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

Human papillomaviruses activate and recruit SMC1 cohesin proteins for the differentiation-dependent life cycle through association with CTCF insulators.

Human papillomaviruses infect stratified epithelia and link their productive life cycle to the differentiation state of the host cell. Productive viral replication or amplification is restricted to highly differentiated suprabasal cells and is dependent on the activation of the ATM DNA damage pathway. The ATM pathway has three arms that can act independently of one another. One arm is centered on p53, another on CHK2 and a third on SMC1/NBS1 proteins. A role for CHK2 in HPV genome amplification has been demonstrated but it was unclear what other factors provided important activities. The cohesin protein, SMC1, is necessary for sister chromatid association prior to mitosis. In addition the phosphorylated form of SMC1 plays a critical role together with NBS1 in the ATM DNA damage response. In normal cells, SMC1 becomes phosphorylated in response to radiation, however, in HPV positive cells our studies demonstrate that it is constitutively activated. Furthermore, pSMC1 is found localized in distinct nuclear foci in complexes with γ-H2AX, and CHK2 and bound to HPV DNA. Importantly, knockdown of SMC1 blocks differentiation-dependent genome amplification. pSMC1 forms complexes with the insulator transcription factor CTCF and our studies show that these factors bind to conserved sequence motifs in the L2 late region of HPV 31. Similar motifs are found in most HPV types. Knockdown of CTCF with shRNAs blocks genome amplification and mutation of the CTCF binding motifs in the L2 open reading frame inhibits stable maintenance of viral episomes in undifferentiated cells as well as amplification of genomes upon differentiation. These findings suggest a model in which SMC1 factors are constitutively activated in HPV positive cells and recruited to viral genomes through complex formation with CTCF to facilitate genome amplification. Our findings identify both SMC1 and CTCF as critical regulators of the differentiation-dependent life cycle of high-risk human papillomaviruses

SMC1 is phosphorylated by both ATM and ATR.

<p>A). ATM inhibitor, KU60019 inhibits CHK2 phosphorylation, but only partially reduces SMC1 phosphorylation in HPV positive cells. HPV 31 positive CIN 612 cells were treated with the ATM kinase inhibitor KU60019 (10uM) in DMSO or DMSO alone for 48 and 72 hours and cell extracts examined by Western blot analysis. pCHK2 levels are inhibited by KU60019 while pSMC1 levels are only partially reduced suggesting another kinase such as ATR is involved in phosphorylation. GAPDH is included as a loading control. B) DNA damage response inhibitor Caffeine (10mM) inhibits both pSMC1 and pATR after 48 hours of treatment.</p

Levels of CTCF are increased in undifferentiated and differentiated HPV positive cells.

<p>A). Whole cell extracts were isolated from undifferentiated normal human foreskin keratinocytes (HFK) and CIN 612 cells grown in monolayer culture and examined by Western blot analysis with antibodies to CTCF. Cytokeratin 10 served as a differentiation control. GAPDH served as a loading control. CTCF knockdown results in a statistically significant reduction in protein levels, p≤.01. B). Knockdown of CTCF with shRNA blocks viral genome amplification. CIN612 were infected with lentiviruses encoding shRNA to CTCF and after 48 hours total cell extracts were isolated and analyzed by Western blot for levels of CTCF. shRNA knockdown of CTCF blocks HPV-31 differentiation-dependent viral amplification as shown by Southern blot. Southern blot analysis of CIN 612 cells infected with lentiviruses encoding shRNAs to CTCF and induced to differentiate in methylcellulose for 48 hours. Total DNA was isolated and examined by Southern analysis for HPV amplification. Similar results were observed in three independent experiments using two different shRNAs against CTCF. Quantification of band intensities represents averages of three independent experiments and were determined by densitometry using FIJI software. CTCF knockdown results in a reduction in amplification, p≤.05.</p

Mutation of 3 CTCF core consensus sequences within the L2 coding region of the HPV 31 genome results in impaired growth, loss of episomes, and rapid integration into the host genome.

<p>A).HFKs were transfected with WT HPV-31 genomes or HPV-31 genomes harboring mutations within three CTCF motifs in the L2 coding region along with a drug selectable marker. Following transfection cells were selected and representative colony sizes as seen by light microscopy are shown three days after initiation of selection. Differences in colony size were reproducibly seen. B). Mutation of CTCF sites impairs genome maintenance resulting in integration and inability to amplify. Following transfection and selection, cells were grown to confluency and differentiated in high calcium media for 72 hours. Total DNA was isolated and examined by Southern blot analysis for HPV amplification. Quantification of band intensity was determined by densitometry using FIJI software. C). Mutation of 3 CTCF core consensus sequences abrogates SMC1 and CTCF binding to the HPV genome. Chromatin Immunoprecipitation analysis of stable cell lines containing the WT HPV-31 genome and the three mutations in CTCF motifs demonstrated a reduction of SMC and CTCF binding in mutant lines. Two separate passages of the mutant cell lines demonstrates loss of SMC-1 and CTCF binding. Data is representative of three independent experiments. Panel i). L2 region in cells with wildtype HPV 31; ii). L2 region in cells with L2 mutant 3X genomes at passage 2 after transfection; iii). L2 region in cells with L2 mutant 3X genomes at passage 3 after transfection.</p

Knockdown of SMC1 with shRNA blocks viral genome amplification.

