Peer review of: Mutant p53 induces Golgi tubulo-vesiculation driving a prometastatic secretome (2020)

Reviewers' Comments: 
 
Reviewer #1: 
Remarks to the Author: 

Capaci et al report that mutant p53 (mut-p53) upregulates the expression of miR-30d in 
cooperation with HIF-1a. This leads to structural changes in the Golgi apparatus, which drive 
augmented protein secretion. They further identify two mRNAs that are targets for direct inhibition 
by miR-30d and confirm their importance in driving the changes in the secretory machinery. 
Additionally, they identify a panel of proteins whose secretion is significantly elevated as a 
consequence of mut-p53/miR-30d action, and show that the altered secretome is responsible for 
augmenting the migratory behavior of cancer cells and of cancer-associated fibroblasts, as well as 
for increased angiogenesis. Through in vivo experiments in mice, they provide compelling evidence 
that the mut-p53/miR-30d axis promotes metastatic spread, by acting both at the primary tumor 
site and at the premetastatic niche. Lastly, they provide data from the analysis of human patient 
samples which support their conclusions. 
Overall, this is a very interesting paper, describing a novel gain-of-function activity of mut-p53 
with high potential clinical relevance. The work is comprehensive and is well performed. Publication 
is recommended after the authors address the points listed below. 
 
Major comments: 
1. The paper gives the impression that ALL the effects of mut-p53 on the secretory machinery are 
mediated by miR-30d. Is this indeed so, or is this an oversimplification of a more complex picture? 
Specifically, does mut-p53 regulate directly the expression of any genes that encode proteins 
involved in the secretory machinery? The authors should compare by RNA-seq the transcriptome 
of miR-30d depleted (dy-30d) cells with and without depletion of mut-p53, and determine whether 
any secretory machinery-related genes are still regulated by mut-p53 even when miR-30d is 
absent. The answer to this question is important in order to place the paper’s findings within a 
more complete picture. 
2. Fig. 1f: This result is a bit puzzling. Is it not surprising that HIF1a protein levels are not higher 
in the hypoxic conditions? Actually, if one compares them to the p53 band intensity in the input 
lanes, they even appear to be lower than in 20%! Moreover, the interaction between mut-p53 and 
HIF1a does not appear to be increased in 2% when compared to 20%. From this analysis, and 
considering the model in Fig. 1h, it is unclear what is driving the increased expression of miR-30d 
under hypoxic conditions. Is it because HIF-1a binds better to the miR-30d chromatin under 
hypoxia? This should be tested by performing a HIF1a ChIP under conditions similar to those used 
in Fig. 1g. Moreover, the model in Fig. 1h predicts that more mut-p53 should be bound to the miR-
30d locus under hypoxia than in normoxia. This should be tested by comparing the p53 ChIP as in 
Fig. 1g between 20% and 2%. 
3. Fig. 5f: the effect of miR-30d on VEGFA secretion by mut-p53-depleted cells is rather marginal, 
suggesting that most (90%?) of the effect of mut-p53 is not due to miR-30d. The authors should 
quantify the band intensities. As a matter of fact, Fig. 5h also shows only a very partial phenotypic 
rescue by miR-30d, which may be consistent with the limited effect of miR-30d on VEGFA 
secretion. Together, this suggests that much of the effect of mut-p53 on VEGFA secretion and 
activity is NOT via miR-30d, unlike what one may conclude from reading the text. The authors 
should discuss this issue. 
 
Minor comments: 
 
1. Fig. 1f: the IgG control lane in the 20% panel seems to have a bubble, making the expected 
negative result less convincing. If will be helpful if the authors can provide a cleaner figure, 
showing clearly that there is no HIF1a band in the IgG control. 
2. Fig. 3a: The PDI staining in the mut-p53 R280K cells look very different from that in the miR-
30d cells. The authors need to comment on this, or provide a more representative image if these 
differences are not representative. 
3. Supplemental Fig. 5c suggests that miR-30d overexpression causes downregulation of mut-p53, 
but this is not seen in Supplemental Fig. 5e. Please explain. 
4. Line 181: 3e should be 2e. 
5. Line 333; 5a should be 5b. 
6. Line 369: ‘ad’ should be ‘and’ 
 
 
 
