Metabolic Silencing via Methionine-Based Amino Acid Restriction in Head and Neck Cancer
Round 1
Reviewer 1 Report
In this study, the authors demonstrate methionine restriction inhibits proliferation of head and neck squamous cell carcinoma cell lines without impacting cell viability. They suggest this is due to cell entering energy-restricted metabolic state and show a dynamic regulation of plasma membrane amino acid transporter mRNA abundances.
Major Comments:
The 3rd paragraph of the Introduction discusses protein expression as it is related to nutrient sensing mechanisms. Nutrient availability and high-throughput measurements of protein expression are reviewed in (Jeong and Vacanti, Systemic vitamin intake impacting tissue proteomes, Nutrition and Metabolism 2020) and it should be cited here.
In the last paragraph of the introduction, the authors acknowledge methionine is an essential amino acid. However they suggest it can be recovered from other intermediates such as those in the methionine cycle. To my knowledge, humans cannot produce any intermediate in the methionine cycle. So where would they get methionine from in methionine restricted conditions? Wouldn't any human cell's proliferation be sensitive to methionine restriction?
Line 261 states f values and response rate to MetR correlate. They should show this correlation in a graph. Perhaps plotting the change in the f value observed upon methionine restriction vs. the f value under control conditions.
Line 283 states homocysteine supplement marginally rescued proliferation due to methionine restriction in FaDu and SCC25 cells. However no significant change was observed so the authors cannot claim any rescue was observed. The authors claims would also be better supported by the statement that no rescue was observed.
In Figure 5 the authors present finding from qPCR experiments where transcript abundances of 4 amino acid transporters were measured. For each transporter in each cell line, triplicate experiments were performed 3 times each. Across replicate experiments, reproducibility is pretty poor. The authors should only make claims that they can support with statistical significance. For example, in Detriot cells, SLC7A5, SLC7A11, and SLC1A5 are dynamically up-regulated before being restored closer to basal levels over a 72 hour period when exposed to methionine restriction.
Author Response
First of all, thank you for the review of our work and for the constructive criticism, which will certainly lead to a qualitative improvement of our publication.
In this study, the authors demonstrate methionine restriction inhibits proliferation of head and neck squamous cell carcinoma cell lines without impacting cell viability. They suggest this is due to cell entering energy-restricted metabolic state and show a dynamic regulation of plasma membrane amino acid transporter mRNA abundances.
Major Comments:
The 3rd paragraph of the Introduction discusses protein expression as it is related to nutrient sensing mechanisms. Nutrient availability and high-throughput measurements of protein expression are reviewed in (Jeong and Vacanti, Systemic vitamin intake impacting tissue proteomes, Nutrition and Metabolism 2020) and it should be cited here.
We have inserted the citation at the appropriate place.
In the last paragraph of the introduction, the authors acknowledge methionine is an essential amino acid. However they suggest it can be recovered from other intermediates such as those in the methionine cycle. To my knowledge, humans cannot produce any intermediate in the methionine cycle. So where would they get methionine from in methionine restricted conditions? Wouldn't any human cell's proliferation be sensitive to methionine restriction?
On the first question - methionine is an essential amino acid. This is because of the sulphur. Most organisms have no way of reducing sulphur (it is energetically very costly) and most of the sulphur comes into nature's cycle via bacteria. However, methionine can be regenerated (salvage pathway) from precursors (also in humans), among other things through the remethylation of homocysteine with the help of methionine synthase. To be on the safe side, we have once again cited a paper in which it is also described that tumour cells often lose this ability and are therefore dependent on “external” methionine.
Chaturvedi, S.; Hoffman, R.M.; Bertino, J.R. Exploiting methionine restriction for cancer treatment. Biochem. Pharmacol. 2018, 154, 170–173; DOI:10.1016/j.bcp.2018.05.003.
Regarding the second question - Wouldn't any human cell's proliferation be sensitive to methionine restriction? A really complete answer is now very complex. But basically yes - every cell responds to methionine restriction. But this is not negative in any way, it is just a reaction to the external circumstances. Cells have no problem responding to the lack (or reduced amount) of energy and mass over a longer period of time. They then go into a “low energy metabolism”. Proliferation is reduced/stopped and autophagy is massively promoted. This state is absolutely fine for the cell. The whole thing is regulated via mTOR. The mechanisms are essentially the same as those of calorie restriction and protein restriction. We would like to mention one more publication, just to clarify that methionine restriction per se is not toxic to cells or equivalent to harmful malnutrition. Additionally, in vivo methionine restriction would be a reduction and not a complete depletion as in our publication. This is only methodological in order to see maximum effects in the experiments as quickly as possible.
