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

  1. James A. Scarth
  2. Christopher W. Wasson
  3. Molly R. Patterson
  4. Debra Evans
  5. Diego Barba-Moreno
  6. Holli Carden
  7. Rosa Cassidy
  8. Adrian Whitehouse
  9. Jamel Mankouri
  10. Adel Samson
  11. Ethan L. Morgan
  12. Andrew Macdonald

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Abstract

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 (K ATP ) 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 K ATP 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 K ATP channels is mediated via the activation of a MAPK/AP-1 signalling axis. A complete characterisation of the role of K ATP 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.

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  1. Version published to 10.1038/s41388-023-02772-w
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    Reply to the reviewers

    Manuscript number: RC-2022-01683

    Corresponding author(s): Prof Andrew Macdonald, Dr Ethan Morgan

    1. General Statements [optional]

    We are very appreciative of the helpful and insightful comments provided by the reviewers, which will greatly aid in improving this manuscript. We were delighted by the many positive comments we received, highlighting the “high quality” data and praising our “detailed and carefully constructed” experiments, which together reveal an “interesting and novel mechanism” of HPV-driven cervical carcinogenesis.

    To summarise our key findings, we identify the host ATP-sensitive potassium ion (KATP) channel as a critical driver of proliferation and HPV oncoprotein expression in HPV+ cervical cancer cells. Use of pharmacological inhibitors and activators of KATP channels revealed that HPV oncoprotein expression correlates with channel activity, findings that were validated via siRNA/shRNA knockdown and overexpression strategies. Indeed, HPV was found to enhance expression of the ABCC8 gene (encoding the SUR1 KATP channel subunit), likely in a manner dependent on the E7 oncoprotein. We also reveal that channel knockdown impeded HPV+ cervical cancer cell proliferation, both in cell culture monolayer and, importantly, in tumour xenograft experiments. This loss of proliferation may be associated with induction of a G1 cell cycle arrest. Finally, we demonstrate that KATP channels are capable of activating ERK1/2 signalling and, in turn, the AP-1 transcription factor, leading to recruitment of AP-1 to the HPV18 promoter. Significantly, this study is the first to our knowledge to explicitly demonstrate modulation of ion channel expression and activity by HPV, and that this can contribute to host cell transformation. We believe the potential for use of the clinically available KATP channel inhibitors as novel therapies for HPV-associated malignancies should therefore be evaluated in future studies.

    Outlined below, in a point-by-point manner, are the changes we have already incorporated to improve the precision and clarity of the manuscript, as well as the additional data we intend to add in order to strengthen our findings.

    2. Description of the planned revisions

    Reviewer #1 (Evidence, reproducibility and clarity (Required)): Addressing the following major points would help to strengthen the impact of the work:

    1. The paper would be greatly strengthened by addressing whether knockdown of SUR1 and knockdown of E6/E7 are affecting cell viability. siRNA depletion of E6 and E7 will cause HeLa and SiHa cells to senesce; at what time point post knockdown were the experiments performed? Is it possible to perform CellTiterGlo or other cell viability assays to confirm that the phenotypes observed upon E6/E7 depletion and upon SUR1 depletion or drug treatment are not the result of cell death/senescence/toxicity?

    We agree that an assessment of cell viability following the treatments/transfections performed will strengthen the manuscript. We will therefore perform the suggested CellTiterGlo assay using both HeLa and SiHa cells after glibenclamide treatment, SUR1 knockdown, and E6/E7 knockdown.

    1. There is a major concern regarding whether SUR1 protein is produced at a biologically relevant level in SiHa and HeLa cells, in which most of the experiments in the paper were conducted. Protein levels are assessed in Fig 2 by immunostaining in raft cultures and in a cervical cancer tissue microarray. However, protein levels are otherwise not examined, especially in SiHa and HeLa cells. Is SUR1 protein produced in these cells? Are its levels reduced by the knockdown approaches? The fold change RNA data presented in figure 2A does not convincingly address this question, since even an 8-fold increase of ABCC8 mRNA over a low background level might not have biological significance. It would be very helpful to measure SUR1 protein in several of the experiments in HeLa and SiHa cells.

    We accept the concern of the reviewer regarding the absence of an assessment of SUR1 protein levels in HeLa and SiHa cells. There is a critical lack of high-quality antibody reagents available for the detection of SUR1, a common phenomenon within the ion channel field. We have therefore been unable to reliably detect SUR1 via immunoblot using the antibodies we have tested thus far. Nevertheless, we would argue that our other experiments in these cell lines demonstrate that not only are KATP channels expressed at biologically relevant levels but, more importantly, are active in these cell lines. Patch clamping electrophysiology, the gold-standard technique for assessing ion channel functionality, was performed in HeLa cells and the changes in current observed following inhibitor/activator application suggests that the channels are active, and by extension, that SUR1 protein must be present. Furthermore, DiBAC4(3) assays were performed following inhibitor treatments, channel stimulation, SUR1 knockdown, and HPV E7 knockdown. Although this involves an assessment of plasma membrane polarisation, and therefore is not a direct measurement of KATP channel activity, the changes observed are consistent with our expectations (e.g. increased DiBAC4(3) fluorescence with glibenclamide treatment, indicative of membrane depolarisation, is consistent with a loss of K+ ion efflux via KATP channels). However, we recognise that providing protein expression data in HeLa and SiHa cells would make our conclusions more convincing. We will therefore continue our search for antibody reagents that will allow us to reliably detect SUR1 protein in HPV+ cell lines by western blot. We will also pursue other detection methods in these cell lines, including immunofluorescence and immunohistochemistry. We hope that we are successful in our optimisation, allowing us to validate that SUR1 protein levels are reduced following our knockdown approaches.

    1. The authors should address the idea of off-target effects, either experimentally or, more feasibly, by discussing the possibility of non-specific effects of SUR1 knockdown. They use a pool of four siRNAs to SUR1 and the risk of off-target effects would be greatly reduced if individual siRNAs were tested and shown to have the same effect as one another. Similarly, several experiments use just one shRNA, limiting the ability to draw conclusions.