Reviewer #2: 
Remarks to the Author: 
NCOMMS-19-40638 
Mutant p53 induces Golgi tubulo-vesiculation driving a prometastatic secretome 
Valeria Capaci, Lorenzo Bascetta, Marco Fantuz, Galina Beznoussenko, Roberta Sommaggio, 
Andrea Bisso, Alexandre Mironov, Valeria Cancila, Jacek Wisniewski, Luisa Ulloa Severino, Denis 
Scaini, Fleur Bossi, Jodi Lees, Noa Alon, Ledia Brunga, David Malkin, silvano Piazza, Licio Collavin, 
Antonio Rosato, Silvio Bicciato, CLAUDIO TRIPODO, Fiamma Mantovani, and Giannino Del Sal 
 
While p53 is one of the most critical tumor suppressor, mutant p53 serves as an oncoprotein in Li-
Fraumeni cancer predisposition syndrome. In this study, Dr. Del Sal and colleagues determined the 
role of mut-p53 in tissue culture cells, animal models and human tissues. They found that p53 
mutants interact with the hypoxia responsive factor HIF1a and induce the expression of miR-30d, 
which reduces the DGKZ and VPS26B protein levels and causes tubulo-vesiculation of the Golgi 
apparatus. Subsequently, this enhances vesicular trafficking and secretion and promotes secretion 
of growth factors such as VEGF and ECM proteins. Thus, the authors have discovered a novel mut-
p53/HIF1a/miR-30d axis that impacts synthesis, trafficking, and secretion of a number of proteins 
essential for tumor growth and metastatic colonization. 
 
Overall the topic is novel and significant, and a large amount of data was provided, covering a 
broad range of aspects of cancer cell biology. However, some of the conclusions are based on 
weak results or speculations. There is also some critical information missing in the manuscript. 
 
Specific points: 
 
1. The interaction of mut-p53 and HIF1 is new, but some critical questions are not addressed. 
1) Figure 1e, can HIF over expression rescue the sip53 effect? 
2) Figure 1f, HIF1 activity is known to be higher at low oxygen level, if the authors’ hypothesis is 
that mut-p53 acts as a partner of HIF1α, why the interaction is lower at low oxygen? 
 
2. The manuscript did not provide an adequate explanation of how the Golgi becomes vesiculated. 
1) Fig. 3a, the shp53+p53 R280K cells have a different phenotype from miR-30d cells for beta-
COP and GM130. What's the explanation? 
2) The authors identified two proteins, DGKZ and VPS26B, that may affect Golgi morphology and 
protein trafficking, but it is not clear how. DGKZ is an enzyme that affects DAG to PA conversion, 
there is no mechanism for how it affects Golgi structure and function. VPS26B is involved in 
retrograde trafficking, no mechanism was provided whether it also affects anterograde trafficking 
and secretion. 
3) The authors should at least test a few key Golgi structural and trafficking proteins (e.g. Sec24) 
by Western blot in cells of the 4 different treatments as in Figure 3A and see which protein is 
affected. Results from tissue culture cells should be confirmed in mouse and human tissues. 
 
3. It is not clear how Golgi vesiculation enhances protein trafficking and secretion. 
1) It is not clear how Golgi vesiculation enhances protein trafficking or secretion. The authors 
speculated that microtubule destabilization or degradation may cause Golgi fragmentation, but this 
is inconsistent with the fact that destabilization of microtubules slows down protein trafficking, 
instead of accelerating it. 

2) Changes in trafficking may not always impact secretion depending on the time when they are 
determined. It is not described how long the cells were incubated before ssGFP was collected in 
the medium, but it was shown that at this time most ssGFP was still inside the cells. Did the 
authors include cycloheximide in this experiment? Since most protein trafficking from the ER to the 
plasma membrane takes about 2 hours, where is ssGFP stuck in cells? How long were the cell 
incubated when the secretome was analyzed? 
3) In general, secretory proteins are either degraded by ERAD if misfolded or secreted. Their 
trafficking speed may be modulated by the trafficking machineries, but they are normally not held 
in the Golgi. The comparison of scretome with the transcriptome from a different study in Figure 
2h is not valid as these two studies may have different experimental conditions or settings. 
4) Is accelerated trafficking and enhanced secretion selective for some proteins but not others? 
This should be confirmed with a few target proteins. 
5) Related to above, it is necessary to figure out whether the reduced secretion of specific proteins 
is because of reduced synthesis or trafficking. 
6) Figure 3d, ManII is a Golgi marker and so can only be used to measure ER-to-Golgi trafficking, 
not intra-Golgi trafficking related to Golgi vesiculation. In this figure, the Golgi does not seem to 
be vesiculated in the miR-30d cells. There is also no quantitation of the movie. 
7) Movie 2 is not convincing. Since E-Cad is N-glycosylated, the authors could use EndoH 
treatment and bandshift to indicate the trafficking speed. 
8) Figure 4g, the pPKD pattern is odd, its localization to the Golgi is not convincing. 
 
