Activated heme synthesis regulates glycolysis and oxidative metabolism in breast and ovarian cancer cells

Pritpal Kaur; Shreya Nagar; Madhura Bhagwat; Mohammad Uddin; Yan Zhu; Ivana Vancurova; Ales Vancura

doi:10.1371/journal.pone.0260400

Peer Review History

Original SubmissionDecember 23, 2020
10 Mar 2021 Decision Letter - Pankaj K Singh, Editor PONE-D-20-40407 Activated heme synthesis regulates glycolysis and oxidative metabolism in breast and ovarian cancer cells PLOS ONE Dear Dr. Vancura, Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process. I believe the findings are very interesting and would certainly be strengthened by testing in additional TNBC cell lines. Please submit your revised manuscript by 1st Sept 2021. If you will need more time than this to complete your revisions, please reply to this message or contact the journal office at plosone@plos.org. 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Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes Reviewer #2: Yes ****** 4. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes Reviewer #2: Yes ****** 5. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: This is an interesting paper that investigates the role of Heme levels and cell metabolism and migration. Key roles for the transcription factor BACH1 and AMPK. The experiments are convincing and the conclusions are supported by the presented experiments. The material and methods are adequately described. The analysis is limited to just two cell lines, MDA-MB-231 and SKOV3 cells which limits impact of these studies and the metabolic analysis is limited to measurements of ECAR and OCR. In addition, the response of the two cell lines to ALA is not the same, leaving one to wonder about the universality of the author’s principal findings. Based on the experiments where AICAR is used to activate AMPK, the authors state several times in the results section that AMPK has certain activities in their different assays. It would be more correct to state that AICAR treatment has a particular activity and corelate that activity with AMPK activation state. In essence, the experiments don’t adequately test AMPK necessity. Reviewer #2: This article demonstrates that enhancing heme synthesis through exogenous supplementation of heme precursor 5-aminolevuliic acid can suppresses oxygen consumption as well as glycolysis and reduces proliferation of ovarian cancer and TNBC cells. Such findings imply a possibility for 5-aminolevuliic acid to treat cancer. The study seems to be an interesting. However, the idea is not so novel as the similar studies with other cancer cell lines have been described. Some justification on the originality of the work is necessary. Specific critiques are provided below: 1. The authors performed extracellular acidification rate (ECAR) approach only to test glycolytic rate. This is limited in that ECAR is highly related to pH value of cell medium and CO2 production. The standard measurements of glycolytic metabolism are the use of 3H labeled glucose and lactate concentration in culture medium of cells treated with or without drugs. 2. In all figures, mRNA levels of HO-1 and Catalase have been tested. However, as important enzymes in the catabolism of heme, the protein levels of these enzymes should be examined as well. 3. Justification of literature citations regarding the dosages of ALA are missing. 4. In figure 1F and 2F, could you explain why you choose these targets (NDUFB5, cox17, cox15, ATP5D and SLC25A15) as ETC and OXPHOS genes, instead of coxⅣ, coxⅤ or any other standard genes? 5. In figure 2I, the area of wound width is not in appropriate position. 6. The data indicated that Bach1 was reduced by ALA supplementation, and silencing Bach1 could reduce ECT and OXPHOS, which is similar to ALA supplementation treatment. However, whether Bach1 is involved in heme for reducing ETC, OXPHOS, and glycolysis, was not discussed. Does any approach (such as chemicals) block the degradation of Bach1 in ALA supplemented cells? And after that, further ETC, OXPHOS, and glycolysis analysis should be performed. 7. The lack of vivo data to validate the results in culture is another limitation of the research, which should be discussed. ****** 6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). 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Revision 1
