Oncogenic functions of the m6A demethylase FTO in breast cancer cells involving translational upregulation of C/EBPβ-LIP

  1. Hidde R. Zuidhof
  2. Christine Müller
  3. Gertrud Kortman
  4. René Wardenaar
  5. Ekaterina Stepanova
  6. Fabricio Loayza-Puch
  7. Cornelis F. Calkhoven

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Abstract

N6-methyladenosine (m6A) is a prevalent posttranscriptional mRNA modification involved in the regulation of transcript turnover, translation, and other aspects of RNA fate. The modification is mediated by multicomponent methyltransferase complexes (so-called writers) and is reversed through the action of the m6A-demethylases fat mass and obesity-associated (FTO) and alkB homolog 5 (ALKBH5) (so-called erasers). FTO promotes cell proliferation, colony formation and metastasis in models of triple-negative breast cancer (TNBC). However, little is known about genome-wide or specific downstream regulation by FTO. Here, we examined changes in the genome-wide transcriptome and translatome following FTO-knockdown in TNBC cells. Unexpectedly, FTO knockdown had a limited effect on the translatome, while transcriptome analysis revealed that genes related to extracellular matrix (ECM) and epithelial-mesenchymal transition (EMT) are being regulated through yet unidentified mechanisms. Differential translation of the CEBPB mRNA into the C/EBPβ transcription factor isoform C/EBPβ-LIP is known to act pro-oncogenic in TNBC cells through regulation of EMT genes. Here we show that FTO is required for efficient C/EBPβ-LIP expression, suggesting that FTO has oncogenic functions through regulation of C/EBPβ-LIP.

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    Reply to the reviewers

    Manuscript number: RC-2023-02199 Corresponding author(s): Cornelis, Calkhoven

    1. General Statements [optional]

    This section is optional. Insert here any general statements you wish to make about the goal of the study or about the reviews.

    We would like to thank the reviewers for their comments that will help to improve the manuscript and the editor(s) for taking care of the process.

    Text revisions and corrections in the manuscript are in red font.

    To keep oversight, see below the distribution of the comments and our replies per category:

    Reviewer 1

    Major comment 1: see 2. planned revisions

    Major comment 2: see 3. already incorporated

    Major comment 3: see 2. planned revisions

    Major comment 4: see 4. prefer not to carry out

    Minor comment 1: see 3. already incorporated

    Minor comment 2: see 4. prefer not to carry out

    Reviewer 2

    Major comment 1: see 4. prefer not to carry out

    Major comment 2: see 4. prefer not to carry out

    Major comment 3: see 3. already incorporated

    Major comment 4: see 4. prefer not to carry out

    Minor comment 1: see 3. already incorporated

    Minor comment 2: see 3. already incorporated

    Minor comment 3: see 3. already incorporated

    Minor comment 4: see 3. already incorporated

    Minor comment 5: see 3. already incorporated

    Reviewer 3

    Major comment 1: see 3. already incorporated

    Major comment 2: see 3. already incorporated

    Major comment 3: see 4. prefer not to carry out

    Minor comment 1: see 4. prefer not to carry out

    Minor comment 2: see 4. prefer not to carry out

    Significance comments by the three reviewers

    2. Description of the planned revisions

    Insert here a point-by-point reply that explains what revisions, additional experimentations and analyses are planned to address the points raised by the referees.

    Reviewer #1

    *Major comment 1: The reviewer is not convinced that FTO-CEBPB regulation is direct (data Figure 5). *

    Reply: We offer to perform immunoprecipitation of FTO from wt cells to examine interaction with CEBPB mRNA to resolve this. HPRT mRNA will be used as negative control (like in the MeRIP-RT-qPCR experiments in the manuscript).

    Major comment 3: FTO effect on proliferation and migration in less aggressive / normal breast epithelial cells (data Figure 1) (reviewer writes “cancer cells” and “MCF-10A”, but these are untransformed epithelial cells).

    Reply: We offer to perform proliferation and migration assays in MCF-10A wt and FTO-knockdown cells.

