ZBTB18 inhibits SREBP-dependent fatty acid synthesis by counteracting CTBPs and KDM1A/LSD1 activity in glioblastoma

  1. R. Ferrarese
  2. A. Izzo
  3. G. Andrieux
  4. S. Lagies
  5. J.P. Bartmuss
  6. A.P. Masilamani
  7. A. Wasilenko
  8. D. Osti
  9. S. Faletti
  10. R. Schulzki
  11. Y. Shuai
  12. E. Kling
  13. V. Ribecco
  14. D.H. Heiland
  15. S.G. Tholen
  16. M. Prinz
  17. G. Pelicci
  18. B. Kammerer
  19. M. Börries
  20. M.S. Carro

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Abstract

Enhanced fatty acid synthesis is a hallmark of tumors, including glioblastoma. SREBF1/2 regulate the expression of enzymes involved in fatty acid and cholesterol synthesis. Yet, little is known about the precise mechanism regulating SREBP gene expression in glioblastoma. Here, we show that a novel interaction between the co-activator/co-repressor CTBP and the tumor suppressor ZBTB18 regulates the expression of SREBP genes. Our study points at CTBP1/2 and LSD1 as co-activators of SREBP genes whose complex functional activity is altered by ZBTB18. ZBTB18 binding to the SREBP gene promoters is associated with reduced LSD1 demethylase activity of H3 active marks leading to increased di-methylation of lysine 4 (H3K4me2). Concomitantly, we observed increased di-methylation of lysine 9 (H3K9me2), and decrease of the active mark H3K4me3 with consequent repression of the SREBP genes. In line with our findings, lipidomic analysis shows a reduction of several phospholipid species upon ZBTB18 expression. Our results outline a new epigenetic mechanism enrolled by ZBTB18 and its cofactors to regulate fatty acid synthesis that could be targeted to treat glioblastoma patients.

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  1. Version published to 10.1101/2020.04.17.046268v3 on bioRxiv
  2. Version published to 10.1101/2020.04.17.046268v2 on bioRxiv
  3. eLife

    ###Reviewer #3:

    The connection between core transcriptional regulation and tumor metabolism is an area of current interest. The reciprocal regulation of ZBTB18 and CTBP2 has potential value in understanding the functional regulation of lipid biology. However, there are substantial concerns with the studies that limit its rigor and value.

    Major concerns:

    1. It is advised that the authors consider referencing the International Cell Line Authentication Committee's Register of Misidentified Cell Lines before investing in experiments. The vast majority of critical experiments used only SNB19 (SNB-19). This is a contaminated line and should not be used for studies. The following is from the ATCC:

    “SNB-19 (ATCC CRL-2219) and U-373 MG (ATCC HTB-17) - STR analysis at ATCC revealed that SNB-19, a human glioblastoma cell line has a STR pattern identical to that for U-373 MG (ATCC HTB-17). SNB-19 and U-373 MG also share derivative chromosomes. These observations were confirmed with the original stock available to ATCC. Since then distribution of SNB-19 was discontinued. U-373 MG (ATCC HTB-17) - As a result of sequencing, the authenticity of ATCC HTB-17 has been questioned by R.F. Petersson in Stockholm and collaborator E.G. Van Meir in Atlanta (personal communication and see Ishii, N., et al. Brain Pathol 9: 469-79, 1999). They report similarities between U-373 MG (ATCC HTB-17) and another glioblastoma, U-251. The cell line U-373 MG, obtained from the original lab in Uppsala has differing genetic properties from the ATCC HTB-17 (U-373 MG). Following further investigations, ATCC stopped distribution of this cell line.”

    It is not only a concern about the naming of the line. The use of a single cell line grown in metabolically artifactual conditions for most of the studies weakens the ability to connect the results to the disease being studied. It also raises concern about global rigor overall. It would have been much better to consider using the BTSC cells for most of these studies. The validation efforts were minimal (sometimes even missing loading controls).

    1. Figure 1A, C, D, F: I assume that EV really was with FLAG alone. If not, the comparison should be between FLAG-ZBTB18 and FLAG alone. In each of these studies, there were no replicates and only a single cell line.

    2. Figure 1B: Why were CTBP1 and CTBP2 prioritized, instead of other molecules with more peptides?

    3. Co-IP of endogenous proteins ZBTB18 and CTBP2 in a panel of cells would be important.

    4. The shRNA experiments are poorly controlled. There is a single shRNA used and no rescue studies to address potential off-target effects. All experiments should include better controls.

    5. As the authors note, ZBTB18 is expressed at different levels in different glioblastomas, with greater expression in mesenchymal tumors. I would suggest that the authors better consider defining the putative reciprocal function of ZBTB18 and CTBP2 with both loss-of-function and gain-of-function studies.

    6. The in vivo studies are limited in scope. There is a single replicate of a single cell line (SNB-19, with the caveats above) with a single shRNA and no rescue studies.

