Propionate reinforces epithelial identity and reduces aggressiveness of lung carcinoma

Abstract The epithelial‐to‐mesenchymal transition (EMT) plays a central role in the development of cancer metastasis and resistance to chemotherapy. However, its pharmacological treatment remains challenging. Here, we used an EMT‐focused integrative functional genomic approach and identified an inverse association between short‐chain fatty acids (propionate and butanoate) and EMT in non‐small cell lung cancer (NSCLC) patients. Remarkably, treatment with propionate in vitro reinforced the epithelial transcriptional program promoting cell‐to‐cell contact and cell adhesion, while reducing the aggressive and chemo‐resistant EMT phenotype in lung cancer cell lines. Propionate treatment also decreased the metastatic potential and limited lymph node spread in both nude mice and a genetic NSCLC mouse model. Further analysis revealed that chromatin remodeling through H3K27 acetylation (mediated by p300) is the mechanism underlying the shift toward an epithelial state upon propionate treatment. The results suggest that propionate administration has therapeutic potential in reducing NSCLC aggressiveness and warrants further clinical testing.

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Herein authors identify an inverse association between short-chain fatty acids, mainly propionate and butanoate, and the EMT program in non-small cell lung cancer (NSCLC) in patients.
By mean of an ample set of experimental approaches, including in vitro experiments -with NSCLC cells -where propionate promotes epithelial transcriptional program and reduces aggressiveness and chemo-resistant EMT phenotype-; and in vivo preclinical models -where propionate decreases the metastatic potential and lymph node spread in nude mice and a genetic NSCLC mouse model, respectively-; the authors nicely highlight the therapeutic potential of propionate administration in reducing NSCLC aggressiveness which merit further investigation in cancer patients.
Interestingly, they reveal that propionate treatment induces a chromatin remodelling epigenetic modification -H3K27 acetylation in the CDH1 promoter-as underlying mechanism, at least, partially implicated in the epithelial reversion from EMT.
Using bioinformatics approaches and public data bases, authors find that low levels of propionate correlate with poor survival outcomes in NSCLC cancer patients.Thus, they identify a SCFA network -mainly propionate-which may be promising to prevent NSCLC recurrence.
In general, I find the findings interesting and that could be relevant and valuable in a clinical setting.
Overall, the results are clear, and the manuscript is well organized and well written.Conclusions are supported by the experimental results where propionate may be proposed as a complementary treatment to avoid tumor recurrence.
Nevertheless, there are minor concerns that should be addressed.
.-Please, indicate why the NSCLC cell lines NCI-H358, HCC827, and A549 are selected for the in vitro experiments.
.-Being metabolic reprogramming one of the hallmarks of cancer, it should be desirable to demonstrate the effect of propionate treatment in cell bioenergetics (oxidative phosphorylation or aerobic glycolysis).Does propionate promote oxphos vs aerobic glycolysis?Does propionate promote increased oxphos with increase on ROS from mitochondria?I am curious if scavenging ROS revert the effect of propionate.
.-Authors indicate propionate pretreatment sensitizes NSCLC cells to cisplatin.Due to the relevance of immunotherapy in lung cancer, is there any link between SCFA and clinical benefits of immune checkpoint inhibitors.This should be discussed in the discussion section.
-It should be interesting to include a final paragraph highlighting the relevance of modifiable risks factors associated to lifestyle, such as diet -(Fibre-microbiome-SCFA) and exercise (that affect systemic lipid metabolism homeostasis, inducing lipolysis to release FAs and Ac-CoA through -oxidation.Thus, Ac-CoA may be available for the synthesis of ketone bodies -such as propionate, acetoacetate and β-OHB-in the liver, being systemically available for other tissues. Referee #2 (Comments on Novelty/Model System for Author): Model systems could be improved, as detailed in the remarks.
Referee #2 (Remarks for Author): In this report, authors describe a new role for short-chain fatty acids in supporting the differentiated epithelial phenotype and opposing EMT/mesenchymal phenotype in lung carcinoma cells.They found that propionate reduced invasiveness and resistance in vitro in lung cancer cell lines and impacted lymph node invasion and metastasis in two mice models.Finally, they identify H3K27 acetylation by p300 as the mediator associated with propionate signaling, chromatin remodeling and EMT suppression.These results are new and significant.EMT pathways controls cell plasticity, an essential feature of cancer cells with a strong clinical relevance.Numerous pathways can induce or inhibit EMT pathways, they typically trigger several families of transcriptional factors involved in all described in vivo EMT situations during development.Chromatin reorganization is also a typical feature of the EMT process, leading to the activation or repression of target genes.
Overall, the manuscript provides new and relevant information concerning lung carcinoma progression.The novelty in this report is the EMT emphasis in vitro and in vivo and the intriguing link to chromatin conformation.These questions are pertinent and important for all cancer researchers.However, several points should be answered to support the strength of the results and conclusions.

1) Literature
Interactions between fatty acid and EMT pathways represent a significant and intriguing new aspect of epithelial differentiation.They have been studied by several authors not cited in this report.In lung cancer, acetate was surprisingly described to inhibit TGFb -induced EMT in the same cell line A549 used in this report (Lyu et al MBC 2022 doi. 10.1091/mbc.E22-02-0066).In different context, palmitate induces EMT in hepatocellular carcinoma (Nath et al Sci Rep, 2015. doi.org/10.1038/srep14752) .Also, fatty acid oxydation has been shown to be critical for endothelial-mesenchymal transition in heart valves (Xiong et al Mol Cell 2018, doi.org/10.1016/j.molcel.2018.01.010.These results should be mentioned in the manuscript. 2) Some problems are linked to the western blotting results shown in Fig 2 and 5. On Fig 2, Ecad expression level appears to be upregulated after 48-72h on the Western blot, even when no treatment is applied.Ecad expression level after 72h without treatment is above the level seen after 24h with 5mM SP, or after 48h with 1 mM SP. Similar range is seen with SB.This suggests that A549 cells modulate their epithelial differentiation with culture time with or without treatment with a comparable extent.Why is Ecad upregulated?On Fig 5, we see the reverse: Ecad expression level appears to be downregulated with time after 3 days.In both cases, Western quantification is missing and it is not clear how many duplicate experiments were performed.
3) All EMT cases during development involve at least one member from the EMT associated trancription factor families (EMT-TF) Snail, Zeb and Twist.However, these genes also have some functions unrelated to the classic EMT process, for example in fibroblast and immune cells.It would appear important to better define putative interactions between SCFA and EMT master genes pathways.Can the SCFA "shortcut" the EMT-TF pathways to target HDAC and impact chromatin configuration?This is suggested by the authors ("SP preferentially promotes an epithelial-specific gene expression program through epigenetic reprogramming") but not supported by data.Results obtained with Zeb1 are rather confusing.Expression pattern of all three families EMT-TF should be gathered to evaluate their potential involvement.4) Authors suggest that SCFA impact on epithelial cell differentiation could represent a general mechanism involving EMT processing (l390:" The effect appears to be a general phenomenon"), but at the same time they mention a specific targeting of " the pulmonary alveolar type-II cell-type markers implying the establishment of epithelial integrity in lungs" (l392) by the propionate treatment that would increase specifically the airway epithelial cell-type genes expression.The text should be consistent and make a stand, mentioning distinct hypothesis.It would be helpful to check how treatment can impact some other markers for lung epithelial cell progenitors (stemness), also linked to EMT pathways 5) Mouse models support the authors finding but do not demonstrate any EMT involvement.The expression of EMT features in the tumors before and after propionate treatment should be checked by classic immunolocalization (or any other approach) to estimate the level of cell depolarization, dissociation and expression of EMT functional markers.6) In Fig 2 .wound healing experiments are conclusive in showing a delay in cell sheet spreading after SP treatment, but they do not indicate a migration halt.It would be important to look at higher magnification to check if the cells are individualized or migrate as an epithelial sheet.Also, the number of repeat experiments should be mentioned.

