Review Reports - Bioinformatics Analysis Reveals E6 and E7 of HPV 16 Regulate Metabolic Reprogramming in Cervical Cancer, Head and Neck Cancer, and Colorectal Cancer through the PHD2-VHL-CUL2-ELOC-HIF-1α Axis

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

This is a well-written paper by Adán Arizmendi-Izazaga and colleagues exploring the possible mechanism by which the E6 and E7 oncoproteins of HPV 16 could be affecting

the PHD2-VHL-CUL2-ELOC-HIF-1α backbone and metabolic reprogramming in CC, HNSC, and CRC cancers.

This is bioinformatics analysis.

The conducted study expands our knowledge about the mechanisms of action of the main HPV16 oncoproteins.

It would be good to add to the discussion whether these results can have any practical application. Apart from that, I have no further comments.

1 Table and 8 figures are very informative and provide great data presentation.

The authors extensively discussed their results in relation to recent scientific literature and clinical data reports.

Paper has 83 references which are relevant to article's subject.

This is a good study, and clinically valuable.

Author Response

Reviewer 1

Comments and Suggestions for Authors

This is a well-written paper by Adán Arizmendi-Izazaga and colleagues exploring the possible mechanism by which the E6 and E7 oncoproteins of HPV 16 could be affecting the PHD2-VHL-CUL2-ELOC-HIF-1α backbone and metabolic reprogramming in CC, HNSC, and CRC cancers.

This is bioinformatics analysis.

The conducted study expands our knowledge about the mechanisms of action of the main HPV16 oncoproteins.

It would be good to add to the discussion whether these results can have any practical application. Apart from that, I have no further comments.

Response: We really appreciate the valuable suggestion of the reviewer 1. In this new version of the manuscript, in the discussion section, the practical use of our results was added.

1 Table and 8 figures are very informative and provide great data presentation.

The authors extensively discussed their results in relation to recent scientific literature and clinical data reports.

Paper has 83 references which are relevant to article's subject.

This is a good study, and clinically valuable.

Response: We appreciate the nice comments of reviewer 1 to the findings described in our manuscript.

Author Response File: Author Response.pdf

Reviewer 2 Report

Comments and Suggestions for Authors

Reviewed Manuscript: "Bioinformatics analysis reveals E6 and E7 of HPV 16 regulate metabolic reprogramming in Cervical Cancer, Head and Neck Cancer, and Colorectal Cancer through PHD2-VHL-CUL2-ELOC-HIF-1α axis"

Strengths:

1. To clarify the impact of HPV 16 E6 and E7 oncoproteins on metabolic pathways across various cancer types, the manuscript employs a comprehensive bioinformatics method. This offers an invaluable multifaceted perspective on viral oncogenesis.

2. Based on thorough data analysis from reliable databases like TCGA and GTEx, it links the changes brought about by E6 and E7 in the PHD2-VHL-CUL2-ELOC-HIF-1α axis to metabolic reprogramming, a crucial cancer characteristic.

3. The manuscript's scientific rigor and repeatability are improved by the methods' clear explanation, especially when it comes to the usage of particular databases and analytical tools (such as the Kaplan-Meier Plotter for survival analysis and the GEPIA for expression analysis).

Recommendations:

Although the bioinformatics predictions are strong, E6 and E7's impact on the identified metabolic pathways would be confirmed by experimental validation using cell models or patient-derived samples.
A few figures, most notably Figure 3, have poor quality, which makes them difficult to read. Improving these figures' clarity and sharpness would greatly enhance the manuscript's ability to communicate results visually.
An examination of any biases present in the bioinformatics datasets utilized (such as population diversity in TCGA and GTEx) might enhance the study's findings.
It would be beneficial to include a discussion of how these findings may be applied in clinical settings, for example, as possible therapeutic targets or biomarkers for early identification.

Overall Assessment: Using a thorough bioinformatics approach, this publication offers important insights into the involvement of HPV 16 E6 and E7 oncoproteins in metabolic reprogramming across several cancer types. The results deepen our knowledge of HPV's function in the biology of cancer. The impact and usefulness of the paper would be increased, though, if experimental validations were included, figure quality was improved, and there was a more thorough discussion of the data' significance for diagnosis and treatment.

