NFAT activation by FKBP52 promotes cancer cell proliferation by suppressing p53

FKBP52 promotes nuclear translocation of NFATc, which activates MDM2 transcription, thus suppressing p53 expression and promoting cell proliferation.

Full guidelines are available on our Instructions for Authors page, https://www.life-science-alliance.org/authorsWe encourage our authors to provide original source data, particularly uncropped/-processed electrophoretic blots and spreadsheets for the main figures of the manuscript.If you would like to add source data, we would welcome one PDF/Excel-file per figure for this information.These files will be linked online as supplementary "Source Data" files.***IMPORTANT: It is Life Science Alliance policy that if requested, original data images must be made available.Failure to provide original images upon request will result in unavoidable delays in publication.Please ensure that you have access to all original microscopy and blot data images before submitting your revision.***- --------------------------------------------------------------------------Reviewer #1 (Comments to the Authors (Required)): This study provides of role for FKBP52 in the regulation of TP53 expression in TP53 wild type cancer cells.The data is clearly presented and the manuscript is for the most part well written.However, additional experiments are needed to define the mechanism of regulation of TP53-MDM2 axis by FKBP52 and translational significance of the study.First, key shRNA knockdown including Figures 1C, 2A, 2B, 3A, 4B, 4C, 4D, 5B and 5C should be repeated using a second targeting sequence.Second, the authors should determine the localization of TP53 and protein expression of other target genes in MCF7 cells.Third, the protein expression of TP53 and p21 in HCT116 cells should be added to the manuscript.Fourth, key experiments should be repeated in these cells (e.g., data from Figures 2, and 5).And fifth, all western blots should include two molecular markers flanking the relevant bands.Also, the quality of some of the blots should be improved including HSP90 from Fig 1C , 5B and EV1; TP53 from EV; and betaActin in Figure 4B and 4D.Finally, the specificity of the FKBP52 antibody should be defined using two shRNA and perhaps a second antibody from a different company.This is particularly important as western blots for this protein show different signal throughout the manuscript Reviewer #2 (Comments to the Authors (Required)): In this manuscript, the authors uncovered the link between NFAT and p53 pathways by revealing the novel role of FKBP52 in regulation of NFAT translocation.The authors also showed that NFAT regulates p53 stability by inducing transcription of Mdm2, a ubiquitin ligase that targets p53.The novelty of this study resides in the role of FKBP52 in regulation of NFAT and p53.FKBP52 is known to be involved in cell proliferation; however, the detailed mechanism remains poorly understood.However, this study's novelty is decreased by the previous findings showing the role of NFAT in regulation of Mdm2 promoter (Xu Zhang et al., JBC, 2012).Also, the current study does not present any in vivo study; hence, the physiological meaning of this finding has not been determined.The conclusion seems reasonable, but more depth needs to be presented in the mechanistic aspect.
1.In Fig. 1A and B, I recommend showing the full analysis of RNA-seq to understand the comprehensive role of FKBP52 and emphasize the significance of the p53 pathway.
2. In Fig. 2A, in addition to the cell culture system, tumor cell growth needs to be shown in a physiological setting.3.In Fig. 4, Mdm2 promoter reporter assay was used to demonstrate the role of NFAT.To clearly demonstrate the direct role of NFAT, it is better to use constitutively active NFAT instead of ionophore.Also, direct Ca2+ measurement is needed to show that these effects are not derived from changes in intracellular Ca2+ levels, especially considering the role of FKBP52 in regulation of Trpc3 channels.4. In Fig. 5, the nuclear fraction does not show any FKBP52 in the nucleus in biochemical assays, but ChIP results showed that it is associated with NFAT in the Mdm2 promoter.These conflicting results need to be resolved.I also recommend using confocal analysis to show the co-localization of NFAT and FKBP52. 5.In the Discussion section, the potential upstream Ca2+ signaling that regulates NFAT activation in their experimental setting needs to be discussed.Also, references indicating the role of the Ca2+-NFAT pathway in cell death and cell cycle arrest need to be included.6.It is a minor point, but the molecular weight should be presented in the immunoblotting analysis throughout the figures.
1st Authors' Response to Reviewers May 1, 2024 Reviewer 1 [Response] We thank the reviewer for the careful review of our manuscript.We also thank the reviewer for the constructive suggestions which have helped us to considerably improve our manuscript.Our specific responses to the points raised can be found below (this letter contains low-resolution thumbnails for clarity; please refer to the manuscript for high-resolution figures).1C, 2A, 2B, 3A, 4B, 4C, 4D, 5B and 5C should be repeated using a second targeting sequence.

