p53 Is Regulated in a Biphasic Manner in Hypoxic Human Papillomavirus Type 16 (HPV16)-Positive Cervical Cancer Cells

Although the effect of hypoxia on p53 in human papillomavirus (HPV)-positive cancer cells has been studied for decades, the impact of p53 regulation on downstream targets and cellular adaptation processes during different periods under hypoxia remains elusive. Here, we show that, despite continuous repression of HPV16 E6/E7 oncogenes, p53 did not instantly recover but instead showed a biphasic regulation marked by further depletion within 24 h followed by an increase at 72 h. Of note, during E6/E7 oncogene suppression, lysosomal degradation antagonizes p53 reconstitution. Consequently, the transcription of p53 responsive genes associated with senescence (e.g., PML and YPEL3) cannot be upregulated. In contrast, downstream genes involved in autophagy (e.g., DRAM1 and BNIP3) were activated, allowing the evasion of senescence under hypoxic conditions. Hence, dynamic regulation of p53 along with its downstream network of responsive genes favors cellular adaptation and enhances cell survival, although the expression of the viral E6/E7-oncogenes as drivers for proliferation remained inhibited under hypoxia

Combined Transcriptome and Proteome Analysis of Immortalized Human Keratinocytes Expressing Human Papillomavirus 16 (HPV16) Oncogenes Reveals Novel Key Factors and Networks in HPV-Induced Carcinogenesis

Human papillomavirus (HPV)-associated cancers still remain a big health problem, especially in developing countries, despite the availability of prophylactic vaccines. Although HPV oncogenes have been intensively investigated for decades, a study applying recent advances in RNA-Seq and quantitative proteomic approaches to a precancerous model system with well-defined HPV oncogene expression alongside HPV-negative parental cells has been missing until now. Here, combined omics analyses reveal global changes caused by the viral oncogenes in a less biased way and allow the identification of novel factors and key cellular networks potentially promoting malignant transformation. In addition, this system also provides a basis for mechanistic research on novel key factors regulated by HPV oncogenes, especially those that are confirmed in vivo in cervical cancer as well as in head and neck cancer patient samples from TCGA data sets.Although the role of high-risk human papillomaviruses (hrHPVs) as etiological agents in cancer development has been intensively studied during the last decades, there is still the necessity of understanding the impact of the HPV E6 and E7 oncogenes on host cells, ultimately leading to malignant transformation. Here, we used newly established immortalized human keratinocytes with a well-defined HPV16 E6E7 expression cassette to get a more complete and less biased overview of global changes induced by HPV16 by employing transcriptome sequencing (RNA-Seq) and stable isotope labeling by amino acids in cell culture (SILAC). This is the first study combining transcriptome and proteome data to characterize the impact of HPV oncogenes in human keratinocytes in comparison with their virus-negative counterparts. To enhance the informative value and accuracy of the RNA-Seq data, four different bioinformatic workflows were used. We identified potential novel upstream regulators (e.g., CNOT7, SPDEF, MITF, and PAX5) controlling distinct clusters of genes within the HPV-host cell network as well as distinct factors (e.g., CPPED1, LCP1, and TAGLN) with essential functions in cancer. Validated results in this study were compared to data sets from The Cancer Genome Atlas (TCGA), demonstrating that several identified factors were also differentially expressed in cervical squamous cell carcinoma and endocervical adenocarcinoma (CESC) and HPV-positive head and neck squamous cell carcinomas (HNSCs). This highly integrative approach allows the identification of novel HPV-induced cellular changes that are also reflected in cancer patients, providing a promising omics data set for future studies in both basic and translational research

The interplay of UV and cutaneous papillomavirus infection in skin cancer development.

