Lipid droplets and ferritin heavy chain: a devilish liaison in human cancer cell radioresistance
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Abstract
Although much progress has been made in cancer treatment, the molecular mechanisms underlying cancer radioresistance (RR) as well as the biological signatures of radioresistant cancer cells still need to be clarified. In this regard, we discovered that breast, bladder, lung, neuroglioma, and prostate 6 Gy X-ray resistant cancer cells were characterized by an increase of lipid droplet (LD) number and that the cells containing highest LDs showed the highest clonogenic potential after irradiation. Moreover, we observed that LD content was tightly connected with the iron metabolism and in particular with the presence of the ferritin heavy chain (FTH1). In fact, breast and lung cancer cells silenced for the FTH1 gene showed a reduction in the LD numbers and, by consequence, became radiosensitive. FTH1 overexpression as well as iron-chelating treatment by Deferoxamine were able to restore the LD amount and RR. Overall, these results provide evidence of a novel mechanism behind RR in which LDs and FTH1 are tightly connected to each other, a synergistic effect that might be worth deeply investigating in order to make cancer cells more radiosensitive and improve the efficacy of radiation treatments.
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Referee #2
Evidence, reproducibility and clarity
Summary:
In the study, the authors found that cancer cells including breast, bladder, lung, neuroglioma, and prostate display an increase of Lipid Droplet (LD) after 6 Gy x-ray (Fig.1). And, the cells containing high LDs showed more radioresistance than the cells with low LDs after irradiated with a dose Gy x-ray (Fig.2). Ferritin Heavy Chain (FTH1), the main intracellular iron storage protein, is found to be upregulated after 6 Gy exposure or in the LDs high cells. FTH1 knockdown decreases LD accumulation and increases radiation sensitivity (Fig.3). Overexpression of FTH1 or DFO (an iron chelator agent) treatment in shFTH1 cells …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
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Referee #2
Evidence, reproducibility and clarity
Summary:
In the study, the authors found that cancer cells including breast, bladder, lung, neuroglioma, and prostate display an increase of Lipid Droplet (LD) after 6 Gy x-ray (Fig.1). And, the cells containing high LDs showed more radioresistance than the cells with low LDs after irradiated with a dose Gy x-ray (Fig.2). Ferritin Heavy Chain (FTH1), the main intracellular iron storage protein, is found to be upregulated after 6 Gy exposure or in the LDs high cells. FTH1 knockdown decreases LD accumulation and increases radiation sensitivity (Fig.3). Overexpression of FTH1 or DFO (an iron chelator agent) treatment in shFTH1 cells rescue the LD accumulation and cancer radioresistance (Fig.4)
Major comments:
- The conclusion has some conflict with some publication (PMC5928893) which shows fatty acid oxidation not LDs lead to cancer radioresistance. So the authors should rule out this possibility through knockdown of CPT1.
- Lipid droplets are dynamic in cells. The sorted cells (10% highest or lowest LD-expressing cells) in Fig.3 may not stand for subpopulation, so the authors should add exogenous lipids or cholesterol to test cancer cell radioresistance.
- It is impossible to overexpress FTH1 in shFTH1 cells (the stable shRNA will target all mRNA of FTH1) (Fig.4 and methods section: cell culture and FTH1 Reconstitution).
- The relationship between the free cytoplasmic iron and LD accumulation is not so convincing. Add exogenous iron to test LD accumulation.
Minor comments:
- Remove fig.1C which is not related to the conclusion;
- Fig.2 label error: LD520 should be LD540;
- Fig.3, A: change loading control HSC70 which not so stable in the cells; D: add quantification of LD number.
- Fig.4, A: change loading control HSC70, and repeat western of MCF7 shFTH1/pcDNA3
- Line 111, "...that general ROS levels resulted not altered..." should be "...that general ROS levels were not altered..."
- Fig.S4 legend: "Figure S2" should be "Figure S4".
Significance
The FTH1 affects Lipid Droplets is novel (some results in this study have published: radiation led to LD accumulation (PMC5928893) and an increase of FTH1 (PMC4688087 and PMID:32937103).
The finding is helpful to improve radiation therapy which may combine with drugs targeting FTH1 or iron metabolism.
The researchers who worked in cancer treatment are interested in this finding.
My expertise is cancer lipid metabolism and cancer therapy.
Referee Cross-commenting
I completely agree the comments from the other Reviewer. The authors need enhance the correlation between the lipid droplets genes (e.g., DGAT1/2, SOAT1, ACAT1) and the iron metabolism (e.g., FTH1), and improve data quality as suggested by reviewers.
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Referee #1
Evidence, reproducibility and clarity
In this work Tirinato and co-authors used different experimental approaches to trace correlations between cancer cell stemness, lipid droplets, iron homeostasis and radiation resistance. Their findings were acquired by using different cancer cell lines from different origin, and using diverse techniques, including cytometry, microscopy, clonogenic assays and shRNAi. In general, the manuscript is well written and organized. It is also easy to be followed and the authors managed to convince the readers about the importance of this important aspect of cancer metabolism. The fact that cell lipid droplets content might condition cancer …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
In this work Tirinato and co-authors used different experimental approaches to trace correlations between cancer cell stemness, lipid droplets, iron homeostasis and radiation resistance. Their findings were acquired by using different cancer cell lines from different origin, and using diverse techniques, including cytometry, microscopy, clonogenic assays and shRNAi. In general, the manuscript is well written and organized. It is also easy to be followed and the authors managed to convince the readers about the importance of this important aspect of cancer metabolism. The fact that cell lipid droplets content might condition cancer survival to radiotherapy poses novelty and therefore it deserves attention in the delimitation of new anticancer therapies or protocols. There are however some issues that should be amended to improve the quality of the manuscript.
Major comments:
The main concern is that all the work is based on cancer cell lines. The use of some cell lines derived from clinical samples or analysis of the clinical data already deposited in bank webs could be useful to support their conclusions. This last could be easy. Exploration of this depositories could help the author to reinforce the correlation between the expression of the lipid droplets genes, as well as that related with the iron metabolism, with the radiotherapy efficacy in patients.
When analyzed in detail I have some comments regarding specific sections:
Lipid droplets detection images (Figs 1, 3 y 4). Why are the nuclei size look so different in both conditions? FTH1 expression. Fig 3A vs 3C and 4A. As depicted is difficult to have a clear picture of the variations in expression in FHT1 in the different cell lines. Why the authors evaluate the success of shRNAi by PCR if the wb works so well? Is there any correlation between the RR and the expression of FTH1 intra cell lines? What happen when FTH1 is downregulated in the rest of the cell lines? More than restore, the authors overexpressed FTH1, what is the result when FTH1 is overexpressed in the different cell lines?
Minor comments:
The correlation between nile red staining and ROS is not clear (Fig S2). The authors may try to graphic the ROS mfi of different subpopulations (lineal in X) vs the mfi of red nile (y, in log scale). Abreviation in introduction ER, is endoplasmic reticulum? A picture with a putative model could be helpful to summarize the findings.
Significance
As stated above the idea that lipid droplets and iron metabolism might be determinants in the cancer survival to radiotherapy poses novelty and therefore it deserves attention in the delimitation of new anticancer therapies or protocols.
Although I have experience in cancer lipid metabolism I am not an expert in the field of lipid droplets.
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