Sirt6 ablation in the liver causes fatty liver that increases cancer risk by upregulating Serpina12

Non-alcoholic fatty liver disease is a chronic liver abnormality that exhibits high variability and can lead to liver cancer in advanced stages. Hepatic ablation of SIRT6 results in fatty liver disease, yet the potential mechanism of SIRT6 deficiency, particularly in relation to downstream mediators for NAFLD, remains elusive. Here we identify Serpina12 as a key gene regulated by Sirt6 that plays a crucial function in energy homeostasis. Specifically, Sirt6 suppresses Serpina12 expression through histone deacetylation at its promoter region, after which the transcription factor, Cebpα, binds to and regulates its expression. Sirt6 deficiency results in an increased expression of Serpina12 in hepatocytes, which enhances insulin signaling and promotes lipid accumulation. Importantly, CRISPR-Cas9 mediated Serpina12 knockout in the liver ameliorated fatty liver disease caused by Sirt6 ablation. Finally, we demonstrate that Sirt6 functions as a tumor suppressor in the liver, and consequently, deletion of Sirt6 in the liver leads to not only the spontaneous development of tumors but also enhanced tumorigenesis in response to DEN treatment or under conditions of obesity.

Thank you for your suggestion.We have changed the term "WT" to "Floxed" accordingly in the revised manuscript.
2. One interesting observation is that SIRT6 LKO mice have reduced immune cells in the liver in both regular condition (Figure S1D) and after breeding into the ob/ob background (Figure S7I).The authors showed that there were reduced abundance of CD4+ and CD8+ T cells in Figure S7I.Have the authors checked Kupffer cells, the liver resident macrophages, in SIRT6 LKO mice under both conditions?Also, can the authors discuss the possible implications of this reduced immunity in promoting hepatic tumorigenesis in SIRT6 LKO mice?
For detecting Kupffer cells, we did IHC with an antibody to IBA1, which is Kupffer cells marker in the liver in Sirt6 LKO mice in both conditions.There were no obvious changes in both conditions (R-Figure 1A and   1B).We observed that the immune response related pathway was decreased in Sirt6 LKO mice using RNA-seq analysis.These pathways were downregulated, suggesting the weaker activity of the immune cells in the Sirt6 LKO mice liver.Although we found that the number of Kupffer cells has no obvious change in the liver, T cells could also affect immune processes.In Sirt6 LKO mice with obesity background, we have found reduced CD4+ and CD8+ T cells in the liver.The reduced immunity could lead to the failure to eliminate cancer cells in the liver, thus promoting the tumor formation.However, the relationship between the immune system and cancer is complex, and enhancing the immune system alone may not be effective to treat liver cancer in clinical condition.Further study is needed to illustrate the effect in this case.The possible implications were discussed in the revised manuscript.We have indicated in the Discussion that the reduced CD4+ and CD8+ T cells in the liver could lead to the failure to eliminate cancer cells in the liver, thus promoting the tumor formation (page 28, line 1-2, line 11-19 and page 29 line 1-6).
3. Figure 3, very nice results!Two questions: does C/EBPa directly interact with SIRT6 so that SIRT6 can be specifically target to the promoter of Serpina12 gene?Could the authors confirm that the binding of C/EBPa is important for SIRT6 to suppress the expression of Serpina12 by generating mutant luciferase reporters that lack functional C/EBPa binding site (site 1 in Figure 3C), then show that SIRT6 repress the WT but not mutant luciferase reporter in hepatocytes?
To determine whether C/EBPa could directly interact with SIRT6, we induced Sirt6-GFP and C/ebpα expression 48h in 293T cells and performed a co-immunoprecipitation assay with antibodies against Sirt6 and C/ebpα.The results showed that CEBPA could directly interact with SIRT6 (R-Figure 2A). 4. Figure 4, does the SIRT6 level change (reduce) during high glucose or oleic acid induced lipogenesis in hepatocytes?
Thank you for your comment.We have observed that Sirt6 level has no significant change in 1g/L (low glucose) compared to 4g/L (high glucose) both at RNA (R-Figure 3A) and protein levels (manuscript Figure 4D).However, the SIRT6 level has significantly decreased after oleic acid treatment in HepG2 cells (R-Figure 3B  Thank you for the suggestion.We checked the expression of SIRT6 in NASH patients in a GEO database and found that it was at a low expression level compared to other genes (revised manuscript Figure 5D).We found a negative correlation between Sirt6 and Serpina12 in the patients with HCC in a GSE25097 database (R-Figure 4A).To understand the role of SIRT6 for patient survival, we analyzed the TCGA database in HCC patients.We defined SIRT6 high or SIRT6 low expression based on the mean value in these HCC patients and the data indicated that overall survival has no significant difference between SIRT6 high and SIRT6 low expression (R-Figure 4B). 4 (A) Pearson correlation analysis of SIRT6 and SERPINA12 mRNA expression in HCC (GSE25097).

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(B) Kaplan-Meier curves for overall survival of HCC patients with high and low SIRT6.