    Reviewer #2 – see below.

    Reviewer #3 – see below.

    3. Description of the revisions that have already been incorporated in the transferred manuscript

    Please insert a point-by-point reply describing the revisions that were already carried out and included in the transferred manuscript. If no revisions have been carried out yet, please leave this section empty.

    Reviewer 1

    Major comment 2: The reviewer asks to discuss the relevance of the findings for m6Am biology.

    Reply: We now discuss this in the discussion session at page 18.

    Here we mention that Sun et al (https://doi.org/10.1038/s41467-021-25105-5) performed m6Am-seq in HEK293T cells and detected no m6Am for C/EBPβ (Supplementary Data 2). These experiments were well controlled using m6A-IP experiments with PCIF1-KO HEK293T cells and compared with m6Am-Exo-seq and miCLIP published data.

    Minor comment 1: Mistake in Figure 5 legend and beyond.

    Reply: Legend of Figure 5 has been updated (text in red) and data for Supplementary Figure 5 has been added, together showing that FTO-CEBPB regulation takes place in MDA-MB-231, MCF-7 and MEFs.

    Reviewer 2

    Major comment 3: The reviewer noticed duplication of figures in Figure 6A and Supplementary Figure 6A.

    Reply: The data presented in the bar graphs of Figure 6 and Supplementary Figure 6 are from two independent experiments. Unfortunately, the pictures of the cells at the bottom for supplementary Figure 6A were the wrong ones and now are replaced by the proper pictures belonging to this experiment.

    *Minor comment 1: Statistic analysis in several panels is missing. *

    Reply: All significant events are clearly marked by *: p0.05).

    *Minor comments 2 and 3: *Issues in Figure 2E.

    Reply: The “E” is removed. We have included the cell type information in the panels D (MDA-MB-231) and E (MDA-MB-231 – shFTO1).

    *Minor comment 4: *Textual description belonging by Figure 2E.

    Reply: We have re-written the text describing the data, at page 6.

    *Minor comment 5: *Missing explanation about WTAP.

    Reply: We have extended the explanation about WTAP function at page 13.

    *Minor comment 5: *Layout of images needs improvement.

    Reply: we have to guess here what the reviewer means, but we have included the requested information in panels 2E and D (see under Minor comments 2 and 3).

    For Figure 4 we matched colours for bars representing shFTO1 and shFTO2 throughout the figure.

    In Figure 1, panel D we now label the cells as MDA-MB-231 – shTFO (instead of FTO-kd), in line with labelling throughout the paper.

    In Figures 1D and 2E we now use +FTO (instead on just FTO) for clearness.

    Concerning the reviewers remark under “significance”:

    *From a translational point of view, it is unclear if the study is significant for the future of therapeutic applications since no experiments have been performed on normal breast cancer cells. *

    Reply: We do not understand what the reviewer means with “normal breast cancer cells”, maybe the reviewer means untransformed breast epithelial cells (MCF-10A), or really breast cancer cells derived from patients?

    We now added supplementary data FTO knockdown decreases C/EBPβ-LIP levels in MCF-7 (luminal A type breast cancer) and in MEFs (Supplementary Figure 5A and B). This indicates that the FTO- C/EBPβ regulation is conserved in different cell lines of different origin. Still, FTO- C/EBPβ could play a more prominent role in breast cancer with high expression of FTO correlated with high C/EBPβ-LIP levels, and the possibility to suppress this.

    Reviewer 3

    Major comment 1: Disconnect between findings presented in Figure 1 and Figure 5.

    Reply: We have improved the reasoning at page 12.

    Major comment 2: In vivo phenotype of FTO deficiency.

    Reply: We already discussed the paper by Niu et al (https://doi.org/10.1186/s12943-019-1004-4) in the discussion, page 18.

    We have now added information about FTO knockout and overexpression mice and how the phenotypes of FTO knockout mice and CEBPB uORF deficient mice relate to each other at page 19.