    To address off-target effects, we will repeat some of the experiments performed in this manuscript using individual siRNAs. HeLa and SiHa cells will be transfected with each of the four siRNAs individually and the impact on HPV E6 and E7 expression examined by RT-qPCR and western blot. Further, we will also repeat colony formation assays and DiBAC4(3) assays to ensure that each siRNA has a similar effect on proliferation and membrane polarisation respectively.

    1. Finally, since many of the experiments rely on knockdown approaches that show similar readouts, a rescue experiment (restore sh or si-resistant SUR1 and assess the impact on the phenotype) would confirm that the effects being observed are due to changes in SUR1 levels and not to off-target effects.

    We agree that rescue experiments would greatly strengthen the manuscript. Our laboratory has significant experience in carrying out this type of experiment and as such we would be very happy to perform them. We will reintroduce siRNA-resistant SUR1 following knockdown of endogenous SUR1 levels and confirm that E6/E7 expression and proliferation are restored.

    CROSS-CONSULTATION COMMENTS I note several areas of common feedback among the reviews. Several reviewers commented on the large number of experiments and that the work is of interest to researchers working on HPV and cancer therapeutics. Several reviewers shared concerns about cell viability upon HPV oncoprotein knockdown and about toxicity in various experiments. Several reviewers also raised concerns about the validation of SUR1 protein levels in several experiments. These concerns seem to me to be critical to address to strengthen the manuscript. I note that Reviewer #3's suggestion of making E7 knockout cells (presumably in HPV+ cancer cell lines) is unlikely to be possible because the cells require E7 for survival.

    We agree that the two main areas of concern shared among the reviewers (cell viability assays and validation of SUR1 protein levels) are issues that should be addressed in a revised version of this manuscript. We will endeavour to perform the experiments described above, which we hope will significantly enhance the impact of our work.

    Reviewer #2 (Evidence, reproducibility and clarity (Required)): Major Comments: Authors did not explain how HPV E7 would upregulate ABCC8 transcription or elevate SUR1 protein (Figure 4). Depletion of E7 is known to produce lethal effect in cervical cancer cell lines. No experiment was done to assess cytotoxicity. Hence it is not clear from the available evidence if the SUR1 is reduced by direct E7 mediated event or indirectly by general cytotoxicity induced by E7 knock down.

    We thank the reviewer for their suggestions. We agree that it would be useful to provide some mechanistic insight into how E7 upregulates *ABCC8 *transcription. We therefore plan to analyse the promoter region of ABCC8 to identify potential transcriptional regulators. ChIP-qPCR and luciferase reporter constructs containing the ABCC8 promoter region will be used to unravel the importance of any candidate TFs identified. An assessment of the impact of E7 on the expression and/or activity of these TFs may also be performed. Finally, we will combine E7 overexpression in a HPV- cell line with knockdown/inhibition of a candidate TF to confirm our findings.

    Experiments to assess cell viability/cytotoxicity will be performed as outlined in our response to Reviewer #1.

    Authors did not analyze expression level and role of p53, pRB proteins, the direct targets of E6 and E7 proteins, on cell cycle regulation following SUR1 siRNA or Glibenclamide-treatment in cervical cancer cell lines.

    We agree that the protein levels of p53 and pRb, the primary targets of E6 and E7 respectively, should be analysed in HeLa and SiHa cells following SUR1 knockdown and glibenclamide treatment and will endeavour to perform this.

    Minor Comments:

    Additional immunofluorescence or histological analysis is necessary to assess the potential cytotoxic effects of E7 siRNA, SUR1 siRNA or KATP inhibitors (Glibenclamide) in cervical cancer cell lines.

    We agree that an assessment of cell viability will strengthen the manuscript and we plan to address this as outlined in our response to Reviewer #1.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    In addition to the points raised by the reviewers, we identified a minor error that occurred during assembly of the manuscript. Incorrect images of colony formation assays were provided in the original version of Figure 5B. This has now been amended in the transferred manuscript.

    __Reviewer #1 (Evidence, reproducibility and clarity (Required)):____ __Overall, the data are of high quality and the individual results are consistent with each other and are convincing. However, the authors have understandably focused on two HPV-positive cancer cell lines (affected by modulating KATP levels) and one HPV-negative cancer cell line (which is not affected in the same way). The ability to extrapolate to conclusions about cervical or HPV-positive cancers in general is therefore limited and many of the authors' statements should be tempered to reflect the experiments they have conducted.

    We thank the reviewer for their positive comments regarding our data. We agree that by mainly focussing our studies on cervical cancer cell lines, we limit the potential for extrapolation to other HPV-associated malignancies. We were careful to qualify many of our statements in the original submission to reflect this: e.g. lines 314-315: “Collectively, these data demonstrate that KATP channels are important drivers of proliferation in HPV+ cervical cancer cells.” Post review, we have altered references to HPV+ cancers in general to more accurately reflect the data presented, as outlined below:

    • Lines 112-113: We hope that the targeting of KATP channels may prove to be beneficial in the treatment of HPV-associated __cervical __neoplasia.____
    • Lines 430-431: As such, we believe that the clinically available inhibitors of KATP channels could constitute a potential novel therapy for HPV-associated malignancies HPV+ cervical cancer.

    Addressing the following major points would help to strengthen the impact of the work:

    The paper would be greatly strengthened by addressing whether knockdown of SUR1 and knockdown of E6/E7 are affecting cell viability. siRNA depletion of E6 and E7 will cause HeLa and SiHa cells to senesce; at what time point post knockdown were the experiments performed? Is it possible to perform CellTiterGlo or other cell viability assays to confirm that the phenotypes observed upon E6/E7 depletion and upon SUR1 depletion or drug treatment are not the result of cell death/senescence/toxicity?

