Peer review of: OTUD5 cooperates with TRIM25 in transcriptional regulation and tumor progression via deubiquity (2020)


RR2
Reviewers' comments:
Reviewer #1 (Remarks to the Author):
  In general, the manuscript has been greatly improved following this round of revisions. Most of my
 points have been duly addressed. That said, the point 3.4 did not convey its desired message.
 The major idea of the manuscript is that OTUD5 deubiquitinates TRIM25 and decreases
 proliferation and tumour growth.
 My comment 3.4 was about generating TRIM25 K117R mutant (incapable of being slef-
 ubiquitinated) cells or mouse model that would support the central idea of the manuscript. As it
 stands, I fully agree with Reviewer #2 who states that 'the role of TRIM25 deubiquitination in the
 hepatocarcinoma cell line xenografts is only correlative'.
 Moreover, the lack of the above experiments highlights some problems with conclusions and data
 interpretation. In Discussion we can read that 'Fittingly, TRIM25 RING domain deletion mutant
 failed to promote cell proliferation because self-ubiquitination of TRIM25 was abolished.' Without,
 TRIM25 K117R mutant cells of mouse model one cannot make such claims as TRIM25 can
 influence cell biology by ubiquitinating many other targets.
 My prediction is that TRIM25 K117R mutant cells or mouse model will lack pathological phenotype.
Reviewer #2 (Remarks to the Author):
 Until I am proven wrong, authors' major idea will only be speculative and should be presented this
 way.
  In the revised version of their manuscript, F. Li et al. present substantial additional data that
 strongly support the proposal: OTUD5 DUB activity contributes to tumor suppression in vivo
 through the negative regulation of TRIM25 and subsequent increase in the PML tumor suppressor.
 The authors improved the quality of the manuscript with new conclusive experiments. In addition
 to mechanistic insights involving OTUD5-mediating debutiquitination in hepatocarcinoma cell line
 growth, the authors have provided important new experiments performed in xenografted mice
 showing crosstalks between OTUD5, TRIM25 and PML. They generalize OTUD5 involvement in
 other solid cancers than hepatocarninoma, notably with longer overall survival of patients with
 lung expressing high level of OTUD5.
 Thus, the authors provided clear responses to the points raised from the initial manuscript and I
 support its publication in Nature Communication after minor modifications, listed below.
 - Contribution of OTUD5-mediated deubiquitination of the TRIM25 targets within the
 OTUD5/TRIM25 complex, in addition to TRIM25 deubiquitination itself, is still not clear. The
 authors did not use denaturing condition to purify ubiquitinated TRIM25, but did elution using
 FLAG or HA peptides that would release TRIM25-associated proteins as well. The results performed
 on the two TRIM25 targets (supplementary fig5) are too weak to conclude in a way or another.
 Thus, as proposed previously, the authors should have tested effect of OTUD5/TRIM25 on global
 ubiquitination: this could be easily done using the samples presented in supplementary fig.4 or 5a
 for PML and MAVS ubiquitination. The His-tagged ubiquitin and denaturing pulldowns may be used
 for blotting with an anti-ubiquitin antibody (as FK2 antibody).
 - More importantly, all ubiquitination assays have been performed from 293T cells. Assessing
 TRIM25 ubiquitination regarding OTUD5 expression in Hep3B or huh7cells would be required to
 definitively link the biochemical mechanism to OTUD5/TRIM25 effects on liver cancer cell line
 growth and tumorogenesis.
 
- It is not completely clear whether the blots presented in fig.2B,C,F-bottom are inputs (after
 centrifugation?) or WCL?
 - Some full blots should be shown as supplementary figures as TRIM25, OTUD5 (fig 3A,E) and PML
 (fig4.E)
 - Legends should be carefully revised. As an example: legend of fig4M and 4N is lacking and 4I,J,K
 are not the good ones.
 - �apoptosis� should be replaced by �cell survival� line 286 and in figure 4J.
 - Some key references are missing in introduction and discussion:
 Papers from R.H. Chen�s lab described PML down-regulation in cancers (line 115-117) through
 another ubiquitin-ligase:
 1Yuan, W.C. et al. A Cullin3-KLHL20 Ubiquitin ligase-dependent pathway targets PML to potentiate
 HIF-1 signaling and prostate cancer progression. Cancer cell 20, 214-228 (2011).
 PML nuclear bodies are now considered as spherical structures more than doughnut shaped
 (line105). Two recent reviews in addition to the cited papers (2007, 2006) highlight new important
 findings on formation/functions of PML NBs notably in cancer, with anti- and pro-tumoral aspects:
 2Hsu, K.S. & Kao, H.Y. PML: Regulation and multifaceted function beyond tumor suppression. Cell
 Biosci 8, 5 (2018).
 3Lallemand-Breitenbach, V. & de The, H. PML nuclear bodies: from architecture to function. Curr
 Opin Cell Biol 52, 154-161 (2018).
 The authors mention the anti-tumoral PML/p53 axis found in cell lines, they might add it is
 relevant in vivo, in cancer mouse models:
 4Ablain, J. et al. Activation of a promyelocytic leukemia-tumor protein 53 axis underlies acute
 promyelocytic leukemia cure. Nature medicine 20, 167-174 (2014).
 Reviewer #3 (Remarks to the Author):
 The authors have addressed essentially all of the comments raised in my previous review. I thinks
 this is an interesting new study that adds to our understanding of how DUBs might promote cancer
 and am therefore fully supportive of its publication.