CREB1 activation promotes human papillomavirus oncogene expression and cervical cancer cell transformation.

Human papillomaviruses (HPVs) infect the oral and anogenital mucosa and can cause cancer. The high-risk (HR)-HPV oncoproteins, E6 and E7, hijack cellular factors to promote cell proliferation, delay differentiation and induce genomic instability, thus predisposing infected cells to malignant transformation. cAMP response element (CRE)-binding protein 1 (CREB1) is a master transcription factor that can function as a proto-oncogene, the abnormal activity of which is associated with multiple cancers. However, little is known about the interplay between HPV and CREB1 activity in cervical cancer or the productive HPV lifecycle. We show that CREB is activated in productively infected primary keratinocytes and that CREB1 expression and phosphorylation is associated with the progression of HPV+ cervical disease. The depletion of CREB1 or inhibition of CREB1 activity results in decreased cell proliferation and reduced expression of markers of epithelial to mesenchymal transition, coupled with reduced migration in HPV+ cervical cancer cell lines. CREB1 expression is negatively regulated by the tumor suppressor microRNA, miR-203a, and CREB1 phosphorylation is controlled through the MAPK/MSK pathway. Crucially, CREB1 directly binds the viral promoter to upregulate transcription of the E6/E7 oncogenes, establishing a positive feedback loop between the HPV oncoproteins and CREB1. Our findings demonstrate the oncogenic function of CREB1 in HPV+ cervical cancer and its relationship with the HPV oncogenes.

Exploitation of ATP-sensitive potassium ion (KATP) channels by HPV promotes cervical cancer cell proliferation by contributing to MAPK/AP-1 signalling.

Persistent infection with high-risk human papillomaviruses (HPVs) is the causal factor in multiple human malignancies, including >99% of cervical cancers and a growing proportion of oropharyngeal cancers. Prolonged expression of the viral oncoproteins E6 and E7 is necessary for transformation to occur. Although some of the mechanisms by which these oncoproteins contribute to carcinogenesis are well-characterised, a comprehensive understanding of the signalling pathways manipulated by HPV is lacking. Here, we present the first evidence to our knowledge that the targeting of a host ion channel by HPV can contribute to cervical carcinogenesis. Through the use of pharmacological activators and inhibitors of ATP-sensitive potassium ion (KATP) channels, we demonstrate that these channels are active in HPV-positive cells and that this activity is required for HPV oncoprotein expression. Further, expression of SUR1, which forms the regulatory subunit of the multimeric channel complex, was found to be upregulated in both HPV+ cervical cancer cells and in samples from patients with cervical disease, in a manner dependent on the E7 oncoprotein. Importantly, knockdown of SUR1 expression or KATP channel inhibition significantly impeded cell proliferation via induction of a G1 cell cycle phase arrest. This was confirmed both in vitro and in in vivo tumourigenicity assays. Mechanistically, we propose that the pro-proliferative effect of KATP channels is mediated via the activation of a MAPK/AP-1 signalling axis. A complete characterisation of the role of KATP channels in HPV-associated cancer is now warranted in order to determine whether the licensed and clinically available inhibitors of these channels could constitute a potential novel therapy in the treatment of HPV-driven cervical cancer.

Vaccination With Recombinant Adenoviruses Expressing the Bluetongue Virus Subunits VP7 and VP2 Provides Protection Against Heterologous Virus Challenge.

Bluetongue virus (BTV) is the causative agent of a disease that affects domestic and wild ruminants and leads to critical economic losses. BTV is an arbovirus from the Reoviridae family that is typically transmitted by the bite of infected Culicoides midges. BTV possesses multiple serotypes (up to 28 have been described), and immunity to one serotype offers little cross-protection to other serotypes. The design of vaccines that provide protection across multiple serotypes is therefore highly desirable to control this disease. We previously reported that a recombinant replication-defective human adenovirus serotype 5 (Ad5) that expresses the VP7 inner core protein of BTV serotype 8 (Ad5VP7-8) induced T-cell responses and provided protection. In the present work, we evaluated as BTV vaccine the combination of Ad5VP7-8 with another recombinant Ad5 that expresses the outer core protein VP2 from BTV-1 (Ad5VP2-1). The combination of Ad5VP2-1 and Ad5VP7-8 protected against homologous BTV challenge (BTV-1 and BTV-8) and partially against heterologous BTV-4 in a murine model. Cross-reactive anti-BTV immunoglobulin G (IgG) were detected in immunized animals, but no significant titers of neutralizing antibodies were elicited. The Ad5VP7-8 immunization induced T-cell responses that recognized all three serotypes tested in this study and primed cytotoxic T lymphocytes specific for VP7. This study further confirms that targeting antigenic determinant shared by several BTV serotypes using cellular immunity could help develop multiserotype BTV vaccines.