7)
To study cell plasticity, an essential feature of EMT, it would be important to insert A549 spheroids into 3D collagen gels to monitor and quantify migration and invasive pattern, with or without treatment.
Minor points 1) In Fig 1F, "Propanoate" is used for propionate found everywhere else in the manuscript.One word should be consistently used.
2) It is not clear what should be seen in Fig 3i .A stronger histological staining and additional higher magnification pictures would be helpful.
3) Fig3H.It is not clear how many mice were used for this survival study.5) Discussion is overall too long and redundant.It should be more focused on this manuscript findings.For example, the section l409-l426 about SP and cisplatin resistance is quite interesting, but does not imply EMT pathways.Similarly, the section about SCFA availability (l428-l445) is rather unclear.

6) In Fig S1, Zeb2
and Axl are found to be downregulated by TGFb treatment.This is not the consensus response described in other reports studying EMT pathways.Any comments?
7) It should be mentioned that most cancer data sets used in this study involve a mix of stroma and tumor cells, an element to consider in the discussion.
8) The statement l409."Our lung experimental metastasis model showed that SP can inhibit metastasis." is too strong.SP treatment has an impact, but does not appear to inhibit metastasis overall.

Referee #3 (Remarks for Author):
The epithelial-to-mesenchymal transition (EMT) is a cellular program that cancer cells can utilize to gain metastatic ability.However, targeting the EMT can be tricky since its regulation is primarily mediated by transcription factors, which are challenging to inhibit.In contrast, metabolic enzymes may represent promising druggable targets for inhibiting EMT.Therefore, investigating the role of metabolic processes in the EMT program could lead to identifying new drug targets.In their study, Ramesh et al. aimed to uncover unknown metabolic processes involved in the EMT program through transcriptome analysis.Their findings revealed that the propionate and butanoate metabolism gene set negatively correlated with the EMT gene set.Further experiments demonstrated that treatment with propionate and butanoate induced the cells to acquire more epithelial characteristics.Additionally, propionate inhibited metastasis and sensitized the cells to cisplatin.Finally, they show that propionate reduces lung cancer EMT by broad chromatin remodeling.However, all of the experiments were conducted by treating epithelial cells with fatty acids, and the role of the endogenous propionate and butanoate in the EMT program is unclear.Additionally, the effect of these fatty acids on mesenchymal-like lung cancer cells needs to be investigated.Thus, the role of endogenous propionate and butanoate in EMT program regulation remains unclear and deserves investigation.
Major comments 1.The authors suggest that the short-chain fatty acids induce the cells to become more epithelial.For example, in Figure 2, they demonstrate that treatment of A549, SK-MES-1, and NCI-H23 cells with propionate and butanoate induces the cells to gain more epithelial characteristics.However, the biological meaning of this finding is unclear as these cells are already in the epithelial state.Therefore, these experiments should also be conducted in lung cancer cell lines in a partially mesenchymal state.Specifically, these experiments will determine whether these short-chain fatty acids can induce the MET program.2. The authors analyzed gene expression databases to identify the metabolic processes.They assumed that short-chain fatty acids are regulated at the transcription level.However, they did not validate or elaborate on this analysis.For example, which enzymes in the short-chain fatty acid biosynthesis pathway are downregulated during the EMT program or in epithelial vs. mesenchymal cells?What will be the outcome of their ectopic expression in epithelial and mesenchymal cells?What will be the outcome of manipulating the enzymes synthesizing these fatty acids?Will the cells induce the EMT program? 3.One of the main themes in this manuscript is that the short-chain fatty acids function as inhibitors of the EMT.However, the physiological relevance of these findings is still missing as they did not validate any changes in the fatty acid intracellular levels.For example, does the intracellular level of propionate and butanoate reduce during the EMT? 4. The role of these short-chain fatty acids as regular of the EMT program is needed to be elaborated.Indeed, Figures 2H-I show that SP and SB inhibit EMT markers expression upon TGFß treatment.However, this was demonstrated only on two markers which is insufficient to conclude.Additionally, showing changes in the marker's expression is not enough to conclude any effect on the EMT program, as the effect on the cell's migratory capabilities is needed to be examined.Minor comments 1.The HR (hazard ratio) value is absent from all of the Kaplan-Meier plots that pertain to survival rates.2. In Figures 2B and 2C, why do E-cadherin levels change over time in samples without the short-fatty acids treatment?Additionally, the ZEB1 blot is problematic.In Figure 2C treatment, its expression levels don't change, and in Figure 2B, there is inconsistency with SP doses.Additional mesenchymal markers are needed to clarify this point.S2).

3rd Aug 2023 1st Authors' Response to Reviewers
For the further in vitro validation, A549, SKMES1, NCI-H520, NCI-H23, CALU-1 and H1299 were used to substantiate and study the molecular level processes of SCFA in lung cancer.These cell lines were used because they represent the two main NSCLC histotypes, adenocarcinoma and squamous cell carcinoma.This has now been better highlighted in the revised version of the manuscript (Page: 7; Line: 156-173).
2. Being metabolic reprogramming one of the hallmarks of cancer, it should be desirable to demonstrate the effect of propionate treatment in cell bioenergetics (oxidative phosphorylation or aerobic glycolysis).Does propionate promote oxphos vs aerobic glycolysis?Does propionate promote increased oxphos with increase on ROS from mitochondria?I am curious if scavenging ROS revert the effect of propionate.Response R1.2This is an interesting suggestion and we have now carried out a Seahorse metabolic flux analysis to investigate cell bioenergetics upon treatment with sodium propionate (SP).A549 cells were treated with SP 5mM for 24 hours, and OCR (oxygen consumption rate) and ECAR (extracellular acidification rate) were analyzed.Interestingly, we found propionate did not increase but rather slightly decreased ATP-linked respiration compared to the control (Fig R1A), while we did not observe changes in ECAR activity (Fig R1B).Previous studies showed that treatment of HCT116 (colorectal cancer) showed an increase in the OCR although minimally at 10 mM propionate concentration (Høgh et al, 2020).In another study, treatment of primary human epidermal keratinocytes (HEK) with butyrate showed a significant reduction in the basal respiration and maximal respiratory capacity (Trompette et al, 2022).We therefore believe the response to propionate could be cell line-and tissue-specific.
Further, we treated A549 cells with SP for 72 hours followed by the measurement of reactive oxygen species level using flow cytometry (DCFDA/H2DCFDA assay).Again, we did not observe any significant increase in the ROS levels (Fig R1C).Cisplatin was used as a positive control for the ROS detection.Thus, ROS is not majorly altered with propionate treatment, and therefore we did not pursue the suggested scavenging experiment.We decided to not add these data to the final version of the manuscript for space limitation.Thanks for the suggestion.

