Peer review of: The REGγ inhibitor NIP30 increases sensitivity to chemotherapy in p53-deficient tumor cells (2020)

Reviewers' comments: 

Reviewer #1 (Remarks to the Author); expert in proteasome regulation: 

This is an interesting and potentially important report that describes regulation of REG gamma 
function via the action of two proteins: NIP30 and CDC25. The authors present data showing that 
NIP30 directly binds to REG gamma and decrease levels of p21, presumably by enhancing 
REGgamma activation of proteasome activity. They further show that NIP30 is phosphorylated 
and that phosphorylation attenuates NIP30/ REGgamma activated p21 degradation. They identify 
CDC25 as a NIP30 phosphatase and provide evidence that this activity may regulate NIP30/ REG - 
mediated levels of p21. In general, the data are internally consistent and offer a model for 
proteasome-dependent control of p21 levels and consequent effects on cell growth. 

A main advance of this manuscript is the identification of CDC25 as a regulator of the NIP30/ 
REGgamma/proteasome axis and the data that support this model. However, many aspects of the 
presentation are premature, descriptive and problematic. 

1. The first section of the results (Figures 1 and 2) describes identification of NIP30 as a 
REGgamma interacting protein and show that the C-terminus of NIP has a conserved site that is 
phosphorylated and required for the REGgamma interaction. Similar analysis and conclusions 
were obtained by Jonik-Nowak et al (ref 32) but are not described in an appropriate context here. 
This is somewhat misleading. Describing those results more completely in the set-up to the Results 
section would allow the authors to streamline the paper, focus on what is new, and perhaps go 
deeper into the mechanisms of the effects reported here. 
2. The role of NIP30 phosphorylation is a key mechanistic part of the manuscript. The authors’ 
data a generally consistent with this but there needs to be more direct evidence. I did not find a 
description of the production of phospho-specific antibodies used in the study – a complete 
description of the production and validation of these antibodies is essential. The use of these 
antibodies is introduced in experiments of Figure 3, but should be used earlier. It would be useful 
to have supporting evidence in the form of direct demonstration of phosphorylation with either 
mass-spec data, or radioactive phosphate incorporation. There must be reference to (32) where 
NIP30 phosphorylation of NIP30 by CKII was demonstrated directly. 
3. The data in Figure 3 are not very completing. The discrimination of co-localization on the scale 
used here is not fine enough to demonstrate physical interactions between proteins or 
assessments of the strengths of these interactions, as claimed in the text. Also, there are many 
proteins whose highest level of expression occurs in the testis – these results are correlative and 
not convincing for the claims. 
4. As the authors note, the molecular mechanisms by which REG gammamight mediate ubiquitin-
independent protein breakdown are not very well understood. In my opinion, this is one of the 
major problems in understanding how REG functions in the degradation of intact proteins. 
Although this manuscript fills in some of this understanding, the results still lack mechanistic 
detail. Steady-state levels of p21 may indeed reflect degradation, but that is not shown directly. 
As noted above, the data are consistent with the authors’ models, but it is possible and likely that 
NIP30/ REGgamma could affect p21 levels indirectly by other mechanisms. In this regard, the 
authors should discuss the results of ref 32 that offer either conflicting or alternative possibilities 
and at least show the complexity of the system. Curiously, others showed that NIP30 inhibited 
REG proteasome activation (although phos-NIP30 increased the degree of inhibition). In any case, 
the authors seem to be in a position to test a direct mechanism with a reconstituted in vitro 
systems. They either have or should be able to prepare pure proteins involved in the proposed 
regulatory axis and directly test the degradation of non-ubiquitinated p21 by it. As noted early in 
the Introduction, but then ignored for the rest of the paper, there is controversy about the relative 
roles of ubiquitin-dependent and ubiquitin-independent degradation of p21. REG gammacould be 
part of a hybrid proteasome that degrades p21 in an ubiquitin dependent mechanism. The 
suggested in vitro constitution experiments would help to sort this out. 
5. The entire manuscript is in need of extensive editing to correct typographical errors and errors 

in English usage and grammar that include the lack of necessary articles, incorrect subject-verb 
agreement, fractured sentences, and odd or inappropriate word choices. The bibliography contains 
many incomplete or incorrectly formatted citations. There are too many instances of each of these 
issues to cite individually here, but in total they make the manuscript distracting and annoying to 
read. I am sensitive to and appreciate English-as-a-second-language challenges, but it is difficult 
to believe that all of the authors read and approved this version of the manuscript or that other 
editing services are not available at Baylor. 

