PINK1-deficiency facilitates mitochondrial iron accumulation and colon tumorigenesis

Abstract

Mitophagy, the process by which cells eliminate damaged mitochondria, is mediated by PINK1 (PTEN induced kinase 1). Our recent research indicates that PINK1 functions as a tumor suppressor in colorectal cancer by regulating cellular metabolism. Interestingly, PINK1 ablation activated the NLRP3 (NLR family pyrin domain containing 3) inflammasome, releasing IL1B (interleukin 1 beta). However, inhibiting the NLRP3-IL1B signaling pathway with an IL1R (interleukin 1 receptor) antagonist or NLRP3 inhibitor did not hinder colon tumor growth after PINK1 loss. To identify druggable targets in PINK1-deficient tumors, ribonucleic acid sequencing analysis was performed on colon tumors from pink1 knockout and wild-type mice. Gene Set Enrichment Analysis highlighted the enrichment of iron ion transmembrane transporter activity. Subsequent qualitative polymerase chain reaction and western blot analysis revealed an increase in mitochondrial iron transporters, including mitochondrial calcium uniporter, in PINK1-deficient colon tumor cells and tissues. Live-cell iron staining demonstrated elevated cellular and mitochondrial iron levels in PINK1-deficient cells. Clinically used drugs deferiprone and minocycline reduced mitochondrial iron and superoxide levels, resulting in decreased colon tumor cell growth in vitro and in vivo. Manipulating the mitochondrial iron uptake protein MCU (mitochondrial calcium uniporter) also affected cell and xenograft tumor growth. This study suggests that therapies aimed at reducing mitochondrial iron levels may effectively inhibit colon tumor growth, particularly in patients with low PINK1 expression.Abbreviation: ANOVA: analysis of variance; APC: adenomatous polyposis coli; cAMP: cyclic adenosine monophosphate; CDX2: caudal type homeobox 2; CGAS: cyclic GMP-AMP synthase; CRC: colorectal cancer; DNA: deoxyribonucleic acid; DFP: deferiprone; DMEM: Dulbecco's modified Eagle medium; DSS: dextran sodium sulfate; ERT2-Cre: Cre recombinase fused to a triple mutant form of the human estrogen receptor; EV: empty vector; GLB: glybenclamide/glyburide; H&E: hematoxylin and eosin; ICP-MS: inductively coupled plasma mass spectrometer; IL1B: interleukin 1 beta; kDa: kilodalton; MCU: mitochondrial calcium uniporter; MKI67: marker of proliferation Ki-67; mRNA: messenger ribonucleic acid; MTT: 3-(4,5-dimethylthiazol-2-Yl)-2,5-diphenyltetrazolium bromide; NLRP3: NLR family pyrin domain containing 3; OE: overexpression; PBS: phosphate-buffered saline; p-CREB: phosphorylated cAMP responsive element binding protein; PINK1: PTEN induced kinase 1; p-PRKAA/AMPK: phosphorylated protein kinase AMP-activated catalytic subunit alpha; qPCR: qualitative polymerase chain reaction; RNA-seq: ribonucleic acid sequencing; ROS: reactive oxygen species; sg: single guide; sh: short hairpin; SLC25A28: solute carrier family 25 member 28; SLC25A37/MFRN: solute carrier family 25 member 37; STING1: stimulator of interferon response cGAMP interactor 1; TP53/p53: tumor protein p53; TUBA: tubulin alpha; µL: microliter; µm: micrometer; µM: micromolar; mm: millimeter.

Keywords: Colorectal cancer; deferiprone; iron chelation; minocycline; mitochondrial iron; mitophagy.

Figure 1.

GLB reduced tumors in PINK1 wild-type but not PINK1-deficient mice. (A) colon tumors under a microscope (bar: 5.2 mm), (B) colon tumor number, (C) colon tumor burden, (D) H&E staining (bar: 1000 µm), (E) histological scoring, (F) MKI67 immunostaining (bar: 200 µm) and (G) quantification of colon tumors from vehicle or GLB treated Pink^+/+; CDX2^ERT2 Apc^F/+ mice and pink^-/-; CDX2^ERT2 Apc^F/+ mice. *p < 0.05, **p < 0.01, ****p < 0.0001. Two-way ANOVA followed by Tukey’s multiple comparisons test for B and C. Unpaired t-test for E and G.

