PTEN-induced kinase PINK1 supports colorectal cancer growth by regulating the labile iron pool

Mitophagy is a cargo-specific autophagic process that recycles damaged mitochondria to promote mitochondrial turnover. PTEN-induced putative kinase 1 (PINK1) mediates the canonical mitophagic pathway. However, the role of PINK1 in diseases where mitophagy has been purported to play a role, such as colorectal cancer, is unclear. Our results here demonstrate that higher PINK1 expression is positively correlated with decreased colon cancer survival, and mitophagy is required for colon cancer growth. We show that doxycycline-inducible knockdown (KD) of PINK1 in a panel of colon cancer cell lines inhibited proliferation, whereas disruption of other mitophagy receptors did not impact cell growth. We observed that PINK KD led to a decrease in mitochondrial respiration, membrane hyperpolarization, accumulation of mitochondrial DNA, and depletion of antioxidant glutathione. In addition, mitochondria are important hubs for the utilization of iron and synthesizing iron-dependent cofactors such as heme and iron sulfur clusters. We observed an increase in the iron storage protein ferritin and a decreased labile iron pool in the PINK1 KD cells, but total cellular iron or markers of iron starvation/overload were not affected. Finally, cellular iron storage and the labile iron pool are maintained via autophagic degradation of ferritin (ferritinophagy). We found overexpressing nuclear receptor coactivator 4, a key adaptor for ferritinophagy, rescued cell growth and the labile iron pool in PINK1 KD cells. These results indicate that PINK1 integrates mitophagy and ferritinophagy to regulate intracellular iron availability and is essential for maintaining intracellular iron homeostasis to support survival and growth in colorectal cancer cells. Mitophagy is a cargo-specific autophagic process that recycles damaged mitochondria to promote mitochondrial turnover. PTEN-induced putative kinase 1 (PINK1) mediates the canonical mitophagic pathway. However, the role of PINK1 in diseases where mitophagy has been purported to play a role, such as colorectal cancer, is unclear. Our results here demonstrate that higher PINK1 expression is positively correlated with decreased colon cancer survival, and mitophagy is required for colon cancer growth. We show that doxycycline-inducible knockdown (KD) of PINK1 in a panel of colon cancer cell lines inhibited proliferation, whereas disruption of other mitophagy receptors did not impact cell growth. We observed that PINK KD led to a decrease in mitochondrial respiration, membrane hyperpolarization, accumulation of mitochondrial DNA, and depletion of antioxidant glutathione. In addition, mitochondria are important hubs for the utilization of iron and synthesizing iron-dependent cofactors such as heme and iron sulfur clusters. We observed an increase in the iron storage protein ferritin and a decreased labile iron pool in the PINK1 KD cells, but total cellular iron or markers of iron starvation/overload were not affected. Finally, cellular iron storage and the labile iron pool are maintained via autophagic degradation of ferritin (ferritinophagy). We found overexpressing nuclear receptor coactivator 4, a key adaptor for ferritinophagy, rescued cell growth and the labile iron pool in PINK1 KD cells. These results indicate that PINK1 integrates mitophagy and ferritinophagy to regulate intracellular iron availability and is essential for maintaining intracellular iron homeostasis to support survival and growth in colorectal cancer cells. Mitochondria are critical metabolic organelles that sustain cellular bioenergetics and biosynthetic needs (1Spinelli J.B. Haigis M.C. The multifaceted contributions of mitochondria to cellular metabolism.Nat. Cell Biol. 2018; 20: 745-754Crossref PubMed Scopus (703) Google Scholar). The electron transport chain (ETC) integrates central carbon metabolism and redox homeostasis to support metabolic demands of the cells. To maintain a healthy mitochondrial network, organellar functions are continuously monitored via quality control mechanisms (2Pickles S. Vigié P. Youle R.J. Mitophagy and quality control mechanisms in mitochondrial maintenance.Curr. Biol. 2018; 28: R170-R185Abstract Full Text Full Text PDF PubMed Scopus (1013) Google Scholar). Dysfunctional mitochondria are turned over by a cargo-specific, lysosomal-dependent, autophagic degradation mechanism termed mitophagy (3Kim I. Rodriguez-Enriquez S. Lemasters J.J. Selective degradation of mitochondria by mitophagy.Arch. Biochem. Biophys. 