Referee #2:
In this study, Li et al demonstrated that depletion of Sirt6 in mice liver resulted in alteration of H3K9 and H3K56 acetylation and gene expression profile.By comparison of the RNA-seq data and ChIP-seq data from wild type and Sirt6-LKO mice live, Serpina12 was identified as the key gene downstream of Sirt6.Ablation of Sirt6 increased the expression level of Serpina12 by enhancement of the level of H3K9ac and H3K56ac and the binding of transcription factor CEBPα. Increased expression of Serpina12 in hepatocytes enhanced insulin signaling and lipid accumulation.In addition, Sirt6 was shown to be a tumor suppressor in liver.Although the work might be interested, several concerns are raised.
1.The authors demonstrated the depletion of Sirt6 in hepatocyte resulted in high level of SERPINA12 to enhance insulin signaling and promote lipid accumulation.Knock out of Sirt6 was shown to cause a lipid rich environment to accelerate hepatocellular carcinoma (HCC).Since insulin signaling was also reported to play important role in tumorigenesis and progression.The authors should evaluate the effect of insulin signaling in HCC.
Thank you for the suggestion.Insulin-dependent signaling pathways are often overactivated in human HCC due to the overexpression of signaling components and loss of negative regulators [1,2] .As a result, hyperinsulinemia can directly promote cancer cell metabolism, proliferation, and survival, potentially contributing to HCC development and progression [3] .We have tested the effect of insulin signaling in HCC and the data indicated that insulin signaling plays a crucial role in the development of hepatocellular carcinoma (HCC) by promoting cell proliferation (R-Figure 5).The data is placed in the revised manuscript Appendix Figure S3, and the corresponding text (page 26, line 6-12).5 Cell proliferation rate with different dose of insulin treatment.

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2. SERPINA12 was reported to be an extracellular serine protease inhibitor which inhibited the activity of serine protease like KLK7 to increase the concentration of insulin in serum and enhance insulin signaling.In this study, Serpina12 was shown to promote expression of genes related to glucose and lipid metabolism.Therefore, the author should demonstrate that SERPINA12 can be transported into nucleus.
Thank you for the suggestion.To understand the location of Serpina12, we isolate cytoplasm extract and nuclear extract in primary hepatocytes.The data that Serpina12 was in cytoplasm in the cells, but not in the nucleus (R-Figure 6).We have also demonstrated that knockdown Sirt6 increased Serpina12 expression in the cytoplasm, which may trigger the lipid accumulated in the cells.Thus, effect of Serpina12 on genes related to glucose and lipid metabolism might be caused other mechanism rather than directly regulates their expression.Sirt6 in primary hepatocytes.
3. An interesting question should be addressed is the mechanism of SERPINA12 be recruited to chromatin.Either the DNA motif or the protein factors targeting SERPINA12 to chromatin should be identified.
In our study, we found that Sirt6 affects the Histone 3 lysine 9 acetylation, and they have increased binding peaks in the Serpina12 locus.As Serpina12 is only in the cytoplasm, but not in the nucleus (R-Figure 6), it is unlikely that Serpina12 could be recruited to chromatin.

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To address the second question, we generated a mutant luciferase reporter that lacks functional C/EBPa binding site.The data indicated that Sirt6 could suppress the increased expression of WT Serpina12-luc activity induced by C/EBPa.In contrast, such suppression was largely abolished after the mutation of the binding site of C/EBPa (R-Figure 2B).This observation indicates that the binding of C/EBPa is important for SIRT6 to suppress the expression of Serpina12.This data was placed in the Figure Extended View 3D and 3E and the corresponding text (page 13 and line 6-11).R-Figure 2 (A) IP experiments on lysates from 293T/17 cells detected by antibodies of Sirt6 and C/ebpα.Sirt6-GFP and C/ebpα expression was induced 48 h prior to IP. (B) Ectopic expression of C/ebpα increased Serpina12 promoter activity, whereas mutation of C/ebpα-binding sites reduced the activity.Ectopic expression of Sirt6 represses the Serpina12 promoter activity but not the mutant C/ebpα-binding sites promoter activity.
and 3C), suggesting that the expression change of Sirt6 might be more sensitive to condition under lipogenesis.R-Figure 3 (A) qPCR analysis of Sirt6 mRNA levels in different glucose concentration.(B) qPCR analysis of Sirt6 mRNA levels in different oleic acid concentration treatment.(C) Western blot analysis of Sirt6 mRNA levels in different oleic acid concentration treatment.5. Figure 5, nice data.Have the authors analyzed the expression of SIRT6 in NASH?How about the correlations between SIRT6 and SERPINA12 as well as between SIRT6 and patient survival in HCC patients?

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Western blot analysis of Serpina12 in cytoplasm extract or nuclear extract with or without knockout ., M. Lequoy, L. Fartoux, et al., Hyperinsulinaemia and insulin signalling in the pathogenesis and the clinical course of hepatocellular carcinoma[J].Liver Int, 2015.

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