Role of Pirin, an Oxidative Stress Sensor Protein, in Epithelial Carcinogenesis
, Gloria M. Calaf 2,6 and Francisco Aguayo 7,*Round 1
Reviewer 1 Report
This review tries to summarize findings that would indicate that the Protein Pirin may act as an intermediate for tumor cells from several causes, including as an oxidative stress sensor, after induction by NRF2 (this is the most common fact reported in the text). It would be an important NF-KB that would promote the oncogenic features of this protein. However, many other factors may be related to these processes, and it is difficult to attribute these features only to Pirin as an intermediate. Few overexpression and KD data seem to support, but there is no demonstration. For example, NRF2 activation by oxidative stress in tumors is a well-known process that may be linked to tumor resistance to chemotherapeutic treatment, and it is not clear the need for Pirin in these cases. Instead, other intermediates are proposed, such as glutathione (see, for example, Silva et al. The balance between NRF2/GSH antioxidant mediated pathway and DNA repair modulates cisplatin resistance in lung cancer cells. Sci Rep. 2019 9(1):17639; Syu et al. Nrf2 is the key to chemotherapy resistance in MCF7 breast cancer cells under hypoxia. Oncotarget. 2016;7(12):14659-72. Zhang L, Wang H. FTY720 inhibits the Nrf2/ARE pathway in human glioblastoma cell lines and sensitizes glioblastoma cells to temozolomide. Pharmacol Rep. 2017 69(6):1186-1193. ).
Also, the manuscript seems to be not correctly revised by the authors, with figures not shown, and the figures numbering completely mistaken. Also the figures deserve better attention and need improvement. Some TCGA data for Pirin expression are presented but badly discussed or presented.
The manuscript also seems to be not correctly revised by the authors, with figures not shown, and the figures numbering completely mistaken. Also, the figures deserve better attention and need improvement. Some TCGA data for Pirin expression are presented but badly discussed or presented.
Line 48: the authors should describe better the association of dioxygenases with neurodegenerative diseases. The citation is only a review on dioxygenases, and the question is if this applies to Pirin. Otherwise, this information is not related to this review. Also, the relation of the human σ1 receptor to Pirin, or even to dioxygenases is not presented.
Line 56 … Homo sapiens. Homo should have a capital letter.
Line 61: The authors state: contains one -helix and four β-strands (Figure 1). However, this is not clear in Figure 1. The protein seems to carry more β-strands!! Please correct either the figure or the information in the text.
Line 68: take off the comma: “inhibitors, are also able to inhibit Pirin activity,”.
Line 92: correct: In Figure 2, and describe better what is the in silico analysis. Are all these proteins associated with tyrosine kinase? Or part is associated with lamellipodia formation. Please specify. Also which of the Pir function can be explained by this finding? Also, why only these genes are represented. According to this review, other genes may interact with Pirin (such as E2F1), and a complete figure (and well commented and discussed) would be necessary for this review.
Line 155: correct NRF2.
Line 158: “The Figure 1 shows immunohistochemically detected Pirin in cervical carcinomas.” Which figure are you talking about? Certainly, not Figure 1!
Line 175: “Altogether, these results indicate that Pirin contributes to negatively controlled cellular senescence in melanomas [63].” In fact, the results mentioned before do not indicate a role of pirin on senescence. This is mentioned on reference 63, but the sentence, as it is, does not make sense! But the results could be in agreement!
Line 223: I do not understand what the results on Figure 2 (or maybe figure 4????) are related to. Is this figure taken from this incomplete website? Is this a publication? Are these unpublished results? Or is it from TCGA databank? By the way, the figure itself (although very simple) is very badly presented. One cannot read the information in any of the coordinates, or even in the inserted legend, without increasing the figure size. Also, if the difference is significant, it should be shown what the difference is? If these results are just taken from database, this should be better described.
Figure 3 (or figure 5???) displays PIR expression from different tumors. It is not clear what that means? Are the expression results related to control cells from the same patient? Otherwise, results from normal tissues should also be shown, as the results may simply represent tissue differences and no meaning for tumors. The letters are better to read in comparison to the previous figure, but they can (should) also be improved.
Table 2 and the other Figure 2 (figure 6- the model) are nice and should be more explored in the text.