Authors indicate propionate pretreatment sensitizes NSCLC cells to cisplatin. Due to the relevance of immunotherapy in lung cancer
, is there any link between SCFA and clinical benefits of immune checkpoint inhibitors.This should be discussed in the discussion section.
Response R1.3This is an important suggestion.In fact, we already pointed out the importance of the positive link between SCFAs and the clinical benefits of immune checkpoint inhibitors in the discussion of the previous version.For instance, "Recently, gut microbiota metabolomics of NSCLC patients undergoing immunotherapy with nivolumab showed that long-term responders were majorly characterized with the presence of SCFA metabolites (propionic, butyric, acetic and valeric acids) with beneficial effects (Botticelli et al, 2020)" (Page: 21; Line: 516-519).However, we have also included few additional references in the discussion to strengthen the importance of these SCFAs in aiding therapeutic efficacy by sensitizing NSCLC to immunotherapy.For example, "Again NSCLC patients responding to programmed cell death protein 1 (PD-1) antibody therapy were found to have higher baseline levels of fecal SCFAs concentration (Zizzari et al, 2020).Similarly, immunotherapy for several solid cancers including lung adenocarcinoma with immune checkpoint inhibitors (nivolumab or pembrolizumab) exhibiting improved PD-1 inhibitor efficacy showed high baseline fecal concentrations of SCFAs mainly propionic, butyric, valeric and acetic acid with longer progression-free survival (Nomura et al, 2020)" (Page: 21; Line: 520-526).All these reports support the notion that SCFAs could improve the therapeutic efficacy of primary line treatment options.

It should be interesting to include a final paragraph highlighting the relevance of modifiable risks factors associated to lifestyle, such as diet -(Fibre-microbiome-SCFA)
and exercise (that affect systemic lipid metabolism homeostasis, inducing lipolysis to release FAs and Ac-CoA through β-oxidation.Thus, Ac-CoA may be available for the synthesis of ketone bodies -such as propionate, acetoacetate and β-OHB-in the liver, being systemically available for other tissues.

Response R1.4
We agree with this view and thank the Reviewer for highlighting the requirement of dietary and physical activity as important factors in cancer mitigation.This is an important suggestion, with many potential beneficial implications for the patients.We have now included these modifiable factors in the discussion of the revised manuscript as follows: "Diet is intimately connected with the microbial community (diversity and abundance) in humans especially with the gut microbiota, and dietary changes could contribute to the microbial profiles during diseased conditions.Epidemiological data shows that high-fiber diets are related to a lower cancer incidence and the anti-cancer effect is significantly due to the formation of SCFAs from high-fiber diet (Mirzaei et al, 2021).It has been shown that mice fed with probiotic supplementation increased the SCFA producing bacteria (propionate and butyrate) in the gut with the inhibition of lung metastasis of melanoma cells (Chen et al, 2021) implicating dietmicrobiome-SCFA link" (Page: 22; Line: 530-537).
"Similarly, evidences showed that physical activity such as exercise can alter the composition of the gut microbiota, and eventually increase the SCFA production as an additional substrate for metabolism (Song & Chan, 2019)" (Page: 22; Line: 538-540).
"Since propionate is a biotic compound and a safe food ingredient with therapeutic ability (Rangan & Mondino, 2022), the beneficial effects reported here make propionate a promising avenue for further clinical exploration as a treatment option for NSCLC patients.Alternative strategies like consuming fiber-enriched diet and physical activities resulting in high SCFA content in the system could offer a realistic approach in cancer prevention and treatment" (Page: 23; Line: 556-561).

Referee #2 (Comments on Novelty/Model System for Author):
Model systems could be improved, as detailed in the remarks.

Referee #2 (Remarks for Author):
In this report, authors describe a new role for short-chain fatty acids in supporting the differentiated epithelial phenotype and opposing EMT/mesenchymal phenotype in lung carcinoma cells.They found that propionate reduced invasiveness and resistance in vitro in lung cancer cell lines and impacted lymph node invasion and metastasis in two mice models.Finally, they identify H3K27 acetylation by p300 as the mediator associated with propionate signaling, chromatin remodeling and EMT suppression.These results are new and significant.EMT pathways controls cell plasticity, an essential feature of cancer cells with a strong clinical relevance.Numerous pathways can induce or inhibit EMT pathways, they typically trigger several families of transcriptional factors involved in all described in vivo EMT situations during development.Chromatin reorganization is also a typical feature of the EMT process, leading to the activation or repression of target genes.
Overall, the manuscript provides new and relevant information concerning lung carcinoma progression.The novelty in this report is the EMT emphasis in vitro and in vivo and the intriguing link to chromatin conformation.These questions are pertinent and important for all cancer researchers.However, several points should be answered to support the strength of the results and conclusions.Response R2.1 As suggested by the reviewer, we have included the mentioned articles in the discussion of the current revised manuscript as follows: 1. "Previous study showed that acetate could inhibit TGFβ1-induced EMT features (Lyu et al, 2022).However, in our study, we did not find any modulation in E-cadherin or ZEB1 levels upon sodium acetate treatment.Further, the study utilized 40 mM of sodium acetate to inhibit TGFβ1-induced EMT features which is 40-fold higher concentration than used in the current study.Therefore, acetate's role in inhibiting EMT in lung cancer cannot be ruled out as higher concentration could have an impact in inhibiting EMT in lung cancer cells potentially through a similar mechanism of propionate and butanoate" (Page: 20; Line: 495-502).We thank the reviewer for highlighting this important reference in the current study.(Nath et al, 2015)" (Page: 17; Line: 415-417).

"Elevation in the long-chain free fatty acid palmitate has been shown to promote EMT in hepatocellular carcinoma via Wnt/β-catenin and TGF-β signaling
3. "On the other hand, a decrease in the fatty acid oxidation was found during endothelialmesenchymal transition by altering the intracellular levels of acetyl-coA (Xiong et al, 2018)" (Page: 17; Line: 417-419).However, for this long-term experiment the cells were split every 3 days using 0.5 mM EDTA with the confluence of around 90-95%, and re-seeded for propagation in a split ratio of 1:3 to 1:5 with the SP treatment.This was necessary because E-cadherin is degraded with the use of trypsin, but not with EDTA.So, the experiment in Fig 5A is not directly comparable to those in Fig 2 , and the observed difference in the level of E-cadherin between 3, 6, 9 and 12 days is most likely due to variability in the confluence during harvesting.Again, we emphasize that the increase in E-cadherin is primarily compared with the control at that specific time point to highlight the reinforcement of E-cadherin or inhibition of EMT, and that the treatment itself does not alter the confluency.We detailed the experimental procedure of EDTA-based harvesting of cells in the method section (Page: 25; Line: 598-607) and discussed about this in the revised manuscript (Page: 18; Line: 425-429).To further confirm the increase of E-cadherin with SP treatment, we performed q-PCR analysis of E-cadherin in A549 cells treated with SP 5mM for 72h and found a similar level of alterations at the transcript level (    We agree that it would be very interesting in future to study the dynamic role of SP in epigenetic reprogramming and EMT-transcription factors modulation in inhibiting EMT.For instance, there could be a cross-regulation between these entities.In addition, it would be essential to elucidate why and how SNAIL up-regulation happens in a cellular environment that is moving towards EMT suppression (another feedback loop?).All these aspects may be object of a future dedicated investigation.This has now been included in the discussion section of the manuscript (Page: 20; Line: 491-493).Thanks a lot for the important input.We apologize for the confusion in the text.We wanted to emphasize the fact that SP's effect could be a general phenomenon as we could observe it with other cancer cell lines such as with pancreatic cancer cell line (Appendix Figs S2A -C).However, specifically in the context of lung, we wanted to highlight the epithelial-inducing phenomenon of SP on the airway epithelial cell-type (pulmonary alveolar type-II cell type) which plays an important role in the alveoli integrity and function.To avoid confusion for the general readers, we have rephrased the sentences in the current revised manuscript (Page: 18; Line: 437-441) as follows:

Some problems
"The epithelial reinforcement could also be observed in other cancer types such as in pancreatic cancer cell line.In the context of lung, propionate treatment increased the genes expression of pulmonary alveolar type-II cell-type implying the establishment of epithelial integrity in lungs" As requested, we have now tested the lung epithelial cell progenitor genes expression (Rawlins, 2008) in the RNA-seq profile of 3 days and 12 days SP treated A549 cells.We observed a significant decrease in the progenitor genes expression at 12 days (Fig R6A).In addition, we also analyzed the expression of cancer stemness marker genes expression related to EMT (ALDH1A1 and ALDH3A1) (Ramesh et al, 2020;Fan et al, 2021).Interestingly, there was a very significant decrease in these cancer stem cell markers genes implying that propionate may also inhibit stemness (    Response R2.7 Thanks for this relevant suggestion.As requested by the Reviewer, we tried to study the cell plasticity of A549 spheroids embedded in the 3D collagen gels with the treatment of SP (at 5 mM).Briefly, A549 spheroids were seeded on a coverslip coated with collagen type I and on the top, a drop of collagen was added forming a 3D milieu.However, we did not find any effect of SP on the migration (Fig R9A).This could be due to weak diffusion of propionate into the 3D collagen scaffolds due to the charge of the polymers.Therefore, we tried few other alternative approaches using 2D culturing of spheroids with collagen wherein the spheroids were anchored to a flat surface by coating the coverslip with collagen type I. Here, we observed a significant reduction in the invasive ability of the cells treated with SP (Fig R9B-C).In another approach, we seeded 2500 cells/well in a 96-well ultra-low adherent plate and allowed cells to form spheroids for 3 days.After 3 days, we treated the spheroids with SP and/or TGF-β1 and layered them with Matrigel.The spheroids were then examined by live-cell imaging.The results showed that TGF-β1-induced invasive ability was strongly inhibited by co-treatment with SP (Fig R10), as also observable on the microscopic pictures.We believe these data are sufficient to indicate a proof-of-concept role of SP on modulating cell plasticity in spheroids settings, while more conditions and spheroid assays could be required in future to fully elucidate the role of SP in inhibiting the cell plasticity.The results of      Response R2.12As suggested, we have now improved the discussion section by highlighting the findings in the study with more focused discussion.We have also rephrased the section of SCFA availability (Page: 22; Line: 540-545), and SP's involvement in cisplatin sensitization (Page: 19; Line: 466-470).However, with the additional revision experiments and additional citations requested the discussion is still long.

In Fig S1, Zeb2
and Axl are found to be downregulated by TGFb treatment.This is not the consensus response described in other reports studying EMT pathways.Any comments?
Response R2.13 Good observation.Indeed, it is well known that ZEB2 and AXL are targets of TGF-β1.The mRNA expression profile of NCI-H358, HCC827 and A549 treated with TGF-β1 used in Appendix Figs S1A-C of the study were obtained from the publicly available database Gene Expression Omnibus (GEO-GSE49644).We utilized this profile to examine the overall changes in the EMT gene signature obtained from (Mak et al, 2016) as a validation.But we are unsure why AXL and ZEB2 are found down-regulated.However, in our own hands we also found AXL and ZEB2 were not increased with TGF-β1 treatment in A549 cell line (Fig R13).In the previous literature, we also found a similar case wherein A549 treated with TGF-β1 did not induce AXL by immunoblotting (Choi et al, 2021).We believe this requires further experiments to justify these EMT markers regulations in A549 cells with the treatment of TGF-β1, although this has no impact on our manuscript.We did not modify the manuscript.8.The statement l409."Our lung experimental metastasis model showed that SP can inhibit metastasis." is too strong.SP treatment has an impact, but does not appear to inhibit metastasis overall.

Response R2.15
As correctly noted by the Reviewer, we have toned down the phrase in line I409: "Our lung experimental metastasis model showed that SP can inhibit metastasis" to "our lung experimental metastasis model showed that SP could reduce the metastasis" (Page: 19; Line: 460-462).

Referee #3 (Remarks for Author):
The epithelial-to-mesenchymal transition (EMT) is a cellular program that cancer cells can utilize to gain metastatic ability.However, targeting the EMT can be tricky since its regulation is primarily mediated by transcription factors, which are challenging to inhibit.In contrast, metabolic enzymes may represent promising druggable targets for inhibiting EMT.Therefore, investigating the role of metabolic processes in the EMT program could lead to identifying new drug targets.In their study, Ramesh et al. aimed to uncover unknown metabolic processes involved in the EMT program through transcriptome analysis.Their findings revealed that the propionate and butanoate metabolism gene set negatively correlated with the EMT gene set.
Further experiments demonstrated that treatment with propionate and butanoate induced the cells to acquire more epithelial characteristics.Additionally, propionate inhibited metastasis and sensitized the cells to cisplatin.Finally, they show that propionate reduces lung cancer EMT by broad chromatin remodeling.However, all of the experiments were conducted by treating epithelial cells with fatty acids, and the role of the endogenous propionate and butanoate in the EMT program is unclear.Additionally, the effect of these fatty acids on mesenchymal-like lung cancer cells needs to be investigated.Thus, the role of endogenous propionate and butanoate in EMT program regulation remains unclear and deserves investigation.