    All experiments involving siRNA-mediated knockdown were performed at 72 hours post-transfection. We apologise for omitting this information, and it has now been added to the ‘Materials and Methods’ section.

    Minor comments: The text and figures are clear and statistics are appropriate. The authors should include at what time point post siRNA transfection the experiments were conducted.

    As above, this information has been added to the ‘Materials and Methods’ section.

    __Reviewer #2 (Evidence, reproducibility and clarity (Required)):____ __E6 and E7 protein bands in DMSO treated HeLa and SiHa cells are not consistent between Figures 1 E, H and J, hence confound the interpretation. There is no information on biological replicates. It is not clear why the data from inhibitor treatments were not corroborated by genetic knock down or knock out experiments.

    Regarding the number of biological replicates, as described under the ‘Statistical analysis’ subheading of the ‘Materials and Methods’ section, “all experiments were performed a minimum of three times, unless stated otherwise”. Where data is presented as bar graphs, this information is also included in the figure legend and each biological replicate is represented by a single data point where possible. In the case of western blots, a statement of the number of biological repeats has been added under the ‘Western blot analysis’ subheading of the ‘Materials and Methods’ section:

    • Lines 614-615: “A minimum of three biological repeats were performed in all cases and representative blot images are displayed.”

    Minor Comments: They did not provide physiological functions of K+ATP channel. I consider this information should be important part of the introduction.

    We agree that this would provide important contextual information and apologise for omitting this in the original submission. Details of some well-characterised functions of KATP channels, outside of their potential role in regulating cell proliferation, have been added to the introduction (lines 99-102).

    The evidence for elevated expression of SUR1 in raft cultures of uninfected and HPV-18 infected HFK, CINs, and HSIL like cultures of W12E cells (Figure 2) is not of good quality. Moreover, in the absence of histological evidence (hematoxylin and eosin staining) and markers for HPV E6 E7 activity it is difficult to interpret about the location of SUR1 signals in spatial relationship to E7 functions.

    In an attempt to resolve the issue highlighted, we have removed a reference in the text to the layers of the epithelium in which SUR1 expression is upregulated, as detailed below:

    • Lines 195-197: This demonstrated a marked increase in SUR1 protein expression in __the suprabasal layers of __HPV18+ rafts in comparison to NHK raft cultures, consistent across both donors (Fig 2C).

    There is no physical evidence that HPV-18 transfected HFK indeed harbored HPV-18 plasmid in this experiment. What is the effect of glibenclamide on HPV-18 episome maintenance or replication?

    The HPV18-containing keratinocytes used in this study are the same model system we have used in previous investigations (Wasson et al. (2017) Oncotarget 8(61): 103581–103600; Morgan et al (2018) PLoS Pathog 14(4): e1006975). In these studies, which focus on the HPV life cycle rather than HPV+ cancer, Southern blot analysis of HPV episomes and western blots for E6/E7 protein levels were performed, providing validation of the presence of HPV in these cells. We have added a reference to our previous work in the text (line 191).

    As this manuscript primarily focusses on HPV+ cancer rather than HR-HPV infection, we believe an assessment of the role of KATP channels on HPV episome maintenance and/or genome replication to be beyond the scope of this study.

    4. Description of analyses that authors prefer not to carry out

    __Reviewer #1 (Evidence, reproducibility and clarity (Required)):____ __Addressing the following major points would help to strengthen the impact of the work:

    The authors should address the idea of off-target effects, either experimentally or, more feasibly, by discussing the possibility of non-specific effects of SUR1 knockdown. They use a pool of four siRNAs to SUR1 and the risk of off-target effects would be greatly reduced if individual siRNAs were tested and shown to have the same effect as one another. Similarly, several experiments use just one shRNA, limiting the ability to draw conclusions.

    Our plan to address potential off-target effects of the siRNAs used is outlined above. Regarding the shRNA data, we use two different shRNAs in the majority of experiments presented. These were found to have highly similar impacts on E6/E7 expression (Figure 3E & 3G) and proliferation (Supp Figure 4). We therefore do not believe that further experiments to eliminate off-target effects of the shRNA are necessary.

    __Reviewer #2 (Evidence, reproducibility and clarity (Required)):____ __Major Comments: Overall, the authors performed many experiments to reveal an interesting and novel mechanism. (1) SUR1 expression and activity is necessary for HPV16 and-18 E6 and E7 expression. (2) HPV-16/18 E7 upregulates expression of ABCC8/SUR1 transcription. (3) SUR1 containing K+ATP channel then phosphorylates ERK. (4) Activated ERK then phosphorylates JUN/AP1. (5) Next, activated JUN/AP1 promotes E7 or E6E7 expression from HPV URR. However, in this cyclic feedforward regulation of these genes there is no control mechanism. Then how is homeostasis maintained in HPV infected lesions?

    We thank the reviewer for praising the “interesting and novel mechanism” we have uncovered. The primary focus of our study is HPV+ cervical cancers, rather than HPV-infected lesions. The data we present in Figure 2 indicates that upregulation of KATP channel expression and/or activity is likely also occurring during HR-HPV infection, given we see e.g. increased SUR1 staining in HPV18-containing organotypic rafts. However, given our primary focus, we believe a more thorough assessment of KATP channels in HR-HPV infection, including any potential homeostatic regulation mechanisms, is outside the scope of our current study and inclusion of additional life cycle data would dilute the main conclusions of this manuscript. It is nonetheless a potentially exciting area for future work, and could form part of a separate, focussed manuscript.

    E6 and E7 protein bands in DMSO treated HeLa and SiHa cells are not consistent between Figures 1 E, H and J, hence confound the interpretation. There is no information on biological replicates. It is not clear why the data from inhibitor treatments were not corroborated by genetic knock down or knock out experiments.

