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.2021 Nov 8;40(1):350.
doi: 10.1186/s13046-021-02168-2.

PGRMC1-dependent lipophagy promotes ferroptosis in paclitaxel-tolerant persister cancer cells

Affiliations

PGRMC1-dependent lipophagy promotes ferroptosis in paclitaxel-tolerant persister cancer cells

Ji Hyeon You et al. J Exp Clin Cancer Res..

Abstract

Background: Progesterone receptor membrane component 1 (PGRMC1) is a heme-binding protein inducing dimerization with cytochrome P450, which mediates chemoresistance. Increased PGRMC1 expression is found in multiple types of resistant cancers, but the role of PGRMC1 in the ferroptosis of cancer cells remains unrevealed. Therefore, we examined the role of PGRMC1 in promoting ferroptosis in paclitaxel-tolerant persister cancer cells (PCC).

Methods: The effects of ferroptosis inducers and PGRMC1 gene silencing/overexpression were tested on head and neck cancer (HNC) cell lines and mouse tumor xenograft models. The results were analyzed about cell viability, death, lipid ROS and iron production, mRNA/protein expression and interaction, and lipid assays.

Results: PCC had more free fatty acids, lipid droplets, and fatty acid oxidation (FAO) than their parental cells. PCC was highly sensitive to inhibitors of system xc- cystine/glutamate antiporter (xCT), such as erastin, sulfasalazine, and cyst(e)ine deprivation, but less sensitive to (1S,3R)-RSL3. PGRMC1 silencing in PCC reduced ferroptosis sensitivity by xCT inhibitors, and PGRMC1 overexpression in parental cells increased ferroptosis by xCT inhibitors. Lipid droplets were degraded along with autophagy induction and autophagosome formation by erastin treatment in PCC. Lipophagy was accompanied by increased tubulin detyrosination, which was increased by SIRT1 activation but decreased by SIRT1 inhibition. FAO and lipophagy were also promoted by the interaction between lipid droplets and mitochondria.

Conclusion: PGRMC1 expression increased FAO and ferroptosis sensitivity from in vivo mice experiments. Our data suggest that PGRMC1 promotes ferroptosis by xCT inhibition in PCC.

Keywords: Fatty acid oxidation; Ferroptosis; Lipophagy; Progesterone receptor membrane component 1; Tubulin detyrosination.

Plain language summary

Paclitaxel-tolerant persister cancer cells (PCC) had PGRMC1 upregulation related to increased free fatty acids, lipid droplets, and fatty acid oxidation. PGRMC1 expression substantially increased ferroptosis by xCT inhibition via lipophagy and tubulin detyrosination, whereas PGRMC1 silencing decreased ferroptosis: this suggests that PGRMC1 expression promotes ferroptosis in PCC.

© 2021. The Author(s).

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Conflict of interest statement

All authors declare no conflict of interests.