    7. It is not surprising that ZBTB18 and CTBP2 have differences in gene regulation, but the current studies make it difficult to fully support the overall model. There are no rescue studies that show the rescue of proliferation or other defects, which would be important for the molecular model.

    8. MTOB is a regulator of the methionine salvage pathway, not simply CTBPs. Why wasn't methionine signaling investigated? The rescue efforts for MTOB with ZBTB18 failed, but it would be important to at least validate CTBP rescues.

    9. It wasn't clear to me why SREBP signaling was not studied in rescue studies? There is largely an effort to show changes in transcription, but few functional studies to show rescue of metabolism, proliferation, and tumor growth.

    10. Figure 4 should include endogenous ZBTB18 IP, as well, with better cells.

    11. Figures 4-7 show that the media used for most studies is not really appropriate to study ZBTB18 and CTBP2 function. These efforts should include more consideration of serum-free conditions and in vivo studies, especially as many studies have shown that standard serum conditions with excess oxygen cause artifacts of metabolism.

    12. The findings of changes in lipid metabolism are interesting, but quite preliminary. Lipid droplets have been strongly linked to aggressiveness in gliomas. The quantification does not show very strong differences. It would be important to show that the differences in lipid biology explain the effects of ZBTB18 and CTBP2 on tumor cell metabolism and proliferation. Are these findings the driver or passenger of effects?

    13. I would suggest that the authors consider deeper in silico efforts to examine target expression and patient outcome or genetic events.

  4. eLife

    ###Reviewer #2:

    In this manuscript, the authors claim that ZBTB18 interacts with CTBP2 and represses SREBP target genes to inhibit fatty acid synthesis in glioblastoma. However, the mechanisms presented in the manuscript are not convincing. This is because there are several major concerns for their conclusions as described below.

    1. It looks that Figure 1D shows almost no endogenous interaction between CTBP2 and ZBTB18 when α-CTBP2 was used. This is perhaps because their cell lines may express very low ZBTB18 levels. Moreover, in reciprocal IP experiments using cells with FLAG-ZBTB-18 overexpression, α-ZBTB18 IP shows weak CTBP2 band that is inconsistent with the CTBP2 band in Figure 1C. In addition, this manuscript relies too much on results that were generated from overexpression for the tumor suppressor candidate gene ZBTB18.Therefore, it is possible that many results in this manuscript may represent artificial results based on FLAG-ZBTB18 overexpression. Of note, knockdown or loss-of-function experiments are generally better for a tumor suppressor genes.

    2. ZBTB18 is a transcriptional repressor. CTBP2 is a transcriptional corepressor that interacts with LSD1 and other repressive proteins, although it may act as a transcriptional activator via the association with certain factors. If ZBTB18 interacts with CTBP2, it is reasonable to think that they would cooperate for gene repression and is also worthy to compare the effect of ZBTB18 knockdown with that of CTBP2 knockdown on gene expression. However, without a good rationale, authors compared the effect of ZBTB18 overexpression with that of CTBP2 silencing on gene expression. In this regard, they should have also compared the effect of ZBTB18 knockdown with that of CTBP2 knockdown on gene expression. If ZBTB18 knockdown is not suitable because of its low expression in their cell lines, they may have to use a different cell line.

    3. LSD1's role: LSD1 can demethylate H3K4me2 and H3K4me1 but not H3K4me3. It may demethylate H3K9me2 in certain contexts (for example, upon the interaction with AR). Authors said "H3K9me2 is a well-established target of LSD1 demethylase activity" and then examined the effect of ZBTB18 overexpression on LSD1, H3K9me2, and H3K4me3 (but not H3K4me2) using quantitative ChIP. Authors should have checked H3K4me2 as well. Nevertheless, their results showed that ZBTB18 overexpression increased LSD1 and H3K9me2 but decreased H3K4me3. Authors then mentioned "a possible explanation is that the recruitment of CTBP2 complex by ZBTB18 to its target sites inhibits LSD1 demethylase activity and might be employed by ZBTB18 to counteract CTBP2-mediated activation.” However, another possibility would be that increased recruitment of ZBTB18 and LSD1, maybe along with CTBP2, would increase the repressive mark H3K9me2 but decrease the active mark H3K4me3. Perhaps, consistent with the latter possibility, authors mentioned that CTBP2 has been linked to the inhibition of cholesterol synthesis in breast cancer cells through direct repression of SREBF2 expression. To clarify this issue, authors need to show the effect of LSD1 knockdown on expression of SREBP target genes as well as on HDAC1/2, H3K4me2 and H3K9me2 levels at these genes.

    Note: authors measured the LSD1 activity in nuclear lysates using a commercial kit. This assay is based on LSD1-mediated H3K4 demethylation but not H3K9 methylation. However, the purpose of this experiment appeared to show the effect of ZBTB18 on LSD1 activity for H3K9me2 demethylation. It is not clear that this was an appropriate use of this assay.