Author Response

Reviewer 2

Strengths:

To clarify the impact of HPV 16 E6 and E7 oncoproteins on metabolic pathways across various cancer types, the manuscript employs a comprehensive bioinformatics method. This offers an invaluable multifaceted perspective on viral oncogenesis.
Based on thorough data analysis from reliable databases like TCGA and GTEx, it links the changes brought about by E6 and E7 in the PHD2-VHL-CUL2-ELOC-HIF-1α axis to metabolic reprogramming, a crucial cancer characteristic.
The manuscript's scientific rigor and repeatability are improved by the methods' clear explanation, especially when it comes to the usage of particular databases and analytical tools (such as the Kaplan-Meier Plotter for survival analysis and the GEPIA for expression analysis).

Recommendations:

Although the bioinformatics predictions are strong, E6 and E7's impact on the identified metabolic pathways would be confirmed by experimental validation using cell models or patient-derived samples.

Response: Although our study did not use cell models or patient-derived samples, here we used repositories that include experimental studies in cell lines from patient-derived samples. All databases have an experimental basis and a description of each database was added in the materials and methods section of the new version of the manuscript.

A few figures, most notably Figure 3, have poor quality, which makes them difficult to read. Improving these figures' clarity and sharpness would greatly enhance the manuscript's ability to communicate results visually.

Response: We appreciate the reviewer's comment about improving the quality of Figure 3. In this new version of the manuscript, Figure 3 was modified to improve the quality.

An examination of any biases present in the bioinformatics datasets utilized (such as population diversity in TCGA and GTEx) might enhance the study's findings.

Response: The reviewer's observation is very interesting. As part of the findings that would improve the research, we considered adding HIF-1α expression in samples obtained from TCGA and GTEx platforms from HPV 16 positive and negative CC, HNSC and CRC. However, only 8 samples were HPV 16 positive and three of the samples were positive for HPV 16, HPV 18 and other HPVs. HNSC and CRC samples are not classified as HPV 16 positive or negative. The figure below shows slightly higher expression of HIF-1α in the HPV 16 positive CRC samples. The data were not statistically significant.

At this time, we are limited by time to analyze HIF-1α expression in CC, HNSC and CRC in TCGA and GTEx, classifying the diversity of the population and cancer types in CC, HNSC and CRC. We will consider performing another manuscript including HIF-1α expression profiling in CC, HNSC and CRC with all the aforementioned biases in the future.

It would be beneficial to include a discussion of how these findings may be applied in clinical settings, for example, as possible therapeutic targets or biomarkers for early identification.

Response: The reviewer's observation is very interesting. Therefore, in this new version of the manuscript, the clinical application of HIF-1α, as potential therapeutic targets or biomarkers, was incorporated in the discussion section. In addition, metabolic pathways regulated by HIF-1α overexpression are proposed as a metabolic signature in the noninvasive diagnosis and identification of CC, HNSC and CRC.

Author Response File: Author Response.pdf

Reviewer 3 Report

Comments and Suggestions for Authors

Arizmendi-Izazaga and colleagues presented a computational research article aimed at exploring the metabolic reprogramming occurring in cancer mediated by HPV E6 and E7 proteins. For this purpose, the authors performed bioinformatics investigations consulting the HIF-1α expression contained in the TCGA database in multiple tumors. Then the authors focused the attention on the interaction between E6/E7, HIF-1α and other factors. Overall, the manuscript is very confusing and the rationale is not clear. Please address the following minor/major issues:

1) The authors emphasized the metabolic reprogramming of cancer cells mediated by E6 and E7 HPV oncoprotein, however, there are no in silico or in vitro investigations on metabolic changes in cancer cells. I suggest to completely revise the focus of your manuscript;

2) In the first sentence of the Introduction section, use brackets for the acronym (HPV) the first time used;

3) Please consider to add a Figure describing all the metabolic changes and modifications of HIF-1α axis mediated by E6 and E7 oncoprotein;

4) It is not clear why the authors analyzed the HIF-1α expression in all TCGA tumors and not in the HPV+ tumors only. A comparison between the expression levels of HIF-1α in HPV- and HPV+ tumors should be presented;

5) Although some interactions between E6/E7 and HIF-1α were established, there are no proves that such interactions determine the overexpression of HIF-1α. Your hypothesis needs further confirmation;

6) It is not clear the link existing among miRNAs, E6 and E7, HIF-1α and PHD2, VHL, CUL2, and ELOC. Please better introduce this concept;

Author Response

Reviewer 3

Comments and Suggestions for Authors

The authors emphasized the metabolic reprogramming of cancer cells mediated by E6 and E7 HPV oncoprotein, however, there are no in silico or in vitro investigations on metabolic changes in cancer cells. I suggest to completely revise the focus of your manuscript;

Response: The observations made by the reviewer are important and interesting. For this reason, it is important to clarify that the databases used in this manuscript are fully supported and experimentally validated. In the materials and methods section of the new version of the manuscript, the description of the databases was incorporated, where we mentioned the number of patient samples, the cell lines and the experimental assays that support the analysis performed.