Figures

Fig. 1
Fig. 1
Paclitaxel-tolerant persister cancer cells (PCC) have a metabolic shift to fatty acid oxidation.A PCC was made from HN3 and HN4 cells using 10 nM paclitaxel for 6 days and then was maintained without paclitaxel for 38 days before the second 6-day drug treatment to get re-derived PCC. Scale bar 10 μm.B Cell viability was measured using cell counting kit-8 (CCK-8) assay after 10 nM paclitaxel treatment for 48 h in parental cells (ctr) and PCC. Data are means and s.d. from three technical replicates. **P < 0.01, ***P < 0.001 relative to DMSO control.C Immunostaining of α-tubulin (green) in HN3 and HN4 parental cells and PCC. Nuclei (blue) were stained with 4′,6-diamidino-2-phenylindole (DAPI). Scale bar 10 μm.D andE Extracellular acidification rate (ECAR) assay in HN3 and HN4 parental cells and PCC. ECAR was measured using a microplate fluorometer at 15 min intervals, and the glycolysis effect was examined from ECAR assay at 120 min. **P < 0.01 relative to parental cells.F Cellular glutamate was quantified in parental cells and PCC. **P < 0.01 relative to parental cells.G-I Free fatty acids and fatty acid oxidation (FAO) were measured in parental cells and PCC. FAO was quantified via assessing changes in oxygen consumption (OCR) and calculated as a formula (sample untreated with etomoxir minus sample treated with 10 μM etomoxir). Data are means and s.d. and from three technical replicates. **P < 0.01 relative to parental cells.J-L Oil red O staining, immunoblotting, and lipid droplet staining in parental cells and PCC. Scale bars 100 μm (J) and 10 μm (L)
Fig. 2
Fig. 2
PCC is vulnerable to xCT inhibitors.A-C Cell death and viability assays in HN4 parental cells and PCC. Cell death was assessed using SYTOX™ Green stain in the cells treated with or without 2 μM ferrostatin-1 plus 1 μM RSL3, 10 μM erastin, 0.5 mM sulfasalazine (SAS), or cyst(e)ine deprivation for 48 h. Dead cells were quantified by counting SYTOX Green positive cells. Cell viability was examined using a CCK-8 assay. Scale bar 100 μm. Data are means and s.d. from three technical replicates. ns, non-significance; *P < 0.05, **P < 0.01, ***P < 0.001 relative to PCC.D Lipid peroxidation was examined using BODIPY™ C11 and fluorescence-activated cell sorting (FACS) in parental cells and PCC after exposure to the ferroptosis inducers of 1 μM RSL3, 10 μM erastin, 0.5 mM SAS, and cyst(e)ine deprivation for 8 h. *P < 0.05, **P < 0.01 relative to parental cells.E andF Relative glutathione (GSH) and free fatty acid contents in parental cells and PCC after treatment with ferroptosis inducers for 24 h; 1 μM RSL3, 10 μM erastin, 0.5 mM SAS, or cyst(e)ine deprivation. *P < 0.05, **P < 0.01 relative to parental cells.G andH FAO in parental cells and PCC with or without 10 μM erastin was quantified via assessing changes in OCR when exposed to etomoxir or not. *P < 0.05, **P < 0.01 relative to parental cells.I andJ Oil red O staining and immunoblotting in parental cells and PCC after treatment with ferroptosis inducers for 24 h; 1 μM RSL3, 10 μM erastin, 0.5 mM SAS, and cyst(e)ine deprivation. Scale bar 100 μm
Fig. 3
Fig. 3
PGRMC1 expression induces ferroptosis.A andB Immunoblotting in HN4PCC with vector or shPGRMC1 transfection (A) and HN4 parental cells with control or PGRMC1 overexpression vector transfection (B).C-E Cell viability, lipid peroxidation, and labile iron pool in the PCC with vector or shPGRMC1. The cells were examined after treatment with ferroptosis inducers: 1 μM RSL3, 10 μM erastin, 0.5 mM SAS, and cyst(e)ine deprivation for 48 h for cell viability and 8 h for lipid peroxidation and labile iron pool. Data are means and s.d. from three technical replicates. *P < 0.05, **P < 0.01, ***P < 0.001 relative to vector control (vtr).F-H Cell viability, lipid peroxidation, and labile iron pool in HN4 parental cells and PGRMC1 overexpression cells were detected after treatment with ferroptosis inducers: 1 μM RSL3, 10 μM erastin, 0.5 mM SAS. *P < 0.05, **P < 0.01, ***P < 0.001 relative to vector control.I Immunoblotting in HN4 parental cells with control or PGRMC1 overexpression vector transfection and HN4PCC with vector or shPGRMC1 transfection
Fig. 4
Fig. 4