Cutaneous human papillomaviruses (HPVs) are considered as cofactors for non-melanoma skin cancer (NMSC) development, especially in association with UVB. Extensively studied transgenic mouse models failed to mimic all aspects of virus-host interactions starting from primary infection to the appearance of a tumor. Using the natural model Mastomys coucha, which reflects the human situation in many aspects, we provide the first evidence that only UVB and Mastomys natalensis papillomavirus (MnPV) infection strongly promote NMSC formation. Using UVB exposures that correspond to UV indices of different geographical regions, irradiated animals developed either well-differentiated keratinizing squamous cell carcinomas (SCCs), still supporting productive infections with high viral loads and transcriptional activity, or poorly differentiated non-keratinizing SCCs almost lacking MnPV DNA and in turn, early and late viral transcription. Intriguingly, animals with the latter phenotype, however, still showed strong seropositivity, clearly verifying a preceding MnPV infection. Of note, the mere presence of MnPV could induce γH2AX foci, indicating that viral infection without prior UVB exposure can already perturb genome stability of the host cell. Moreover, as shown both under in vitro and in vivo conditions, MnPV E6/E7 expression also attenuates the excision repair of cyclobutane pyrimidine dimers upon UVB irradiation, suggesting a viral impact on the DNA damage response. While mutations of Ras family members (e.g. Hras, Kras, and Nras) were absent, the majority of SCCs harbored-like in humans-Trp53 mutations especially at two hot-spots in the DNA-binding domain, resulting in a loss of function that favored tumor dedifferentiation, counter-selective for viral maintenance. Such a constellation provides a reasonable explanation for making continuous viral presence dispensable during skin carcinogenesis as observed in patients with NMSC

Post-translational control of IL-1β via the human papillomavirus type 16 E6 oncoprotein: a novel mechanism of innate immune escape mediated by the E3-ubiquitin ligase E6-AP and p53.

Infections with high-risk human papillomaviruses (HPVs) are causally involved in the development of anogenital cancer. HPVs apparently evade the innate immune response of their host cells by dysregulating immunomodulatory factors such as cytokines and chemokines, thereby creating a microenvironment that favors malignancy. One central key player in the immune surveillance interactome is interleukin-1 beta (IL-1β) which not only mediates inflammation, but also links innate and adaptive immunity. Because of its pleiotropic physiological effects, IL-1β production is tightly controlled on transcriptional, post-translational and secretory levels. Here, we describe a novel mechanism how the high-risk HPV16 E6 oncoprotein abrogates IL-1β processing and secretion in a NALP3 inflammasome-independent manner. We analyzed IL-1β regulation in immortalized keratinocytes that harbor the HPV16 E6 and/or E7 oncogenes as well as HPV-positive cervical tumor cells. While in primary and in E7-immortalized human keratinocytes the secretion of IL-1β was highly inducible upon inflammasome activation, E6-positive cells did not respond. Western blot analyses revealed a strong reduction of basal intracellular levels of pro-IL-1β that was independent of dysregulation of the NALP3 inflammasome, autophagy or lysosomal activity. Instead, we demonstrate that pro-IL-1β is degraded in a proteasome-dependent manner in E6-positive cells which is mediated via the ubiquitin ligase E6-AP and p53. Conversely, in E6- and E6/E7-immortalized cells pro-IL-1β levels were restored by siRNA knock-down of E6-AP and simultaneous recovery of functional p53. In the context of HPV-induced carcinogenesis, these data suggest a novel post-translational mechanism of pro-IL-1β regulation which ultimately inhibits the secretion of IL-1β in virus-infected keratinocytes. The clinical relevance of our results was further confirmed in HPV-positive tissue samples, where a gradual decrease of IL-1β towards cervical cancer could be discerned. Hence, attenuation of IL-1β by the HPV16 E6 oncoprotein in immortalized cells is apparently a crucial step in viral immune evasion and initiation of malignancy

Molecular analyses of tumor-bearing animals.

