Inhibition of kinase IKKβ suppresses cellular abnormalities induced by the human papillomavirus oncoprotein HPV 18E6

Human papillomavirus (HPV) is the leading cause of cervical cancer and has been implicated in several other cancer types including vaginal, vulvar, penile, and oropharyngeal cancers. Despite the recent availability of a vaccine, there are still over 310,000 deaths each year worldwide. Current treatments for HPV-mediated cancers show limited efficacy, and would benefit from improved understanding of disease mechanisms. Recently, we developed a Drosophila ‘HPV 18 E6’ model that displayed loss of cellular morphology and polarity, junctional disorganization, and degradation of the major E6 target Magi; we further provided evidence that mechanisms underlying HPV E6-induced cellular abnormalities are conserved between humans and flies. Here, we report a functional genetic screen of the Drosophila kinome that identified IKK\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document}β—a regulator of NF-κB—as an enhancer of E6-induced cellular defects. We demonstrate that inhibition of IKK\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document}β reduces Magi degradation and that this effect correlates with hyperphosphorylation of E6. Further, the reduction in IKK\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document}β suppressed the cellular transformation caused by the cooperative action of HPVE6 and the oncogenic Ras. Finally, we demonstrate that the interaction between IKK\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document}β and E6 is conserved in human cells: inhibition of IKK\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document}β blocked the growth of cervical cancer cells, suggesting that IKK\documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document}$$\beta$$\end{document}β may serve as a novel therapeutic target for HPV-mediated cancers.

Immunohistochemistry. For immunolabeling pupal eyes, 40-42 h after puparium formation, were dissected in PBS and fixed in 4% formaldehyde. Fixed tissues were washed three times in PBS solution containing 0.1% Triton-X-100 and blocked in 5% normal goat serum for 1 h before incubation with primary antibodies. The primary antibodies used in this study were rabbit anti-Magi 1:200 39 , rabbit anti-Baz 1:1000, rat anti-DE-cadherin 1:50 (Developmental Studies Hybridoma Bank). The appropriate secondary antibodies were conjugated Alexa488, Alexa594, and Alexa 647 (Invitrogen).

Results
Kinome screen identified IKKβ as a mediator of E6 + hUBE3A-mediated defects. The fly eye is a compound eye consisting of 750 unit eyes. These 'ommatidia' are clusters of sensory neurons arranged in a precisely repeated hexagonal pattern formed by the precise arrangement of supporting pigment cells. Formation of this highly organized pattern requires precisely regulated cell proliferation, cell differentiation and programmed cell death; disruption of any of these processes leads to a disorganized, rough eye phenotype that is readily scored under a light microscope 40 .
We have previously shown that co-expression of E6 and human UBE3A (hUBE3A) in the developing fly eye leads to a disorganized, rough eye phenotype. To identify loci that modify the E6 + hUBE3A-induced eye defects, we used mutations in the kinome to perform a dominant genetic modifier screen. Flies with stable integration of the transgenes GMR-Gal4, UAS-E6, and UAS-hUBE3A (referred to as GMR>E6/hUBE3A) were crossed to flies heterozygous for a mutant kinase. Comparing GMR>E6/hUBE3A; kinase +/− to GMR> E6/hUBE3A flies, we screened 195 kinases and identified IKKβ as the strongest suppressor of the E6 + hUBE3A-mediated rough eye  www.nature.com/scientificreports/ phenotype (Fig. 1D, compare with Fig. 1B; Fig. 1A,C are controls). Rescue was evident in 100% of flies (n = 40) with GMR>E6/hUBE3A; IKKβ +/− genotype and to the same extent shown in Fig. 1D. The rescue by reduced IKKβ activity was further confirmed with an RNA-interference transgene targeting IKKβ (Fig. 1E).

