Cutaneous Papillomaviruses and Non-melanoma Skin Cancer: Causal Agents or Innocent Bystanders?

There is still controversy in the scientific field about whether certain types of cutaneous human papillomaviruses (HPVs) are causally involved in the development of non-melanoma skin cancer (NMSC). Deciphering the etiological role of cutaneous HPVs requires - besides tissue culture systems - appropriate preclinical models to match the obtained results with clinical data from affected patients. Clear scientific evidence about the etiology and underlying mechanisms involved in NMSC development is fundamental to provide reasonable arguments for public health institutions to classify at least certain cutaneous HPVs as group 1 carcinogens. This in turn would have implications on fundraising institutions and health care decision makers to force - similarly as for anogenital cancer - the implementation of a broad vaccination program against "high-risk" cutaneous HPVs to prevent NMSC as the most frequent cancer worldwide. Precise knowledge of the multi-step progression from normal cells to cancer is a prerequisite to understand the functional and clinical impact of cofactors that affect the individual outcome and the personalized treatment of a disease. This overview summarizes not only recent arguments that favor the acceptance of a viral etiology in NMSC development but also reflects aspects of causality in medicine, the use of empirically meaningful model systems and strategies for prevention.Fil: Hasche, Daniel. German Cancer Research Center; AlemaniaFil: Vinzon, Sabrina Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Rösl, Frank. German Cancer Research Center; Alemani

Growth Arrest of HPV-Positive Cells after Histone Deacetylase Inhibition Is Independent of E6/E7 Oncogene Expression

AbstractInhibitors of histone deacetylase (HDAC) are capable of arresting growth in cervical carcinoma cells in the G1 phase of the cell cycle. Although HPV E6/E7 mRNA steady-state levels appeared to be constant after prolonged treatment, time-course experiments revealed that viral transcription was transiently down-regulated between 7–10 h prior to cdk2 suppression. To test whether transitory suppression was a prerequisite for the biological outcome after HDAC inhibition, we took advantage of two immortalized human keratinocyte cell lines in which E6/E7 oncogene expression was controlled by different regulatory regions. After treatment with sodium butyrate (NaB) or trichostatin A (TSA), HPV16 upstream regulatory region (URR)-directed transcription was down-regulated, showing kinetics similar to those in cervical carcinoma cells. In contrast, β-actin promoter controlled E6/E7 transcription was even temporarily increased and finally declined to levels initially detected in the untreated controls. Both cell lines, however, were arrested in G1 and showed complete suppression of cdk2 activity that was preceded by a strong up-regulation of the cdk2 inhibitors p21CIP1 and p27KIP1. These results demonstrate that growth of HPV16/18-positive cells can be arrested by HDAC inhibitors despite ongoing HPV transcription and thus independently of any potential position effects uncoupling URR-directed gene expression by adjacent cellular promoters or by downstream 3′-polyadenylation sites after viral integration into the host genome during multistep carcinogenesis

Regulation of MCP-1 chemokine transcription by p53

<p>Abstract</p> <p>Background</p> <p>Our previous studies showed that the expression of the monocyte-chemoattractant protein (MCP)-1, a chemokine, which triggers the infiltration and activation of cells of the monocyte-macrophage lineage, is abrogated in human papillomavirus (HPV)-positive premalignant and malignant cells. <it>In silico </it>analysis of the MCP-1 upstream region proposed a putative p53 binding side about 2.5 kb upstream of the transcriptional start. The aim of this study is to monitor a physiological role of p53 in this process.</p> <p>Results</p> <p>The proposed p53 binding side could be confirmed <it>in vitro </it>by electrophoretic-mobility-shift assays and <it>in vivo </it>by chromatin immunoprecipitation. Moreover, the availability of p53 is apparently important for chemokine regulation, since TNF-α can induce MCP-1 only in human keratinocytes expressing the viral oncoprotein E7, but not in HPV16 E6 positive cells, where p53 becomes degraded. A general physiological role of p53 in MCP-1 regulation was further substantiated in HPV-negative cells harboring a temperature-sensitive mutant of p53 and in Li-Fraumeni cells, carrying a germ-line mutation of p53. In both cases, non-functional p53 leads to diminished MCP-1 transcription upon TNF-α treatment. In addition, siRNA directed against p53 decreased MCP-1 transcription after TNF-α addition, directly confirming a crosstalk between p53 and MCP-1.</p> <p>Conclusion</p> <p>These data support the concept that p53 inactivation during carcinogenesis also affects immune surveillance by interfering with chemokine expression and in turn communication with cells of the immunological compartment.</p