    1. Some results are not entirely novel. For example, previous studies from authors and other groups showed that ZBTB18 negatively affected proliferation of cancer cells (Figure S2). In addition, other previous studies have reported that CTBP2 promotes tumorigenesis for hepatoma and may be a glioma prognostic marker (PMID: 27698809) (Figures 2I & 2J). LSD1-interacting proteins (Figures 4A-4C) have been known.

    2. Many labels and legends for the figures should have been better described as they are often confusing and difficult to read. Along with this, many figures should have been better presented. Some examples are as follows:

    • What is the protein number in Figure 1B?

    • For multiple figures (Figures 2H, 3H, 3G & 3H, 4D-4I, 5C, etc), there are no statistical analysis.

    • Authors should have better labelled to present their figures. For example, to present transfection and ChIP in Figure 3G, authors may want to use the labels as follows: EV + IgG; EV + α-FLAG; FLAG-ZBTB18 + IgG; FLAG-ZBTB18 + α-FLAG (instead of IgG_EV; FLAG_EV; IgG_ ZBTB18; FLAG_ ZBTB18, respectively).

    • In Figure 7E, SREBP target genes would be better than SREBP genes

  5. eLife

    ###Reviewer #1:

    This manuscript explores the mechanism by which ZBTB18 regulates the expression of SREBP genes in glioblastomas. The authors use IP and MS experiments to identify CTBP2 as a new ZBTB18 binding protein. ChIP-seq shows some overlaps of CTBP2 with ZBTB18 largely on gene promoters. CTBP2 activates, while ZBTB represses the expression of some SREBP genes. ZBTB18 disrupts the CTBP2/LSD1 complex leading to increased H3K9me2, decreased H3K4me3, and gene silencing. SREBP proteins are transcription factors that control the expression of enzymes involved in fatty acids and cholesterol biosynthesis. Consequently, ZBTB18 expression leads to reduction of several phospholipid species. Overall, although this manuscript demonstrates the role of ZBTB18 in suppressing lipid synthesis and storage and a potential oncogenic role of CTBP2 in glioblastoma cells, the mechanism underlying its regulation of gene expression is still not clear.

    1. According to the model, CTBP2 binds at SREBP gene promoters to maintain active transcription; expression of ZBTB18 enhances its binding to other LSD1 complex components and their chromatin association, however, on the contrary, ZBTB18 inhibits the enzymatic activity of LSD1 thus to repress gene expression. This model itself is seemingly paradoxical. Why does CTBTP18 recruit a corepressor (such as LSD1) and then inhibits its repressive function? Does LSD1 indeed function as a co-repressor or co-activator? Is its enzymatic function required?

    2. LSD1 is well-known for its demethylation activity against H3K4 mono- and di-methylation; its demethylase activity on H3K9 is far from clear. The data as presented does not rule out the possibility that LSD1 is a co-repressor of ZBTB18.

    3. The enzymatic assay in Figure 4J is preliminary. In vitro enzymatic assays using pure proteins with proper controls are necessary.

    4. The analysis of ChIP-seq data is preliminary. In Figure 3B, there are close to 12K peaks of CTBP2 binding sites (EV-CTBP2 only) that are lost upon co-expression of ZBTB18, and these peaks are not bound by ZBTB18. How does this happen? Also, there are close to 10K of gained CTBP2 binding sites upon coexpression of ZBTB18, half of which are bound by ZBTB18. What are these peaks? I did not find information on how many repeats are done for each ChIP. If only once, this may simply reflect huge variations between experiments. Basic analysis to access the quality of ChIP-seq is also not shown.

    5. Supplementary Figure 6A does not tell whether there is a good overlap between ZBTB18 bound peaks and the bindings of CTBP2 interactors (NCOR1, ZNF217 and LSD1). Vann diagrams need to be used to show overlaps with P-values.

    6. The entire study relies on overexpression of ZBTB18. Complementary knockouts using CRISPR in cells expressing ZBTB18 are needed.

    7. All Western blots miss protein standard markers. Percentage of input is also not labelled making it difficult to judge how strong the ZBTB18 and CTBP2 protein-protein interaction is.

  6. eLife

    ##Preprint Review

    This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript.

    ###Summary:

    This manuscript describes the mechanism by which the transcriptional repressor ZBTB18 regulates the expression of sterol regulatory-element binding proteins (SREBP), transcription factors that control the expression of enzymes involved in fatty acid and cholesterol biosynthesis,in glioblastomas. The connection between core transcriptional regulation and tumor metabolism is an area of current interest. This manuscript uses immunoprecipitation and mass spectrometry to identify the well-characterized transcriptional corepressor CTBP2 as a new ZBTB18 interacting protein, which the authors show overlap in binding at many gene promoters. They further show that CTBP2 activates, while ZBTB represses the expression of some SREBP genes. The reciprocal regulation of ZBTB18 and CTBP2 has potential value in understanding the functional regulation of lipid biology. However, reviewers raised substantial concerns with the studies, as described below.

  7. Version published to 10.1101/2020.04.17.046268v1 on bioRxiv