Concerted functions of HDAC1 and microRNA-574-5p repress alternatively spliced ceramide synthase 1 expression in human cancer cells

Histone deacetylases (HDACs) and microRNAs (miRs) have pro-survival roles, but the mechanism behind this is unclear. Repression of ceramide synthase 1 (CerS1), altering C18-ceramide generation, was linked to drug resistance and metastasis. Here we report that the CerS1 promoter was repressed by HDAC1-dependent inhibition of Sp1 recruitment to two specific GC-boxes spanning the −177 and −139 region. Moreover, an alternatively spliced variant CerS1 mRNA (CerS1-2) was detected mainly in cancer cells or primary tumour tissues compared to controls, which was targeted by miR-574-5p for degradation. A specific 3′UTR-targeting site, localized within the retained intron between exons 6 and 7, was identified, and its mutation, or miR-574-5p knockdown prevented the degradation of CerS1-2 mRNA. Interference with HDAC1 and miR-574-5p reconstituted CerS1-2 expression and C18-ceramide generation in multiple human cancer cell lines, which subsequently inhibited proliferation and anchorage-independent growth. Accordingly, knockdown of CerS1 partially protected cancer cells from MS-275/miR-574-5p siRNA-mediated growth inhibition. Thus, these data suggest that the HDAC1/miR-574-5p axis might provide a novel therapeutic target to reconstitute tumour suppressor CerS1/ceramide signalling.

Thank you for the submission of your manuscript to EMBO Molecular Medicine. We have now heard back from the three referees whom we asked to evaluate your manuscript. Although the referees find the study of potential interest, they also raise a number of concerns that should be addressed in a major revision.
As you will see below, all three referees are rather positive about your work. Nevertheless, particularly referees #2 and 3 have raised some substantial issues mainly about the significance of some experiments presented, which will need to be further clarified and completed as suggested.
Given the balance of these evaluations, we feel that we can consider a revision of your manuscript if you can address the issues that have been raised within the time constraints outlined below. Please note that it is EMBO Molecular Medicine policy to allow only a single round of revision and that, as acceptance or rejection of the manuscript will depend on another round of review, your responses should be as complete as possible.
Revised manuscripts should be submitted within three months of a request for revision; they will otherwise be treated as new submissions, except under exceptional circumstances in which a short extension is obtained from the editor.
I look forward to seeing a revised form of your manuscript as soon as possible. This is a well designed and presented work.
There are several minor errors the authors should address in the revised version. 1. It is not clear what the effect of microRNA-574-5p has on activities of CerS1-1 and CerS1-2 isoforms. Clarifying this will help readers to understand the role of miRNA-574-5p in alternative splicing and C18-ceramide generation. Does CerS1-2 isoform generate different species of ceramide or does it not have any activity in ceramide synthase? 2. Meyers-Needham et al. have performed an elegant series of experiments to demonstrate that CerS1 transcription is enhanced by Sp1, repressed by HDAC1 and post-transcriptionally degraded in a miR-574-5p-dependent manner in HNSCC cells. Their extensive analysis includes the cloning of the CerS1 promoter, identification and analysis of the core region, determination of its transcription factor regulation/association (principally Sp-1) and inhibition by HDACs (HDAC1 implicated). CerS1 mRNA was suggested to be reduced in HNSCC compared to non-cancerous immortalized keratinocytes and primary keratinocytes (which contrasts with CerS6 mRNA that appears not to differ) and coincides with reduced C18Cer levels (whereas C16Cer levels, derived from CerS6, are unaffected). Subsequently, the study addresses the expression of variant form of CerS1, CerS1-2, which is a minor form in keratinocytes but of significance in HNSCC cells. A binding site for miR-574-5p, located between exons 6 and 7, is identified and shown to be important in the degradation of mRNA transcribed from a luciferase construct containing the 3'UTR of CerS1-2. A combination of miR-574-5p knockdown and HDAC1 inhibition enhances CerS1-2 mRNA expression, CerS1 expression and C18Cer levels whilst reducing cell growth. This is a comprehensive, novel set of data albeit the significance of some of the observations requires clarification/consolidation. Specific comments: 1. Whilst mRNA levels for CerS1 are reduced in HNSCC vs. non-cancerous keratinocytes and primary keratinocytes (Fig 1A and 1C), how does this relate to protein expression? Are CerS1 mRNA/protein levels also reduced in UM-SCC-1 and UM-SCC-22A relative to primary keratinocytes ( Fig 1C) - Fig 1A suggests this might not be the case. How significant is the reduction in CerS1 mRNA/protein expression in HNSCC vs. primary keratinocytes given that the major change appears to be an elevation of mRNA in immortalized non-cancerous keratinocytes compared to primary keratinocytes ( Fig 1A vs Fig 1C)? Similarly, is the reduced t1/2 for CerS1 mRNA (UM-SCC-1 and UM-SCC-22A) relative to immortalized non-cancerous keratinocytes or primary keratinocytes?