Inhibiting IKKβ in cervical cancer cells blocked their growth.
IKKβ is a serine/threonine kinase that is highly conserved between flies and humans. It regulates the innate immune pathway by activating NF-κB, which in turn activates the expression of antimicrobial peptides to fight against pathogens [41][42][43] . To determine whether IKKβ also has a functional link to E6 + UBE3A in human cells, we assessed the effect of the synthetic IKKβ inhibitor IMD 0354 (N-(3,5-Bis-trifluoromethylphenyl)-5-chloro-2-hydroxybenzamide) 44 on the growth of HaCat (HPV−ve), HeLa (HPV 18 +ve), CasKi (HPV 16 +ve), and SiHa (HPV 16 +ve) cells. IMD 0354 is a non-adenosine triphosphate-binding (ATP-binding) competitive selective IKKβ inhibitor that prevents ATP attachment to IKKβ 38 . Different concentrations of the inhibitor were tested ranging from 250, 500, 750, 1000, to 1250 nM. IMD 0354 reduced growth of all four cell types; HeLa cells were most strongly affected, with a significant effect starting at the lowest concentration of 250 nM. While IMD 0354 had a significant effect on the HPV 18 and 16 positive cells, it had a minor effect on the growth of HaCat cells that lack HPV ( Fig. 2A). This result suggested that the IKKβ-mediated mechanism of E6 + UBE3A-induced cellular abnormalities is conserved between humans and fruit flies. To understand whether the reduced cell number resulting from inhibition of IKKβ is due to arrest in cell cycle or to apoptosis, we treated these cells with 500 nM of IMD 0354 and analyzed their cell cycle profile 3 h post treatment. We found no increase in apoptotic cell death for HeLa and CaSki cells in comparison with HaCat cells. However, the majority of cells were found to exhibit a G1 cell cycle arrest (Fig. 2B,C). This finding indicated that the reduction in cell numbers upon IKKβ inhibition is due to a halt in cell growth and not to an increase in apoptotic cell death.
Reducing IKKβ suppressed the cellular defects caused by co-expression of E6 and hUBE3A. As reduction in IKKβ suppressed the rough eye phenotype caused by co-expression of E6 and hUBE3A, we examined the eye tissue of these flies 40 h after puparium formation, the time point at which the E6 + hUBE3A effect becomes apparent. In the normal pupal eye, each ommatidium consists of eight photoreceptor cells covered by four glial-like cells or cone cells and two primary pigment cells. Neighboring ommatidia are separated from each other by a lattice of secondary and tertiary pigment cells and the sensory bristle cells. This interweaving lattice of pigment cells organizes the ommatidial array into a precise pattern of repeated hexagons 45 . Pupal eyes expressing E6 and hUBE3A exhibit severe morphological defects, including fusion of neighboring ommatidia, increase in the number of pigment cells and cone cells, and a severe alteration in the stereotyped pattern of ommatidia. In comparison, removing a single genomic copy of IKKβ (GMR>E6/hUBE3A; IKKβ +/− ) led to a strong phenotypic rescue: pigment and cone cell defects were reduced and the overall organization of the ommatidial array improved ( Fig. 3A-C). These observations suggest that E6 interferes with a molecular mechanism involving IKKβ and that this mechanism plays an important role in E6-induced cellular abnormalities.
Reducing IKKβ suppressed the junctional and polarity defects caused by co-expression of E6 and hUBE3A. We have previously shown that E6, in cooperation with UBE3A, perturbs the integrity of junctional and polarity complexes 35 . Therefore, we hypothesized that reducing IKKβ activity might suppress these defects. Immunolabeling for junctional marker E-cadherin and polarity marker Bazooka (the homolog of human Par-3) revealed that, in comparison with pupal eyes expressing E6 and UBE3A, in which both the junctional and polarity complexes were perturbed in ommatidia ( Fig. 3 E-H′), GMR>E6/hUBE3A; IKKβ +/− pupal eyes showed no disruption of junctional and polarity complexes and the integrity of these complexes was restored to the extent seen in control eye tissues ( Fig. 3F-I′ compared to D-G′). These observations suggest that alterations in IKKβ significantly contribute to the E6-induced cellular junctional and polarity disorganization.