Figure 2.

GLB activated PRKAA-CREB signaling and reduced tumor growth in a xenograft mouse model of CRC in a PINK1-dependent manner. (A) Representative images, (B) tumor weight, (C) western blot analysis and qPCR analysis for Prkaa1 (D) and Creb1 (E) of xenograft tumor tissues from MC38 sgEV or sgpink1 cells inoculated C57BL/6 mice. *p < 0.05 vs sgEV control, ***p < 0.01 vs sgEV GLB, ###p < 0.001 vs sgpink1 control, two-way ANOVA followed by Uncorrected Fisher’s LSD test.

Figure 3.

PINK1 deficiency promoted upregulation of mitochondrial iron transporters in CRC cells. qPCR analysis for (A) Pink1, (B) Slc25a28, (C) Slc25a37 and (D) Mcu, and (E) western blot analysis in MC38 sgEV and sgpink1 cells. **p < 0.01, ***p < 0.001, ****p < 0.0001. Unpaired t-test.

Figure 4.

PINK1 deficiency promoted upregulation of mitochondrial iron transporters in xenograft colon tumor tissues. qPCR analysis for (A) Pink1, (B) Slc25a28, (C) Slc25a37 and (D) Mcu, and (E) western blot analysis in MC38 sgEV or sgpink1 cells derived xenograft colon tumors. *p < 0.05, ***p < 0.001. Unpaired t-test.

Figure 5.

pink1 deletion increased cellular and mitochondrial iron levels. (A) Representative images of single-cell mitoFerro green staining (bar: 10 µm), (B) histogram and (C) positive-cell percentage quantification in MC38 sgEV and sgpink1 cells. (D) Representative confocal images of FerroOrange staining (bar: 10 µm) and (E) quantification in MC38 sgEV and sgpink1 cells. *p < 0.05, **p < 0.01. Unpaired t-test.

Figure 6.

Mitochondrial iron restriction decreased mitochondrial iron and mitochondrial superoxide levels. (A) Representative images of single cell mitoFerro green staining (bar: 10 µm), (B) histogram and (C) positive cell percentage quantification in MC38 sgEV and sgpink1 cells treated with vehicle control (con), mitochondrial iron chelator DFP or MCU inhibitor minocycline (mino). (D) Representative images of single cell mitoSOX staining (bar: 10 µm), (E) histogram and (F) positive cell percentage quantification in MC38 sgEV and sgpink1 cells treated with vehicle con, mitochondrial iron chelator DFP or MCU inhibitor mino. ****p < 0.0001. Two-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 7.

Mitochondrial iron chelation reduced colon tumor cell growth in vitro and in vivo. Cell survival rate in (A) mitochondrial iron chelator DFP treated and (B) MCU inhibitor minocycline treated MC38 sgEV and sgpink1 cells. (C) Representative images of gross tumor, (D) tumor weight for mice treated with DFP or minocycline. *p < 0.05, **p < 0.01, ****p < 0.0001. Two-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 8.

Mitochondrial iron restriction suppressed colon tumor development in a colitis-associated CRC mouse model. (A) colon lengths, (B) tumor number, (C) tumor burden, (D) tumor number at size of 3-4 mm, (E) tumor number at size of >4 mm, (F) tumor tissue iron levels from vehicle, mitochondrial iron chelator DFP treated or MCU inhibitor minocycline treated Pink^+/+; CDX2^ERT2 Apc^F/+ mice and pink^-/-; CDX2^ERT2 Apc^F/+ mice. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Two-way ANOVA followed by Tukey’s multiple comparisons test.

Figure 9.

MCU was necessary and sufficient for driving colon cell growth. Immunoblot analysis in MC38 cells with stable transfected (A) shEV and shMcu cells, and (B) EV and MCU OE cells. MTT assay in MC38 cells with stable transfected (C) shEV and shMcu cells, and (D) EV and MCU OE cells. Crystal violet staining in MC38 cells with stable transfected (E) shEV and shMcu cells, and (F) EV and MCU OE cells. Crystal violet staining quantification in MC38 cells with stable transfected (G) shEV and shMcu cells, and (H) EV and MCU OE cells. **p < 0.01, ***p < 0.001, and ****p < 0.0001. Unpaired Student t test for A, B, G and H. Two-way ANOVA followed with Sidak’s multiple comparisons test for C and D.