4 Aug 2021 Author Response Response to Reviewers Academic Editor: 1. “I believe the findings are very interesting and would certainly be strengthened by testing in additional TNBC cell lines.” Response: We agree; we have included additional four cell lines, including another TNBC cell line (MDA MB 468) and another breast cancer cell line (MCF7) (Fig. 8 of the revised manuscript). Reviewer 1: 1. “This is an interesting paper that investigates the role of Heme levels and cell metabolism and migration. Key roles for the transcription factor BACH1 and AMPK. The experiments are convincing and the conclusions are supported by the presented experiments. The material and methods are adequately described. The analysis is limited to just two cell lines, MDA‐MB‐231 and SKOV3 cells which limits impact of these studies and the metabolic analysis is limited to measurements of ECAR and OCR. In addition, the response of the two cell lines to ALA is not the same, leaving one to wonder about the universality of the author’s principal findings.” Response: We have included additional four cell lines with different activities of oxidative metabolism (Fig. 8 of the revised manuscript). The general conclusion of these studies is that proliferation of cells with a high activity of oxidative metabolism, such as H1299 and MCF7, is not affected by low concentrations of ALA (0.1 and 1 mM). In contrast, proliferation of cells with a lower activity of oxidative metabolism, such as SKOV3 and OVCAR3, is affected even by low (0.1 and 1 mM) ALA concentrations. The inverse correlation between the OCR and ALA sensitivity is particularly interesting for cells with the same origin, as shown for MCF7, MDA MB 231, and MDA MB 468 (Fig. 2 and 8 of the revised manuscript). 2. “Based on the experiments where AICAR is used to activate AMPK, the authors state several times in the results section that AMPK has certain activities in their different assays. It would be more correct to state that AICAR treatment has a particular activity and corelate that activity with AMPK activation state. In essence, the experiments don’t adequately test AMPK necessity.” Response: We agree; we have corrected the Results section “AMPK represses oxidative metabolism and glycolysis in both ovarian cancer and TNBC cells” (p. 12 of the revised manuscript). Reviewer 2: “This article demonstrates that enhancing heme synthesis through exogenous supplementation of heme precursor 5‐aminolevuliic acid can suppresses oxygen consumption as well as glycolysis and reduces proliferation of ovarian cancer and TNBC cells. Such findings imply a possibility for 5‐aminolevuliic acid to treat cancer. The study seems to be an interesting. However, the idea is not so novel as the similar studies with other cancer cell lines have been described. Some justification on the originality of the work is necessary.” Response: We agree; we have modified the last paragraph of the Introduction to better explain the originality of the work. (“In this study, we explored the role of cellular heme level in regulation of Warburg effect and expression of pro-metastatic genes in ovarian cancer and TNBC cells. Additional motivation for this study was to determine whether supplementation with heme precursor 5-aminolevulinic acid (ALA) downregulates Bach1. We show that ALA supplementation leads to downregulation of glycolysis, mitochondrial respiration, as well as cell migration. The underlying mechanisms differ in ovarian and TNBC cells, but likely involve destabilization of Bach1, activation of AMP-activated protein kinase (AMPK), and induction of anti-oxidant response.”) 1. “The authors performed extracellular acidification rate (ECAR) approach only to test glycolytic rate. This is limited in that ECAR is highly related to pH value of cell medium and CO2 production. The standard measurements of glycolytic metabolism are the use of 3H labeled glucose and lactate concentration in culture medium of cells treated with or without drugs.” Response: We agree; we have determined the lactate concentration in the culture medium. The new results are included in Fig. 1, 2, 4, and 5. 2. “In all figures, mRNA levels of HO‐1 and Catalase have been tested. However, as important enzymes in the catabolism of heme, the protein levels of these enzymes should be examined as well.” Response: Since heme is known to regulate HO-1 and catalase at the transcriptional level, we believe that measuring mRNA levels of HO-1 and catalase is the most direct and informative way to test the effect of ALA. 4. “In figure 1F and 2F, could you explain why you choose these targets (NDUFB5, cox17, cox15, ATP5D and SLC25A15) as ETC and OXPHOS genes, instead of coxⅣ, coxⅤ or any other standard genes?” Response: These ETC and OXPHOS genes (NDUFB5, cox17, cox15, ATP5D and SLC25A15) encode components of electron transport chain and mitochondrial ATP synthase. Cox15, ATP5D and SLC25A15 were identified as targets of Bach1 (Lee et al., Effective breast cancer combination therapy targeting Bach1 and mitochondrial metabolism. Nature 568, 254-258, 2019). Since one of the possible mechanisms of ALA supplementation and heme levels is mediated by Bach1, we believe that selecting these genes is appropriate. NDUFB5 and cox17 were identified as targets of LKB1-AMPK-PGC1α axis and were designed on the basis of the corresponding paper (Li et al., CAB39L elicited an anti-Warburg effect via a LKB1-AMPK-PGC1α axis to inhibit gastric tumorigenesis. Oncogene 37:6383–6398, 2018). 