Myoferlin targeting triggers mitophagy and primes ferroptosis in pancreatic cancer cells

Abstract

Myoferlin, an emerging oncoprotein, has been associated with a low survival in several cancer types including pancreas ductal adenocarcinoma where it controls mitochondria structure and respiratory functions. Owing to the high susceptibility of KRAS-mutated cancer cells to iron-dependent cell death, ferroptosis, and to the high iron content in mitochondria, we investigated the relation existing between mitochondrial integrity and iron-dependent cell death. We discovered that myoferlin targeting with WJ460 pharmacological compound triggered mitophagy and ROS accumulation culminating with lipid peroxidation and apoptosis-independent cell death. WJ460 caused a reduction of the abundance of ferroptosis core regulators x_c^- cystine/glutamate transporter and GPX-4. Mitophagy inhibitor Mdivi1 and iron chelators inhibited the myoferlin-related ROS production and restored cell growth. Additionally, we reported a synergic effect between ferroptosis inducers, erastin and RSL3, and WJ460.

Keywords: Ferroptosis; Mitochondria; Mitophagy; Myoferlin; Pancreas cancer.

Fig. 1

Pharmacological targeting of myoferlin exhibits the same effects than myoferlin gene silencing. (A) Treatment of PDAC cell lines (MiaPaCa-2, BxPC-3, Panc-1 and PaTu 8988T) with WJ460 ranging from 1 to 100 nM for the indicated times causes cell growth reduction. (B) WJ460 half maximal inhibitory concentration (IC50) determination in PDAC cell lines after 24 h exposition. (C) Treatment of pancreas normal epithelial cell line (HPNE) with WJ460 ranging from 1 to 100 nM for the indicated times. (D) Visualization of mitochondrial network shape of PDAC cell lines after 50 nM WJ460 treatment during 24 h. Scale bar = 10 μm, except for HPNE where scale bar = 7.5 μm. (E–F) Oxygen consumption rate (OCR) in PDAC cell lines and HPNE after 50 nM WJ460 treatment for 24 h. Kinetic oxygen consumption rate response of PDAC cells to 1 μM oligomycin (O), 1 μM FCCP (F), rotenone and antimycin A mix (RA, 0.5 μM each). Cell number was estimated using Hoechst incorporation (arbitrary unit, A.U.). Each data point represents mean ± SEM, n = 3. ****P < 0.0001, ***P < 0.001, *P < 0.05. (G) Reactive oxygen species (ROS) accumulation in PDAC cell lines and HPNE treated for 24 h with 50 nM WJ460. Each data point represents mean ± SEM, n = 3. ****P < 0.0001, ***P < 0.001. (H) Kinetic of Panc-1 cell tumor development in NOD-SCID mice upon WJ460 administration (10 mg/kg daily). ****P < 0.0001, **P < 0.01.

Fig. 2

WJ460 triggers a cell cycle arrest in G2/M phase and mitophagy in PDAC cell lines. (A) Relative apoptosis measured by annexin-V staining in PDAC cell lines (BxPC-3, MiaPaCa-2, Panc-1 and PaTu 8988T) and HPNE treated for 24 h with 50 nM WJ460. Puromycin was used as a positive control. Each data point represents mean ± SEM, n = 5. ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05. (B) Proportion of PDAC cells in each cell cycle phase was measured 24 h after 50 nM WJ460 treatment. Each data point represents mean ± SEM, n = 3. ****P < 0.0001. (C) Ultrastructure of mitochondria in PDAC cell lines treated for 24 h with 50 nM WJ460. (D) Immunofluorescence of autophagosome (LC3-II – green) and mitochondria (mitochondrial-specific 60 kDa protein - red) in PDAC cell lines and HPNE treated for 24 h with 50 nM WJ460. Arrows point to autophagosome and mitochondria colocalization. (E) Decrease of mitochondrial reactive oxygen species (Mito. ROS) accumulation in Panc-1 cell lines treated for 16 h or 24 h with 50 nM WJ460. (For interpretation of the references to colour in this figure legend, the reader is referred to the Web version of this article.)

Fig. 3

WJ460 induces lipid peroxidation in PDAC cell lines. (A–B) Malondialdehyde (MDA) concentration in PDAC (BxPC-3, MiaPaCa-2, Panc-1, PaTu 8988T) and pancreas normal epithelial (HPNE) cell lines exposed to 50 nM WJ460 for 24 h. Each data point represents mean ± SEM, n = 4. ***P < 0.001, **P < 0.01, *P < 0.05. (C) Trolox 5 mM mitigates the WJ460-induced reduction of cell confluency. Each data point represents mean ± SEM, n = 3. (D) Western-blot showing the x_c^- transporter SLC7A11, and glutathione peroxidase (GPX)-4 in PaTu 8988T and HPNE cells exposed to 50 nM WJ460 for 8–24 h. HSC70 was used as a loading control. Associated charts represent the relative abundance of SLC7A11 and GPX-4.

Fig. 4

WJ460 induces an iron-dependent cell death in PDAC cell lines. (A) Intracellular iron concentration in PDAC (BxPC-3, MiaPaCa-2, Panc-1, PaTu 8988T) and pancreas normal epithelial (HPNE) cell lines exposed to 50 nM WJ460 for 24 h. Each data point represents mean ± SEM, n = 3. ****P < 0.0001, ***P < 0.001, *P < 0.05. (B) Treatment of PDAC cell lines with 50 nM WJ460 and iron chelators, 1 mM deperiprone (DFP) or 1 mM deferoxamine (DFX). Each data point represents mean ± SEM, n = 3. (C) Western-blot showing the transferrin receptor CD71, heavy-chain ferritin, and nuclear receptor coactivator 4 (NCOA4) in Panc-1 cells exposed to 50 nM WJ460 for 8–24 h. HSC70 was used as a loading control. (D) Relative reactive oxygen species (ROS) accumulation in Panc-1 cell line treated for 24 h with 50 nM WJ460 and iron chelators, 1 mM DFP or 1 mM DFX, or mitophagy inhibitor 10 μM Mdivi-1. Each data point represents mean ± SEM, n = 3. ***P < 0.001, **P < 0.01, *P < 0.05.

Fig. 5

WJ460 sensitizes Panc-1 cells to ferroptosis. Cell growth inhibition of Panc-1 cells exposed for 48 h to (A) 10-50 nM WJ460, (B) 1-100 μM erastin, (C) 1-50 nM RSL3, (D) 25 nM WJ460 and 1-100 μM erastin, (E) 25 nM WJ460 and 1-50 nM RSL3. Each data point represents mean ± SEM, n = 3. (F) Combination index (CI) according to drug combination effect (Fa).

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