Vitamin D induces SIRT1 activation through K610 deacetylation in colon cancer

Reviewer #1 (Public Review):

This study demonstrates that vitamin D-bound VDR increased the expression of SIRT1 and that vitamin D-bound VDR interacts with SIRT1 to cause auto-deacetylation on Lys610 and activation of SIRT1 catalytic activity. This is an important finding that is relevant to the actions of VDR on colorectal cancer. The data presented to support the presented conclusion is convincing.

A strength of the study is that it is focused on a narrow group of conclusions.

The major weakness of the study is that the site of SIRT1 regulatory lysine acetylation is defined by mutational analysis rather than by direct biochemical analysis. This issue is partially mitigated by previous reports of K610 acetylation using mass spec (https://www.phosphosite.org/proteinAction.action?id=5946&showAllSites=true). However, Fig. 4E is reassuring because it shows that the apparent acetylation of the K610 mutant SIRT1 appears to be lower than WT SIRT1

A second weakness of the study relates to the use of shRNA-mediated knockdown of VDR for some studies in which a previously reported cell line was employed. The analysis presented would be more compelling if similar data was obtained using more than one shRNA. Similarly, only a single siRNA for SIRT1 is presented in Table 1.

A third weakness of the study is that the conclusion that the VDR interaction with SIRT1 is the cause of auto-deacetylation rather than an associated event mediated by another mechanism would be more strongly supported by mutational analysis of SIRT1 and VDR residues required for the binding interaction. Will VDR increase SIRT1 activity when mutations are introduced to block the interaction? While the finding that catalytically inactive SIRT1 does not interact with VDR is helpful, this does not address the role of the binding surface.

A fourth weakness of the study is that it would be improved by testing the proposed hypothesis through in vitro reconstitution with purified proteins. Does VDR cause auto-deacetylation and activation of Sirt1 in vitro?

Reviewer #2 (Public Review):

The authors decipher the signaling between vitamin D and proteins that are downstream of SIRT1. The importance of vitamin D in physiology is clear. However, the link between vitamin D and cancer is less clear. This study provides very interesting and solid information on the link between vitamin D and colorectal cancer. It is likely that this study will provide insight into the importance of vitamin D in other types of cancer. It may also lead to new therapeutic strategies for specific cases.

The authors focus on vitamin D-mediated signaling through VDR, SIRT1 and Ace H3K9. They highlight the importance of K610 in SIRT1 in this process. This article is convincing, although the authors can improve their study as outlined below:

* The authors should specify which cell line was used to perform the experiment in Figure 1E,F. What would be the result in the presence/absence of 1,25(OH)2D3? In Figure 1G, what is the meaning of # and ###?

* Figure 2C, it would have been ideal to show the VDR-SIRT1 interaction after a Sirt1 IP.

* I understand the authors' overall message for this figure, but it is far from clear. This section needs to be improved. For example, in Figure 3G, does this mean that the level of AceH3K9 is independent of the level of SIRT1? Is there a contradiction? The authors should indicate the color of the different stainings for Figure 3D. Do the authors mean that the secondary antibody marks in brown/red? If so, these results are inconsistent with the text considering that hematoxylin was used for non-tumor tissue. This part needs to be clarified. What about the level of FOXO3A in these tissues/tumors? What is the level of 1,25(OH)2D3 in these patients? In Figure 3D, the following information is missing: "A detailed amplification is shown in the lower left of each micrograph." In Figure 3E, it says p=0.325, in the legend p<0.01, and in the text there is a trend. Which is the correct version?

* Figure 4F. The quality of the presented blots is not optimal. It needs to be improved. In addition, the number of independent biological experiments is not indicated. In general, the authors should better indicate the number of independent biological experiments performed, at least for some of them. For example, see Figure 1G. Regarding Figure 2C, we understand that the WB was performed 3 times. Is this the case for the PI? etc...