5. “In figure 2I, the area of wound width is not in appropriate position.” Response: We agree and we corrected Fig. 2I. 6. “The data indicated that Bach1 was reduced by ALA supplementation, and silencing Bach1 could reduce ECT and OXPHOS, which is similar to ALA supplementation treatment. However, whether Bach1 is involved in heme for reducing ETC, OXPHOS, and glycolysis, was not discussed. Does any approach (such as chemicals) block the degradation of Bach1 in ALA supplemented cells? And after that, further ETC, OXPHOS, and glycolysis analysis should be performed.” Response: The only known approach to stabilize Bach1 is inhibition of the proteasome by MG132 (Zenke-Kawasaki et al., Heme induces ubiquitination and degradation of the transcription factor Bach1. Mol. Cell. Biol. 27, 6962-6971, 2007) or other proteasome inhibitors, such as Bortezomib. However, inhibition of proteasome (in ALA treated or control cells) would produce a pleiotropic effect that would be difficult to interpret. 7. “The lack of vivo data to validate the results in culture is another limitation of the research, which should be discussed.” Response: We modified the Discussion section of the manuscript to indicate the need for in vivo studies of ALA supplementation. I would like to thank the Reviewers for the useful critiques and suggestions. Ales Vancura Attachments Attachment Submitted filename: Response to Reviewers.docx https://doi.org/10.1371/journal.pone.0260400.r002
10 Nov 2021 Decision Letter - Pankaj K Singh, Editor Activated heme synthesis regulates glycolysis and oxidative metabolism in breast and ovarian cancer cells PONE-D-20-40407R1 Dear Dr. Vancura, We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements. Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication. An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org. If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org. Kind regards, Pankaj K Singh, Ph.D. Academic Editor PLOS ONE Additional Editor Comments (optional): Reviewers' comments: Reviewer's Responses to Questions Comments to the Author 1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation. Reviewer #1: All comments have been addressed ******** 2. Is the manuscript technically sound, and do the data support the conclusions? The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented. Reviewer #1: Yes ****** 3. Has the statistical analysis been performed appropriately and rigorously? Reviewer #1: Yes ****** 4. Have the authors made all data underlying the findings in their manuscript fully available? The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified. Reviewer #1: Yes ****** 5. Is the manuscript presented in an intelligible fashion and written in standard English? PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here. Reviewer #1: Yes ****** 6. Review Comments to the Author Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters) Reviewer #1: Thank you for addressing my concerns. No further comments. In my opinion this manuscript makes an interesting contribution to the literature ****** 7. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files. If you choose “no”, your identity will remain anonymous but your review may still be made public. Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy. Reviewer #1: Yes:** Don Ayer https://doi.org/10.1371/journal.pone.0260400.r003
Formally Accepted
12 Nov 2021 Acceptance Letter - Pankaj K Singh, Editor PONE-D-20-40407R1 Activated heme synthesis regulates glycolysis and oxidative metabolism in breast and ovarian cancer cells Dear Dr. Vancura: I'm pleased to inform you that your manuscript has been deemed suitable for publication in PLOS ONE. Congratulations! Your manuscript is now with our production department. If your institution or institutions have a press office, please let them know about your upcoming paper now to help maximize its impact. If they'll be preparing press materials, please inform our press team within the next 48 hours. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information please contact onepress@plos.org. If we can help with anything else, please email us at plosone@plos.org. Thank you for submitting your work to PLOS ONE and supporting open access. Kind regards, PLOS ONE Editorial Office Staff on behalf of Dr. Pankaj K Singh Academic Editor PLOS ONE https://doi.org/10.1371/journal.pone.0260400.r004

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