1) First, key shRNA knockdown including Figures
[Response] As suggested by the reviewer, we performed shRNA knockdown experiments using the second or third target sequence.
Figure 1C: We have shown the effect of shFKBP52-2 on p53 and p21 expression in the original manuscript (Figure 1C).Similar to the effect of shFKBP52-1, depletion of FKBP52 by shFKBP52-2 increased the abundance of p53 and p21 in MCF7 cells.We have now addressed this point in the revised manuscript (page 5, lines 89-91).
Figure 2A: Given that it was technically difficult, for unknown reasons, to deplete FKBP52 by shFKBP52-2 in addition to shRNA-mediated depletion of p53 in MCF7 cells, we decided to deplete FKBP52 by one of three independent shRNAs (shFKBP52-1, shFKBP52-3, and shFKBP52-4) and CRISPR-Cas9-mediated deletion of the p53 gene in HCT116 cells (clone 10) (new Supplementary Figure S2A and new Supplementary Figure S2B).These experiments revealed that the attenuation of cell proliferation caused by FKBP52 deletion was restored by the additional deletion of p53 in HCT116 cells (new Supplementary Figure S2C).We have now addressed these points in the revised manuscript (page 6, lines 110-115).
Figure 2B: This experiment was repeated using shFKBP52-3.Similar to the results with shFKBP52-1, depletion of FKBP52 resulted in an increase in the sub-G1 population, which was restored by the additional depletion of p53 (new Supplementary Figure S2E).We have now addressed this point in the revised manuscript (page 6, lines 117-119).
Figure 3A: This experiment was repeated using shFKBP52-3.Similar to the results obtained with shFKBP52-1, p53 was stabilized by FKBP52 depletion (new Supplementary Figure S3A).We have now addressed this point in the revised manuscript (page 7, lines 135-137).
Figure 4B: The original results with shNFATc1, shNFATc3, and shNFATc2 showed that the abundance of MDM2 was reduced by the depletion of NFATc1 and NFATc3, but not NFATc2.Thus, we repeated these experiments with other sets of shRNAs (shNFATc1-2, shNFATc3-2, and shNFATc2-2) and obtained similar results (new Supplementary Figure S4A).We have now addressed this point in the revised manuscript (page 8, lines 164-166).
Figure 4C: We also used shNFATc1-2 and shNFATc3-2, and obtained similar results (new Supplementary Figure S4B).We have now addressed this point in the revised manuscript (page 8, lines 164-165).
Figure 4D: CaN depletion by shCaN Aα-1 reduced the abundance of MDM2 in the original manuscript.We repeated this experiment with another shRNA (shCaN Aα-2), and obtained similar results (new Supplementary Figure S4C).We have now addressed this point in the revised manuscript (page 8, lines 168-170).
Figure 5B: The original experiments revealed that the nuclear translocation of NFATc1 and NFATc3 was inhibited by FKBP52 depletion mediated by shFKBP52-1.As suggested by the reviewer, we have performed the same experiments using shFKBP52-3.The effect of shFKBP52-3 was similar to that of shFKBP52-1 (new Supplementary Figure S5A).We have now addressed this point in the revised manuscript (page 9, lines 184-188).

2) Second, the authors should determine the localization of TP53 and protein expression of other target genes in MCF7 cells.
[Response] The subcellular localization of p53 was examined by the biochemical fractionation of FKBP52depleted cells.These results revealed that the abundance of p53 was increased by FKBP52 depletion; however, its localization was not affected (new Figure 5B).We have now addressed this point in the revised manuscript (page 9, lines 190-191).
[Response] The protein expression of GADD45A and PUMA, which are other targets of p53, was examined in MCF7 cells depleted of FKBP52.We found that the abundance of GADD45A and PUMA was increased in FKBP52-depleted cells mediated by two independent shRNA sequences (shFKBP52-1 and shFKBP52-2) (new Supplementary Figure S1A).We have now addressed this point in the revised manuscript (page 5, lines 91-92).

3) Third, the protein expression of TP53 and p21 in HCT116 cells should be added to the manuscript.
[Response] We have already shown the data requested by the reviewer in the original manuscript (Supplementary Figure S1B).In HCT116 cells, FKBP52 depletion increased the abundance of p53 and p21.
We have now addressed this point in the revised manuscript (page 5, lines 94-95).2, and 5).

4) Fourth, key experiments should be repeated in these cells (e.g., data from Figures
[Response] As suggested by the reviewer, we conducted key experiments using the HCT116 cells.LSA now encourages authors to provide a 30-60 second video where the study is briefly explained.We will use these videos on social media to promote the published paper and the presenting author (for examples, see https://twitter.com/LSAjournal/timelines/1437405065917124608).Corresponding or first-authors are welcome to submit the video.Please submit only one video per manuscript.The video can be emailed to contact@life-science-alliance.orgTo upload the final version of your manuscript, please log in to your account: https://lsa.msubmit.net/cgi-bin/main.plexYou will be guided to complete the submission of your revised manuscript and to fill in all necessary information.Please get in touch in case you do not know or remember your login name.
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Figure 2A :
Figure 2A: FKBP52 depletion inhibited the proliferation of HCT116 cells, similar to that of MCF7 cells.In addition, combined depletion of FKBP52 and p53 recovered the proliferation of HCT116 cells compared with FIGURE CHECKS-we encourage you to arrange FigureS2so that the panels are introduced in alphabetical order, and update the legend and callouts accordingly If you are planning a press release on your work, please inform us immediately to allow informing our production team and scheduling a release date.
Thank you for this interesting contribution, we look forward to publishing your paper in Life Science Alliance.
Thank you for submitting your Research Article entitled "NFAT Activation by FKBP52 Promotes Cancer Cell Proliferation by Suppressing p53".It is a pleasure to let you know that your manuscript is now accepted for publication in Life Science Alliance.Congratulations on this interesting work.