BAG2 promotes tumorigenesis through enhancing mutant p53 protein levels and function

Despite overall positive reviews on your paper, there were two issues that were raised during consultation between the reviewers and the member of the board that is handling this paper.

First, with the exception of Figure 2–figure supplement 1 where the authors do a co-IP in SK-BR-3 cells, all other experiments in human cells are in either H1299 and Saos2 p53 null cells or HCT116 cells each of which came from tumors that did not have mutant p53. It is felt that the authors should examine more cancer cell lines with endogenously expressed mutant p53 to determine the generality of their findings that BAG2 and mutant p53 interact.

Thanks for this very good suggestion. We agree with the reviewers that additional data obtained from more cancer cell lines with endogenous mutp53 can provide important information about the generality of BAG2-mutp53 interaction. We have employed human colorectal cancer cell lines HT-29 (mutp53 R273H), SW480 (mutp53 R273H), human breast cancer cell line MDA-MB-468 (mutp53 R273H) and human hepatocellular cancer cell line Huh-7 (mutp53 Y220C) to examine the interaction between the endogenous BAG2 and mutp53 proteins by co-IP assays. In the revised manuscript, we have presented new data showing that the BAG2-mutp53 interaction was observed in all these cell lines. These data in revised Figure 2–figure supplement 1 strongly suggest the generality of BAG2-mutp53 interaction in human tumor cells.

Second, the authors should examine normal tissues (or cells therefrom) expressing mutant p53 where there is a lot of BAG2 but no stabilization of p53 to see if in this setting BAG2 binds to mutant p53. This might provide insight into reviewer#3's cogent comments.

This is a very good suggestion. The reviewers raised a very good point that while normal thymus tissues from mutp53^R172H/R172H mice express a lot of BAG2, there is no apparent effect of BAG2 on mutp53 accumulation in normal tissues (Figure 1C) (see detailed response to Reviewer 3). The reviewers provided a very good suggestion to carefully analyze the BAG2-mutp53 interaction in normal tissues from mutp53^R172H/R172H expressing mice. We re-analyzed the raw data for Figure 1C. We observed lower levels of mutp53 protein and weak interaction between BAG2 and mutp53 in normal thymus tissues from mutp53^R172H/R172H expressing mice after longer exposure. We have presented the film with longer exposure time in revised Figure 1C. In addition, we examined the interaction between BAG2 and mutp53 in normal kidney and spleen tissues in addition to thymus tissues collected from mutp53^R172H/R172H expressing mice. In all these tissues, we observed weak interaction between BAG2 and mutp53. These data are presented in Figure 1–figure supplement 1, which suggest that some additional tumor-specific changes might contribute to the effect of BAG2 on mutp53 accumulation. We have added the following to the Discussion: “It is also worth noting that while normal tissues from p53^R172H/R172H mice express a lot of BAG2, there is a limited amount of interacted BAG2-mutp53 protein complex and no clear accumulation of mutp53 proteins in the normal tissues (Figure 1C and Figure 1–figure supplement 1). These results suggest that some tumor-specific events might contribute to the effect of BAG2 on mutp53 accumulation.”

Minor points:

1) The authors are encouraged to test whether BAG2 binds to p53 directly in an in vitro binding assay, or to state clearly in the Discussion that whether BAG2 binds to p53 directly, or is brought by Hsc70, is not yet clear.

Thanks for this very good suggestion. It is unclear whether BAG2 binds to mutp53 directly or indirectly. As suggested by the reviewer, we have stated this in the Discussion: “It remains unclear whether BAG2 interacts with mutp53 directly or interacts with mutp53 through other protein, such as Hsc70, which will be of interest to investigate in future studies.”

2) In the subsection “BAG2 Is A Novel Mutp53-interacting Protein in p53^R172H/R172H Mouse Tumors and Human Cells”, please tell us how many thymic lymphomas were used for the IP/LC-MS/MS experiments.

We have added the number (n = 3) of thymic lymphomas used for the IP/LC-MS/MS assays in the subsection “BAG2 Is A Novel Mutp53-interacting Protein in Trp53^R172H/R172H Mouse Tumors and Human Cells”.

3) In the subsection “BAG2 Promotes Mutp53 GOF in Chemoresistance”, the authors indicate that BAG2 greatly increased 5-FU induced apoptosis. It was only 50%, so this statement needs to be toned down a bit.

As the reviewer suggested, we have changed the statement to: “knockdown of BAG2 increased 5-FU-induced apoptosis in Saos2-R175H…”.

4) Figure 7A needs to be described a little better in the legend. I do not know what the horizontal line at 0 stands for. Shouldn't the normal controls be at zero?

This is a very good suggestion. We have provided the explanation for the calculation of the intensity of BAG2 expression presented in Figure 7A in the corresponding legend. The BAG2 expression levels for the normal controls may have a value less than zero when BAG2 is expressed at a level less than the median and may have a value greater than zero when BAG2 is expressed at a level higher than the median.

5) The Discussion was simply a retelling of the experiments performed and not really a discussion. The authors need to discuss their findings. Why, for example, do some tumors have increased BAG but no accumulation of mutant p53.

This is a very good suggestion. We further discussed our findings in the Discussion. In particular, we stated that not all tumors with BAG2 overexpression showed the accumulation of mutp53 protein: “It is unclear why not all tumors with BAG2 overexpression showed the accumulation of mutp53 protein […]. It will be interesting to examine whether BAG2 has weak or no interaction with mutp53 protein in this subgroup of tumor samples in future studies.”

Reviewer #3:

This is an interesting paper and the data presented are exceptionally clear and convincing. I don't have any criticisms of the work as it stands. My questions are more around the mechanism. Why has BAG2 evolved to specifically bind mutant p53 and how can it discriminate between the DNA binding domains of a diverse range of mutant p53 s compared to wild type? How does BAG2 interfere with MDM2 binding? I don't think the authors can answer all of these questions but they might pose them and speculate.

These are very good questions. While currently we don’t have final answers for these questions, we added following to the Discussion: “In this study, we found that BAG2 preferentially binds to mutp53 at the DBD domain […]. It remains unclear whether BAG2 interacts with mutp53 directly or interacts with mutp53 through other protein, such as Hsc70, which will be of interest to investigate in future studies.” Also: “It is unclear how the BAG2-mutp53 interaction interferes with the MDM2-mutp53 interaction since MDM2 binds to the N- terminus of mutp53 whereas BAG2 binds to the DBD of mutp53. Future studies are needed to further understand its mechanism.”