Short-Chain Fatty Acids Suppress mTOR Signaling in Colon Cancer Cells via Long Non-Coding RNA RMST

Round 1

Reviewer 1 Report

Short chain fatty acids (SCFAs) including acetate, propionate and butyrate are produced by fiber fermentation in the colon.  They have been implicated in modulating a variety of colonic processes such as cell differentiation, proliferation, apoptosis, that eventually may lead to various effects on colonic functions including oncogenesis. The molecular mechanisms that are involved in the induction a regulation of these processes is not clear yet. The group has shown in the past SCFAs induce autophagy in colon cancer cells via downregulating mTOR signaling, and in the current study they extent the research to identify the mechanisms that lead to mTOR suppression. They identified the lncRNA rhabdomyosarcoma 2 associated transcript (RMST), as an important regulator of the SCFAs’ effects on mTOR phosphorylation. Its expression was significantly induced by SCFAs and its depletion reduced the abilities of SCFAs to induce autophagic responses, which is likelyTSC2- mTOR dependent. 

This is an interesting study that demonstrates a novel SCFAs-induced RMST/TSC2/mTOR cellular pathway that may lead to autophagy and eventually also colon cancer. However, the duration and timing of the effects are somewhat problematic, as mTOR usually responses to extracellular signals within minutes and not two days. This point as well as a few other comments should be addressed prior to publications. The points are as follows:

1.   The response of cells to extracellular stimuli is mediated by a variety of intracellular signaling pathway such as AKT/mTOR or Ras/ERK that respond to the stimuli within several minutes. initiated within a few minutes. After their stimulation, the signaling pathways orchestrate a large number of cellular processes that are induced within hours or even days. During these times, the original cascades are desensitized, and later may response to other signaling components that are initiated at later stages. In this study, the authors studied the effect of SCFAs on mTOR after days, and this is clearly not a direct effect. In fact, it is likely due to changes in basal signaling activities that may participate in the SCFAs-induced cell fate. In their 2011 study the authors have shown that mTOR phosphorylation is reduced much earlier (even in 1 hour). Therefore, it is strongly suggested to follow the role of RMST in mTOR dephosphorylation at earlier stages (hours and not days). 

2.   The results in figure 2 are very minimal. Usually, such small changes in phosphorylation of mTOR do not have physiological effects. The only significant change that I see is siRNA7 effect on pS6K. This point should be explained. 

3.   In continuation of the comments to figure 2, there are significant differences in effects between the three SiRNAs. In some cases, The SiRNA7 gives the highest effect, although its effect on RMST expression is the lowest. Is it possible that there is an off-target effect on some of them? It is not clear why the results with and without propionate are not presented in 2C. Also, it would be important to show the amount of general S6K. Finally, the blot results of 2A should be shown.

4.   In figure 4A, it is suggested to show the effects with and without propionate. How do the author know how specific are the SiRNAs? 

5.   The effect of propionate on mTOR phosphorylation varies between the experiments. In Fig. 5D the propionate reduces pmTOR Ser 2481), while in figure 2 the effect is minimal. Please explain the diversity. 

6.   In all figures the legends of the y-axis should be more informative and include the term changed (not just the units).

The English is fine

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Reviewer 2 Report

I've been invited to review the research paper from Wang et al, dealing with the suppression of mTOR signaling by SCFA in colon cancer cells. In the present study, lncRNA RMST expression was stimulated by SCFAs, while and RMST was in turn required for SCFAs to suppress mTOR signaling inducing autophagy. As SCFA were unable to elicit mTOR suppression without RMST, data collectively suggest that RMST plays a significant role in SCFA modulation of mTOR signaling.

Authors share with readers of Kinases and Phosphatases a well written and organized paper, whose content could be appreciated even by readers not particularly skilled in basic science. BEfore its potential acceptance, some issues could and should be addressed, and more precisely:

1) Figure 3b, Figure 5c, 5d; Figure 6b: please provide, either as annex or supplementary material, higher resolution specimens;

2) Because of the required organization of the paper (with materials and methods section posticipated to the results section), Authors were forced to move to the results section some content that would more properly fit materials and methods. For example, but not limited to: rows 56-57, rows 74-75, 87-88, etc. Authors should reorganize introduction in order to provide concise information about the aforementioned topics and by limiting the results section to a very objective reporting of results.

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Reviewer 3 Report

A manuscript "Short chain fatty acids suppress mTOR signaling in colon cancer cells via long non-coding RNA RMST" by Wang et al. identified rhabdomyosarcoma 2 associated transcript (RMST), a long non-coding RNA, as a key mediator for SCFAs to suppress mTOR activation in colon cancer cells. RMST could be significantly induced by SCFA in a time and dose dependent manner. RMST, by itself, was sufficient to suppress mTOR signaling and augment autophagosome formation. Depletion of RMST, through siRNA or CRISPR knockdown, reduced the abilities of SCFAs to suppress mTOR activation or to induce autophagic responses. RMST increased the expression level of TSC2, a negative regulator of mTOR signal pathway. Our data delineate a novel RMST/TSC2 cellular pathway, enlisted by SCFAs, to modulate mTOR activities in colon cancer cells.

The results are interesting and well presented. The results are technically valid.

Specific comments:

1. Fig. 1C, D - why only propionate was chosen?

2. Figure legend for 2C is unclear. In addition, Western blot images are shown with too much background subtraction in all figures.

3. Fig. 4A - scale bars are missing.

4. Western blots should be quantified.

Discussion of the results is sufficient. References cited are appropriate.

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Round 2

Reviewer 1 Report

Unfortunately, the authors did not properly answer some of the comments.

1.     In comment 1, the authors did not check the short-term effects. This should be performed. As presented, the results only show that there is a change in equilibrium after 48 hours. This can not be considered activation of a signaling pathway. 

2.     The district results obtained with the different SiRNAs are not well explained. The authors write that “the use of siRNA technology to knock down the expression of a gene is well established, for caveats including potential off-target effects. That’s why we used three siRNAs, designed and synthesized by a commercial company (IDT). The idea of using three distinct SiRNAs is OK only if all of them give similar results”, which is not the case here. 

3.     The response to point 5 is not clear to me. If the results change from experiment to experiment, they are not reliable and can’t be published.

4.     The legends to the X axis in the figure was not properly changed. For example, Fig. 3A the authors do not mention what expression was detected. 

NA

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