Chemotherapy resistance due to epithelial-to-mesenchymal transition is caused by abnormal lipid metabolic balance

Reviewer #1 (Public review):

The authors focus on the molecular mechanisms by which EMT cells confer resistance to cancer cells. The authors use a wide range of methods to reveal that overexpression of Snail in EMT cells induces cholesterol/sphingomyelin imbalance via transcriptional repression of biosynthetic enzymes involved in sphingomyelin synthesis. The study also revealed that ABCA1 is important for cholesterol efflux and thus for counterbalancing the excess of intracellular free cholesterol in these snail-EMT cells. Inhibition of ACAT, an enzyme catalyzing cholesterol esterification, also seems essential to inhibit the growth of snail-expressing cancer cells.

However, It seems important to analyze the localization of ABCA1, as it is possible that in the event of cholesterol/sphingomyelin imbalance, for example, the intracellular trafficking of the pump may be altered.
The authors should also analyze ACAT levels and/or activity in snail-EMT cells that should be increased. Overall, the provided data are important to better understand cancer biology.

Reviewer #2 (Public review):

Summary:

In this study, the authors discovered that the chemoresistance in RCC cell lines correlates with the expression levels of the drug transporter ABCA1 and the EMT-related transcription factor Snail. They demonstrate that Snail induces ABCA1 expression and chemoresistance, and that ABCA1 inhibitors can counteract this resistance. The study also suggests that Snail disrupts the cholesterol-sphingomyelin (Chol/SM) balance by repressing the expression of enzymes involved in very long-chain fatty acid-sphingomyelin synthesis, leading to excess free cholesterol. This imbalance activates the cholesterol-LXR pathway, inducing ABCA1 expression. Moreover, inhibiting cholesterol esterification suppresses Snail-positive cancer cell growth, providing potential lipid-targeting strategies for invasive cancer therapy.

Strengths:

This research presents a novel mechanism by which the EMT-related transcription factor Snail confers drug resistance by altering the Chol/SM balance, introducing a previously unrecognized role of lipid metabolism in the chemoresistance of cancer cells. The focus on lipid balance, rather than individual lipid levels, is a particularly insightful approach. The potential for targeting cholesterol detoxification pathways in Snail-positive cancer cells is also a significant therapeutic implication.

Weaknesses:

The study's claim that Snail-induced ABCA1 is crucial for chemoresistance relies only on pharmacological inhibition of ABCA1, lacking additional validation. The causal relationship between the disrupted Chol/SM balance and ABCA1 expression or chemoresistance is not directly supported by data. Some data lack quantitative analysis.

Author response:

Response to Reviewer 1

We will investigate the intracellular localization of ABCA1 in both EpH4 and EpH4-Snail cells. We will also examine the changes in ACAT expression levels within these cell lines.

Response to Reviewer 2

We will first investigate whether the chemoresistance exhibited by EpH4-Snail cells can be abolished not only through pharmacological inhibition of ABCA1 but also by knocking out the ABCA1 gene. Regarding causality, as demonstrated in Figure 2, we have already shown that reducing cholesterol levels in EpH4-Snail cells decreases ABCA1 expression. To further explore this relationship, we will assess whether increasing sphingomyelin levels by adding ceramide to the culture medium, thereby correcting the sphingomyelin-to-cholesterol ratio, would reduce ABCA1 expression. Furthermore, we will evaluate whether lowering cholesterol levels in EpH4-Snail cells via simvastatin treatment, along with normalization of the sphingomyelin-to-cholesterol ratio, attenuates their resistance to the anticancer drug nitidine chloride. Additionally, we will incorporate quantitative analyses for several experiments, as suggested in the reviewers’ comments, to enhance the robustness of our findings.