<p><b>A)</b> Viral load in tissue samples from UV-irradiated and control animals from the MnPV-infected colony analyzed by qPCR and normalized to a plasmid standard. Samples were grouped according to their origin as indicated (ctrl skin: skin from unirradiated animals; ui skin/UV skin: unirradiated or UV-irradiated skin from irradiated animals; KSCC/nKSCC: UV-induced SCCs; non-UV tumor: tumors from non-UV sites of irradiated animals and spontaneous tumors from unirradiated animals). UV^+/- indicates whether the animal was UV-exposed or not (Kruskal-Wallis test, *p<0.05, ***p<0.001, ^nsp>0.05). <b>B)</b> Southern blot analysis of unirradiated and UV-irradiated skins, a KSCC and a non-UV tumor. DNA was digested with ApaI (no cleavage site in MnPV), XbaI (one site) or XhoI (two sites) as indicated (Form I: supercoiled; Form II: relaxed circular; Form III: linear form of MnPV). <b>C)</b> Semi-quantitative RT-PCR for the most abundant MnPV <i>E1^E4</i> transcript in non-UV tumors and UV-induced SCCs or the control <i>GAPDH</i>. <b>D)</b> Semi-quantitative RT-PCR for MnPV <i>E6</i>, <i>E7</i> and <i>L1</i> transcripts in non-UV tumors and UV-induced SCCs or the control <i>GAPDH</i>.</p

Histological analyses of a non-UV tumor and UV-induced tumors.

<p><b>A)</b> Tumors from unirradiated sites show papilloma-like growth of well-differentiated neoplastic squamous cells (H&E). Especially the basal layers are hyperproliferative as indicated by strong Ki-67 staining. Throughout all layers of the lesion, neoplastic cells strongly express cytokeratins (pan-Cytokeratin). <b>B)</b> A UV-induced KSCC with well-differentiated exoendophytic proliferations of squamous cells expressing Ki-67 throughout all neoplastic squamous layers. <b>C and D)</b> In some cases well-differentiated KSCCs <b>(C)</b> further developed into more aggressive poorly differentiated nKSCCs <b>(D)</b>. Proliferating altered squamous cells thereby invaded deeper layers and often changed to a spindle-like phenotype (H&E). The Ki-67 staining becomes diffuse in this process (compare insets) and cytokeratin expression is reduced. (d: dermis; e: epidermis; f: fat; k: keratin; m: muscle; u: ulceration; t: tumor. Scale bars: macroscopic: 10 mm, overviews: 1 mm, insets 100 μm).</p

Dedifferentiation correlates with positive p53 staining.

<p>Consecutive sections of a poorly differentiated nKSCC were stained with antibodies against E-cadherin, vimentin, pan-Cytokeratin and p53. DAPI was used as nuclear counter stain. Note that in this tumor, only mutation R266C could be detected (Scale bars: 100 μm).</p

Transactivating capacity of <i>Mastomys</i> p53 in the presence of MnPV E6.

<p><b>A)</b> The capacity of p53 to transactivate a p53-responsive firefly luciferase gene measured in H1299 cells transfected with reporter plasmids and expression vectors for <i>Mastomys</i> p53 and MnPV E6 or human p53 and HPV16 E6 as a control. Transactivation activity was measured by luminescence (RLU, relative light units). Cells transfected only with p53 served as control and their RLU levels were arbitrarily set to 1 (Mean ± SEM; n = 7; 1way-ANOVA, ***p<0.0001). <b>B)</b> Western blots showing protein levels of p53 and E6 in the lysates of the transactivation assay. Actin served as an internal loading control.</p

Collagen IV staining on tissue sections reveals invasion of keratinocytes through the basal membrane (BM).

<p>The BM was stained against collagen IV (green). Nuclei were counterstained with DAPI (blue). Consecutive sections stained for pan-cytokeratin are shown in comparison. <b>A)</b> In normal skin, the BM (white arrows) marks the barrier between epidermis and dermis. <b>B)</b> Early stage carcinoma formation in UV-irradiated skin. A lack of collagen IV expression indicates the disruption of the BM (orange arrows) accompanied by downward migrating cells (black arrow). <b>C)</b> In the edge region of a UV-induced KSCC, the BM is lost and invading altered keratinocytes are detectable. <b>D)</b> In nKSCC, invasion of neoplastic cells is advanced as indicated by pan-cytokeratin staining. The discontinuous staining of the BM marks transition zones where invading neoplastic squamous cells acquire a spindle cell phenotype (Scale bars: 100 μm).</p