PGRMC1 promotes ferroptosis via lipophagy.A andB Lipid droplets in HN4 parental cells and PCC with or without erastin treatment. HN4 cells were transfected with vector (vtr) or PGRMC1 overexpression (O/E) vector or treated with 100 nM progesterone (P4). HN4PCC were transfected with vector or shPGRMC1 or treated with 20 μM AG205, a PGRMC1 antagonist. The cells were treated with or without 30 nM Wortmannin plus DMSO or 10 μM erastin for 24 h. Lipid droplets (green) were quantified using ImageJ, displayed as a heatmap relative to PCC vector control. Scale bar 10 μm.C Immunoblotting in HN4 parental cells and PCC with or without PGRMC1 overexpression or inhibition and with DMSO or 10 μM erastin treatment for 24 h.D Co-staining of Lysotracker™ Deep Red and LC3-GFP (green) in HN4 parental cells and PCC with or without PGRMC1 overexpression or inhibition and with DMSO or 10 μM erastin for 4 h. Nuclei (blue) were stained with DAPI. Scale bar 10 μm.E andF Quantification of cellular lipid contents by gas chromatography and mass spectrometer (GC-MS) and of free fatty acids in HN4 parental cells and PCC with or without PGRMC1 overexpression or inhibition. The GC-MS data were normalized to HN4 vector control. O/E, PGRMC1 overexpression vector; sh, shPGRMC1; SFA, saturated fatty acids; MUFA, monounsaturated fatty acids; PUFA, polyunsaturated fatty acids. Data are means and s.d. from three technical replicates. ***P < 0.001 relative vector control
Fig. 5
Fig. 5
PGRMC1 promotes lipophagy via increased tubulin detyrosination.A Co-staining of lipid droplet (LD, green) and detyrosinated α-tubulin (red) in HN4 parental cells and PCC with or without PGRMC1 overexpression or inhibition. HN4 cells were transfected with control or PGRMC1 overexpression vector (vtr) or treated with 100 nM P4. HN4PCC were transfected with vector or shPGRMC1, or 20 μM AG205. The cells were also treated with DMSO, 10 μM erastin, or 10 μM erastin plus 20 μM parthenolide (PTN), a sesquiterpene lactone inhibiting the activity of tubulin carboxypeptidase (TCP) [24], for 24 h. Nuclei (blue) were stained with DAPI. Scale bar 5 μm.B An illustration showing the relation between tubulin detyrosination and lipophagy.C andD mRNA expression (C) and genomic DNA methylation levels (D) in HN4 parental cells and PCC with or without PGRMC1 overexpression or inhibition. Data are means and s.d. from three technical replicates. **P < 0.01 relative to vector control.E andF Immunoblotting in HN4 parental cells and PCC with or without PGRMC1 overexpression or inhibition and with DMSO or 10 μM erastin for 24 h. HN4 cells with control or PGRMC1 overexpression vector transfection were also transfected with scrambled or SIRT1 siRNA and treated with DMSO or 10 μM erastin for 24 h (F).G Co-staining for PGRMC1 (magenta), LC3-GFP (green), and nuclei (blue) in the cells treated with 10 μM EX527 (a selective SIRT1 inhibitor), 5 μM SRT1720 (a selective SIRT1 activator), 10 μM erastin, 20 μM PTN, or their combinations for 24 h. Scale bar 5 μm
Fig. 6
Fig. 6
PGRMC1 increases FAO by anchoring lipid droplets to mitochondria.A Co-staining of mitotracker (orange), lipid droplets (LD, green), and PGRMC1 (red) in HN4 parental cells and PCC with or without 10 μM erastin treatment for 24 h. HN4 cells were transfected with vector (vtr) or PGRMC1 overexpression vector or treated with 100 nM P4. HN4PCC were transfected with vector or shPGRMC1 or treated with 20 μM AG205. Scale bar 5 μm.B Mitochondrial ROS was examined using incubation with 5 μM mitoSOX™ Red and FACS in HN4 parental cells and PCC with or without PGRMC1 overexpression or inhibition and with DMSO or 10 μM erastin for 8 h. Data are means and s.d. from three technical replicates. *P < 0.05, **P < 0.01, ***P < 0.001 relative to DMSO control or between different groups.C-F Quantification of FAO in HN4 parental cells and PCC with or without PGRMC1 overexpression or inhibition and with DMSO or 10 μM erastin. *P < 0.05, **P < 0.01 relative to DMSO control.G Immunoblotting in HN4 parental cells and PCC with or without PGRMC1 overexpression or inhibition and with DMSO or 10 μM erastin for 24 h. O/E, PGRMC1 overexpression vector; sh, shPGRMC1; P4, progesterone; AG, AG205
Fig. 7
Fig. 7
PGRMC1 expression increases ferroptosis sensitivity in vivo.A-E Representative images, tumor weights, and tumor volumes in HN4 parental cells and PCC with or without vector (vtr), PGRMC1 overexpression, or shPGRMC1 transfection, and vehicle (ctr) or SAS treatment. *P < 0.05, **P < 0.01 relative to vehicle control or between different groups.F andG Lipid droplet staining of tumors. Lipid droplets (green) were quantified using ImageJ and were normalized to HN4PCC vector control. Scale bar 100 μm.H Comparison of PGRMC1 mRNA expression between normal mucosa and tumor samples in the HNC patient cohort from TCGA datasets.t-test,P < 0.001.I Kaplan-Meier curves estimating overall survival (OS) according to patients with low and high expression levels of tumor PGRMC1 mRNA (cutoff = 34.57) from the TCGA datasets. Log-rank test,P < 0.001
Fig. 8
Fig. 8
An illustration shows that PGRMC1 expression promotes ferroptosis in PCC. PCC has increased PGRMC1 expression related to increased free fatty acid acids (FFA), lipid droplets (LD), and fatty acid oxidation (FAO). PGRMC1 expression increased FAO and ferroptosis sensitivity by xCT inhibitors via lipophagy and tubulin detyrosination
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