2. The predicted sites of the transcription factor recognition sequences are not clear ( Fig 1D) and therefore why regulation by other transcription factors (E2A, CREB, HIF-1, STAT) was not addressed. Have these been investigated and what are the possible relative contributions of each to CerS1 expression? Whilst modulation of Sp1 by siRNA or over-expression affects the CerS1luciferase reporter assay (Fig 2), what effect does this have on CerS1 protein and C18Cer levels? Similarly, for HDAC1 RNAi knockdown or inhibition by MS-275? Can the authors comment of why HDAC1 RNAi has a minimal, yet significant, effect (~0.3 fold) in the CerS1-luciferase reporter assay ( Fig 5A) whereas MS-275 enhances activity by (~7 fold, Fig 5B)? How do these treatments affect Sp1 association with the CerS1 promoter ( Fig 4A/B), where HDAC1 is suggested to be inhibitory? 3. Data (described by the authors as 'important') relating to the relative expression of mRNA for CerS1-1 and Cers1-2 (3' UTR #1 and 3'UTR #5) should be presented (e.g. as supplementary data) to consolidate the claim that the latter is expressed in all primary tumor tissue whereas the former two are mainly found in normal tissue.
4. The significance of the miR-574-5p levels of patients ( Fig 7F) is not clear given that this is higher in only 5 of the 10 samples. Presumably, other factors contribute -please clarify.
5. Trypan blue exclusion is a poor assay to measure proliferation ( Fig 9C). Could this account for the poor reversal of the effects of miR-574-5p knockdown/HDAC1 inhibition by CerS1 RNAi. An alternative technique should be used to address this important validation.
6. The study would be significantly enhanced if it could be shown that targeting CerS1 using the approaches outlined here was efficacious in vivo.
Minor comments: 1. Data in supplementary Fig 3A relating to HDAC1 RNAi appears incorrect, with the expected reduction in mRNA levels indicated for HDAC2.
2. Should data for Box1 mutant be included in Fig 3B? The text suggests that this should be the case, albeit it has no significant effect on the CerS1 promoter activity. This manuscript makes a major contribution towards the understanding of how CerS1 is regulated, and relates the findings to cancer where CerS1 expression is abnormal.
The majority of the authors' conclusions are substantiated by the data, which are extensive. In the view of this reader, the manuscript could be accepted if the problematic conclusions are removed (or adequately tempered) because the paper would remain worth publishing without them.
The conclusions that are of concern deal with the combined effects of HDAC inhibition and miR-574-5p suppression. The data in Fig. 9A show that MS-275 elevates CerS1 but there does not seem to be an effect of miR-574-5p RNAi in the UM-SCC-22A cells (there does seem to be a combined effect in the UM-SCC-1 cells, but these are not used for the studies in the other panels of Fig. 9). In Fig. 9B, there does not appear to be an effect of MS-275 on C18-Cer levels, but there is an effect of miR-574-5p RNAi. Therefore, these results are opposite from each other. Likewise, the effects of the combined agents on cell number looks very small (Fig. 9C) and only does a bigger effect seem to appear when the number of cells in soft agar is shown (Fig. 9D), however, the magnitude of the difference in Fig. 9D is far out of proportion to any of the changes seen in the other panels for this cell and these reagents, which seems strange. Therefore, the authors have made a good case that each of these are relevant to CerS1 expression and C18-Cer biosynthesis, but the evidence for a greater effect from the combination is weak. The combined effect appears to be greater for the UM-SCC-1 cells (Fig. 9A), however, these were not used for the other studies, or at least the results with them have not been shown. Without more conclusive results, the authors should relegate their comments about the HDAC1/miR-574-5p axis to a speculation rather than a conclusion. Some more minor concerns are that the authors state on p. 5 that the promoter region spans nucleotides +1 to -1556, but this is more accurately described as a region that contains promoters since there might be important promoters for this gene elsewhere (this is a minor semantic point).
On p. 9 the authors say that GC boxes 1 and 4 do not play roles in activation of the CerS1 promoter, which should be qualified by saying "under basal expression conditions." On p. 14 the authors say "Importantly, while CerS1-1 and CerS1-2 with the A1 poly(A)...etc." followed by (data not shown). If the point is important, the authors should include these data in the Supplemental figures.