Reviewer #2 (Remarks to the Author); expert in cell cycle, DNA damage: 

The authors build on the work of another group (Beata Jonik-Nowak et al, Proc Natl Acad Sci, 
2018, 115:E6477; PMC6048556) who showed that PIP30/FAM192A (here called NIP30) is 
phosphorylated by CKII, and that phosphorylated PIP30/NIP30 binds and regulates the function of 
REGgamma. Here Gao et al confirm those findings and further: 
1) Show that overexpression of wild-type and phosphomimetic (4A) PIP30/NIP30 
posttranscriptionally affects the levels of p21, which is a substrate of REGgamma. 
2) Show that overexpression of nonphosphorylatable (4D) PIP30/NIP30 reverses the 
antiproliferative effects of REGgamma knockout 
3) Claim that CDC25A dephosphorylates PIP30/NIP30, which in turn regulates p21 levels in a p53-
independent manner. 
4) Identify that PIP30/NIP30 is altered in a low percentage of tumors 
5) Show that overexpression of wild-type and phosphophomimetic PIP30/NIP30 increases tumor 
sensitivity to etoposide, cisplatin, 5-fluorouracil, and doxorubicin 
6) Claim that PIP30/NIP30 is a tumor suppressor 
7) Present a model in which CDC25A regulates NIP30/PIP30, which in turn regulates REGgamma 
that then regulates p21 levels and cell cycle progression. 

This is an interesting study. However, there are several serious concerns. 

1) Although the results showing that altering CDC25A levels affects PIP30/NIP30 phosphorylation 
in cells is solid, the data showing that CDC25A directly de-phosphorylates PIP30/NIP30 is very 
weak (Fig. 3E); this analysis used immunopurified PIP30/NIP30 and immunopurified CDC25A. It is 
very possible that a contaminating phosphatase is responsible for the in vitro dephosphorylation 
that is observed. 
2) It is not clear that CDC25A’s effects on p21 levels are driven by CDC25A-mediated 
dephosphorylation of PIP30/NIP30. They could be through another unknown pathway. Does 
expression of phosphomimetic PIP30/NIP30 prevent CDC25A-induced changes in p21? 
3) Most of the experiments rely on PIP30/NIP30 overexpression. This is a serious concern because 
overexpression studies are prone to artefacts. Additionally, given that the authors propose that 
PIP30/NIP30 is a tumor suppressor, a serious analysis of the effects of losing PIP30/NIP30 would 
be important to understanding its functions. 
4) PIP30/NIP30 mutations found in tumors are very infrequent and the effects mutations that are 
shown in the supplemental figure are not known. Accordingly, there are no data to support the 
contention that PIP30/NIP30 is a tumor suppressor. 
5) Although p21 levels are affected by CDC25A and PIP30/NIP30, it is not clear that changes in 
p21 levels are actually driving cell cycle changes induced by CDC25A and PIP30/NIP30 
manipulation. 

Minor comments: 
1) There are many grammatical errors. 
2) It is not always clear how many times an experiment was done. 

3) Abbreviations are not always defined. 
4) Figures are too small to read, especially annotations and symbols on graphs, even when 
zoomed in on. 
5) Some figures are not appropriately labeled. For example, in Fig. 6B, the x-axes are not labeled 
and the y-axes are labeled as “survival rate.” If an MTT assay was used, this is not a survival rate. 

Reviewer #3 (Remarks to the Author); expert in p53, mouse models: 

The manuscript by Gao, et al. focuses on NIP30 as an inhibitor of the REG proteasome, which is 
oncogenic and distinct from the 26S proteasome. The authors uncovered a tumor suppressor role 
for NIP30 by its interactions with REGmapping the amino acids of interaction and uncovering a 
phosphorylation switch for this interaction. CDC25A was identified as the major regulatory 
phosphatase for this inhibitory switch during cell cycle and in response to DNA damage. Cell cycle 
inhibitor p21 is a major target of the CDC25A-NIP30- REG axis, which induces regulation in a 
p53-dependent manner. Multiple approaches were employed in these studies: IP/mass spec; yeast 
two-hybrid; co-IP; domain and amino acid interaction mapping; xenograft tumor analyses of 
expressing, mutated and KD of key players; screening of a phosphatase library; cell cycle and DNA 
damage studies and UV-response of the skin taken from newborn REG WT, KO, Tpr53 KO and 
REG;Tpr53 double KO; and chemosensitivity studies with cell lines and xenografts. 

Overall, this is a very thorough analysis that pinpoints a specific phosphorylation switch control of 
proteasome regulation of p21 during cell cycle and DNA damage response. The studies are 
primarily cell line-based but are also complemented by xenograft, genetically modified mice and 
TCGA data. This work suggests a new target and approach toward effectively treating p53-
dysfunctional cancers with chemotherapeutics, a major problem in the clinic. This finding is likely 
to be of considerable interest to the community. 

The major problem with the manuscript is poor preparation, improper use of English and inexact 
terminology. The authors should employ an English editor and science writer to resubmit a better 
manuscript. 

Specific examples of inexact terminology include “tight binding”, found in the Abstract and within 
the manuscript. How is “tight binding” measured and what are the values for binding constants 
that would indicate “tight”? Quantifying this binding is needed for such terms to be used. Showing 
co-localization in immunofluorescence microscopy is insufficient and non-robust (Suppl. Fig. 3A). 

Examples of poor manuscript preparation are the numerous grammatical errors, incorrect word 
usage that obscures the likely intended meaning (a couple of examples: “difference cells”? “Hiked 
levels”?), and some references improperly formatted. Additionally, some figure legends lack 
sufficient detail (Fig. 1A, what is N1 and S1, for example). References are sometimes lacking. A 
major example of this is the human Ser/Thr phosphatase library screen. There is no reference for 
this library or any data for the screen shown. These data should be included as supplemental data 
or referenced.