    4. Description of analyses that authors prefer not to carry out

    Please include a point-by-point response explaining why some of the requested data or additional analyses might not be necessary or cannot be provided within the scope of a revision. This can be due to time or resource limitations or in case of disagreement about the necessity of such additional data given the scope of the study. Please leave empty if not applicable.

    *Reviewer #1 (Evidence, reproducibility and clarity (Required)): *

    Major comment 4: To support the data on the FTO and WTAP regulation of C/EBPβ isoform expression analysis of the correlation in expression between FTO/ C/EBPβ and WTAP/ C/EBPβ could be performed using the cancer cell encyclopedia (CCLE) dependency map portal.

    Reply: The CCLE map uses transcript expression data. Our observation is that mRNA-stability is not affected, but the protein C/EBPβ-LIP/-LAP ratio is. Therefore, CCLE analysis is not informative since it does not provide information on C/EBPβ-LIP/LAP protein ratios.

    *Minor comment 2: For the C/EBPβ-LIP overexpression-mediated rescue of the migration phenotype of figure 6 an additional replicate for shFTO1 may be helpful as only one of the two replicates presented is statistically significant. *

    Reply: Taken together the results presented in the main and supplementary figure show:

    • significant downregulation of migration for all shFTO1 and shFTO2 knockdowns (EV vs. EV-shFTO1 or EV-shFTO2).

    • Significant upregulation of migration in FTO-knockdown cells by ectopic LIP expression in three of the four FTO knockdown samples (shFTO1/2-EV vs. LIP).

    • Significant further upregulation of migration in one of the control (scr) cells by ectopic LIP expression (scr cells EV vs. LIP).

    • Throughout the experiments the observed changes in migration are consistent with shFTO knockdown and LIP expression.

    Reviewer #2 *(Evidence, reproducibility and clarity (Required)): *

    Major comment 1: 1. The authors focused on FTO and its function of RNA demethylation but the m6A-seq is not used at all. It is not likely to find the direct down stream effector of FTO without m6A-seq if you believe demethylation is the key function of FTO.

    Reply: In Figure 5A we show collective m6A sites based on experiments of others by miCLIP, DART-seq, and SRAMP prediction software. In combination with MeRIP-RT-qPCR in response to FTO KD this shows regulation of CEBPB-mRNA m6A modification by FTO, but without knowledge of the exact m6A sites affected. This study aimed to examine the way FTO affects cell proliferation and migration in different breast cancer cell lines. We revealed the FTO-C/EBPβregulation and will further examine the FTO-C/EBPβ interaction as proposed under session 2, planned revisions. Determination of specific m6Am sites in relation to the translational mechanism involved would be subject of a future study.

    *Major comment 2: Related to above concerns, the authors claimed that FTO regulated C/EBPβ-LIP. But how FTO regulated the protein expression is missing. As is known to all, FTO mainly affect RNA m6A. There is a gap between C/EBPβ-LIP protein and the RNA of this gene. *

    Reply: It is true we do not understand the mechanism of how m6A modification affects the differential translation of the CEBPB mRNA in the different protein isoforms. Examining this is a major effort and would take considerable time, which is beyond this study, but would be subject of further study.

    *Major comment 4: The authors claimed that EMT related genes are affected upon FTO knock down, but Figure 2C do not seem to align with the EMT process. Also, if the authors believe EMT process is regulated, another GSEA enrichment panel like Figure 2B should be included. *

    Reply: we have difficulty in understanding this comment. Relevance of the EMT-process is clearly shown by GSEA in panel 2B. GSEA uses a ranked list of gene expression (including all genes in the RNA-seq data set, also the ones not significantly changed in expression) and is thereby a powerful and comprehensive technique to identify relevant pathways since all data in the dataset is used. The GO-term analysis in Figure 2C looks at significantly regulated genes and maps these to GO-terms for biological process (BP), molecular function (MF) or cellular component (CC). Here we used BP, and find many GO-terms involved in ECM/cell membrane/cell adhesion/cell migration (linked to cell migration and thereby EMT since the cells are of epithelial origin). GO-term analysis only requires a list of genes that were found to be significantly regulated (It doesn’t know where your list of genes has been derived from and does not take into account the p-value of a specific gene or the fold change in expression observed between SCR and shFTO cells, in contrast to GSEA). GSEA and GO-term analysis are therefore complementary analyses based on different principles to help indicate what biological processes are changed upon FTO knock-down; their results cannot be directly compared.

