Mad1 destabilizes p53 by preventing PML from sequestering MDM2

Mitotic arrest deficient 1 (Mad1) plays a well-characterized role in the mitotic checkpoint. However, interphase roles of Mad1 that do not impact mitotic checkpoint function remain largely uncharacterized. Here we show that upregulation of Mad1, which is common in human breast cancer, prevents stress-induced stabilization of the tumor suppressor p53 in multiple cell types. Upregulated Mad1 localizes to ProMyelocytic Leukemia (PML) nuclear bodies in breast cancer and cultured cells. The C-terminus of Mad1 directly interacts with PML, and this interaction is enhanced by sumoylation. PML stabilizes p53 by sequestering MDM2, an E3 ubiquitin ligase that targets p53 for degradation, to the nucleolus. Upregulated Mad1 displaces MDM2 from PML, freeing it to ubiquitinate p53. Upregulation of Mad1 accelerates growth of orthotopic mammary tumors, which show decreased levels of p53 and its downstream effector p21. These results demonstrate an unexpected interphase role for Mad1 in tumor promotion via p53 destabilization.

The paper addresses an open question in the field, unveiling the role of Mad1 protein in interphase, as opposed to the well characterized role in chromosome segregation. In detail, they describe a model -appropriately supported by experimental data-in which upregulated Mad1, as commonly occurs in human breast cancer, interferes with p53 signalling, by binding PML through its CTD (binding enhanced by sumoylation) displacing MDM2 from PML, which in turn sequesters Mad1 into nucleoli. MDM2 is thus "free" to bind p53 and initiate its degradation, ultimately promoting tumorigenesis. The subject of the paper is novel and of interest to the field, as it demonstrates the existence a novel mechanism of regulation/de-regulation of the p53 pathway. Being p53 the most commonly altered tumor suppressor in human cancer, having a comprehensive understanding of the related molecular mechanisms is indeed critical. Though the finding is unlikely to represent a breakthrough in the field and radically change the thinking in the field, it adds an important piece of information, supported by well-designed, convincing experimental data; a large body of results, indeed, fully support the assertions made by the authors. Methods section is exhaustive, providing all the required information to potentially reproduce the experiments, even though additional details on the percentage of SDS-PAGE gel used for the western blot might be useful, as well as the composition of the blocking solution (as these may affect antibody efficiency). Minor points: In figure legend of figure 1, scale bar of panel D is missing. Panel G-H, the rationale for the experiments is provided in the main text, thus unnecessary in the figure legend. It can be removed. Figure 2 panel B. Authors claim only fragments of Mad1 containing CTD domain (aa 597-718, and so also all the fragments containing the 597-718 aminoacidic stretch) co immunoprecipitate HA PML, but a signal seems slightly visible also in the lane co immunoprecipitating the fragment 1-178. It would be nice to see another blot. Figure 2 panel F, having the acronym (NIS, NES, CTD …) on the figure rather than the aminoacid sequence might be helpful for a more straightforward look of the data. Figure 4 panel A, is the labelling of the second half of the blot correct ( (+) (-) tet) ?
Overall, some details about the procedure should be moved from the figure legend to the methods or results section where it is more appropriate (see also figure 4E).
Reviewer #2 (Remarks to the Author): In this manuscript Wan et al investigate the consequences of overexpressing Mad1, a known spindle checkpoint protein. The authors report here that when overexpressed Mad1 has not only a function in mitosis, but also changes the behavior of interphasic cells. This is potentially relevant as Mad1 has been often reported to be overexpressed in cancer cells. Specifically the authors show with biochemical and cell biology experiments that overexpressed Mad1 accumulates in PML bodies via Sumoylation interaction motif in the C-terminal part of the protein. This interaction displaces Mdm2 from PML bodies preventing the accumulation of p53 in the presence of DNA damage. In the last part of the manuscript the authors show with murine models that Mad1 overexpression can transform non-cancerous MCF10A cells and accelerate cancer formation when overexpressed in cancerous cells expressing a mutated version of p53. Based on these experiments they infer that the Mad1-overexpression seen in human cancer patients can participate to cancer formation.
Overall, this study addresses an interesting and original biological question. The results are novel and of high technical quality, in particular the protein-protein interaction experiments. The study has the potential for an excellent publication in Nature Communication, yet at this stage suffers from several conceptual weaknesses that should be addressed before publication.
Major points: 1) the entire study is based on the strong overexpression of Mad1 in different cell lines. It is however, unclear whether Mad1 is really overexpressed to such high levels in cancer cells, raising the question whether the observed effects ever occur in a human cancer. The most convincing way to address this point would be for the patient to identify a tissue culture cell line that overexpresses Mad1 into PML bodies, and to quantify the p53 response after control or Mad1 depletion. Such an experiment would go a long way to prove that the Mad1 overexpression seen in cancer patients does participate to p53-regulation, and that the observed results are physiologically relevant.
2) the other important aspect is that the authors assume that Mad1 overexpression participates to cancerogenesis because it targets p53. This is plausible based on their data, but not directly proven in the manuscript. In fact Mad1 overexpression also has an effect in a cell line (MDA-MB-231) in which the p53 response is seriously attenuated, since it expresses a potentially dominantnegative p53 mutant. An important control would have been to also overexpress Mad1 in a cancer cell line bearing a p53 deletion, to show that in such case Mad1 overexpression has no effect on cancerogenesis, as p53 is missing. Since such an experiment would take a long time, I don't think it is necessary for the revision of this manuscript. Nonetheless if the authors had data that would address this point, this would significantly strengthen the conclusions. Otherwise the authors should at least comment on this question in their discussion.
Minor point: 1) since MDA-MB-231 cells express a mutant version of p53 that is strongly attenuatef in its function, one would expect high levels of p53, due to low p53-dependent transcription of the MDM2 gene, which is what has been reported in the past. It is therefore surprising that the authors see a similar p53 accumulation in MDA-MB-231 and non-cancerous MCF10A cells after DNA damage, since p53 is supposed to be only very partially active and expressed at high levels in MDA-MB-231 cells.