Reduced IKKβ activity suppressed E6 + hUBEA-induced degradation of PDZ domain proteins.
Proteasomal degradation of PDZ domain-containing proteins, including Magi, Dlg, and Scribble, was shown to be crucial for the cancerous effect of HPV 16 and 18 E6 25 . We have previously shown that HPV 18 E6, with the addition of human UBE3A, targets the fly counterparts of these proteins for ubiquitin-mediated proteasomal destruction 35 . As a reduction in IKKβ levels suppressed the cellular defects caused by E6 plus hUBE3A, we asked whether the E6-mediated degradation of PDZ domain proteins was altered. To address this question we examined the level of Magi, as Magi has been identified as a major degradation target of HPV18 E6 in human and Drosophila 19,35 . Immunolabeling of pupal eyes for Magi revealed that, whereas GMR>E6/hUBE3A eyes exhibited a complete loss of Magi, GMR>E6/hUBE3A; IKKβ +/− pupal eyes exhibited no detectable loss of Magi ( Fig. 4A-C). This result suggests that reducing IKKβ activity suppresses the E6-induced degradation of Magi, and that rescue of Magi degradation is likely to play a role in suppression of E6-induced cellular defects.

Reducing IKKβ resulted in hyperphosphorylation of E6. Phosphorylation of the HPV 18 E6 PBM
was previously demonstrated to block its interaction with PDZ domain proteins Dlg and Magi 37,46,47 . To assess whether phosphorylation-mediated regulation of E6 plays a role in suppressing Magi degradation, we treated cells expressing HPV 18 E6 with the IKKβ inhibitor IMD 0354 (at 100 and 500 nM) and compared with untreated cells for E6 phosphorylation. Western blot analysis was performed using an antibody to detect phosphorylated E6. We found that inhibition of IKKβ resulted in extensive phosphorylation of E6, which was absent in untreated cells (Fig. 4D). This result is consistent with previous findings, and suggests that the lack of Magi degradation in cells expressing E6 + hUBE3A with mutated IKKβ could be due to the loss of PDZ recognition by E6. www.nature.com/scientificreports/   www.nature.com/scientificreports/ the cellular transformation caused by the cooperation of oncogenic Ras and E6. Co-expression of oncogenic Ras, Ras64B V14 with E6, and hUB3A in eye imaginal discs (GMR>E6/hUBE3A/Ras64B V14 ) at 25 °C resulted in overgrowth and pupal lethality (Fig. 5A). However, when the level of IKKβ was reduced (GMR>E6/hUBE3A/ Ras64B V14 ; IKKβ +/− ), the lethality was rescued and flies developed to adulthood. These adult flies, however, still exhibited significant abnormalities in eye morphology, suggesting incomplete rescue (Fig. 5B). Gal4 activity is reduced at lower temperatures 51 . At 22 °C, GMR>E6/hUBE3A/Ras64B V14 flies developed to adulthood, exhibiting enhanced transformed eye morphology in comparison to expression of oncogenic Ras alone (Fig. 5D compared to 5C). Reducing IKKβ activity significantly suppressed the transformed eye morphology in these conditions (Fig. 5E), further demonstrating that IKKβ is important for the cooperative action of E6 and oncogenic Ras.