    Reviewer #3 (Evidence, reproducibility and clarity (Required)):

    Major comment 3: The lack of clear impact of FTO KD on the translatome is surprising. Maybe the reverse experiment (over expression) would lead to more relevant findings. Likewise, use of the mutant FTO could differentiate between the demethylase function of FTO vs other non-enzymatic functions.

    Reply: Although we agree the proposed experiments are interesting, we believe them to be beyond the scope and time of this manuscript – Major point 3.

    Minor comment 1: baseline FTO level in MDA MB231 seems inconsistent (1A vs 1D--controls). It would be useful to see FTO expression across breast cancer cell lines and normal mammary cells to justify choice of cell lines in which experiments were carried out.

    Reply: It is due to unfortunate choice of blots with different exposures: The immunoblot in Figure 1A shows wild-type MDA-MB-231 cells with scr-shRNA with expected expression of FTO, compared to shFTO knockdowns with low expression of FTO. Figure 1D are MDA-MB-231 cells with knockdown of FTO and therefore very low FTO expression, compared to FTO knockdown cells with re-expression of FTO. The exposure was a bit unnecessary low, and we changed the blot with a higher exposure blot.

    Niu et all (https://doi.org/10.1186/s12943-019-1004-4) have analysed breast cancer samples form patients and breast cancer cell lines and showing a general upregulation of FTO in breast cancer compared to healthy tissue or other cancer cell lines, respectively.

    Minor comment 2: lack of data from human specimens.

    Reply: Not available. Very difficult to realize since immunostaining techniques cannot discriminate between C/EBPβ-LAP and -LIP isoforms because LIP shares all possible antibody epitopes with LAP. This means that one needs enough material for western blotting which is difficult to arrange.

    Significance

    Reviewer #1:* Despite the interest in the finding that FTO knockdown influences cellular proliferation and migration, the authors do not have enough mechanistic insights on how FTO regulates this process, leaving uncertainty on the study's relevance.*

    Reply: We agree, but we feel that performing detailed mechanistic study would take considerable time and therefore should be part of future work.

    *From a translational point of view, it is unclear if the study is significant for the future of therapeutic applications since no experiments have been performed on normal breast cancer cells. *

    Reply same as under section 3: We do not understand what the reviewer means with “normal breast cancer cells”, maybe the reviewer means untransformed breast epithelial cells (MCF-10A), or really breast cancer cells derived from patients?

    We now added supplementary data FTO knockdown decreases C/EBPβ-LIP levels in MCF-7 (luminal A type breast cancer) and in MEFs (Supplementary Figure 5A and B). This indicates that the FTO- C/EBPβ regulation is conserved in different cell lines of different origin. Still, FTO- C/EBPβ could play a more prominent role in breast cancer with high expression of FTO correlated with high C/EBPβ-LIP levels, and the possibility to suppress this.

    *From a molecular point of view, FTO can influence m6A and m6Am. The authors do not mention the relevance of their findings in terms of m6Am biology. *

    Reply: See above under 3. Description revisions already incorporated - Major comment 2.