Overall, however, this is a very impressive study and the findings will be of considerable interest to many readers of the paper. Please find below our point-by-point response to the comments raised by the Reviewers: Reviewer 1: We first thank the Reviewer 1 for stating that "This is a very important work for CerS1 gene regulation, and has significant impact on cancer cell biology. This is a well designed and presented work".
We have addressed the points raised by the Reviewer as follows: 1. The Reviewer has raised an important point regarding the possible effects of miR-574-5p on the functions of CerS1-1 and CerS1-2 for C18-ceramide generation. This important point was addressed by data presented in Fig. 9B. The data showed that siRNA-mediated knockdown of miRNA-574-5p alone had no statistically significant effect on C18-ceramide generation. It is also not very clear whether miRNA-574-5p plays any role in the alternative splicing of CerS1 to generate CerS1-1 or CerS1-2. Our data presented in Figs. 6B-D and 7A-C suggest that miRNA-574-5p is involved in reduced stability of CerS1-2 mRNA in multiple human cancer cells and tumor tissues.
The Reviewer also raised an important point to know whether CerS1-2 generates different species of ceramide or it does not have any activity in ceramide synthase. This important question was answered directly by cloning and expression of CerS1-1 and CerS1-2 containing N-terminal FLAG tags in UM-SCC-22A cells followed by LC/MS/MS for endogenous ceramide measurements. The data showed that expression of CerS1-2 as well as CerS1-1, both were confirmed by Western blotting, selectively and almost equally induced C18-and C18:1-ceramide generation, but had no effect on the generation of other ceramide species, compared to vector-transfected controls. These new data are now presented in Supporting Information Fig. S6A-C.
3. HNSCC was spelled out when it first appeared in the text (p. 3, lines 19-20).
Reviewer 2: We thank the Reviewer 2 for stating that "Meyers-Needham et al. have performed an elegant series of experiments", and that "this is a comprehensive, and novel set of data".
We have addressed the points raised by the Reviewer as follows: 1. As suggested, CerS1 mRNA was measured using RT-qPCR in UM-SCC-1 and UMSCC-22A cells compared to primary keratinocytes (NHEK) and immortalized skin keratinocytes. These data are now presented in Figs. 1A and 1B showed that CerS1 mRNA is down-regulated in HNSCC cells when compared to non-cancerous keratinocytes (both primary and immortalized). This was also confirmed by lower levels of C18-ceramide measured in HNSCC cells compared to immortalized keratinocytes (as shown in Fig. 1C), suggesting that lower CerS1 mRNA is associated with decreased C18-ceramide generation in these cancer cells compared to non-cancerous keratinocytes.
Therefore, we focused on defining the mechanisms that regulate repression and stability of CerS1 mRNA, which seems to play key roles for its suppression and decreased C18-ceramide generation in multiple human cancer cells compared to non-cancerous keratinocytes. Then, effects of reconstitution of CerS1-2 mRNA in response to MS-275 plus miR-574-5p knockdown on CerS1 protein expression and ceramide generation in cancer cells have been examined, and the data are presented in Figs. 9A and 9B, respectively.
The Reviewer also wanted to know if t1/2 for CerS1 mRNA in HNSCC cells was measured relative to immortalized or primary keratinocytes. Our previous data were obtained from HNSCC and immortalized keratinocytes (Fig. 5D). To address this important point, we have measured t1/2 of CerS1 mRNA in HNSCC versus primary keratinocytes (NHEK), and similar data were obtained as they are now presented in Fig. 5E.
2. We agree with the Reviewer that other transcription factors (E2A, CREB, HIF-1, STAT) that have recognition sites on CerS1 promoter DNA might affect CerS1 expression. However, our deletion mutations, site-directed mutagenesis and functional analyses showed that Sp1 plays an important role for the regulation of the minimal functional CerS1 promoter. However, other transcription factors at the minimal promoter site, or at distant sites might have additional effects on the CerS1 promoter/expression in other cell models. This point was also raised by the Reviewer 3, and now mentioned in the text (p. 9, lines 8-12).
In addition, Sp1 or HDAC1 knockdown or inhibition induced CerS1 promoter activity, but not CerS1 mRNA levels in our experiments (data not shown, and Supporting Information Fig. S3B respectively). These data suggested that there was another layer of regulation for CerS1 transcription controlling CerS1 mRNA stability by miR-574-5p, in addition to repression of the CerS1 promoter by HDAC1 via inhibition of Sp1 recruitment to the promoter.