Neither of them are mentioned in the text before the conclusion, although Table 2 summarizes the main articles for each type of epithelial cancer in the text. The model in figure 6 should be presented and discussed as a summary of this review's ideas.
Author Response
Reviewer. This review tries to summarize findings that would indicate that the Protein Pirin may act as an intermediate for tumor cells from several causes, including as an oxidative stress sensor, after induction by NRF2 (this is the most common fact reported in the text). It would be an important NF-KB that would promote the oncogenic features of this protein. However, many other factors may be related to these processes, and it is difficult to attribute these features only to Pirin as an intermediate. Few overexpression and KD data seem to support, but there is no demonstration. For example, NRF2 activation by oxidative stress in tumors is a well-known process that may be linked to tumor resistance to chemotherapeutic treatment, and it is not clear the need for Pirin in these cases. Instead, other intermediates are proposed, such as glutathione (see, for example, Silva et al. The balance between NRF2/GSH antioxidant mediated pathway and DNA repair modulates cisplatin resistance in lung cancer cells. Sci Rep. 2019 9(1):17639; Syu et al. Nrf2 is the key to chemotherapy resistance in MCF7 breast cancer cells under hypoxia. Oncotarget. 2016;7(12):14659-72. Zhang L, Wang H. FTY720 inhibits the Nrf2/ARE pathway in human glioblastoma cell lines and sensitizes glioblastoma cells to temozolomide. Pharmacol Rep. 2017 69(6):1186-1193).
Answer. Many thanks for these comments. We agree with the reviewer because many other processes and biomolecules are involved in oxidative stress response. As stated in the abstract, the relationship between Pirin and cancer is a new area of research. In this review, we address the suggested role of Pirin in cancer, including the evidence from us and others. However, we understand that many other protagonists are involved in oxidative stress and cancer, NRF2 among them. A sentence related to lung cancer chemoresistance by NRF2 activation/addiction was added to the manuscript (lines 144-146) and the corresponding references.
Reviewer. Also, the manuscript seems to be not correctly revised by the authors, with figures not shown, and the figures numbering completely mistaken. Also, the figures deserve better attention and need improvement. Some TCGA data for Pirin expression are presented but badly discussed or presented.
Answer. Many thanks for these observations. Our apologies, the manuscript was extensively reviewed, and these mistakes were corrected.
Reviewer. Line 48: the authors should describe better the association of dioxygenases with neurodegenerative diseases. The citation is only a review on dioxygenases, and the question is if this applies to Pirin. Otherwise, this information is not related to this review. Also, the relation of the human σ1 receptor to Pirin, or even to dioxygenases is not presented.
Answer. Many thanks for this observation. There is no published information on the possibility that Pirin is directly involved in neurodegenerative diseases, so the information related to dioxygenases was deleted from the manuscript.
Reviewer. Line 56 … Homo sapiens. Homo should have a capital letter.
Answer. This was corrected.
Reviewer. Line 61: The author’s state: contains one alpha-helix and four β-strands (Figure 1). However, this is not clear in Figure 1. The protein seems to carry more β-strands!! Please correct either the figure or the information in the text.
Answer. Many thanks for this observation. We clarified that the crystal structure of Pirin shows two antiparallel beta-barrel domains. These domains are composed of beta-strands, as shown in Figure 1. Please, note that Figure 1 shows the secondary Pirin structure.
Reviewer. Line 68: take off the comma: “inhibitors, are also able to inhibit Pirin activity,”.
Answer. This was corrected.
Reviewer. Line 92: correct: In Figure 2, and describe better what is the in silico analysis. Are all these proteins associated with tyrosine kinase? Or part is associated with lamellipodia formation. Please specify. Also which of the Pir function can be explained by this finding? Also, why only these genes are represented. According to this review, other genes may interact with Pirin (such as E2F1), and a complete figure (and well commented and discussed) would be necessary for this review.
Answer. Many thanks for these observations. Additional sentences were added concerning the functions of these proteins and the suggested association with Pirin (lines 96-113). This is a bioinformatics analysis showing Pirin functional associations (STRING 11.0).
Reviewer. Line 155: correct NRF2.
Answer. This was corrected
Reviewer. Line 158: “The Figure 1 shows immunohistochemically detected Pirin in cervical carcinomas.” Which figure are you talking about? Certainly, not Figure 1!