Major comments
1.The authors suggest that the short-chain fatty acids induce the cells to become more epithelial.For example, in Figure 2, they demonstrate that treatment of A549, SK-MES-1, and NCI-H23 cells with propionate and butanoate induces the cells to gain more epithelial characteristics.However, the biological meaning of this finding is unclear as these cells are already in the epithelial state.Therefore, these experiments should also be conducted in lung cancer cell lines in a partially mesenchymal state.Specifically, these experiments will determine whether these short-chain fatty acids can induce the MET program.
Response R3.1 As rightly pointed out, it is true that investigating the propionate's and butanoate's ability to increase the epithelial features should be done in a mesenchymal or in a partial-EMT cell (highly-aggressive and highly-metastatic state (Pastushenko et al, 2018)).
However, previous study on scoring EMT in both clinical samples and in cancer cell line encyclopedia (CCLE) across several cancer types, showed that NSCLC is situated in between epithelial and mesenchymal state implying a partial EMT mode (Tan et al, 2014).Similarly, another study predicted and validated the partial EMT state of A549 cell line where it was observed to co-express both epithelial and mesenchymal markers (George et al, 2017), data also confirmed by our own single cell RNA-seq (Page: 13; Line: 301-308).
Anyway, based on this relevant comment, in the current revised manuscript we have now included treatment with SCFAs (SP and SB) of additional mesenchymal-like NSCLC cells CALU-1 (Thomson et al, 2008) and H1299 (Tan et al, 2018).As a result, although we observed an increase in the epithelial markers expression with the SP treatment (E-cadherin, GRHL1 and EPCAM), less to no difference in the mesenchymal markers (ZEB1, BCAT1 and NNMT) was found in CALU-1.The epithelial reinforcement effect was not observed with SB except for Ecadherin (Fig R14A-B).In case of H1299, we did not observe any increase of E-cadherin with SP/SB, but only a moderate EPCAM up regulation.Similarly, we did not observe any changes in the mesenchymal markers (ZEB1 and BCAT1) in this cell line (Fig R14C-D).Overall, there was a no or lower effect on mesenchymal-like NSCLC cell lines compared to partial EMT cell line A549.The results have been now appended in the current revised manuscript (Page: 7; Line: 164-173; Figs EV1D and EV1E).
It also needs to be considered that in the previous version of the manuscript we also included an experiment wherein we induced A549 cells to become fully mesenchymal by treating with TGF-β1, and then treated the cells with SP or SB (Figs EV1I and EV1J in the revised manuscript).Those experiments also revealed that SP and SB could not revert the EMT program when the same A549 cells were already induced in the mesenchymal state.Altogether, our data seem to indicate that SCFA SP and SB could better inhibit the partial-EMT state while have no or less effect when the cells are transformed to full mesenchymal state.This could be in the future better mechanistically elucidated, and this observation could be important in the selection of the patients to be enrolled in clinical trials testing SCFA and has been now added in the discussion (Page: 18; Line: 429-434).Thanks for the important input.S6C).Interestingly, it has recently been shown that deregulation in the propionate metabolism can contribute to an anaplerotic reaction in the accumulation of methylmalonic acid (MMA) in breast and lung cancer leading to EMT and metastasis (Gomes et al, 2022).In apparent contrast to this, we observe that treatment with propionate results in the epigenetic involvement in inducing epithelial identity implying a dual and opposing effect on EMT.This has been detailed in the discussion section of the manuscript (Page: 19; Line: 448-455).
Following this comment, we now tested if the treatment of propionate has any increase in the metabolic pathway enzymes.We treated A549 cell line with SP and found a relative increase in the PCCA and PCCB although only at later time points (Fig R15), implicating that probably epigenetic involvement overrides the metabolic counterpart and reinforces the epithelial identify of the cells.However, these may be crucial aspects in the cellular EMT control in lung cancer and beyond, which would require a dedicated effort in the future, including, for instance, the genetic and pharmacological manipulation of these enzymes in vitro and in vivo, as mentioned by the Reviewer.Our lab is working on it, for instance with the employment of custom lung tumor-specific knockout models via CRISPR/Cas9.We have highlighted about this in the discussion   R16B-C).Since the basal level of propionate in the cells was below or around the blank estimate, we were technically unable to quantify intracellular amount of propionate (Fig R16D).However, we tested if the treatment of A549 cell line with sodium propionate could increase the intracellular propionate.As expected, a very significant increase (Folds=~27; p=0.0006) was detected (Fig R16E), implying that the quantification method is working and that the physiological origin of propionate may not be intracellular.
We believe that propionate in NSCLC might be contributed by the tumor microenvironment, and lung resident microbiota are a possible suspect due to the fact that bacteria are the natural source of propionate in the body (in the gut, for instance).4. The role of these short-chain fatty acids as regular of the EMT program is needed to be elaborated.Indeed, Figures 2H-I show that SP and SB inhibit EMT markers expression upon TGFß treatment.However, this was demonstrated only on two markers which is insufficient to conclude.Additionally, showing changes in the marker's expression is not enough to conclude any effect on the EMT program, as the effect on the cell's migratory capabilities is needed to be examined.

Response R3.4
As suggested by the Reviewer, we have included additional EMT-specific marker NNMT (Shaul et al, 2014;Yang et al, 2022) to demonstrate SP's or SB's action on EMT inhibition upon treating the A549 cell line both by pre-treatment of cells with SCFA followed by TGF-β1 treatment (  Western blot analysis of E-cadherin, ZEB1 and NNMT protein levels in A549 cells pre-treated with sodium propionate (SP, 5 mM) or sodium butanoate (SB, 1 mM) for 48 hours followed by TGF-β1 (2 ng/ml) for 24 hours.β-Actin was used as an internal control.B. Western blot analysis of E-cadherin, ZEB1 and NNMT protein levels in A549 cells co-treated with sodium propionate (SP, 5 mM) or sodium butanoate (SB, 1 mM) in combination with TGF-β1 (2 ng/ml) for 72 hours.β-Actin was used as an internal control.
We also agree that markers alone are anyway not sufficient and that functional assays like in vitro migration are more indicative of EMT.We have also now performed cell migration analysis with A549 pre-treated or co-treated with SP and TGF-β1, as suggested.As expected, we found a significant reduction in the migratory ability of the cells between SP, and SP with TGF-β1 (Fig R18A and R18B).A similar level of inhibitory effect of SB on TGF-β1 induced migration was also observed (   EV3I) along with the corresponding confidence intervals in the revised manuscript.2B and 2C, why do E-cadherin levels change over time in samples without the short-fatty acids treatment?Additionally, the ZEB1 blot is problematic.In Figure 2C treatment, its expression levels don't change, and in Figure 2B, there is inconsistency with SP doses.Additional mesenchymal markers are needed to clarify this point.Response R3.6 A similar concern was raised by the Reviewer 2 (please see more detailed response R2.2), and the increase in E-cadherin over the time is due to the culture confluence as the cells were harvested from low confluence to high confluence over the course of treatment for 72h.In case of ZEB1, which is normally a hard protein to detect, this was found downregulated at 72h with SCFA (SP and SB) treatment in all 3 independent repeats (Fig R2).The down-regulation of ZEB1 in the SP treated cells is also consistent with longer treatment from 3 days to 12 days in all 3 independent repeats (Fig R3 Figure R19.Western blot analysis of E-cadherin, TWIST1 and NNMT protein levels in A549 cells treated with sodium propionate (SP, 5 mM) in the indicated time-dependent manner.Β-Actin was used as an internal control.

Other changes:
This resubmission work has required the involvement of new external and internal collaborators for the mass-spec quantification of propionate (3 authors from the University of Southern Denmark), the spheroid assays (2 authors from the University of Heidelberg) and for the immunohistochemistry on mouse samples (2 authors from the University of Turin), which have been added to the list of co-authors and their specific contribution has been mentioned in the dedicated section.
31st Aug 2023 1st Revision -Editorial Decision 31st Aug 2023 Dear Dr. Ceppi, Thank you for the submission of your revised manuscript to EMBO Molecular Medicine, and please accept my apologies for the delay in getting back to you as the referees reports came back while I was out of the office.We have now received the reports from the 2 referees who re-reviewed your manuscript.As you will see, they are supportive of publication, and we will therefore be able to accept your manuscript once the following editorial points will be addressed: 1/ Referees: please address the remaining minor point from referee #2.