<p>A). CIN612 were infected with lentiviruses encoding shRNAs to SMC1 and after 48 hours total cell extracts were isolated and analyzed by Western blot for levels of SMC1. Quantitation of band intensity revealed an approximate 50% knockdown of SMC1 protein levels on average across three experiments. The knockdown is statistically significant between mock and shSMC and shCTRL and shSMC, p≤.0001, and p≤.001 respectively. B). shRNA knockdown of SMC1 blocks HPV-31 differentiation-dependent viral amplification. Southern blot analysis of CIN 612 cells infected with lentiviruses encoding shRNAs to SMC1 and induced to differentiate in methylcellulose for 48 hours. UD stands for undifferentiated while D stands for differentiated. Total DNA was isolated and examined by Southern analysis for HPV amplification. Similar results were seen in four independent experiments using two different shRNAs as well as the combination of the two. Quantification of band intensity was determined by densitometry using Image J software and represents an average of three independent experiments. SMC knockdown results in a reduction in amplification that is statistically significant, p≤.05</p

Levels of total SMC1 and pSMC1 are increased in HPV positive cells and remain high upon calcium-induced differentiation.

<p>A). Whole cell extracts were isolated from undifferentiated normal human foreskin keratinocytes (HFK) and HPV 31 positive CIN 612 cells, and HFKs stably transfected to maintain HPV-31 genomes grown in monolayer culture and examined by Western blot analysis with antibodies to total SMC1, pSMC1 and pCHK2. GAPDH served as a loading control. B). CIN612 cells and HFKs were induced to differentiate by addition of high calcium media and cell extracts harvested at 0, 24, 48 and 72 hours. Western blot analysis was performed with antibodies against total SMC1 and pSMC1. GAPDH served as a loading control. Quantitation of band intensity revealed an approximate 2-fold, differentiation-independent increase of SMC1 protein levels in HPV-positive cells.</p

pSMC1 and pCHK2 as well as pSMC1 and γ-H2AX co-localize in distinct foci in nuclei of HPV positive cells following differentiation.

<p>CIN 612 cells were induced to differentiate by addition of high calcium media for 72 hours and examined by immunofluorescence for co-localization of A). pSMC1 and pCHK2; B), pSMC1 and γ-H2AX using the corresponding antibodies. Red signal identifies pCHK2 or γ-H2AX in top or bottom panels as shown. Green identifies pSMC1 (s957). Blue represents nuclear DAPI staining. Yellow signal in merged views demonstrates co-localization. The antibody to pCHK2 Thr68 (Cell Signalling) may be responsive to DNA damage factors in general.</p

On-slide co-immunoprecipitations for pSMC1 in HFKs and HPV positive cells shows a direct interaction between pSMC1 and γ-H2AX as well as pSMC1 and pCHK2 in HPV positive cells but not HFKs.

<p>A). HFKs and CIN 612 cells grown in monolayer cultures were treated with 4% Methanol-free formaldehyde in PBS. Cells were permeabilized in PBS +. 1% Triton X-100 (PBT) and blocked in Normal Goat Serum (NGS+T) (Invitrogen) with. 1% Triton X-100. Primary antibodies were added to the NGS+T and incubated for one hour. Secondary antibodies, covalently linked with plus/minus oligos (OLINK DuoLink, Upsalla Sweden) were added and incubated for an additional 45 minutes. Ligations, and polymerase amplifications, and washes were performed as per the manufacturer’s instructions. All incubations were performed in a humidity chamber. The appearance of red foci indicates complex formation in distinct nuclear puncta. B). Quantitation of number of red interaction foci from on-slide co-immunoprecipitation analyses. For the interaction between pSMC1 and γ-H2AX 132 HFKs and 71 CIN612 cells were examined. For the interaction between pSMC1 and pCHK2 187 HFKs and 92 CIN612 cells were examined. Foci counts are from three individual experiments. Interactions of these factors with pSMC1 in HPV positive cells is statistically significant as indicated by the students t-test, p≤.0001.</p

pSMC1 is localized to nuclear foci in HPV positive cells that become more numerous upon differentiation.

<p>A) Immunofluorescence analysis of undifferentiated HFKs and CIN612 cells for pSMC1 and total SMC1 demonstrates localization to the nucleus in HPV positive cells. Green indicates either pSMC1 (s957) or total SMC1 as indicated. Blue represents nuclear DAPI staining. B). Immunofluorescence of undifferentiated and 72-hour calcium-induced differentiated CIN612 cells identifies large discrete foci of pSMC1 in HPV positive cells. The left panel represents undifferentiated cells while the right panel represents differentiated cells. Green identifies either pSMC1 (s957) or total SMC1 as indicated. Blue represents nuclear DAPI staining. C). The number of pSMC1 foci greater than 3 per nucleus increases upon differentiation of HPV 31 positive cells. The number of pSMC1 nuclear foci in 559 individual undifferentiated and 569 differentiated cells obtained from 3 separate experiments was determined. The graph demonstrates the percentage of total nuclei with greater than 3 foci per nucleus increasing to 80% of cells upon differentiation in high calcium media for 72 hours. A standard students t-test was used to determine statistical significance of <.0001.</p