Answer. Many thanks for this observation. The sentence “Figure 1 shows immunohistochemically detected Pirin in cervical carcinomas” was deleted. In a previous draft of this manuscript, we included the expression of Pirin in cervical carcinomas from a Latin American country (only considering a few cases). However, in the final submitted version of the manuscript, the TCGA analysis shows Pirin expression in 10,071 tumors, including cervical carcinomas and other epithelial and non-epithelial cancers.
Reviewer. Line 175: “Altogether, these results indicate that Pirin contributes to negatively controlled cellular senescence in melanomas [63].” In fact, the results mentioned before do not indicate a role of pirin on senescence. This is mentioned on reference 63, but the sentence, as it is, does not make sense! But the results could be in agreement!
Answer. Many thanks for this comment. The sentence was replaced by the following: “Altogether, these results suggest that Pirin contributes to the metastatic properties of melanoma cells” (lines 191-192).
Reviewer. Line 223: I do not understand what the results on Figure 2 (or maybe figure 4????) are related to. Is this figure taken from this incomplete website? Is this a publication? Are these unpublished results? Or is it from TCGA databank? By the way, the figure itself (although very simple) is very badly presented. One cannot read the information in any of the coordinates, or even in the inserted legend, without increasing the figure size. Also, if the difference is significant, it should be shown what the difference is? If these results are just taken from database, this should be better described.
Answer. Many thanks for this very important observation. Figures 3 and 4 were prepared by the authors from the TCGA databank (head and neck carcinomas, N=604) by the Xena bioinformatic approach. Figure 3 was improved and additional information was added to the legend.
Reviewer. Figure 3 (or figure 5???) displays PIR expression from different tumors. It is not clear what that means? Are the expression results related to control cells from the same patient? Otherwise, results from normal tissues should also be shown, as the results may simply represent tissue differences and no meaning for tumors. The letters are better to read in comparison to the previous figure, but they can (should) also be improved.
Answer. Many thanks for this very important observation. Figure 4 was prepared by the authors from the TCGA databank by Xena bioinformatic approach. Thus, this figure shows PIR (Pirin) transcripts expression in 10, 071 patients with different epithelial and non-epithelial tumors (public free access). Besides, Figure 3 was improved, and additional information was added to the legend of Figures 3 and 4 including the corresponding reference.
Reviewer. Table 2 and the other Figure 2 (figure 6- the model) are nice and should be more explored in the text.
Answer. Many thanks. Additional comments were included in the text
Reviewer. Neither of them are mentioned in the text before the conclusion, although Table 2 summarizes the main articles for each type of epithelial cancer in the text. The model in figure 6 should be presented and discussed as a summary of this review's ideas.
Answer. Many thanks for these observations. Both Table 2 and the suggested model were discussed in the text.
Reviewer 2 Report
The manuscript by Perez-Dominguez et al., is aimed to summarize the evidence about Pirin in epithelial carcinogenesis, the manuscript is well written and certainly represents a topic of interest for those in the area. Although, the manuscript presents very nice data, authors need to address several aspects before considering this manuscript for publication.
Major comments
- The order of the figures is messy, authors need to pay attention to the presented images. This need revision.
- The carcinogenic role of Pirin in lung cancer is merely speculative, authors could enhance this part in order to provide more data regarding the potential actions of Pirin in cancer development.
- Lines 183-184: What do the authors mean with: Pirin was recently identified as a potential target in antitumor therapies? This must be explained and supported with relevant bibliography
- Figure 2 (Pirin expression in HNC from TCGA): authors need to declare where that information came from, was it obtained by them (unpublished data)? or was this obtained from an already published material? This needs revision and an appropriate presentation.
- The main weakness of the work is that Pirin role in epithelial carcinogenesis is not completely understood, authors need to include more data that, directly or indirectly, support that idea. Also, they must discuss about lack of animal models for proving the participation of Pirin in cancer establishment.
Minor comments
- Figure 1 seems blurred, authors need to get a better resolution of the figure, mainly in the upper part of the figure.
- What do the authors mean when Yellow: Protein data bank?
- Lines 92-95: authors declare that figure 1 shows Pirin association with several tyrosine kinases but this is not clearly displayed in the figure, probably authors could enhance figure 1.