2/ Main manuscript file:
-Please address the queries from our data editors in the related Data Edited manuscript file.Remove the yellow highlights in the text and only keep in track changes mode any new modification.
-We noted a minor discrepancy in an author's name: Nils Joakim Faergeman in the manuscript vs. Nils Faergeman in the submission system.
-The manuscript details on p. 2-3 should be removed from the manuscript -Materials and methods: o Mice: please indicate the gender and age of the mice at the time of experiment.o Statistics: please include information on blinding, randomization, inclusion/exclusion criteria and adjust the checklist accordingly.
-Data Availability: Please place this section after the Materials and Methods, before the Acknowledgements.
-Acknowledgements: All funding sources should be added to the submission system, and match the information provided in the manuscript (currently missing in the submission system: Interdisciplinary Center for Clinical Research of the University of Erlangen-Nuremberg).
-Please remove the Abbreviations from p. 53 (we do not have abbreviations list; abbreviations should be defined in the text the first time they are used).

3/ Figures:
-Please clearly mention in the legend of Figure EV2K that the image is re-used from Figure 5E.
-Please make sure that all figures and figure panels are referenced in the manuscript text.Currently, callouts are missing for Fig. 1G and 1H.4/ The Paper Explained: I introduced minor modifications in your text, please let me know if you agree with the following or amend as you see fit: Problem Epithelial-to-mesenchymal transition (EMT), a phenotypic developmental process, contributes to various highly aggressive features in cancer, including metastasis and chemoresistance with poor patient survival.However, pharmacological targeting of EMT in malignancies has proven very challenging.In recent years, targeting deregulated metabolic processes in cancer emerged as a realistic therapeutic strategy, though identification of precise EMT metabolic processes remains difficult.

Results
Taking advantage of hundreds of thousands of non-small cell lung cancer (NSCLC) patient-derived gene expression profiles and of numerous metabolic processes gene-sets, we performed an EMT-focused functional genomic analysis and identified negative association between EMT and short-chain fatty acids, especially propionate.Strikingly, treatment of lung cancer cell lines with propionate reinforced the epithelial transcriptional program, promoting cell-cell contact features along with the inhibition of aggressive EMT process, and sensitized the cells to cisplatin treatment.In addition, propionate reduced metastasis ability in a lung experimental metastasis model and limited lymph node metastatic spread in a genetic NSCLC mouse model.Finally, propionate was involved in chromatin remodeling with increased histone acetylation.Impact In this study, we identify in pre-clinical models the therapeutic relevance of the short-chain fatty acid propionate in reducing EMT-induced aggressive features of lung cancer.

5/ Synopsis:
-Thank you for providing a nice synopsis image.Please make sure that the width is 550 pixels (currently 548px) and that the text is legible.
-I added minor modifications to your text, let me know if you agree or amend as you see fit: An EMT-centric investigation of metabolic processes in a comprehensive lung cancer transcriptome profiles collection identified an inverse association between EMT and SCFAs propionate and butyrate.
• Propionate enhances the epithelial features in vitro both at the molecular and cellular levels • Pre-treatment of cells with propionate inhibits EMT associated processes and sensitizes the cells to the chemotherapeutic drug cisplatin • Oral administration of propionate inhibits EMT-mediated lung colonization ability of NSCLC cells, and lymph node metastasis in a genetic mouse NSCLC model • Molecular mechanistic investigation revealed chromatin remodelling through p300-mediated histone acetylation in E-cadherin gene regulation along with epithelial features reinforcement 6/ As part of the EMBO Publications transparent editorial process initiative (see our Editorial at http://embomolmed.embopress.org/content/2/9/329),EMBO Molecular Medicine will publish online a Review Process File (RPF) to accompany accepted manuscripts.This file will be published in conjunction with your paper and will include the anonymous referee reports, your point-by-point response and all pertinent correspondence relating to the manuscript.Let us know whether you agree with the publication of the RPF and as here, if you want to remove or not any figures from it prior to publication.Please note that the Authors checklist will be published at the end of the RPF.This completed version answers most points satisfactorily.Some new results bring up new questions, that will be ground for future works hopefully.One minor remark is that the new Fig R9 could be improved with higher contract pics to show better the crown of spreading and migrating cells in the 3D gel and on the 2D collagen substrate.The Fig R9B methodology helps to solve the difficulty of a true 3D setting, but the cell migration pattern is very similar to the mode used in wound healing assay.Based on these two pictures, it actually appears that the SP treated cells express less scattering and full cell-cell dissociation than the control cells in both situations.Because of the poor focus, it is difficult to assume, only based on the figure.This relevant data could probably be quantitated using relevant microscopy methods and/or image processing.

Referee #3 (Remarks for Author):
The manuscript is significantly improved I have no more comments.

Referee #3 (Remarks for Author):
The manuscript is significantly improved I have no more comments.

Response R3.1
We thank the reviewer for the positive comment on our manuscript.

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4) Fig 5E higher magnification pictures would be useful to assert ZO-1 and Epcam localization and organization pattern.
We obtained the gene expression profiles of three NSCLC cell lines mentioned by the Reviewer (NCI-H358, HCC827 and A549) treated with TGF-β from a publicly available database (GEO -GSE49644) as part of a validation of the EMT gene signature obtained fromMak et al., 2016 employed in the current study (Appendix Fig S1A-C, Appendix Table

1.
Literature: Interactions between fatty acid and EMT pathways represent a significant and intriguing new aspect of epithelial differentiation.They have been studied by several authors not cited in this report.In lung cancer, acetate was surprisingly described to inhibit TGFb -induced EMT in the same cell line A549 used in this report (Lyu et al MBC 2022 doi.10.1091/mbc.E22-02-0066).In different context, palmitate induces EMT in hepatocellular carcinoma (Nath et al Sci Rep, 2015.doi.org/10.1038/srep14752) .Also, fatty acid oxydation has been shown to be critical for endothelial-mesenchymal transition in heart valves (Xiong et al Mol Cell 2018, doi.org/10.1016/j.molcel.2018.01.010.These results should be mentioned in the manuscript.
are linked to the western blotting results shown in Fig 2 and 5. On Fig 2, Ecad expression level appears to be upregulated after 48-72h on the Western blot, even when no treatment is applied.Ecad expression level after 72h without treatment is above the level seen after 24h with 5mM SP, or after 48h with 1 mM SP. Similar range is seen with SB.This suggests that A549 cells modulate their epithelial differentiation with culture time with or without treatment with a comparable extent.Why is Ecad upregulated?On Fig 5, we see the reverse: Ecad expression level appears to be downregulated with time after 3 days.In both cases, Western quantification is missing and it is not clear how many duplicate experiments were performed.Response R2.2This is an important observation, which gives us opportunity to explain.Cells in Fig 2A-C were harvested from low to high confluence every 24h until 72h and show quite consistently an increase in the E-cadherin levels in a time-dependent manner without treatment, as noted, primarily due to the increase in the cell-cell contact establishment during the confluence attainment.We therefore consider changes in the E-cadherin levels and EMT markers by SP or SB with respect to the control of the specific time point.By contrast, in Fig 5A, we did not observe any up-regulation of E-cadherin over 12 days treatment in the controls.
Moreover, as requested by the Reviewer, we have included the Image J quantifications for the figures (Figs 2B, 2C and 5A) and the level of E-cadherin or ZEB1 is compared to the respective time point controls after normalizing to β-Actin (Figs R2 and R3), and quantifications have been added as fold changes in the revised manuscript (Fig 2B, 2C, 5A).
Fig R4).The result is included in the revised manuscript (Page: 8; Line: 180-182 and Fig EV1G).This indicates the robustness of the E-Cadherin increase induced by propionate with an independent method.Regarding the repeats, all the western blots in the manuscript were done at least 3 independent times, as shown below for these specific experiments (FigR2 and R3) and the legend has been now updated with the number of repeats performed in the revised manuscript (Page: 57; Line: 1458-1460).