    *Reviewer #2: The study's significance is somewhat unclear. The initial sections in the main body present primarily negative or statistically insignificant results. While the explanations in later sections are insightful, they may give the impression of an attempt to justify these negative findings, leaving the study's significance somewhat ambiguous. *

    Reply: This raises the issue whether we as a scientific community should present “negative” results. In our opinion this is important since it informs about what is probably not involved (regulation of translation efficiency by FTO) and it leads the way to other hypothesis and explanations of a biological phenomenon. How can – in the reviewers’ words - insightful explanations by labelled as just justifying? They are what they are; insightful and contributing to the topic. Currently the role of FTO in cancer and/or translation regulation is not clear and different views exist (see for example our review https://doi.org/10.1158/0008-5472.CAN-21-3710). In this manuscript we provide evidence that FTO might not be significantly involved in global regulation of mRNA translation efficiency in MDA-MB-231 cells, these results seem quite relevant to share. Furthermore, we provide a possible mechanism that could explain part of the effects observed, and already indicate ourselves future work is needed to further strengthen these findings.

    *Reviewer #3: * Well written report on the functions of FTO in breast cancer pointing to an oncogenic role and regulation of transcripts related to EMT. Data are well done and well presented, 2 shRNA transfected cell lines are included in the experiments; methodology is clear.

    Reply: we are grateful for this assessment and hope the reviewer will find the manuscript further improved after revision.

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    Referee #3

    Evidence, reproducibility and clarity

    The strengths are the well written manuscript, robust and well presented data, study of the effects of FTO on both transcriptome and translatome.

    Major weaknesses:

    1. There is a big disconnect between the findings presented in Figures 1-3 and the focus on CEBP in Figure 5. This needs to be reconciled either by demonstrating a link between CEBP and the transcripts found to be dysregulated in Fig 2 or some other way.
    2. There are no in vivo data. Does the phenotype caused by FTO KD lead to an in vivo phenotype?
    3. The lack of clear impact of FTO KD on the translatome is surprising. Maybe the reverse experiment (over expression) would lead to more relevant findings.Likewise, use of the mutant FTO could differentiate between the demethylase function of FTO vs other non-enzymatic functions.

    Minor weaknesses:

    1. baseline FTO level in MDA MB231 seems inconsistent (1A vs 1D--controls). It would be useful to see FTO expression across breast cancer cell lines and normal mammary cells to justify choice of cell lines in which experiments were carried out.
    2. lack of data from human specimens.

    Significance

    Well written report on the functions of FTO in breast cancer pointing to an oncogenic role and regulation of transcripts related to EMT. Data are well done and well presented, 2 shRNA transfected cell lines are included in the experiments; methodology is clear.

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    Referee #2

    Evidence, reproducibility and clarity

    The authors are trying to demonstrate the oncogenic role of FTO in breast cancer. RNA-seq was used to explore the effect of FTO on gene transcription and translation. And it turned out that the protein expression ratio of LIP/LAP is the most prominent effect of FTO on breast cancer development. While the oncogenic roles of FTO in breast cancer cell lines look well supported, several key questions remain to be answered before it can be considered to publish.

    major comment:

    1. The authors focused on FTO and its function of RNA demethylation but the m6A-seq is not used at all. It is not likely to find the direct down stream effector of FTO without m6A-seq if you believe demethylation is the key function of FTO.
    2. Related to above concerns, the authors claimed that FTO regulated C/EBPβ-LIP. But how FTO regulated the protein expression is missing. As is known to all, FTO mainly affect RNA m6A. There is a gap between C/EBPβ-LIP protein and the RNA of this gene.
    3. Data quality is not convincing, the transwell migration assay image in figure 6 and Supplementary Figure 6 is identical, which is unacceptable.
    4. The authors claimed that EMT related genes are affected upon FTO knock down, but Figure 2C do not seem to align with the EMT process. Also, if the authors believe EMT process is regulated, another GSEA enrichment panel like Figure 2B should be included.

    Minor concerns

    1. Statistic analysis in several panels is missing. Normally, every date should include statistic analysis, even its not significant.
    2. Issues in Figure 2E, there is a "E" above the first column, which is not supposed to be there.
    3. Also in Figure 2E, these results conducted in FTO-knocked down cells, but the panel did not show clearly.
    4. In Figure 2E, the textual description does not entirely correspond with the image results, and there is a lack of information about the expression levels of COL12A1, FN1, MMP1, and TNC.
    5. Furthermore, while the article conveys the meaning of WTAP, it lacks a thorough textual explanation. Additionally, the layout of the article's images needs improvement.