McIsaac, R.S.; Lewis, K.N.; Gibney, P.A.; Buffenstein, R. From yeast to human: exploring the comparative biology of methionine restriction in extending eukaryotic life span. Ann N Y Acad Sci 2016, 1363, 155-170; DOI:10.1111/nyas.13032
In the case of a tumour, the "differential stress resistance" comes into play - as described in the discussion. The normal cells of the body go into low energy metabolism and can, for example, compensate for methionine restriction (no proliferation, autophagy, reduction of protein synthesis, prolongation of the half-life of the proteome etc. etc.). The neoplastic cells do not. They want to continue dividing with all their might and therefore become more vulnerable. We have analysed this phenomenon in more detail in a separate publication in murine cells and would just like to briefly refer to this publication. Here, the complex situation is described in more detail.
Koderer, C.; Schmitz, W.; Wünsch, A.C.; Balint, J.; El-Mesery, M.; Volland, J.M.; Hartmann, S.; Linz, C.; Kübler, A.C.; Seher, A. Low Energy Status under Methionine Restriction Is Essentially Independent of Proliferation or Cell Contact Inhibition. Cells 2022, 11, 551.
Line 261 states f values and response rate to MetR correlate. They should show this correlation in a graph. Perhaps plotting the change in the f value observed upon methionine restriction vs. the f value under control conditions.
In principle, we find the representation proposed by the reviewer very reasonable and also logical. But there are two arguments why we cannot perform the representation in this way. First, we collected much more time points for the determination of the f-values than for the representation of the proliferation rate of the cells under methionine restriction, so we cannot correlate the measurements 1:1. The experiments could of course be performed under the same conditions, but would not provide any additional information gain for methionine restriction and would only be performed for the representation requested by the reviewer. For this, we would need approximately 1-2 months as a review period.
Second, under complete methionine restriction, we see that the cell number under methionine restriction decreases slightly in the long run. Over time, of course, complete methionine restriction can no longer compensate and some of the cells die. However, when calculating the f-values under methionine restriction, this would lead to negative values. And isn't a comparison of the values in a plot more confusing? Basically, we wanted to present a possible and also probable interpretation with the results. In principle, a much higher number of cell lines would be necessary for a statistically significant interpretation. We hope that the reviewer agrees with our argumentation.
Line 283 states homocysteine supplement marginally rescued proliferation due to methionine restriction in FaDu and SCC25 cells. However no significant change was observed so the authors cannot claim any rescue was observed. The authors claims would also be better supported by the statement that no rescue was observed.
We have amended the paragraph in line with the Revierwer's comments.
In Figure 5 the authors present finding from qPCR experiments where transcript abundances of 4 amino acid transporters were measured. For each transporter in each cell line, triplicate experiments were performed 3 times each. Across replicate experiments, reproducibility is pretty poor. The authors should only make claims that they can support with statistical significance. For example, in Detriot cells, SLC7A5, SLC7A11, and SLC1A5 are dynamically up-regulated before being restored closer to basal levels over a 72 hour period when exposed to methionine restriction.
We have changed the text as follows in accordance with the intention of the reviewer:
The results show no clearly significant strictly reproducible results for the genes studied. However, in most cases it can be observed that within the first 24h the receptors are upregulated, the cells respond to the amino acid restriction with an increased expression compared to the control, while after 72h the expression then decreases again. This can be seen very well in the cell line HeLa for the receptor SLC7A11. For the cell line SSC9, on the other hand, the results for all analysed genes are very heterogeneous and do not show a clear trend.
Author Response File: 
Author Response.pdf
Reviewer 2 Report
Overall, the authors have well-presented certain interesting results on a novel strategy of exploration of the role of amino acid restriction on cell proliferation in head and neck cancer cell lines. Although the mechanism needs to be further supported by other experiments like viability, colony forming assay etc, the preliminary results still show interesting facts. The manuscript can be accepted for publications in Current Issues in Molecular Biology. The major concern is authors need to provide focused and good resolution images and also mention magnification in the figure legend. Secondly, authors mentioned “described under the Pico Assay section 4.3”. There’s no 4.3 section in the manuscript.
English language is fine and understandable.
Author Response
First of all, thank you for the review of our work and for the constructive criticism, which will certainly lead to a qualitative improvement of our publication.
Overall, the authors have well-presented certain interesting results on a novel strategy of exploration of the role of amino acid restriction on cell proliferation in head and neck cancer cell lines. Although the mechanism needs to be further supported by other experiments like viability, colony forming assay etc, the preliminary results still show interesting facts. The manuscript can be accepted for publications in Current Issues in Molecular Biology. The major concern is authors need to provide focused and good resolution images and also mention magnification in the figure legend. Secondly, authors mentioned “described under the Pico Assay section 4.3”. There’s no 4.3 section in the manuscript.
We mentioned in the figure legend, that the magnification was 10x. And for better resolution we will add the figures as a power point file in the supplement. And we changed 4.3 to 2.3.
Author Response File: Author Response.pdf
Round 2
Reviewer 2 Report
Authors have responded well to the comments. The manuscript may be considered for publication.
English language and grammar have been corrected accordingly.