Figure R2 .
Figure R2.In vitro treatment effect of SCFAs, propionate or butanoate, on EMT markers gene expression.A-F.Western blot analysis of E-cadherin and ZEB1 protein levels in A549 cells treated with sodium propionate (SP) (A-C) or sodium butanoate (SB) (D-F) in the indicated dose-and time-dependent manner for 3 independent times.Left panel shows the blot while middle and right panel shows the quantification for E-Cadherin and ZEB1 levels, respectively.β-Actin was used as an internal control and used it to normalize the protein levels of EMT markers.Relative expression is indicated in comparison to the control of respective time points.

Figure R3 .
Figure R3.In vitro treatment effect of propionate for 12 days on the EMT markers gene expression.A-C.Western blot analysis of E-cadherin and ZEB1 protein expression in time series in A549 cell line treated with sodium propionate (SP) at 5 mM for 12 days.The experiment was performed for 3 independent times.Left panel shows the blot while middle and right panel shows the quantification for E-cadherin and ZEB1 levels, respectively.β-Actin was used as an internal control and used to normalize the protein levels.Relative expression is indicated in comparison to the control of respective time points.

Figure R4 .
Figure R4.In vitro treatment effect of propionate for 3 days on the E-cadherin gene expression.Real-time quantitative PCR analysis of epithelial gene (CDH1) in A549 cell line treated with sodium propionate (SP) at 5 mM for 3 days.GAPDH was used as an internal control.Red dotted line represents the fold change cut-off at 1.5.Significance was calculated using unpaired t-test.*** -p<0.001,**** -p<0.0001.3.All EMT cases during development involve at least one member from the EMT associated transcription factor families (EMT-TF) Snail, Zeb and Twist.However, these genes also have some functions unrelated to the classic EMT process, for example in fibroblast and immune cells.It would appear important to better define putative interactions between SCFA and EMT master genes pathways.Can the SCFA "shortcut"

Figure R5 .
Figure R5.In vitro treatment effect of propionate for 3 days on the EMT-associated transcription factors expression.Western blot analysis of E-cadherin and EMT-transcription factors (ZEB1, ZEB2, TWIST1, SNAIL and SLUG) in A549 cells treated with sodium propionate (SP) at 5 mM in the indicated time-dependent manner.β-Actin was used as an internal control.4.Authors suggest that SCFA impact on epithelial cell differentiation could represent a general mechanism involving EMT processing (l390:" The effect appears to be a general phenomenon"), but at the same time they mention a specific targeting of " the pulmonary alveolar type-II cell-type markers implying the establishment of epithelial integrity in lungs" (l392) by the propionate treatment that would increase specifically the airway Figs R6B-C).Nevertheless, further experiments are needed in future to understand the mechanistic basis of propionate in inhibiting the stemness property of lung cancer.The results are included in the revised manuscript (Page: 13; Line: 318-322; Fig 5K, Figs EV3F and EV3G).

Figure R6 .
Figure R6.Propionate's effect on lung epithelial progenitor and cancer stemness gene expression.A-C.Box plot visualization of the distal lung epithelial progenitor genes (A), ALDH1A1 (B) and ALDH3A1 (C) in RNA-seq profile of SP treated A549 cell line for 3 days and 12 days.Significance was calculated using un-paired t-test between the SP treated cells and the control.ns -non-significant, *** -p<0.001,**** -p<0.0001. 5. Mouse models support the authors finding but do not demonstrate any EMT involvement.The expression of EMT features in the tumors before and after propionate treatment should be checked by classic immunolocalization (or any other approach) to

Figure R7 .
Figure R7. A. Immunohistochemistry staining of EPCAM in lung tissue of KPL-mouse administered with SP.Significance was calculated using un-paired t-test between the SP treated cells and the control.B. Principal component analysis of RNA-seq expression profile of lung tissue samples from Cas9-C57BL/6 mice with lung tumorigenesis intubated with AAV-KPL virus and administered with sodium propionate (SP) in drinking water.C. M-A plot of differential gene expression between sodium chloride and sodium propionate administered Cas9-C57BL/6 mice with lung tumorigenesis intubated with AAV-KPL virus.D. Gene-set ontology of differentially expressed genes from RNA-seq expression profile of lung tissue samples from Cas9-C57BL/6 mice with lung tumorigenesis intubated with AAV-KPL virus and administered with sodium propionate (SP) in drinking water.6.In Fig 2. wound healing experiments are conclusive in showing a delay in cell sheet spreading after SP treatment, but they do not indicate a migration halt.It would be

Figure R8 .
Figure R8.Cell migration analysis in A549 cell line treated with sodium propionate for 3 days.A. Magnified images represent the wound width (highlighted by red region) at the indicated time points in A549 cells treated with SP for 3 days for migration assay.B-D.Line plots depict the rate of wound healing, expressed as relative wound density in A549 cells treated with SP over the course of 72 hours from making the wound for migration assay.7.To study cell plasticity, an essential feature of EMT, it would be important to insert Fig R10 have been included in the revised manuscript (Page: 9; Line: 205; Fig EV1M).

Figure R9 .
Figure R9.Spheroid assay in A549 cells treated with sodium propionate for 3 days.A. Images representing the A549 spheroids inserted into 3D collagen gels treated with sodium propionate (5 mM) for 3 days (n=5).B. Images representing the A549 spheroids anchored to the collagen type I (n=5) treated with SP (5 mM).C. Quantification of migratory pattern of A549 spheroids by anchoring to the collagen type I treated with SP (5 mM).*** p-value < 0.001.

Figure R11 .
Figure R11.Representative H&E images of lung cancer metastasis to axial lymph nodes in KPL mice of control (LN-Control) condition while no metastasis in the sodium propionate (LN-SP) condition.Scale bar represents 200 μm.Red arrows point to the metastatic cells in the lymph nodes.3. Fig3H.It is not clear how many mice were used for this survival study.Response R2.10 We thank the reviewer for pointing out this.In Fig 3H, we have used n=7 mice per group.This is now appended to the revised manuscript in Fig 3H, and also in the corresponding legend of the revised manuscript text (Page: 58; Line: 1493-1495).
We have now provided higher magnification pictures of ZO-1 and EPCAM staining to visualize the localization and organization pattern of the epithelial markers.We detect an increase in the expression of ZO-1 and EPCAM in the membrane regions with SP treatment, indicating the functional level process of epithelial feature establishment with SP (FigR12).We have also provided the magnified images in the revised manuscript (FigEV2K).