- Lines 158-159: authors indicate that Figure 1 shows Pirin expression in cervical carcinomas but such information is missing. Authors could add that particular data to improve the manuscript. Also, a description will be helpful in understanding why Pirin levels/localization in cervical cancer could be associated to carcinogenesis.
- Figure 2 in lines 233-234 is difficult to assess, authors need to provide a better version of the image.
Author Response
Reviewer. The manuscript by Perez-Dominguez et al., is aimed to summarize the evidence about Pirin in epithelial carcinogenesis, the manuscript is well written and certainly represents a topic of interest for those in the area. Although, the manuscript presents very nice data, authors need to address several aspects before considering this manuscript for publication.
Answer. Many thanks for these observations. The manuscript was extensively reviewed, and aspects were corrected.
Reviewer. Major comments. The order of the figures is messy, authors need to pay attention to the presented images. This need revision.
Answer. The figures were corrected and additional information was added.
Reviewer. The carcinogenic role of Pirin in lung cancer is merely speculative, authors could enhance this part in order to provide more data regarding the potential actions of Pirin in cancer development.
Answer. We agree with this observation. A sentence respect the suggested role of Pirin in lung cancer was included. Additional studies are warranted (lines 154-155).
Reviewer. Lines 183-184: What do the authors mean with: Pirin was recently identified as a potential target in antitumor therapies? This must be explained and supported with relevant bibliography
Answer. Many thanks for this observation. The sentence “Pirin was recently identified as a potential target in antitumor therapies” was deleted from lines 183-184 because it is out of context. However, the following sentence “Pirin has been reported as a molecular target of metastatic suppressors”, was modified adding the word “potential”, including the reference 43 (Identification of Pirin as a Molecular Target of the CCG-1423/CCG-203971 Series of Antifibrotic and Antimetastatic Compounds, Lisabeth EM, ACS pharmacol Transl Sci, 2019).
Reviewer. Figure 2 (Pirin expression in HNC from TCGA): authors need to declare where that information came from, was it obtained by them (unpublished data)? or was this obtained from an already published material? This needs revision and an appropriate presentation.
Answer. Many thanks for this very important observation. Figures 3 and 4 were prepared by the authors from TCGA databank by the Xena bioinformatic approach. The Figure 3 was improved and additional information was added to the legend.
Reviewer. The main weakness of the work is that Pirin role in epithelial carcinogenesis is not completely understood, authors need to include more data that, directly or indirectly, support that idea. Also, they must discuss about lack of animal models for proving the participation of Pirin in cancer establishment.
Answer. We agree with this observation. The role of Pirin in epithelial cancers is not clear, thus, in this review we present the published evidence. Additional information was added respect functionally associated proteins, which will help to understand the role of Pirin in cell migration, invasion and metastasis. In addition, we added a sentence respect the necessity of including animal models in future studies as a gold standard for Pirin-mediated tumorigenesis.
Reviewer. Minor comments. Figure 1 seems blurred, authors need to get a better resolution of the figure, mainly in the upper part of the figure. What do the authors mean when Yellow: Protein data bank?
Answer. Many thanks for this observation. The Figure quality was improved and PDB was defined in the legend.
Reviewer. Lines 92-95: authors declare that figure 1 shows Pirin association with several tyrosine kinases but this is not clearly displayed in the figure, probably authors could enhance figure 1.
Answer. This sentence was corrected. The Figure 2 shows Pirin interactions. In particular, Pirin functionally interacts with tyrosine kinases. These descriptions were improved for a better understanding of this Figure.
Reviewer. Lines 158-159: authors indicate that Figure 1 shows Pirin expression in cervical carcinomas, but such information is missing. Authors could add that particular data to improve the manuscript. Also, a description will be helpful in understanding why Pirin levels/localization in cervical cancer could be associated to carcinogenesis.
Answer. The sentence was deleted, because Pirin expression in cervical carcinomas (from TCGA database) is included in Figure 4.
Reviewer. Figure 2 in lines 233-234 is difficult to assess, authors need to provide a better version of the image.
Answer. The quality of Figure 2 was improved.
Round 2
Reviewer 1 Report
The manuscript was revised according to our criticisms. Thank you.
Reviewer 2 Report
Authors improved the manuscript, This now fits is suitable for publication