4. Some critical controls or details are missing. 
1) miR-30d was previously identified as a negative regulator of p53 (Kumar et al., Oncogene, 
2011). This was not discussed in this study. 
2) On the other hand, several known microRNAs regulated by p53 such as miR-34 were not 
included in the screening. 
3) In addition to the p53 mutants, WT p53 should be tested in parallel in some of the critical 
experiments. 
4) The authors collected conditioned medium from MDA-MB-231 and H1299 cells, but did not 
mention how long the cells were incubated before the conditioned medium was collected. In 
addition, MDA-MB-231 cells were cultured in DMEM while H1299 cells were cultured in RPMI1640; 
the two different media have different compositions. 
5) Molecular weight markers are missing for some gels, scale bars are missing in some 
micrographs. 
6) For the proteomic and bioinformatic studies it would be helpful to indicate what proteins/genes 
are included in which categories and how much they have changed under deferent conditions. 
Some of the tables include the protein/gene names but did not show which categories they belong 
to. 
 
 
 
Reviewer #3: 
Remarks to the Author: 
This is an interesting manuscript that documents a novel gain of function activity of mutant p53 
proteins in breast cancer. The authors intriguingly show that the transcriptional axis mutp53/HIF1a 
promotes aberrant activation of miR-30d that provokes the release of a pro-metastatic secretome. 
The listed below comments need to be fully addressed to render the manuscript more logically 
organized and more balanced between in vitro and vivo reported findings. 
 
Specific comments: 
1. Most of the reported in vivo experiments refer to the mutp53-miR30d axis lacking any evidence 
that includes HIF1a. This is not consistent with the transcriptional regulatory role that 
mutp53(HIF1a exerts on miR-30d expression; thereby providing a set of data lacking the main 
transcriptional and functional player such as HIF1a. 
2. Do breast cancer patients carrying TP53 mutations in Metabric and TGCA or other datasets 
exhibit higher levels of miR-30d? What about the reported and investigated miR-30d targets? Is 
their expression anti-correlated to miR-30d? 
3. TP53 sequencing and LOH analyses of the 12 analyzed human breast cancers is required to 
confirm that they are representative of specific mutant p53 gain of function tumors. P53 
immunostaining isn't sufficient for making the conclusion proposed by the authors. 
4. It appears rather unclear why adult fibroblasts from Li-Fraumeni represent a unique genetic 
model to study the impact of mutp53/miR-30d axis on breast tumorigenesis. If any, it's difficult to 
believe that fibroblasts can recapitulate the complexity of established breast cancer tumors. One 
intriguing possibility could be that of IHC of breast cancer tissues from LFS patients. 
5. Does HIF1a depletion affect the mutp53-miR30d dependent secretome? While the authors 
initially showed that miR30d expression is controlled by the transcriptional crosstalk 
mutp53/HIF1a, most of the in vitro and vivo biological experiments have been performed lacking 
any evidence on the role of HIF1a in mutp53-miR30d axis. 
6. How the authors explain that mutap53/HIF1a bind miR30d regulatory regions in both normoxic 
and hypoxic conditions? The described transcriptional complex is active in both conditions? What 
about the analysis of markers of chromatin modifications or the recruitment of acetylase or 
deacetylase in both conditions to address this issue. 
7. Recruitment of HIF1A on miR-30d promoter needs to be analyzed as well, to conclude that mut-
p53 acts as a partner of HIF1α, leading to transcriptional upregulation of miR-30d expression. 
8. Effect on secretion after HIF1A stabilization in presence/absence of miR-30d inhibition needs to 
be analyzed to conclude that the mutp53/HIF1A complex impinges on secretion through miR-30d 
induction and activity. 
9. Golgi vesiculation has been mainly analyzed in non-transformed cells throughout the manuscript 
with the majority of results obtained in MCF10A cells and fibroblasts. The relevance of the 
identified network in cancer cells could be strengthened by the inclusion of additional analyses 
performed in cancer cells after modulation of the key players of the identified network. 
 
 
Minor comments: 
 
1. Figure 2, was GSEA performed only considering the protein secretion pathway? Information 
provided in the results/legend for GSEA analysis is incomplete (q-val? Enrichment?). 
2. Does miR-30d impact on proliferation? What about mutp53? If this is the case, secretion should 
be analyzed also after additional cell cycle arresting stimuli. 
3. Fig2g, the description is not clear, please include in the matrix the control samples siSCR and 
miR-CTR 
4. Fig2. Venn diagram of proteins controlled by mp53 and by miR-30 would help in understanding 
results