Figure R12 .
Figure R12.Magnified images of immunofluorescence staining of EPCAM and ZO-1 in A549 cell line treated with sodium propionate (SP) at 5 mM for 3 days.DAPI was used as a nuclear stain.Scale bar represents 10 µm.5.Discussion is overall too long and redundant.It should be more focused on this manuscript findings.For example, the section l409-l426 about SP and cisplatin resistance is quite interesting, but does not imply EMT pathways.Similarly, the section about SCFA availability (l428-l445) is rather unclear.

Figure R13 .
Figure R13.Western blot analysis of AXL and ZEB2 in A549 cells treated with TGF-β1 (2 ng/ml) for 72 hours.β-Actin was used as an internal control.7. It should be mentioned that most cancer data sets used in this study involve a mix of stroma and tumor cells, an element to consider in the discussion.Response R2.14As suggested by the Reviewer, we have included a statement in the discussion section (Page: 17; Line: 404-409) that "tumor gene expression profiles used in the study from the publicly available database could involve a mix of stroma and tumor cells, and the potential contribution of the tumor microenvironment remains to be investigated".

Figure R14 .
Figure R14.A-D.Western blot analysis of epithelial (E-Cadherin, GRHL1 and EPCAM) and mesenchymal (ZEB1, BCAT1 and NNMT) markers in CALU-1 (A and B) and in H1299 (C and D) cell lines treated with sodium propionate (SP) or sodium butanoate (SB) in the indicated dose-dependent manner for 48 hours.β-Actin was used as an internal control.2.The authors analyzed gene expression databases to identify the metabolic processes.They assumed that short-chain fatty acids are regulated at the transcription level.However, they did not validate or elaborate on this analysis.For example, which enzymes in the short-chain fatty acid biosynthesis pathway are downregulated during the EMT program or in epithelial vs. mesenchymal cells?What will be the outcome of their ectopic expression in epithelial and mesenchymal cells?What will be the outcome of manipulating the enzymes synthesizing these fatty acids?Will the cells induce the EMT program?Response R3.2 Very good point.We previously carried out knock down of metabolic enzymes involved in main (PCCB, MCEE and MUT) and shunt (HIBCH and ALDH6A1) propionate metabolism and did not observe any change in E-cadherin/ ZEB1 in cells treated with SP (Appendix FigsS6B and S6C).Interestingly, it has recently been shown that deregulation in the propionate metabolism can contribute to an anaplerotic reaction in the accumulation of methylmalonic acid (MMA) in breast and lung cancer leading to EMT and metastasis(Gomes et al, 2022).In apparent contrast to this, we observe that treatment with propionate results in the epigenetic involvement in inducing epithelial identity implying a dual and opposing effect on EMT.This has been detailed in the discussion section of the manuscript (Page: 19; Line: 448-455).

Figure R15 .
Figure R15.Western blot analysis of epithelial (E-cadherin), mesenchymal (ZEB1), and propionate metabolic genes (PCCA and PCCB) in A549 cell line treated with sodium propionate (SP) in the indicated dose-and time-dependent manner.β-Actin was used as an internal control.3.One of the main themes in this manuscript is that the short-chain fatty acids function as inhibitors of the EMT.However, the physiological relevance of these findings is still missing as they did not validate any changes in the fatty acid intracellular levels.For example, does the intracellular level of propionate and butanoate reduce during the EMT? Response R3.3 Very important point.As suggested by the Reviewer, we have now performed an experiment to quantify intracellular propionate during EMT.We induced EMT by treating a large amount of A549 cells (around 7.5 million cells per replicate) with TGF-β1 for 4 days (FigR16A), and performed mass spectrometry analysis to quantify the intracellular propionate at different time points (48h, 72h and 96h) using 13C labelled standards (Figs R16B-C).Since the basal level of propionate in the cells was below or around the blank estimate, we were technically unable to quantify intracellular amount of propionate (FigR16D).However, we tested if the treatment of A549 cell line with sodium propionate could increase the intracellular propionate.As expected, a very significant increase (Folds=~27; p=0.0006) was detected (FigR16E), implying that the quantification method is working and that the physiological origin of propionate may not be intracellular.
[...]  [...]   We have added the quantification of the levels of propionate upon treatment to the revised manuscript (Page: 8; Line: 188-191; Fig EV1H)because we believe it nicely confirms the high cellular uptake for potential treatment purpose (Page: 22; Line: 543-551).We did not address butanoate as the main focus of the manuscript was on propionate.Thanks for the input.

Figure
Figure for reviewers (and associated text above) removed Fig R17A) or by co-treatment of cells with SCFA and TGF-β1 (Fig R17B).In both cases, we observed NNMT to be down-regulated by SCFA's action.

Figure
FigureR17. A. Western blot analysis of E-cadherin, ZEB1 and NNMT protein levels in A549 cells pre-treated with sodium propionate (SP, 5 mM) or sodium butanoate (SB, 1 mM) for 48 hours followed by TGF-β1 (2 ng/ml) for 24 hours.β-Actin was used as an internal control.B. Western blot analysis of E-cadherin, ZEB1 and NNMT protein levels in A549 cells co-treated with sodium propionate (SP, 5 mM) or sodium butanoate (SB, 1 mM) in combination with TGF-β1 (2 ng/ml) for 72 hours.β-Actin was used as an internal control.

Figure R18 .
Figure R18.A-C.Line plot depicts the rate of wound healing, expressed as relative wound density from making the wound for migration assay in pre-treated A549 cells with SP for 48 hours followed by TGFβ1 for 24 hours (A), in co-treated SP and TGFβ1 A549 cells (B), and in co-treated SB and TGFβ1 A549 cells (C) over the course of 72 hours.Significance was calculated using Two-way ANOVA followed by inter-group comparison using Tukey's multiple comparison test.**** p-value < 0.0001.Minor comments 1.The HR (hazard ratio) value is absent from all of theKaplan-Meier plots that pertain to survival rates.Response R3.5 We thank the reviewer for the suggestion, we have provided the hazard ratios for all the survival figures (Fig 1F, Fig 3H, and Figs EV3H and EV3I) along with the corresponding confidence intervals in the revised manuscript.
).We have included the quantification in the Fig R2 and Fig R3 for E-cadherin and ZEB1.In addition, as suggested by the Reviewer, we have also shown that SP inhibits EMT by few additional EMT-specific markers such TWIST1 and NNMT wherein these markers were downregulated with the SP treatment (Fig R19).The result is included in the revised manuscript (Page: 8; Line: 186-188; Appendix Fig S2E).
treated with sodium propionate for 3 days.A. Left panel, Images representing the A549 spheroids inserted into 3D collagen gels treated with sodium propionate (5 mM) for 3 days (n=5).Right panel, Quantification of migratory pattern of A549 spheroids by anchoring to the collagen type I treated with SP (5 mM).B. Left panel, Images representing the A549 spheroids anchored to the collagen type I (n=5) treated with SP (5 mM).Right panel, Quantification of migratory pattern of A549 spheroids by anchoring to the collagen type I treated with SP (5 mM).
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