    We believe that bands for E6 and E7 protein in DMSO-treated samples in Figure 1E and 1J are broadly consistent. However, we appreciate that E6 and E7 protein expression appears to be lower in DMSO samples in Figure 1H. All experiments involving diazoxide stimulation were performed under conditions of serum starvation, as indicated in the legends for Figures 1 and 8. Oncoprotein expression is known to be regulated by a series of host transcription factors, many of which become active in response to growth factor stimulation, such as AP-1 and SP1 (see: Tan et al. (1992) Nucleic Acids Res. 20(2):251-6; Hoppe-Seyler et al. (1992) Nucleic Acids Res. 20(24):6701-6; Butz K et al. (1993) J Virol. 67(11):6476-86). Serum starvation was therefore used to disentangle the upregulation of HPV gene expression by KATP channels from the myriad of other host signalling pathways demonstrated to drive E6/E7 expression. A side effect of this was a reduction in basal E6 and E7 protein levels in DMSO-treated cells. In addition, shorter exposure times were deliberately selected to prevent overexposure of protein bands corresponding to 50uM diazoxide treated cells. We apologise for any confusion caused.

    Data from inhibitor treatments has been corroborated by knockdown experiments throughout the manuscript. The loss of E6/E7 expression with glibenclamide treatment was confirmed by SUR1 siRNA and shRNA knockdowns (Figure 3D-G) and Kir6.2 knockdown (Supp Figure 3C-D). The glibenclamide-induced loss of proliferation observed in HeLa and SiHa cells was also validated using the same approaches (Figure 5D-F, Supp Figure 3E-G, Supp Figure 4). Indeed, the cell cycle dysregulation experiments in Figure 7 were all performed with both inhibitor treatments and SUR1 siRNA knockdown.

    The increase of G1 population, determined by flow cytometry, of HeLa cells treated with Glib or SUR1 siRNA is relative to controls appears to be small and not supported by similar study on other HPV+ or HPV_ vervical cancer cell lines. Importantly the mechanism of this increased G1 in HeLa cell line is not clear. The immunoblot data about the role of cyclins are not sufficient.

    The flow cytometry experiments with glibenclamide treatment and SUR1 knockdown in HeLa cells were also performed with HPV16+ SiHa cells (Figure 7A-B) and the effects are highly consistent between the two cell lines. We believe that elucidating a more detailed mechanism of the observed G1 arrest is beyond the scope of this manuscript. Analysis of the mRNA and protein expression of cyclins was intended to provide corroboration of our findings via flow cytometry (i.e. a specific reduction in G1-phase cyclins corresponds to an accumulation of cells in G1 phase), rather than provide mechanistic insight.

    What is the physiological effect of cyclin D1 in the context of HR-HPV infection (Figure 7)? In the event of HPV E7 mediated pRB degradation in cervical cancer cell lines, the inactivation of pRB by cyclin D1 does not appear to be physiologically relevant, may not account for difference in growth. It is known in literature that Cyclins A2 and B1 are often elevated by E7 activity. If SUR1 siRNA reduces E7-transcription and protein levels as shown in earlier results, why cyclinB1 and A2 protein level did not change?

    As discussed, this manuscript primarily focusses on the role of KATP channels in HPV+ cervical cancer cells. An investigation into the effects on cyclin D1 expression in the context of HR-HPV infection, whilst an important question, is beyond the scope of this study and should form part of a standalone manuscript.

    Regarding the expression of cyclins A2 and B1, we repeatedly observed very little impact following glibenclamide treatment and SUR1 knockdown in both cell lines examined. As highlighted above, given we observe a G1 arrest after KATP channel knockdown/inhibition, it would perhaps be unusual to observe changes in the expression of cyclins which drive G2 and M phase progression, respectively.

    If activated ERK1/2 and c-Jun is required for URR activity, why are not they detectable in DSO or scrRNA treated HeLa cells (Fig 8A, B)? Why there is no 18 E7 in DMSO treated HeLa cells (Fig. 8A)? Authors also did not explain how inhibition of KATP channel regulates ERK phosphorylation in cervical cancer cell lines. There is no data from additional cervical cancer cell lines or HSIL mimicking W12E.

    As mentioned above and as referred to in the figure legend, the experiment presented in Figure 8A was performed under serum starved conditions. Thus, the levels of phosphorylated ERK1/2 and cJun are much reduced in the unstimulated, DMSO-treated cells. Importantly, cJun/AP-1 is capable of activating its own expression, explaining the low total protein level of cJun in this sample. Further, as has widely been reported, MAPK signalling and AP-1 activity are critical drivers of HPV URR-driven transcription (e.g., see: Morgan et al. (2021) Cell Death Differ. 28(5):1669-87), so the reduced signalling activity resulting from serum starvation will consequently lower 18E7 expression. Shorter exposure times were selected for the pERK1/2, p-cJun and cJun western blots in Figure 8B to highlight the dramatic increase in pathway activation following KATP channel overexpression and for consistency with Figure 8A. We apologise for any confusion caused but do not believe that the potential issue raised here significantly alters any of the conclusions drawn.

    We believe that an explanation of the mechanism by which KATP channel activity contributes to the activation of ERK1/2 signalling in HPV+ cervical cancer cells is beyond the scope of this initial publication. In the course of acquiring the data presented here, we aimed to provide some evidence of how KATP channels regulate proliferation in these cells, and therefore decided to investigate what impact they had on host cell signalling pathways known to be critical for proliferation. This led to us identifying enhanced ERK1/2 activity in response to KATP channel stimulation/overexpression. We agree that the critical next step will be to elucidate how channels promote ERK1/2 signalling, but this would require a significant body of work and as such would warrant being in a standalone or follow-up publication.

