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


RR2
 Reviewers' Comments:
 Reviewer #1:
 Remarks to the Author:
 Overall, the authors have done an impressive job in revising the MS. Almost all my concerns have
 been satisfactorily addressed. The only concern that remains unaddressed is the request to
 perform RNA-seq in response to my comment 1. The authors justify this by the severe restrictions
 imposed by COVID-19, and I am willing to accept this explanation.
 Therefore, I recommend acceptance after the minor comments below are properly addressed.
 BTW: the rebuttal refers to Fig. 1e as a Western showing HIF1a stabilization. This seems to be a
 mistake in figure number.
 1.The paper by Amelio et al (reference 65 in this MS) is mentioned only once, in a rather vague
 context of mutant p53 and ECM. However, the Amelio paper mainly focuses on the transcriptional
 effects of the interaction between mutant p53 and HIF1a, a theme that is closely related to the
 present MS. For the sake of fairness, this should be mentioned in the Discussion.
 2. Line 324: please remove “and”
 3. Line 441: Figure number (7) missing.
 Reviewer #2:
 Remarks to the Author:
 The authors adequately addressed some of the major comments and made appropriate changes in
 the manuscript by including additional supplemental data as well as updating panels to the main
 figures. Good effort was made to perform some of the suggested experiments. Satisfactory new
 experiments were performed to demonstrate increased HIF1 interaction with mut-p53 under
 hypoxic conditions, and recruitment of mut-p53 to the miR-30d promoter. Notably, the authors
 were able to show that in tissue culture cells and in both human and mouse tissues, the protein
 levels of PDIA5, Sec24A, and GM130 were affected by miR-30d expression. Convincing new
 evidence in human breast tissue expressing mut-p53 stained for Sec24A and β-COP sufficiently
 supports the authors’ findings that the mut-p53/miR-30d axis affects the secretory machinery.
 Discussion of the data and interpretation of the results has improved.
 Experiments which were not performed due to COVID-19 lockdown, such as - the EndoH
 experiment for E-cad and other experiments to interrogate trafficking rate to plasma membrane,
 or identification of specific cargoes for which secretion rates are reduced – would have been
 insightful but less critical to the author’s overall conclusions in the paper. This work opens up
 many avenues to pursue in understanding of the mechanistic pathway in which mut-p53 and HIF1
 oncogenes regulate Golgi structure and function.
 Overall, the data describes a novel p53/HIF1a/miR-30d axis that impacts synthesis, trafficking,
 and secretion of numerous proteins essential for tumor growth and metastatic colonization.
 There still remains a few specific points that should be addressed:
 1. Although the authors speculate that miR-30d stabilization of microtubules as mechanism for
 enhanced Golgi vesiculation and accelerated protein trafficking, the data to support this is limited
 and perhaps probing for difference in Ac-Tubulin in the conditions presented in Supplementary Fig
 6l would strengthen the immunofluorescence observations from Fig 3a.
 2. If local activation of PKD is central to the mechanistic hypothesis for the role of DGKZ in
 affecting Golgi structure, then Fig 4g is not sufficient. An additional method of detecting activated
 PKD1 at the Golgi would strengthen the author’s claim. A simple IB for Golgi localized phospho-
 PKD1 for the same conditions tested in the IF is recommended.
 3. The quantitation for the RUSH assay (Fig 3d) which measures trafficking from ER to Golgi is
 appreciated. Statistical significance demarcation in the graph is missing.
 4. New Fig 1-h has a bar for statistical analysis from control normoxia to control hypoxia but is

 missing asterisk(s) to denote significance.
 5. Fig. 5b has some overlapping words on the label.
 Reviewer #3:
 Remarks to the Author:
 The revised version of the manuscript has been significantly improved and is compelling with the
 concerns raised within my previous evaluation. Based on this, the manuscript is suitable for
 publication.