Lipid Droplets and Ferritin Heavy Chain: a Devilish Liaison in Cancer Radioresistance

Although much progress has been made in cancer treatment, the molecular mechanisms underlying cancer radioresistance (RR) as well as the biological characteristic 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 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 restoration 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 molecular mechanism behind RR in which LDs and FTH1 are tightly connected to each other, a synergistic effect which might be worth deeply investigating in order to make cancer cells more radiosensitive and improve the efficacy of radiation treatments.

eventually induce cell death. The molecular mechanisms activated by cancer cells in response to 43 ionizing radiation are extensively investigated and many advances have been so far made, but 44 considerably many questions are still unanswered and much remains poorly understood. Cancer 45 cell radioresistance (RR) makes different tumor types difficult to treat. In this regard, the presence 46 within the tumor mass of a small cell subpopulation called Cancer Stem Cells (CSCs) or Cancer Initiating-Cells (CICs) seems to represent one of the driving forces contributing to tumor resistance 48 and recurrence after radiotherapy treatments 1 .

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Recently, lipid metabolic reprogramming in cancer cells has become a central aspect of cancer 50 aggressiveness 2,3 . In particular, an increase of small lipid organelles inside cancer cells, namely 51 lipid droplets (LDs), has been shown to correlate with a CSC phenotype in colon 4 ovary 5 , breast 6 52 and glioblastoma 7 .

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Cell survival upon radiation treatment is also modulated by several tumor parameters such as 54 hypoxia, oxidative stress, inflammation, acidic stress, and low glucose, all of which have been 55 reported to mediate their effects through iron metabolism 8 .

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To date, altered expression and activity of many iron-related proteins in cancer cells have been 57 reported and associated to cancer progression and metastasis 9,10 . In fact, an uncontrolled balance 58 of iron results in the free radical production through the Fenton reaction (Fe 2+ + H2O2 → Fe 3+ + •OH 59 + OH -) and free radicals are considered strong contributors to tumor proliferation and 60 aggressiveness 11 . Among all molecules involved in iron metabolism, ferritin is responsible for the 61 cytoplasmic iron storage and the maintenance of the redox homeostasis. Ferritin is a protein 62 complex composed of two chains, light (FTL) and heavy (FTH), and its clinical importance has been 63 demonstrated in many cancers through multiple roles: the contribution to tumorigenesis, the 64 restoration of tumor-dependent vessel growth and the association with tissue invasion 8,12 .

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Moreover, high levels of ferritin are often found in patients with various advanced cancers which 66 could potentially be treated with radiotherapy 13 , although iron homeostasis is still poorly 67 investigated in the context of radiation oncology.

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A recently published paper highlighted a very intriguing relationship between iron balance and LDs.

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The Authors showed that iron depletion caused ER expansion and, as a consequence, LD

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This study demonstrates that radioresistant cancer cells of different origin (neuroglioma, lung, 75 breast, bladder and prostate) were characterized by a higher expression of LDs. The subpopulation 76 containing the highest amount of LDs (LD High ) showed a higher clonogenic potential compared to 77 the LD Low counterpart. Interestingly, the number of cytoplasmic LDs was directly correlated with the 78 amount of FTH1. In fact, FTH1 knockdown in lung H460 (H460 shFTH1 ) and breast MCF7 79 (MCF7 shFTH1 ) reduced the LD amount and increased the sensitivity to ionizing radiation. FTH1 80 restoration as well as the treatment with an iron chelating agent in MCF7 shFTH1 and H460 shFTH1 81 restored the LD amount and increased their resistance to radiation treatment.

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Altogether, these data provide evidence of a new pivotal role for LDs in cancer RR linking their 83 expression with iron metabolism and specifically to FTH1 expression.

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To verify whether LD content was affected by ionizing radiation treatment, H4 (neuroglioma), H460 89 (lung), MCF7 (breast), PC3 (prostate) and T24 (bladder) cancer cells were treated with 6 Gy X-ray 90 and left in culture for 72 hours (hrs) in order to select only resistant/surviving cells. Treated and 91 untreated cancer cells were stained with LD540 and imaged at the confocal microscope for the 92 detection of LDs.