Point-by-point response to the referees' comments
The paper addresses an open question in the field, unveiling the role of Mad1 protein in interphase, as opposed to the well characterized role in chromosome segregation during mitosis. In detail, they describe a model -appropriately supported by experimental data-in which upregulated Mad1,as commonly occurs in human breast cancer, interferes with p53 pathway, by binding PML through its CTD (binding enhanced by sumoylation) displacing MDM2 from PML, which in turn sequesters Mad1 into nucleoli. MDM2 is thus "free" to bind p53 and initiate its degradation, ultimately promoting tumorigenesis. The subject of the paper is novel and of interest in the field, as it demonstrates the existence and the role of a protein in controlling the p53 pathway. Being p53 the most commonly altered tumor suppressor in human cancer, having a comprehensive understanding of the related molecular mechanisms is important. Though the finding is unlikely to represent a breakthrough in the field and radically change the thinking in the field, it adds an important piece of information, supported by well-designed, convincing experimental data; a large body of results, indeed, fully support the assertions made by the authors. Methods section is exhaustive, providing all the required information to potentially reproduce the experiments, even though additional details on the percentage of SDS-PAGE gel used for the western blot might be useful, as well as the composition of the blocking solution (as these may affect antibody efficiency).
Minor points: In figure legend 1, scale bar of panel D is missing. Panel G-H, the rationale for the experiments is provided in the main text, thus unnecessary in the figure legend. It can be removed. Figure 2 panel B. Authors claim only fragments of Mad1 containing CTD domain (aa 597-718, and so also all the fragments containing the 597-718 aminoacidic stretch) co immunoprecipitate HA PML, but a signal seems slightly visible also in the lane co immunoprecipitating the fragment 1-178. It would be nice to see another blot. Figure 2 panel F, having the acronym (NIS, NES, CTD …) on the figure rather than the aminoacid sequence might be helpful for a more straightforward look of the data. Figure 4 panel A, the second half of the blot should be (+) tet rather than (-) tet Reviewer #2 (Remarks to the Author): The authors have addressed the reviewers concerns in a convincing manner. I therefore support publication of this interesting study.