Cutaneous HPV23 E6 Prevents p53 Phosphorylation through Interaction with HIPK2

Ultraviolet irradiation (UV) is the major risk factor for the development of skin cancer. Moreover, increasing evidence suggests cutaneotropic human papillomaviruses (HPV) from the beta genus to play a causal role as a co-factor in the development of cutaneous squamous cell carcinoma. Homeodomain-interacting protein kinase 2 (HIPK2) operates as a potential suppressor in skin tumorigenesis and is stabilized by UV-damage. HIPK2 is an important regulator of apoptosis, which forms a complex with the tumor suppressor p53, mediating p53 phosphorylation at Ser 46 and thus promoting pro-apoptotic gene expression. In our study, we demonstrate that cutaneous HPV23 E6 protein directly targets HIPK2 function. Accordingly, HPV23 E6 interacts with HIPK2 both in vitro and in vivo. Furthermore, upon massive UVB-damage HPV23 E6 co-localizes with endogenous HIPK2 at nuclear bodies. Functionally, we demonstrate that HPV23 E6 inhibits HIPK2-mediated p53 Ser 46 phosphorylation through enforcing dissociation of the HIPK2/p53 complex. In addition, HPV23 E6 co-accumulates with endogenous HIPK2 upon UV damage suggesting a mechanism by which HPV23 E6 keeps HIPK2 in check after UV damage. Thus, cutaneous HPV23 E6 prevents HIPK2-mediated p53 Ser 46 phosphorylation, which may favour survival of UV-damaged keratinocytes and skin carcinogenesis by apoptosis evasion

A novel rodent papillomavirus isolated from anogenital lesions in its natural host

AbstractIn the present work we describe both the prevalence and the histopathologic features of a novel papillomavirus (referred as McPV2) that naturally infects the rodent Mastomys coucha. Viral DNA could be isolated not only from anogenital wart-like lesions but also from healthy tissues (e.g. liver, kidney, spleen and intestine) without apparent signs of infection. Our finding of a second papillomavirus infecting M. coucha, phylogenetically very distant from the previously known MnPV, reinforces the growing view of warm-blooded vertebrates as being hosts for a number of different papilloma virus types that are not necessarily closely related. The histological descriptions of McPV2-associated anogenital lesions provided here, together with earlier knowledge on MnPV-associated skin carcinogenesis, define M. coucha as an excellent system where the link between infection towards malignancy can be studied in molecular, histochemical and immunological terms in immunocompetent animals. The availability of such an in vivo model also offers the unique opportunity to address defined questions about prophylactic and therapeutic strategies against different papillomavirus infections in their natural host. To date, McPV2 is the first rodent papillomavirus found in anogenital lesions

Expression of different L1 isoforms of Mastomys natalensis papillomavirus as mechanism to circumvent adaptive immunity

Although many high-risk mucosal and cutaneous human papillomaviruses (HPVs) theoretically have the potential to synthesize L1 isoforms differing in length, previous seroepidemiological studies only focused on the short L1 variants, co-assembling with L2 to infectious virions. Using the multimammate mouse Mastomys coucha as preclinical model, this is the first study demonstrating seroconversion against different L1 isoforms during the natural course of papillomavirus infection. Intriguingly, positivity with the cutaneous MnPV was accompanied by a strong seroresponse against a longer L1 isoform, but to our surprise, the raised antibodies were non-neutralizing. Only after a delay of around 4 months, protecting antibodies against the short L1 appeared, enabling the virus to successfully establish an infection. This argues for a novel humoral immune escape mechanism that may also have important implications on the interpretation of epidemiological data in terms of seropositivity and protection of PV infections in general.Fil: Fu, Yingying. German Cancer Research Center; AlemaniaFil: Cao, Rui. German Cancer Research Center; AlemaniaFil: Schäfer, Miriam. German Cancer Research Center; AlemaniaFil: Stephan, Sonja. German Cancer Research Center; AlemaniaFil: Braspenning Wesch, Ilona. German Cancer Research Center; AlemaniaFil: Schmitt, Laura. German Cancer Research Center; AlemaniaFil: Bischoff, Ralf. German Cancer Research Center; AlemaniaFil: Müller, Martin. German Cancer Research Center; AlemaniaFil: Schäfer, Kai. German Cancer Research Center; AlemaniaFil: Vinzon, Sabrina Eugenia. Consejo Nacional de Investigaciones Científicas y Técnicas. Oficina de Coordinación Administrativa Parque Centenario. Instituto de Investigaciones Bioquímicas de Buenos Aires. Fundación Instituto Leloir. Instituto de Investigaciones Bioquímicas de Buenos Aires; ArgentinaFil: Rösl, Frank. German Cancer Research Center; AlemaniaFil: Hasche, Daniel. German Cancer Research Center ; Alemani