    Throughout the manuscript, we endeavoured to validate our discoveries in the HPV18+ HeLa cell line in an additional cervical cancer cell line (HPV16+ SiHa cells). We provide evidence that the requirement for KATP channel activity is shared by both cell lines, and the impacts on oncoprotein expression, proliferation and cell cycle progression are highly concordant between the two cell lines. Thus, it is reasonable to assume that the mechanism by which KATP channels drive proliferation and HPV URR activity, elucidated in Figure 8 using HeLa cells, will be common to other HPV+ cervical cancer cell lines. Given the wealth of evidence supporting our proposal in Figure 8, and the highly concordant data presented earlier in the manuscript, we do not believe repeating all of the experiments in Figure 8 in a HPV16+ cell line is warranted. Similarly, as this manuscript focusses on HPV+ cancer, we believe that a study of the activation of MAPK/AP-1 signalling by KATP channels in HSIL mimicking W12E cells is beyond the scope of this paper and should constitute part of a standalone manuscript.

    Minor Comments: In introduction, the authors mentioned that high risk HPV E6 and E7 deregulate cell cycle in host cells, and current limitations in cervical cancer treatments. Then they introduced importance of K+ ion channels in cell cycle regulation by sighting published literature not related to HPV, immediately followed by their proposed study on role of K+ATP channels in HPV infection. However, authors did not sufficiently clarify the rational of taking up a study on K+ channels in the context of HPV infection or E6 and E7 expression. If K+ATP channel proteins are elevated by E7, it is highly likely that there are some prior information on status of these transporters in the published literature or data from RNAseq analyses.

    As the reviewer acknowledges, we described in the introduction the importance of K+ channels in regulating cell proliferation and the absence of effective HPV-specific therapies. We also stated that “ion channels may represent ideal candidates for … novel therapies given the abundance of licensed and clinically available drugs targeting the complexes which could be repurposed…”. Thus, the rationale for studying K+ channels in HPV+ cancers was to see if any of the available inhibitors of K+ channels could potentially be repurposed to be used in treating HPV-driven cervical cancer. We believe this logic is explained with sufficient clarity in the original draft.

    Regarding prior analysis of KATP channel expression, in Figure 2H of the manuscript we analyse a publically available microarray dataset, which provides mRNA expression data for 128 cervical tissue specimens (24 normal, 14 CIN1 lesions, 22 CIN2 lesions, 40 CIN3 lesions, and 28 cancers specimens). *ABCC8 *expression was found to be significantly higher in the CIN3 and cervical cancer specimens, compared to normal samples. Details of the dataset used are provided in the ‘Materials and Methods’ section.

    The evidence for elevated expression of SUR1 in raft cultures of uninfected and HPV-18 infected HFK, CINs, and HSIL like cultures of W12E cells (Figure 2) is not of good quality. Moreover, in the absence of histological evidence (hematoxylin and eosin staining) and markers for HPV E6 E7 activity it is difficult to interpret about the location of SUR1 signals in spatial relationship to E7 functions.

    Organotypic raft culture data was included to demonstrate increased SUR1 expression in primary keratinocytes containing HR-HPV to show broader upregulation of the host factor and strengthen data from cell lines and clinical samples. Our data shows an upregulation of SUR1 in the HPV-containing rafts compared to controls. However, raft culture is a highly complex, time-consuming and expensive technique to perform. Therefore, whilst we agree, in principal, that being able to more closely correlate the increase in SUR1 protein levels to specific layers of the epithelium (e.g. via H&E staining and for markers of E6/E7 activity) would be of value, we would not be able to perform these assays within a reasonable time frame. Moreover, we feel that it would not add significant new knowledge to the study of SUR1 in the context of HPV-driven cancers.

    There is no physical evidence that HPV-18 transfected HFK indeed harbored HPV-18 plasmid in this experiment. What is the effect of glibenclamide on HPV-18 episome maintenance or replication?

    As this manuscript primarily focusses on HPV+ cancer rather than HR-HPV infection, we believe an assessment of the role of KATP channels on HPV episome maintenance and/or genome replication to be beyond the scope of this study.

    __Reviewer #2 (Significance (Required)):____ __ (1) General Assessment: Strengths and limitations This study identified KATP channel components as novel regulators of transcriptional activity of high-risk HPV-18 URR through ERK1/2-c-Jun/AP1 pathway. Authors revealed that HPV E7 regulates expression of ABCC8, the gene for channel component SUR1 protein.

    There are important limitations. (1) Lack of any information about homeostatic regulation of SUR1 in HPV infection, (2) Lack of sufficient evidence about potential confounding cytotoxic effects of SUR1 inhibition or E7 down regulation on cervical cancer cells. (3) Immunoblot experiments are not consistent. (4) There is no mechanism of how E7 regulates ABCC8 transcription. (5) What is the mechanism for SUR1 regulate cell cycle in HPV+ cells? (6) There is no mention of effect of SUR1 on cell cycle regulators p53 and pRB, which are direct targets of HPV E6 and E7 proteins. (7) There is no evidence for the role of Kir6.2 /SUR1 in the regulation of HPV-16 URR, which causes most of HPV-attributed cancers. (8) Authors did not analyze spatial relationship between HPV E6 and E7 activity and expression of SUR1 protein in raft cultures of human foreskin keratinocytes with or without E6E7 expression and in cervical cancer tissue.

    We thank the reviewer for summarising in a concise manner the areas of this manuscript they believe could be improved. Our responses to points 1-6 and point 8 have been detailed above. Regarding (7), we present data showing that KATP channel inhibition/knockdown negatively affects E6 and E7 expression in HPV16+ SiHa cells (Figure 1D-E, Figure 3D and F, Supp Figure 3C-D). Furthermore, we present evidence that SUR1 knockdown in SiHa cells correlates with a reduction in HPV16 URR-driven luciferase activity (Figure 3H). We therefore believe that this issue was adequately addressed in the original manuscript.

    __Reviewer #3 (Evidence, reproducibility and clarity (Required)):____ __Minor comments:

    Knockout SUR1 stable cell lines and knockout HPV E7 stable cell lines should be established to test all the related data.