Moreover, we agree with the Reviewer that HDAC1 siRNA has a minimal, yet significant effect on the CerS1 promoter (Fig 5C), whereas MS-275 enhances the promoter activity by (~7 fold, Fig 5A). We believe that this might be due to efficacy of siRNA versus small molecule inhibitor for the regulation of HDAC1 activity in UMSCC-22A cells. It is also possible that HDAC1 knockdown by itself might not be sufficient to induce CerS1 promoter activity, and it might require the inhibition of HDAC1 and/or HDAC1-class 1 HDAC complexes by MS-275. These points are now discussed in the text (p. 20, lines 22-23, and p. 21, lines 1-3).
Also, as suggested by the Reviewer, we performed new Q-ChIP assays to measure the effects of MS-275 on Sp1 recruitment to the CerS1 promoter in UM-SCC-22A cells (Fig. 5B).
3. Data relating to the relative expression of mRNA for CerS1-2 in human tumor versus normal head and neck tissues are now presented (Supporting Information Fig. S4B), as suggested by the Reviewer.
4. The Reviewer raised an important point regarding the data shown in Fig. 6F, which show that miR-574-5p levels of patients are higher in only 5 of the 10 tumour samples compared to normal adjacent tissues. The significance of these data is unknown, but these data suggest that presumably, other factors might also contribute to the repression of CerS1 mRNA expression in tumor tissues.
Interestingly, these data also show that 10/10 of the tumors express miR-574-5p, and since tumor tissues seem to have preference for the expression of CerS1-2, which is a target for miR-574-5p, the overexpression of miR might not be needed, and its steady state expression should be sufficient for targeting CerS1-2 in tumor tissues. However, in normal tissues, CerS1-1 is preferentially expressed, which is not targeted by miR-574-5p. These points are now discussed in the text (p. 21, lines 14-22).

5.
As suggested by the Reviewer, we have used an alternative method to measure cell growth/survival using CyQUANT Cell proliferation Assay kit (Invitrogen) in another cancer cell line , UM-SCC-1 (Fig. 9D), in addition to trypan blue exclusion assay in UMSCC-22A cells (Fig.  9C).
6. We agree with the Reviewer that the study would be significantly enhanced if it could be shown that targeting CerS1 using the approaches outlined here was efficacious in vivo.
However, these in vivo studies require specialized tools, such as inducible shRNAs against miR-574-5p for stable/inducible knockdown in cells to conduct in vivo studies. Moreover, delivery of siRNAs against miR-574-5p in vivo might be difficult, and siRNA-mediated knockdown of miR-574-5p for long-term in vivo studies might not be optimal. Therefore, in vivo studies will be the focus of our future studies after development of necessary molecular and/or pharmacologic tools to inhibit miR-574-5p effectively for long-term xenograft studies in SCID mice.
Nevertheless, to address this important point, in addition to trypan blue exclusion and CyQUANT cell proliferation assays, we have measured growth on soft agar to detect the effects of MS-275 and miR-574-5p siRNA treatment on anchorage-independent growth of UM-SCC-22A cells (Fig. 9E).
Minor points mentioned by the Reviewer were also addressed in the revised manuscript as suggested: 1. Fig. 3A  Reviewer 3: We thank the Reviewer for stating that "this manuscript makes a major contribution towards the understanding of how CerS1 is regulated, and relates the findings to cancer where CerS1 expression is abnormal". We also appreciate that the Reviewer mentioned that "this is a very impressive study and the findings will be of considerable interest to many readers of the paper".
We have addressed the points raised by the Reviewer as follows: 1. We agree with the Reviewer that the combined effects of MS-275 and knockdown of miR574-5p appears to be greater for the UM-SCC-1 cells (Fig. 9A), however, these were not used for the growth studies. This was addressed with new set of data obtained using UM-SCC-1 cells, which are presented now in Fig. 9D (as suggested by the Reviewer 3, see below), in addition to UM-SCC-22A cells ( Fig. 9C and 9E). Nevertheless, we agree that our discussion about these data appeared to be overstated as concrete conclusions in the previous version of the text, which are now corrected throughout the revised manuscript, as suggested.
2. The Reviewer raised an important point regarding how the promoter region of CerS1 was mentioned in page 5 of the previous version of the text. This is now corrected as suggested (p. 5, lines 11-15).
3. The effects of GC boxes 1 and 4, which had no significance, on activation of the CerS1 promoter were mentioned as "under basal expression conditions", as suggested by the Reviewer in the revised text (p. 9, line 8).