PIK3CA-mediated PI3-kinase signalling is essential for HPV-induced transformation in vitro

<p>Abstract</p> <p>Background</p> <p>High-risk human papillomavirus (hrHPV) infections are causally related to cervical cancer development. The additional (epi)genetic alterations driving malignant transformation of hrHPV-infected cells however, are not yet fully elucidated. In this study we experimentally assessed the role of the PI3-kinase pathway and its regulator PIK3CA, which is frequently altered in cervical cancer, in HPV-induced transformation.</p> <p>Methods</p> <p>Cervical carcinomas and ectocervical controls were assessed for PIK3CA mRNA and protein expression by quantitative RT-PCR and immunohistochemical staining, respectively. A longitudinal <it>in vitro </it>model system of hrHPV-transfected keratinocytes, representing the immortal and anchorage independent phenotype, was assayed for PI3-kinase activation and function using chemical pathway inhibition i.e. LY294002 treatment, and PIK3CA RNA interference. Phenotypes examined included cellular viability, migration, anchorage independent growth and differentiation. mRNA expression of hTERT and HPV16 E6E7 were studied using quantitative RT-PCR and Northern blotting.</p> <p>Results</p> <p>Cervical carcinomas showed significant overexpression of PIK3CA compared to controls. During HPV-induced transformation <it>in vitro</it>, expression of the catalytic subunit PIK3CA as well as activation of downstream effector PKB/AKT progressively increased in parallel. Inhibition of PI3-kinase signalling in HPV16-transfected keratinocytes by chemical interference or siRNA-mediated silencing of PIK3CA resulted in a decreased phosphorylation of PKB/AKT. Moreover, blockage of PI3-kinase resulted in reduced cellular viability, migration, and anchorage independent growth. These properties were accompanied with a downregulation of HPV16E7 and hTERT mRNA expression. In organotypic raft cultures of HPV16- and HPV18-immortalized cells, phosphorylated PKB/AKT was primarily seen in differentiated cells staining positive for cytokeratin 10 (CK10). Upon PI3-kinase signalling inhibition, there was a severe impairment in epithelial tissue development as well as a dramatic reduction in p-PKB/AKT and CK10.</p> <p>Conclusion</p> <p>The present data indicate that activation of the PI3-kinase/PKB/AKT pathway through PIK3CA regulates various transformed phenotypes as well as growth and differentiation of HPV-immortalized cells and may therefore play a pivotal role in HPV-induced carcinogenesis.</p

Nucleolin as Activator of Human Papillomavirus Type 18 Oncogene Transcription in Cervical Cancer

High risk human papillomaviruses (HPVs) are central to the development of cervical cancer and the deregulated expression of high risk HPV oncogenes is a critical event in this process. Here, we find that the cell protein nucleolin binds in a sequence-specific manner to the HPV18 enhancer. The DNA binding activity of nucleolin is primarily S phase specific, much like the transcription of the E6 and E7 oncoproteins of HPV18 in cervical cancer cells. Antisense inactivation of nucleolin blocks E6 and E7 oncogene transcription and selectively decreases HPV18+ cervical cancer cell growth. Furthermore, nucleolin controls the chromatin structure of the HPV18 enhancer. In contrast, HPV16 oncogene transcription and proliferation rates of HPV16+ SiHa cervical cancer cells are independent of nucleolin activity. Moreover, nucleolin expression is altered in HPV18+ precancerous and cancerous tissue from the cervix uteri. Whereas nucleolin was homogeneously distributed in the nuclei of normal epithelial cells, it showed a speckled nuclear phenotype in HPV18+ carcinomas. Thus, the host cell protein nucleolin is directly linked to HPV18-induced cervical carcinogenesis

Repression of human papillomavirus oncogene expression under hypoxia is mediated by PI3K/mTORC2/AKT signaling