    Significance

    The study's significance is somewhat unclear. The initial sections in the main body present primarily negative or statistically insignificant results. While the explanations in later sections are insightful, they may give the impression of an attempt to justify these negative findings, leaving the study's significance somewhat ambiguous.

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    Referee #1

    Evidence, reproducibility and clarity

    In this study, the authors investigated the effect of FTO knockdown in triple-negative breast cancer (TNBC) cells. They show that FTO knockdown reduces the proliferation and migration of two breast cancer cell lines using clonogenic and transwell migration assays. Through transcriptome analysis of FTO-depleted cells, they showed a negative enrichment of the epithelial-to-mesenchymal transition (EMT) signature and showed that this decrease is not mediated at a transcript stability level. Through ribosome profiling experiments they demonstrated that the translatome is only mildly affected by FTO depletion and using differential ribosomal codon reading (diricore) analysis showed that FTO knockdown does not cause specific tRNA or amino acid shortages. They show that FTO and WTAP knockdown reciprocally regulate the expression of C/EBPβ isoform. Finally, through rescue experiments, the authors showed that C/EBPβ-LIP overexpression rescues the decreased migration phenotype observed upon FTO knockdown.

    Major comments:

    The authors showed that upon FTO knockdown, the C/EBPβ-LIP protein levels decrease, and conversely, upon WTAP knockdown, the levels of C/EBPβ-LIP protein increase (figure 5b and 5d) and suggest that a reversible WTAP- and FTO-controlled m6A modification of CEBP mRNA alters its translation. Upon FTO knockdown, however, the stability of CEBP mRNA is not affected, and despite a clear trend in the increase of the CEBP transcript m6A levels from MeRIP-RT-qPCR (figure 5E), this is not statistically significant. It remains unclear whether the regulatory effect of FTO on the CEBP mRNA transcript is a direct, m6A-mediated effect or an indirect effect. For this reason, further experiments may help to strengthen this result. It would be helpful to clarify if m6A levels increase in the CEPB mRNA level with more replicates of the MeRIP-RT-qPCR or using other quantitative techniques. Moreover, to clarify whether this is directly mediated by FTO a RIP-qPCR for the FTO protein and CEPB mRNA in wild-type cells could be performed.

    Additionally, the authors never consider the possibility that FTO could regulate CEBP because of a possible m6Am site. Does CEBP have an m6Am site? Would PCIF1 knockdown cause the same effect that FTO knockdown? Also, the authors should consider classifying mRNAs based on the number of m6A sites or m6Am sites and determine if mRNAs with an m6Am site or an m6A site show a difference in their translation or expression levels compared to non-methylated sites.

    It would be interesting to see if the effect of FTO on proliferation and migration impact less aggressive form of cancer or normal breast cancer cells, such as MCF-10A.

    To support the data on the FTO and WTAP regulation of C/EBPβ isoform expression analysis of the correlation in expression between FTO/ C/EBPβ and WTAP/ C/EBPβ could be performed using the cancer cell encyclopedia (CCLE) dependency map portal.

    Minor comments:

    The figure 5 legend is missing the description of the MeRIP-RT-qPCR experiment.

    For the C/EBPβ-LIP overexpression-mediated rescue of the migration phenotype of figure 6 an additional replicate for shFTO1 may be helpful as only one of the two replicates presented is statistically significant.

    Significance

    Despite the interest in the finding that FTO knockdown influences cellular proliferation and migration, the authors do not have enough mechanistic insights on how FTO regulates this process, leaving uncertainty on the study's relevance.

    From a translational point of view, it is unclear if the study is significant for the future of therapeutic applications since no experiments have been performed on normal breast cancer cells.

    From a molecular point of view, FTO can influence m6A and m6Am. The authors do not mention the relevance of their findings in terms of m6Am biology.

  5. Version published to 10.1101/2023.09.21.558784v1 on bioRxiv