    We have performed experiments using two well characterised inhibitors of KATP channels (glibenclamide and tolbutamide) and both siRNA- and shRNA-mediated knockdown of SUR1, all of which result in similar reductions in HPV E6/E7 expression. Further, the reductions in proliferation observed in HPV+ cell lines following glibenclamide treatment or siRNA/shRNA knockdown of SUR1 are also highly concordant. Thus, we do not believe the establishment of knock__out__ cell lines, using e.g. CRISPR/Cas9 technology, would significantly enhance the manuscript, particularly given the time and expense involved in this.

    As noted by Reviewer #1 following cross-consultation, the establishment of E7 knockout cells lines is “unlikely to be possible because the cells require E7 for survival”. It has been previously demonstrated that in the absence of E7 expression, HeLa cells cease to proliferate and undergo senescence within 10 days (see: DeFelippis et al. (2003) J.Virol 77(2): 1551–1563). We therefore agree with Reviewer #1 and believe that, unfortunately, we would be unable to carry out the suggested experiment.

    Tumor weights of the in vivo experiment should be indicated.

    Tumour weights were not collected following the conclusion of the in vivo experiment, so we are unable to provide this information. The experiment was designed such that animals would be sacrificed upon the tumours reaching a set measurement (15 mm in either direction), rather than concluding the experiment at a set end point. Therefore, many of the tumours would have been of similar weight upon sacrifice, but critically the SUR1-depleted tumours took significantly longer to reach that size. We therefore believe that, given the experimental set-up, adding the tumour weights would not add significant value, even if we were able to provide this information.

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    Referee #3

    Evidence, reproducibility and clarity

    Summary:

    Provide a short summary of the findings and key conclusions (including methodology and model system(s) where appropriate).

    Authors present the evidence that HPV can target ATP-sensitive potassium ion (KATP) channels of a host to promote cervical carcinogenesis. They indicate that these channels are active in HPV-positive cells and that this activity is required for HPV oncoprotein expression by using activators and inhibitors of KATP channels. Furthermore, they verified SUR1 was upregulated in both HPV+ cervical cancer cells and in clinical samples in a manner dependent on the E7 oncoprotein. Knockdown of SUR1 or KATP channel inhibition significantly impeded cell proliferation via induction of a G1 cell cycle phase arrest. They propose that tumorgenesis effect of KATP channels is mediated via the activation of a MAPK/AP-1 signalling axis. Overall, It is an interesting research to unveil the mechanism how HPV promote cervical carcinogenesis through ATP-sensitive potassium ion channels. However，some major concerns should be addressed.

    Major comments:

    • Are the key conclusions convincing?

    I think the key conclusions are convincing.

    • Should the authors qualify some of their claims as preliminary or speculative, or remove them altogether?

    NO

    • Would additional experiments be essential to support the claims of the paper? Request additional experiments only where necessary for the paper as it is, and do not ask authors to open new lines of experimentation.

    NO

    • Are the suggested experiments realistic in terms of time and resources? It would help if you could add an estimated cost and time investment for substantial experiments.
    • Are the data and the methods presented in such a way that they can be reproduced?

    Yes

    • Are the experiments adequately replicated and statistical analysis adequate?

    The experiments are adequately replicated and statistical analysis

    Minor comments:

    • Specific experimental issues that are easily addressable.

    Yes

    • Are prior studies referenced appropriately?

    Yes

    • Are the text and figures clear and accurate?

    yes

    • Do you have suggestions that would help the authors improve the presentation of their data and conclusions?

    Knockout SUR1 stable cell lines and knockout HPV E7 stable cell lines should be established to test all the related data.

    Tumor weights of the in vivo experiment should be indicated.

    Significance

    • Describe the nature and significance of the advance (e.g. conceptual, technical, clinical) for the field.

    The author explain for the first time that HPV can upregulate host ATP-sensitive potassium ion channels, consequently activate MAPK/AP-1 signalling contribute to cervical carcinogenesis. This is a new mechanism how HPV cause cervical carcinogenesis. However, the MAPK/AP-1 signalling contributes to carcinogenesis is well known. The technical in the experiment is commonly used.

    • Place the work in the context of the existing literature (provide references, where appropriate).

    HPV can promote cervical carcinogenesis through different pathway including MAPK/AP-1(1: Wang M, Qiao X, Cooper T, Pan W, Liu L, Hayball J, Lin J, Cui X, Zhou Y,Zhang S, Zou Y, Zhang R, Wang X. HPV E7-mediated NCAPH ectopic expression regulates the carcinogenesis of cervical carcinoma via PI3K/AKT/SGK pathway. Cell Death Dis. 2020 Dec 11;11(12):1049. doi: 10.1038/s41419-020-03244-9. PMID:33311486; PMCID: PMC7732835. 2. Singh T, Chhokar A, Thakur K, Aggarwal N, Pragya P, Yadav J, Tripathi T, Jadli M, Bhat A, Gupta P, Khurana A, Chandra Bharti A. Targeting Aberrant Expression of STAT3 and AP-1 Oncogenic Transcription Factors and HPV Oncoproteins in Cervical Cancer by Berberis aquifolium. Front Pharmacol.2021 Oct 28;12:757414. doi: 10.3389/fphar.2021.757414. PMID: 34776976; PMCID: PMC8580881.). However, the detail mechanism is still elusive. Here, authors indicated E7 can upregulate SUR1, one component of ATP-sensitive potassium ion channels and activate MAPK/AP-1. SUR1 can also upregulat E7 levels. They set up a positive feedback loop to contribute cervical cancer.

    • State what audience might be interested in and influenced by the reported findings.

    cervical carcinogenesis researchers and cancer drug researches.

    • Define your field of expertise with a few keywords to help the authors contextualize your point of view. Indicate if there are any parts of the paper that you do not have sufficient expertise to evaluate.

    I familiar with cancer related signaling pathway and cancer chemoprevention research. Especially MAPK signaling pathway and related drugs.