Oncogenic HPV types are major human carcinogens. Under hypoxia, HPV-positive cancer cells can repress the viral E6/E7 oncogenes and induce a reversible growth arrest. This response could contribute to therapy resistance, immune evasion, and tumor recurrence upon reoxygenation. Here, we uncover evidence that HPV oncogene repression is mediated by hypoxia-induced activation of canonical PI3K/mTORC2/AKT signaling. AKT-dependent downregulation of E6/E7 is only observed under hypoxia and occurs, at least in part, at the transcriptional level. Quantitative proteome analyses identify additional factors as candidates to be involved in AKT-dependent E6/E7 repression and/or hypoxic PI3K/mTORC2/AKT activation. These results connect PI3K/mTORC2/AKT signaling with HPV oncogene regulation, providing new mechanistic insights into the cross talk between oncogenic HPVs and their host cells.Hypoxia is linked to therapeutic resistance and poor clinical prognosis for many tumor entities, including human papillomavirus (HPV)-positive cancers. Notably, HPV-positive cancer cells can induce a dormant state under hypoxia, characterized by a reversible growth arrest and strong repression of viral E6/E7 oncogene expression, which could contribute to therapy resistance, immune evasion and tumor recurrence. The present work aimed to gain mechanistic insights into the pathway(s) underlying HPV oncogene repression under hypoxia. We show that E6/E7 downregulation is mediated by hypoxia-induced stimulation of AKT signaling. Ablating AKT function in hypoxic HPV-positive cancer cells by using chemical inhibitors efficiently counteracts E6/E7 repression. Isoform-specific activation or downregulation of AKT1 and AKT2 reveals that both AKT isoforms contribute to hypoxic E6/E7 repression and act in a functionally redundant manner. Hypoxic AKT activation and consecutive E6/E7 repression is dependent on the activities of the canonical upstream AKT regulators phosphoinositide 3-kinase (PI3K) and mechanistic target of rapamycin (mTOR) complex 2 (mTORC2). Hypoxic downregulation of E6/E7 occurs, at least in part, at the transcriptional level. Modulation of E6/E7 expression by the PI3K/mTORC2/AKT cascade is hypoxia specific and not observed in normoxic HPV-positive cancer cells. Quantitative proteome analyses identify additional factors as candidates to be involved in hypoxia-induced activation of the PI3K/mTORC2/AKT signaling cascade and in the AKT-dependent repression of the E6/E7 oncogenes under hypoxia. Collectively, these data uncover a functional key role of the PI3K/mTORC2/AKT signaling cascade for viral oncogene repression in hypoxic HPV-positive cancer cells and provide new insights into the poorly understood cross talk between oncogenic HPVs and their host cells under hypoxia

Induction of apoptosis in myeloid leukaemic cells by ribozymes targeted against AML1/MTG8

The translocation (8;21)(q22;q22) is a karyotypic abnormality detected in acute myeloid leukaemia (AML) M2 and results in the formation of the chimeric fusion gene AML1/MTG8. We previously reported that two hammerhead ribozymes against AML1/MTG8 cleave this fusion transcript and also inhibit the proliferation of myeloid leukaemia cell line Kasumi-1 which possesses t(8;21)(q22;q22). In this study, we investigated the mechanisms of inhibition of proliferation in myeloid leukaemic cells with t(8;21)(q22;q22) by ribozymes. These ribozymes specifically inhibited the growth of Kasumi-1 cells, but did not affect the leukaemic cells without t(8;21)(q22;q22). We observed the morphological changes including chromatin condensation, fragmentation and the formation of apoptotic bodies in Kasumi-1 cells incubated with ribozymes for 7 days. In addition, DNA ladder formation was also detected after incubation with ribozymes which suggested the induction of apoptosis in Kasumi-1 cells by the AML1/MTG8 ribozymes. However, the ribozymes did not induce the expression of CD11b and CD14 antigens in Kasumi-1 cells. The above data suggest that these ribozymes therefore inhibit the growth of myeloid leukaemic cells with t(8;21)(q22;q22) by the induction of apoptosis, but not differentiation. We conclude therefore that the ribozymes targeted against AML1/MTG8 may have therapeutic potential for patients with AML carrying t(8;21)(q22;q22) while, in addition, the product of the chimeric gene is responsible for the pathogenesis of myeloid leukaemia. © 1999 Cancer Research Campaig