Discussion
In this study we identify IKKβ as a mediator of the HPV 18 E6 and hUBE3A-induced cellular defects in both fly and human cancer models. This is consistent with previous studies demonstrating that HPV 16 E6 interacts with components of the innate immune pathway, including IKKβ, and it activates the NF-κB transcription factor 52,53 . The role of the innate immune system in HPV infection and cancer progression is not well understood. Activation of the innate immune system, particularly the Toll pathway, has been associated with regression and clearance of HPV 16 infection 54 . This is consistent with other studies demonstrating that HPV 16 oncogenes repress the expression of a key innate immune sensor, Toll like receptor 9 (TLR9) 55 , suggesting that persistent infection may reflect a defect in the host innate immune system. Conversely, activation of the innate immune system, and consequent inflammation, contributes to HPV-induced tumor progression 56 . Several studies have shown an increase in the level of inflammatory cytokines in individuals with reduced HPV clearance [57][58][59] . Additionally, upregulation of several TLRs and components of the NF-κB signaling pathway have been implicated in HPV-related cancers. It has been suggested that the malignant phenotype of cervical cancer cells requires the function and activation of IKKβ/NF-κB signaling 60,61 . IKKβ phosphorylates the inhibitor of NF-κB, resulting in its ubiquitination and www.nature.com/scientificreports/ proteasomal degradation. This action of IKKβ frees the NF-κB, which in turn enters the nucleus and activates the transcription of pro-inflammatory, pro-cell proliferation and anti-apoptotic genes 62,63 . Increased expression of IKKβ and its association with an aggressive phenotype has been reported in several types of cancers including head-and-neck, ovarian and liver cancers [64][65][66] . It is notable that IKKβ's role in cancer is not only limited to its function in regulation of the NF-κB pathway; IKKβ can also phosphorylate p53, resulting in its ubiquitination and subsequent degradation. Inactivation or loss of p53 has been identified in more than 50% of cancers, including HPV-induced cancers: high-risk HPV E6s target p53 for ubiquitination and proteasomal degradation 14,67 . IKKβ-mediated loss of p53 can be suppressed by inhibition of IKKβ in cancer cells 68,69 . The inhibitory function of IKKβ on p53 was further found in HPV38E6E7 human keratinocytes. IKKβ phosphorylates and stabilizes a dominant-negative inhibitor of p53, ∆Np73α, resulting in repression of p53-regulated genes such as p21. This inhibitory effect of IKKβ on p53 can be suppressed upon its inactivation, which results in destabilization and degradation of ∆Np73α 70 . Another phosphorylation target of IKKβ is fork-head transcription factor FOXO3a. IKKβ has been found to stimulate cell cycle progression and proliferation of breast cancer cells, through phosphorylation of FOXO3a, leading to its exclusion from nucleus and subsequent degradation in the proteasome. This function of IKKβ could be further overridden by the overexpression of FOXO3a 71 . All these findings suggest that IKKβ may contribute to HPV-induced cellular abnormalities in several ways, some through the innate immune pathway, and some independent of it. IKKβ inhibitors have served as potent anti-tumor agents inhibiting cell proliferation and invasiveness, as well as inducing cell death. Their activity has been demonstrated in several cancer types including breast, colon, ovarian, oral, prostate, liver, melanoma, and leukemia 65,66,[72][73][74][75][76] . One such inhibitor is IMD 0354, which selectively inhibits IKKβ by preventing ATP attachment to IKKβ. This will in turn block phosphorylation of IκBα, thus preventing nuclear translocation and activation of NF-κB 38 . Although IMD 0354 was initially designed to inhibit inflammation 77 , it has recently proved to have strong anticancer properties 78,79 in several cancers. IMD 0354 inhibits breast cancer cell proliferation, progression of mast cells, and it induces apoptosis in prostate, lung and colon cancers 44,[80][81][82] . In our study we found that IMD 0354 inhibited the growth of HPV 16+ and 18+ cervical cancer cell lines. This result is consistent with previously reported function of IMD 0354 in cancer cells and hence suggests a therapeutic potential for IKKβ inhibitors to treat HPV-induced cancers. Although both HPV16+ and HPV18+ cervical cancer cell lines responded to IMD 0354, however, IMD 0354-mediated inhibition of IKKβ exhibited greater effectiveness on HeLa cells (HPV 18+) compared with SiHa and CaSki cells (HPV 16+). It has previously been demonstrated that these cell lines differ in a number of molecular pathways, and in their response to treatments such as therapeutic agents that induce cell death [83][84][85] . HeLa cells exhibit greater apoptotic cell death in response to cisplatin than SiHa and CaSki cells do, a difference that is due to the higher levels of p53 and p21 in HeLa cells 84 . Additionally, these cell lines show distinct proteomic profiles for pathways connected to p53 activation, mitochondrial function and oxidative stress 85,86 . These findings suggest that the stronger effect of IMD 0354 in HeLa relative to SiHa and CaSki cells could in part be due to their difference www.nature.com/scientificreports/ in molecular pathways affected by IKKβ. Additionally, IMD 0354 caused an extensive phosphorylation of E6, which was absent in untreated cells. This finding is intriguing, as phosphorylation of E6 on the PDZ binding motif results in disruption of its interaction with PDZ domain-containing proteins 46,47 . This is consistent with our results, as degradation of Magi was inhibited when IKKβ was reduced. It would be of interest to understand the mechanism by which inhibition of IKKβ results in E6 hyperphosphorylation. In summary this study suggests an important role for IKKβ in E6-induced cellular abnormalities and that targeting IKKβ could be a potential therapeutic option for cervical cancer and, perhaps, for other HPV-related cancers for which there is currently no effective treatment. www.nature.com/scientificreports/