The deubiquitinase (DUB) USP13 promotes Mcl-1 stabilisation in cervical cancer.

Protein ubiquitination is a critical regulator of cellular homeostasis. Aberrations in the addition or removal of ubiquitin can result in the development of cancer and key components of the ubiquitination machinery serve as oncogenes or tumour suppressors. An emerging target in the development of cancer therapeutics are the deubiquitinase (DUB) enzymes that remove ubiquitin from protein substrates. Whether this class of enzyme plays a role in cervical cancer has not been fully explored. By interrogating the cervical cancer data from the TCGA consortium, we noted that the DUB USP13 is amplified in ~15% of cervical cancer cases. We confirmed that USP13 expression was increased in cervical cancer cell lines, cytology samples from patients with cervical disease and in cervical cancer tissue. Depletion of USP13 inhibited cervical cancer cell proliferation. Mechanistically, USP13 bound to, deubiquitinated and stabilised Mcl-1, a pivotal member of the anti-apoptotic BCL-2 family. Furthermore, reduced Mcl-1 expression partially contributed to the observed proliferative defect in USP13 depleted cells. Importantly, the expression of USP13 and Mcl-1 proteins correlated in cervical cancer tissue. Finally, we demonstrated that depletion of USP13 expression or inhibition of USP13 enzymatic activity increased the sensitivity of cervical cancer cells to the BH3 mimetic inhibitor ABT-263. Together, our data demonstrates that USP13 is a potential oncogene in cervical cancer that functions to stabilise the pro-survival protein Mcl-1, offering a potential therapeutic target for these cancers.

E6-mediated activation of JNK drives EGFR signalling to promote proliferation and viral oncoprotein expression in cervical cancer.

Human papillomaviruses (HPV) are a major cause of malignancy worldwide, contributing to ~5% of all human cancers including almost all cases of cervical cancer and a growing number of ano-genital and oral cancers. HPV-induced malignancy is primarily driven by the viral oncogenes, E6 and E7, which manipulate host cellular pathways to increase cell proliferation and enhance cell survival, ultimately predisposing infected cells to malignant transformation. Consequently, a more detailed understanding of viral-host interactions in HPV-associated disease offers the potential to identify novel therapeutic targets. Here, we identify that the c-Jun N-terminal kinase (JNK) signalling pathway is activated in cervical disease and in cervical cancer. The HPV E6 oncogene induces JNK1/2 phosphorylation in a manner that requires the E6 PDZ binding motif. We show that blockade of JNK1/2 signalling using small molecule inhibitors, or knockdown of the canonical JNK substrate c-Jun, reduces cell proliferation and induces apoptosis in cervical cancer cells. We further demonstrate that this phenotype is at least partially driven by JNK-dependent activation of EGFR signalling via increased expression of EGFR and the EGFR ligands EGF and HB-EGF. JNK/c-Jun signalling promoted the invasive potential of cervical cancer cells and was required for the expression of the epithelial to mesenchymal transition (EMT)-associated transcription factor Slug and the mesenchymal marker Vimentin. Furthermore, JNK/c-Jun signalling is required for the constitutive expression of HPV E6 and E7, which are essential for cervical cancer cell growth and survival. Together, these data demonstrate a positive feedback loop between the EGFR signalling pathway and HPV E6/E7 expression, identifying a regulatory mechanism in which HPV drives EGFR signalling to promote proliferation, survival and EMT. Thus, our study has identified a novel therapeutic target that may be beneficial for the treatment of cervical cancer.