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As shown by z-projection confocal microscopy images, surviving cancer cells were characterized 95 by a significant increase of LDs for all the aforementioned cell lines (Figure 1A). Although the LD 96 increase was a common feature observed in all cell lines, the relative LD ratio between treated and 97 untreated cells showed little differences, with H460 exhibiting the highest amount ( Figure 1B). LD 98 Figure 1: Lipid Droplet detection in neuroglioma (H4), lung (H460), breast (MCF7), prostate (PC3) and bladder (T24) 6 Gy X-ray resistant cancer cells. Cancer cells have been irradiated with 6 Gy X-ray and left in culture for 72 hrs. Afterwards, surviving and untreated cells have been stained with LD540 and imaged at the fluorescence confocal microscope. Zprojection of the z-stack acquisitions for untreated and 6 Gy treated cells are reported in column A (Scale bar, 20 M). B) For each cell line, 50 cells have been randomly imaged, and their LD number counted by using FiJi software. C) qPCR analysis of the PLIN genes in the indicated cell lines. PLIN5 in the H4 6 Gy treated cells is not reported in the graph because it was not expressed. Error bars represent the means  SD from three independent experiments. *  0.05; **  0.01; ***  0.001 and **** 0.0001.
for the proteins associated with LD surface and they are involved in their biogenesis as well as in several other roles 15 . Differences in tissue expression have been reported for all the PLIN genes 101 (PLIN 1-5). Accordingly, we observed that, after 6 Gy radiation treatment, PLIN1 was up-regulated 102 in H460, MCF7, PC3 and T24; PLIN2 was down-regulated in H460; PLIN3 showed mRNA 103 increased expression in H4 and MCF7; PLIN4 expression was incremented in MCF7 and T24;

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PLIN5 resulted down-regulated in MCF7 and up-regulated in PC3 and T24.

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It is well known that photon radiation acts, at the molecular level, producing reactive oxygen species 106 (ROS) 8 . In this regard, we found that cytoplasmatic ROS, measured by means of fluorogenic CM-107 H2DCFDA probe, were significantly upregulated in H4, H460, MCF7 and PC3, while no differences 108 were detected in T24, after radiation ( Figure S1). Moreover, H4, H460 and PC3 showed 109 upregulated levels of SOD1, SOD2 and catalase, respectively. SOD2 mRNA was also upregulated 110 in T24, despite the fact that general ROS levels resulted not altered 72 hrs after radiation treatment, 111 while it was downregulated in radiation treated MCF7.

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In order to deal with this ROS increase, cancer cells need to tune their ROS scavenging systems    One of the main cellular ROS sources is the Fenton reaction, in which the Fe 2+ reacts with hydrogen 141 peroxide (H2O2) to produce Fe 3+ and highly reactive radicals, such as the hydroxyl radical (•OH).

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Since the Ferritin is the main intracellular iron storage protein, we investigated the FTH1 role in 143 radiation resistance.

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We found that FTH1 protein was upregulated in all resistant cell lines after 72 hrs from 6 Gy 145 exposure, as reported in Figure 3A. Moreover, H460 and MCF7, sorted on the basis of their LD 146 content, were characterized by an increase in the mRNA level of FTH1 in the LD high subpopulation 147 compared to the LD low cells ( Figure 3B).

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To better clarify this link, FTH1 was silenced in H460 and MCF7 (H460 shFTH1 and MCF7 shFTH1) , the 149 efficiency of which is shown in Figure 3C.  PCR results showed that in H460 shFTH1 and MCF shFTH1 there was a clear FTH1 mRNA reduction compared with their relative controls. D) LD content was measured in H460 shFTH1 and MCF7 shFTH1 by confocal microscopy. LD540 staining revealed that the FTH1 gene silencing caused a LD decrease in both cell systems. (Scale bars 20 M). E) Cellular irradiation response in H460 and MCF7 silenced for FTH1 was investigated by radiobiological clonogenic assay and compared with H460 shRNA and MCF7 shRNA respectively. Survival fraction (in log-linear scale) is reported in the panel E. Error bar represents the means  SD from three independent experiments. *  0.05; **  0.01; ***  0.001 and **** 0.0001. decreased, LDs were also reduced and this significantly impaired cancer RR.