In the first sentence of the Introduction section, use brackets for the acronym (HPV) the first time used;

Response: In the new version of the manuscript the acronym of HPV was already included in brackets.

Please consider to add a Figure describing all the metabolic changes and modifications of HIF-1α axis mediated by E6 and E7 oncoprotein;

Response: We appreciate the reviewer's observation. In this new version of the manuscript, the figure 9 was incorporated and we described all the metabolic changes and modifications of HIF-1α axis mediated by E6 and E7 oncoproteins.

It is not clear why the authors analyzed the HIF-1α expression in all TCGA tumors and not in the HPV+ tumors only. A comparison between the expression levels of HIF-1α in HPV- and HPV+ tumors should be presented;

Response: We appreciate the reviewer's observations. In this regard, we analyzed HIF-1α expression in all TCGA tumors and not only in HPV+ tumors. Unfortunately, in TCGA platform the classification of CC, HNSC and CRC samples is limited. Only 8 samples were positive for HPV 16 and three of the samples were positive for HPV 16, HPV 18 and other HPV. The HNSC and CRC samples are not classified as HPV 16 positive or negative.

The figure depicts a slightly higher expression of HIF-1α not significant in the HPV 16 positive samples, although the number of samples in the two groups limits the requested analysis.

Although some interactions between E6/E7 and HIF-1α were established, there are no proves that such interactions determine the overexpression of HIF-1α. Your hypothesis needs further confirmation;

Response: We appreciate the reviewer's comment. In the new version of the manuscript, in the introduction section we described the importance of the elongation and ubiquitination complex (VCB-CR) constituted by von Hippel Lindau (VHL), Elongin C (ELOC), Elongin B (ELOB), Cullin 2 (CUL2) and ring box protein 1 (RBX1) in the stability of HIF-1α. Inhibiting VCB-CR complex formation enables HIF-1α stability.

It is not clear the link existing among miRNAs, E6 and E7, HIF-1α and PHD2, VHL, CUL2, and ELOC. Please better introduce this concept;

Response: In this new version of the manuscript in the results and discussion section, the relationship of miRNA, E6 and E7, HIF-1α and PHD2, VHL, CUL2, and ELOC was added.

Author Response File: Author Response.pdf

Round 2

Reviewer 3 Report

Comments and Suggestions for Authors

Dear Authors,

almost all of my previous comments were not correctly addressed. Specifically, you fail in correct answer to my previous comment N.1. In my previous comment, I asked for validation of metabolic changes mediated by E6 and E7 HPV oncoprotein on cancer cells. However, you have not provided data on it (nor in silico nor wet data).

As regards my previous comment N.4, the lack of information on the HPV status of the TCGA tumors analyzed is a severe limitation of the study. If you do not know the HPV status of cancer samples your analyses are not focused on HPV.

Similarly, my previous comments N.5 and N.6 were not correclty addressed.

For all these reasons, the manuscript is not suitable for publication.

Author Response

RESPONSE LETTER

We thank reviewer number 3 for his generosity for his valuable contributions, suggestions and questioning of our manuscript. All the comments have undoubtedly improved our manuscript considerably. In this new revised version of our manuscript we have incorporated, to the extent possible, the responses to the reviewer's concerns. We hope that with these modifications, the reviewer will find our manuscript suitable for publication in the journal CIMB.

Reviewer 3

Almost all of my previous comments were not correctly addressed. Specifically, you fail in correct answer to my previous comment N.1. In my previous comment, I asked for validation of metabolic changes mediated by E6 and E7 HPV oncoprotein on cancer cells. However, you have not provided data on it (nor in silico nor wet data).