  4. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #2

    Evidence, reproducibility and clarity

    Summary: Authors explored the role of k+ channel transporters in HPV induced cervical cancers. They used several inhibitors and gene knockdowns in biochemical and cell biology experiments to show that SUR1 and Kir6.2 components of K+ ATP channel, via activating pERK1/2 and c-Jun/AP1, up-regulate HPV URR promoter mediated expression of E6 and E7 proteins. They showed that SUR1 knockdown inhibited growth of HeLa cells in vivo.

    Major Comments:

    Overall, the authors performed many experiments to reveal an interesting and novel mechanism. (1) SUR1 expression and activity is necessary for HPV16 and-18 E6 and E7 expression. (2) HPV-16/18 E7 upregulates expression of ABCC8/SUR1 transcription. (3) SUR1 containing K+ATP channel then phosphorylates ERK. (4) Activated ERK then phosphorylates JUN/AP1. (5) Next, activated JUN/AP1 promotes E7 or E6E7 expression from HPV URR. However, in this cyclic feedforward regulation of these genes there is no control mechanism. Then how is homeostasis maintained in HPV infected lesions?

    E6 and E7 protein bands in DMSO treated HeLa and SiHa cells are not consistent between Figures 1 E, H and J, hence confound the interpretation. There is no information on biological replicates. It is not clear why the data from inhibitor treatments were not corroborated by genetic knock down or knock out experiments.

    Authors did not explain how HPV E7 would upregulate ABCC8 transcription or elevate SUR1 protein (Figure 4). Depletion of E7 is known to produce lethal effect in cervical cancer cell lines. No experiment was done to assess cytotoxicity. Hence it is not clear from the available evidence if the SUR1 is reduced by direct E7 mediated event or indirectly by general cytotoxicity induced by E7 knock down.

    The increase of G1 population, determined by flow cytometry, of HeLa cells treated with Glib or SUR1 siRNA is relative to controls appears to be small and not supported by similar study on other HPV+ or HPV_ vervical cancer cell lines. Importantly the mechanism of this increased G1 in HeLa cell line is not clear. The immunoblot data about the role of cyclins are not sufficient.

    What is the physiological effect of cyclin D1 in the context of HR-HPV infection (Figure 7)? In the event of HPV E7 mediated pRB degradation in cervical cancer cell lines, the inactivation of pRB by cyclin D1 does not appear to be physiologically relevant, may not account for difference in growth. It is known in literature that Cyclins A2 and B1 are often elevated by E7 activity. If SUR1 siRNA reduces E7-transcription and protein levels as shown in earlier results, why cyclinB1 and A2 protein level did not change?

    Authors did not analyze expression level and role of p53, pRB proteins, the direct targets of E6 and E7 proteins, on cell cycle regulation following SUR1 siRNA or Glibenclamide-treatment in cervical cancer cell lines.

    If activated ERK1/2 and c-Jun is required for URR activity, why are not they detectable in DSO or scrRNA treated HeLa cells (Fig 8A, B)? Why there is no 18 E7 in DMSO treated HeLa cells (Fig. 8A)? Authors also did not explain how inhibition of KATP channel regulates ERK phosphorylation in cervical cancer cell lines. There is no data from additional cervical cancer cell lines or HSIL mimicking W12E.

    Minor Comments:

    In introduction, the authors mentioned that high risk HPV E6 and E7 deregulate cell cycle in host cells, and current limitations in cervical cancer treatments. Then they introduced importance of K+ ion channels in cell cycle regulation by sighting published literature not related to HPV, immediately followed by their proposed study on role of K+ATP channels in HPV infection. However, authors did not sufficiently clarify the rational of taking up a study on K+ channels in the context of HPV infection or E6 and E7 expression. If K+ATP channel proteins are elevated by E7, it is highly likely that there are some prior information on status of these transporters in the published literature or data from RNAseq analyses. They did not provide physiological functions of K+ATP channel. I consider this information should be important part of the introduction.

    The evidence for elevated expression of SUR1 in raft cultures of uninfected and HPV-18 infected HFK, CINs, and HSIL like cultures of W12E cells (Figure 2) is not of good quality. Moreover, in the absence of histological evidence (hematoxylin and eosin staining) and markers for HPV E6 E7 activity it is difficult to interpret about the location of SUR1 signals in spatial relationship to E7 functions.

    Additional immunofluorescence or histological analysis is necessary to assess the potential cytotoxic effects of E7 siRNA, SUR1 siRNA or KATP inhibitors (Glibenclamide) in cervical cancer cell lines

    There is no physical evidence that HPV-18 transfected HFK indeed harbored HPV-18 plasmid in this experiment. What is the effect of glibenclamide on HPV-18 episome maintenance or replication?

    Significance

    (1) General Assessment: Strengths and limitations

    This study identified KATP channel components as novel regulators of transcriptional activity of high-risk HPV-18 URR through ERK1/2-c-Jun/AP1 pathway. Authors revealed that HPV E7 regulates expression of ABCC8, the gene for channel component SUR1 protein.

    There are important limitations. (1) Lack of any information about homeostatic regulation of SUR1 in HPV infection, (2) Lack of sufficient evidence about potential confounding cytotoxic effects of SUR1 inhibition or E7 down regulation on cervical cancer cells. (3) Immunoblot experiments are not consistent. (4) There is no mechanism of how E7 regulates ABCC8 transcription. (5) What is the mechanism for SUR1 regulate cell cycle in HPV+ cells? (6) There is no mention of effect of SUR1 on cell cycle regulators p53 and pRB, which are direct targets of HPV E6 and E7 proteins. (7) There is no evidence for the role of Kir6.2 /SUR1 in the regulation of HPV-16 URR, which causes most of HPV-attributed cancers. (8) Authors did not analyze spatial relationship between HPV E6 and E7 activity and expression of SUR1 protein in raft cultures of human foreskin keratinocytes with or without E6E7 expression and in cervical cancer tissue.