In summary, we thank all three Reviewers for their positive review and constructive comments. We are very excited about our data presented in this manuscript regarding the regulation of CerS1 transcription and mRNA stability of its alternatively spliced variant CerS1-2 by the coordinated functions of HDAC1 and microRNA-574-5p in multiple cancer cells and primary tissues. We believe that both clinicians and scientists will be interested in this manuscript. We hope that the revised manuscript will meet the criteria for publication in EMBO Molecular Medicine.
2nd Editorial Decision 14 October 2011 Thank you for the submission of your revised manuscript to EMBO Molecular Medicine. We have now received the enclosed reports from the referees that were asked to re-assess it. As you will see the reviewers are now supportive and I am pleased to inform you that we will be able to accept your manuscript pending the following final amendments: 1/ We have small concerns with some of your figures: - Figure 2E, could you please provide a less dark image as after printing, the whole panel is black; - Figure 4A and B, could you please reduce the contrast settings as the blots are too white and we do not see the limit of the gel. You could also add a black delineating line around the blots; -all blots cut and assembled should provide at least 5 band-widths up and down of the band of interest; move the "Figure x" label outside of the figure pannels for Production purposes.
Please see below for more information regarding image processing and correct handling of electrophoretic gels and blots: "Processing (such as changing brightness and contrast) is appropriate only when it is applied equally across the entire image and is applied equally to controls. Contrast should not be adjusted so that data disappear. Excessive manipulations, such as processing to emphasize one region in the image at the expense of others (for example, through the use of a biased choice of threshold settings), is inappropriate, as is emphasizing experimental data relative to the control. When submitting revised final figures upon conditional acceptance, authors may be asked to submit original, unprocessed images." "The display of cropped gels and blots in the main paper is permitted if it improves the clarity and conciseness of the presentation. Cropped gels in the paper must retain all important bands, and space (at least 5 band-widths) should be retained above and below the relevant band(s). Vertically sliced images that juxtapose lanes that were non-adjacent in the gel must have a clear separation or a black line delineating the boundary between the gels." 2/ I have noticed that within the Materials and Methods section, an ethical statement regarding the obtention of primary tissues and appropriate consent from patients is missing.
3/ The Paper Explained: EMBO Molecular Medicine articles are accompanied by a summary of the articles to emphasize the major findings in the paper and their medical implications for the nonspecialist reader. Thank you for your recent review of our manuscript MS#: EMM-2011-00713, and invitation for submission of a revised manuscript. We are very happy that the Reviewers were satisfied with our revisions, and our manuscript will be acceptable upon required editorial amendments.
We have revised the manuscript to address these editorial points, and are now submitting the amended manuscript entitled "Concerted functions of HDAC1 and microRNA-574-5p repress alternatively spliced ceramide synthase 1 expression in human cancer cells" to be considered for publication in EMBO Molecular Medicine.
We have amended the manuscript as follows: 1. Fig. 2E is now provided with brighter image, as requested.
Figs. 4A and 4B are now provided with black lines around the blots, as suggested. Some of the blots in some figures were cut in smaller sizes due to space limitations in our figures, which contain multiple panels and maximum number of total (9) figures. If required or needed, we will be pleased to provide the actual blots for these figures.
2. We now include the consent and institutional review board-approved protocol statement for our studies involving patients specimens in Supplementary materials and methods. This was also added to the main text under Materials and Methods.
3. We are now providing The Paper Explained section, as required.
4. Since appropriate web links were mentioned in the text where necessary, we do not have any additional links For More Information.We hope that the amended manuscript will meet the criteria for publication in EMBO Molecular Medicine.
Thank you for your consideration.

Additional Editorial Correspondence 19 October 2011
Thank you very much for resubmitting your manuscript and amending it as we suggested.
I very much appreciate that you followed our recommendations for the Figures and I would like to accept your kind offer to provide the actual raw blots.
EMBO encourage submission of source data and we highly appreciate your willingness at participating in this process.
In addition, I attach here the Paper Explained where I have edited only few words, for your approval.
You can return both blots and TPE by return email.
I am looking forward to hearing from you soon.

Editor EMBO Molecular Medicine
Additional Author Correspondence 25 October 2011 Please find attached PDF that contains larger Western blot scans of original figures to be consistent with the journal's format, as an additional information.
The corrected TPE file is also attached, as your edits were accepted, and some minor additional corrections are also made in this final form.
I hope that these will be sufficient for the publication of our MS in EMM. We are extremely excited about this work, and we think that this MS will attract a lot of attention from investigators with broad interests.