Response: Previously, our laboratory reports that high expression of enzymes and transporters involved in glycolytic metabolism such as SLC2A1 (GLUT1), LDHA, SLC16A3 (MCT4), CA9 (CAIX) and BSG (Basigin or CD147) are related to the histological grade of cervical cancer, including cervical intraepithelial neoplasia (CIN), invasive cervical carcinoma (ICC) and HPV 16 infection (DOI: 10. 1369/00221554221101662, 10.3390/pathogens12010033). In addition, a review published by our lab reports the role of HPV 16 E6 and E7 oncoproteins and their variants in metabolic reprogramming (DOI: 10.3390/pathogens10030347). These findings discussing the role of HPV E6 and E7 through the VHL ubiquitin-ligase axis, CUL2, ELOC and ELOB were addressed in the discussion section of the current version of the manuscript.

We report below all reports of the role of E6 and E7 oncoproteins in metabolic reprogramming requested in order to respond to the reviewer.

Table 1. Effect of HPV 16 E6 and E7 variants on metabolic reprogramming in cancer.

Up or down arrows indicate the increase or decrease, respectively.

Figure 2. Relative expression of genes involved in metabolic reprogramming. The relative expression level of HIFA1 (a), SLC2A1 (b), LDHA (c), CA9 (d), SLC16A3 (e), and BSG (f) in the SiHa and Ca SKi cell compared to C33-A. A value of p < 0.05 was considered statistically significant through a one-way ANOVA test and using mean and standard error. Data were measured in three independent experiments in triplicate in RT-qPCR and calculated by the 2−ΔΔCT method. Expression of the six transcripts was normalized to endogenous GAPDH. Relative expression levels were analyzed in GraphPad Prism software. * p < 0.05; ** p < 0.001.

As regards my previous comment N.4, the lack of information on the HPV status of the TCGA tumors analyzed is a severe limitation of the study. If you do not know the HPV status of cancer samples your analyses are not focused on HPV.

Response: Thank you for your comment. In this new version of the manuscript, HIF1A expression in HPV-, HPV+ tumors and HR-HPV genotypes was added in the section on results. Expression was assessed through GSE9750 GSE67522 and GSE73761 datasets obtained from GEO repository, Array- and sequence-based functional genomic data.

Similarly, my previous comments N.5 and N.6 were not correclty addressed.

Although some interactions between E6/E7 and HIF-1α were established, there are no proves that such interactions determine the overexpression of HIF-1α. Your hypothesis needs further confirmation;

Response:We appreciate the reviewer's comments. In this manuscript we propose that E6 and E7 favor HIF1A stability through attenuating their interaction with VHL, CUL2, ELOC and ELOB ubiquitin ligase axis proteins. Data demonstrating the interactions of E6-VHL, E7-CUL2, E7-ELOC and E7-ELOB were obtained from fully supported and experimentally validated databases. In the discussion section of the new manuscript version, we mention some of the interaction assays that were performed that support the interaction of E6 and E7 with the ubiquitin-ligase axis VHL, CUL2, ELOC and ELOB (Affinity capture-MS assays, FRET, Affinity capture-Western, Two-hybrid, co-localization, PCA a protein fragment complementation assay, protein-peptide interaction).

It is not clear the link existing among miRNAs, E6 and E7, HIF-1α and PHD2, VHL, CUL2, and ELOC. Please better introduce this concept;

Response: Thank you for the suggestion. In the new version of the manuscript in the introduction and discussion section we explain the link between miRNAs, E6 and E7, HIF-1α and PHD2 axis, VHL, CUL2 and ELOC. Furthermore, in this new version of the manuscript we add results showing the expression of miRNAs transcriptionally targeting the PHD2, VHL, CUL2 and ELOC axis in HPV+ and HPV- cell lines. The expression data of miRNAs were obtained from the GSE158659 dataset of the GEO repository. We also analyzed the expression of PHD2, VHL, CUL2 and ELOC axis transcripts in HPV+ and HPV- samples, in order to correlate the high expression of HPV E6 and E7 positively regulated miRNAs and the low expression levels of PHD2, VHL, CUL2 and ELOC transcripts. PHD2, VHL, CUL2 and ELOC expression data were obtained from the GSE9750 dataset of the GEO repository.

Author Response File: Author Response.pdf

Round 3

Reviewer 3 Report

Comments and Suggestions for Authors

The addition of new analyses considering the data contained in the GEO DataSets database is an added value to the manuscript. However, the results reported in Figure 3 are very conflicting as only the HPV45 strain is associated with HIF1a overexpression.

Therefore the first hypothesis of your manuscript is not so strong.