/E7-Tg mice were highly susceptible to upper digestive tract carcinogenesis upon initiation with 4-nitroquinoline 1-oxide (4NQO). This was a selective effect, as the same mice did not exhibit any skin lesions after chronic UV irradiation. Opposite results were observed in an analogous Tg model expressing the ß-2 HPV38 E6 and E7 oncogenes at the same anatomic sites. While these mice were highly susceptible to UV-induced skin carcinogenesis, as previously shown, they were little affected by 4NQO treatment. Overall, our findings highlight important differences in the biologic properties of certain ß-type HPV that affect their impact on carcinogenesis in an anatomic site-specific manner. Cancer Res; 76(14); 4216-25. ©2016 AACR.

Thioredoxin-Displayed Multipeptide Immunogens.

Fusion to carrier proteins is an effective strategy for stabilizing and providing immunogenicity to peptide epitopes. This is commonly achieved by cross-linking of chemically synthesized peptides to carrier proteins. An alternative approach is internal grafting of selected peptide epitopes to a scaffold protein via double stranded-oligonucleotide insertion or gene synthesis, followed by recombinant expression of the resulting chimeric polypeptide. The scaffold protein should confer immunogenicity to the stabilized and structurally constrained peptide, but also afford easy production of the antigen in recombinant form. A macromolecular scaffold that meets the above criteria is the redox protein thioredoxin, especially bacterial thioredoxin. Here we describe our current methodology for internal grafting of selected peptide epitopes to thioredoxin as tandemly arranged multipeptide repeats ("Thioredoxin Displayed Multipeptide Immunogens"), bacterial expression and purification of the recombinant thioredoxin-multipeptide fusion proteins and their use as antigens for the production of anti-peptide antibodies for prophylactic vaccine as well as diagnostic purposes.

The pentose phosphate pathway plays an essential role in supporting human sperm capacitation.

In swim-up-selected spermatozoa of 38 normozoospermic patients, capacitated spermatozoa exhibited enhanced pentose phosphate pathway (PPP) activity and increased expression of glucose-6-phosphate dehydrogenase (G6PD). The G6PD inhibitor DHEA and the inhibitors of NADPH oxidase apocynin and diphenylene iodonium (DPI) prevented both superoxide generation and capacitation in human spermatozoa, but whereas DPI and DHEA inhibited PPP, apocynin did not influence it, suggesting that PPP activation during capacitation is not a response to increased oxidative stress but exerts a role by supplying reducing equivalents to oxygen.

Na+/H+ exchanger activity is increased in doxorubicin-resistant human colon cancer cells and its modulation modifies the sensitivity of the cells to doxorubicin.

Multidrug resistant (MDR) tumor cells exhibit an altered pH gradient across different cell compartments, which favors a reduced intracellular accumulation of antineoplastic drugs and a decreased therapeutic effect. In our study, we have observed that the activity and expression of Na+/H+ exchanger (NHE), which is involved in the homeostasis of intracellular pH (pHi), are increased in doxorubicin-resistant (HT29-dx) human colon carcinoma cells in comparison with doxorubicin-sensitive HT29 cells. The pH(i) was significantly higher in HT29-dx cells, which accumulated less doxorubicin than HT29 cells. The NHE inhibitor 5-(N-ethyl-N-isopropyl)amiloride (EIPA) significantly reduced the pHi value and increased the intracellular accumulation of doxorubicin in both cell populations: in the presence of EIPA HT29-dx cells accumulated as much drug as control HT29 cells. On the other hand, monensin, a Na+/H+ ionophore mimicking NHE activation, and phorbol 12-myristate 13-acetate (PMA), which stimulates NHE, significantly increased the pHi and decreased the drug accumulation in HT29 cells to values similar to those observed in control HT29-dx cells. EIPA potentiated the cytotoxic effect of doxorubicin in HT29 cells, and made HT29-dx cells as sensitive to the cytotoxic effect of the drug as control HT29 cells. Instead, PMA and monensin made HT29 cells as insensitive to doxorubicin as HT29-dx cells. These results suggest that in MDR cells the higher cytosolic pH is likely to decrease drug accumulation, and that such resistance can be reverted by inhibiting the NHE activity. This result opens the possibility to revert MDR with the clinical use of NHE inhibitors.