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Iron Imbalance as well as FTH1 reconstitution re-establish LD expression and radiation 165 resistance 166 As well known, the FTH1 role is crucial for the iron storage within the cell and the maintenance of 167 the redox homeostasis. When its expression is downregulated, the 168 balance between the iron uptake and release is compromised. By consequence, the free cellular 169 iron amount becomes critical for the correct cellular functions 10,21 . Here we found that this iron 170 Figure 4: FTH1 reconstitution as well as DFO treatment restore the LD content re-establishing cancer radioresistance. A) Western Blotting analysis of FTH1 expression in MCF7 shFTH1/pcDNA3FTH1 and H460 shFTH1/pcDNA3FTH1. HSC70 was used as loading control. B) Z-stack representative confocal fluorescence images of LD detection in MCF7 shFTH1/pcDNA3FTH1 and H460 shFTH1/pcDNA3FTH1 cells and their H460 shFTH1/pcDNA3 and MCF7 shFTH1/pcDNA3 controls. (Scale bars 20 M). (C) Survival fractions (in log-linear scale) after FTH1 reconstitution in MCF7-and H460-shFTH1 cells. The iron chelator agent Deferoxamine (DFO), whose chemical structure is reported in D, was used for treating MCF7 shFTH1 and H460 shFTH1 for 24 hrs. (E) In both cell lines, DFO treatment increased the LD numbers, as showed by confocal microscopy images (Scale bar 20 M). (F) Survival curves (in log-linear scale) of FTH1-silenced MCF7 and H460 cells after DFO treatment. F. Error bar represents the means  SD from three independent experiments. *  0.05; **  0.01; ***  0.001 and **** 0.0001. deficiency on LD content and radiosensitivity, we reconstituted the FTH1 expression by full length 172 FTH1 cDNA transfection to further verify such connection. Figure 4A shows that FTH1 gene 173 restoration successfully raised FTH1 protein expression up in both MCF7 shFTH1 + pcDNA3FTH1 and 174 H460 shFTH1 + pcDNA3FTH1.

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Moreover, such a reconstitution was sufficient to fully restore the LD pool ( Figure 4B) and to 176 reacquire a higher RR in both cell lines ( Figure 4C).

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To further elucidate the connection between iron and LDs, we used an iron chelator agent,

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Deferoxamine (DFO), to cope with the iron imbalance due to the FTH1 silencing. DFO is a high-179 affinity Fe 3+ chelator and an FDA approved drug used to treat patients with iron overload.

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H460 shFTH1 and MCF7 shFTH1 were treated with DFO for 24 hrs and their LD content was analyzed.

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LD540 staining on both treated H460 shFTH1 (H460 shFTH1 + DFO) and MCF7 shFTH1 (MCF7 182 shFTH1 + DFO) univocally showed that the iron chelation was able to induce LD accumulation and 183 this, in turn, conferred higher survival ability to cells after radiation treatment, as shown by   In support of that, we demonstrated that a higher LD content was a feature already present in the 221 heterogeneous populations, as pre-sorted (LD high and LD low ) cells displayed differential survival 222 capacity after radiation, with the LD high subpopulation displaying the highest clonogenic response.

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This indicates that the presence of a higher LD amount was an intrinsic feature of the cells and 224 may represent a selective advantage which might allow resistant cells to survive damages, 225 including oxidative stress induced by ROS production following exposure to ionizing radiation.

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Many intracellular mechanisms participate in ROS production and the Fenton reaction is one of 227 them. In this reaction, ferrous ion is used as a catalyst to convert H2O2 into the highly oxidative 228 hydroxyl radical (OH•). Iron is an important player in normal cells because it is involved in many 229 processes and therefore its homeostasis is tightly regulated. However, in cancer cells iron

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In this work, we investigated the effects of X-ray radiation on RR cancer cells in order to determine

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Although the data reported here need to be validated in more physiologically complex systems,

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Irradiation has been carried out using a Multi Rad 225kV irradiator. Cells, seeded at a density of

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(Thermo Fischer Scientific). Cells were then stained with LD540 for 10 min at 37°C in the incubator.

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The excess of dye was washed away with PBS and cells were resuspended in sorting buffer (PBS 324 Ca/Mg-free, BSA 0,5%, EDTA 2 mM and Hepes 15mM).

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Cells were sorted in two populations (LD High and LD Low ) using a FACSAria Fusion (BD Bioscience).

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Sorting gates were established based on the 10% most bright and 10% most dim subpopulation.

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Student's t-test with unequal variances was used for the calculation of statistical significances.

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Differences of two groups with P values below 0.05 were considered statistically significant.

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MCF7 shFTH1 and H460 shFTH1 cells were seeded in six-well plates at 3 × 10 5 cells/well and 407 grown overnight prior to transfection.