    (2) Advance: This study identifies SUR1/Kir6.2 as new targets to intervene HPV-18 URR activity and demonstrates potential to inhibit growth of cervical cancer tumors using HeLa xenograft model. This study did not develop any new methodology, novel mutation or model system.

    (3) Audience: This study is aimed at basic scientists involved in the field of HPV research.

    (4) Describe your expertise. I have long experience in HPV research. I study regulation of HR-HPV18 life cycle in 3D organotypic raft cultures of HPV-18 infected neonatal foreskin keratinocytes. A major part of my research is focused on identification of novel therapeutics against cervical cancers using in vitro 3D organoids and in vivo PDX models.

  5. Review Commons

    Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.

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    Referee #1

    Evidence, reproducibility and clarity

    In this manuscript by Scarth and colleagues, the authors investigate the relationship between ATP-sensitive potassium ion channels (KATP) and the viability and growth of certain HPV-positive cancer cell lines. In a series of detailed and carefully conducted experiments, they determine that there is a correlation between KATP channel activity and levels of certain HPV E6 and E7 RNA and protein. KATP channels are active in HeLa cells. In HeLa and SiHa cells, inhibiting KATP activity decreases HPV oncoprotein levels. HPV-positive status in the cell lines examined is found to be associated with an upregulation of the ABCC8 gene, which encodes the SUR1 KATP subunit, and some of the data supports that SUR1 protein levels increase with cervical cancer CIN grade. Depleting SUR1 with shRNA or siRNA reduces KATP activity and decreases HPV E6 and E7 levels in HeLa and SiHa cells. The opposite is also true; depleting ABCC8 reduces the levels of HPV E6 and E7 transcripts. The effect on ABCC8 appears to be due mainly to the effects of HPV E7. Decreased KATP activity is associated with decreased growth of HeLa and SiHa cells in monolayer and in anchorage independent growth assays, perhaps not surprising given that E6/E7 levels are reduced in the cells under the treatment conditions. SUR1 overexpression itself promotes cell growth even in the absence of HPV oncoproteins. The growth defect upon KATP inhibition or siSUR1 is associated with some modest cell cycle dysregulation and, impressively, with reduced tumor growth in a mouse model. Finally, the authors present evidence that increased KATP activity is associated with increased recruitment of the transcription factor AP-1 to the HPV18 promoter and enhancer.

    Overall, the data are of high quality and the individual results are consistent with each other and are convincing. However, the authors have understandably focused on two HPV-positive cancer cell lines (affected by modulating KATP levels) and one HPV-negative cancer cell line (which is not affected in the same way). The ability to extrapolate to conclusions about cervical or HPV-positive cancers in general is therefore limited and many of the authors' statements should be tempered to reflect the experiments they have conducted.

    Addressing the following major points would help to strengthen the impact of the work:

    1. The paper would be greatly strengthened by addressing whether knockdown of SUR1 and knockdown of E6/E7 are affecting cell viability. siRNA depletion of E6 and E7 will cause HeLa and SiHa cells to senesce; at what time point post knockdown were the experiments performed? Is it possible to perform CellTiterGlo or other cell viability assays to confirm that the phenotypes observed upon E6/E7 depletion and upon SUR1 depletion or drug treatment are not the result of cell death/senescence/toxicity?
    2. There is a major concern regarding whether SUR1 protein is produced at a biologically relevant level in SiHa and HeLa cells, in which most of the experiments in the paper were conducted. Protein levels are assessed in Fig 2 by immunostaining in raft cultures and in a cervical cancer tissue microarray. However, protein levels are otherwise not examined, especially in SiHa and HeLa cells. Is SUR1 protein produced in these cells? Are its levels reduced by the knockdown approaches? The fold change RNA data presented in figure 2A does not convincingly address this question, since even an 8-fold increase of ABCC8 mRNA over a low background level might not have biological significance. It would be very helpful to measure SUR1 protein in several of the experiments in HeLa and SiHa cells.
    3. The authors should address the idea of off-target effects, either experimentally or, more feasibly, by discussing the possibility of non-specific effects of SUR1 knockdown. They use a pool of four siRNAs to SUR1 and the risk of off-target effects would be greatly reduced if individual siRNAs were tested and shown to have the same effect as one another. Similarly, several experiments use just one shRNA, limiting the ability to draw conclusions.
    4. Finally, since many of the experiments rely on knockdown approaches that show similar readouts, a rescue experiment (restore sh or si-resistant SUR1 and assess the impact on the phenotype) would confirm that the effects being observed are due to changes in SUR1 levels and not to off-target effects.

    It is recognized that some of these experiments would be lengthy and technically challenging to perform. Measuring cell viability and SUR1 protein levels in SiHa and HeLa cells should be relatively straightforward. The experiments to address off-target effects (rescue experiment, deconvolving siRNA pool) are more involved. If it is not possible to complete such experiments, the possibility of off-target effects should be discussed in the text.

    Minor comments:

    The text and figures are clear and statistics are appropriate. The authors should include at what time point post siRNA transfection the experiments were conducted.

    Referees cross-commenting

    I note several areas of common feedback among the reviews. Several reviewers commented on the large number of experiments and that the work is of interest to researchers working on HPV and cancer therapeutics. Several reviewers shared concerns about cell viability upon HPV oncoprotein knockdown and about toxicity in various experiments. Several reviewers also raised concerns about the validation of SUR1 protein levels in several experiments. These concerns seem to me to be critical to address to strengthen the manuscript. I note that Reviewer #3's suggestion of making E7 knockout cells (presumably in HPV+ cancer cell lines) is unlikely to be possible because the cells require E7 for survival.

    Significance

    The work connects the biology of certain cervical cancer cell lines to KATP channels. It will be of interest to HPV researchers and to cancer researchers whose interests involve KATP signaling. As a reviewer, I have expertise in HPV biology but not in KATP signaling.

  6. Version published to 10.1101/2022.09.22.508991v1 on bioRxiv