2007; 462: 245-253Crossref PubMed Scopus (1284) Google Scholar). Dysregulation of mitophagy is associated with progression of several cancers (4Vara-Perez M. Felipe-Abrio B. Agostinis P. Mitophagy in cancer: a tale of adaptation.Cells. 2019; 8: 493Crossref PubMed Scopus (120) Google Scholar). Parkin-induced protein kinase 1 (PINK1) is a sensor of mitochondrial health, and activation of PINK1 regulates one of the most well-defined mitophagy pathways (5Jin S.M. Youle R.J. PINK1- and parkin-mediated mitophagy at a glance.J. Cell Sci. 2012; 125: 795-799Crossref PubMed Scopus (412) Google Scholar, 6Lazarou M. Sliter D.A. Kane L.A. Sarraf S.A. Wang C. Burman J.L. et al.The ubiquitin kinase PINK1 recruits autophagy receptors to induce mitophagy.Nature. 2015; 524: 309-314Crossref PubMed Scopus (1654) Google Scholar). However, the role of PINK1 as a tumor suppressive surveillance mechanism for enhancing survival and proliferation are context dependent (7O'Flanagan C.H. O'Neill C. PINK1 signalling in cancer biology.Biochim. Biophys. Acta. 2014; 1846: 590-598Crossref PubMed Scopus (48) Google Scholar, 8Dai K. Radin D.P. Leonardi D. Deciphering the dual role and prognostic potential of PINK1 across cancer types.Neural Regen. Res. 2020; 16: 659-665Google Scholar, 9Berthier A. Navarro S. Jiménez-Sáinz J. Roglá I. Ripoll F. Cervera J. et al.PINK1 displays tissue-specific subcellular location and regulates apoptosis and cell growth in breast cancer cells.Hum. Pathol. 2011; 42: 75-87Crossref PubMed Scopus (48) Google Scholar). Induction of PINK1-mediated mitophagy is triggered by the loss of membrane potential from uncoupling the proton or potassium gradient (10Amo T. Sato S. Saiki S. Wolf A.M. Toyomizu M. Gautier C.A. et al.Mitochondrial membrane potential decrease caused by loss of PINK1 is not due to proton leak, but to respiratory chain defects.Neurobiol. Dis. 2011; 41: 111-118Crossref PubMed Scopus (62) Google Scholar). Recent results illustrated that chelation of mitochondrial iron potently induced mitophagy (11Allen G.F.G. Toth R. James J. Ganley I.G. Loss of iron triggers PINK1/Parkin-independent mitophagy.EMBO Rep. 2013; 14: 1127-1135Crossref PubMed Scopus (361) Google Scholar).The role of mitochondria in iron metabolism is well established. The synthesis of iron sulfur cluster (Fe-S) and heme is initiated in the mitochondria. Moreover, mitochondria ETC require Fe-S cluster and heme-containing protein such as cytochrome c to facilitate electron transfer. However, the exact mechanism linking the loss of mitochondrial iron and mitophagy is not yet fully understood (11Allen G.F.G. Toth R. James J. Ganley I.G. Loss of iron triggers PINK1/Parkin-independent mitophagy.EMBO Rep. 2013; 14: 1127-1135Crossref PubMed Scopus (361) Google Scholar, 12Hara Y. Yanatori I. Tanaka A. Kishi F. Lemasters J.J. Nishina S. et al.Iron loss triggers mitophagy through induction of mitochondrial ferritin.EMBO Rep. 2020; 21e50202Crossref PubMed Scopus (49) Google Scholar). In excess, iron is cytotoxic to cells. Cellular iron levels are balanced by an intricate network of regulatory mechanisms. A central protein in iron handling is the iron storage protein ferritin (FTN). A study demonstrates that following uptake, iron goes through a transient FTN intermediate to prevent oxidative damage (13Das N.K. Jain C. Sankar A. Schwartz A.J. Santana-Codina N. Solanki S. et al.Modulation of the HIF2α-NCOA4 axis in enterocytes attenuates iron loading in a mouse model of hemochromatosis.Blood. 2021; 139: 2547-2552Crossref Scopus (13) Google Scholar). Subsequently, FTN-bound iron release is mediated by autophagic degradation of nuclear receptor coactivator 4 (NCOA4) in a process termed ferritinophagy. Following FTN degradation, lysosomal/endosomal iron can be distributed in the cell via direct lysosome-organelle contacts or chaperones (14Barra J. Crosbourne I. Wang L. Nelson I. Goldwag J. Jourd’heuil F. et al.DMT1-mediated endosome-mitochondria interactions regulates iron homeostasis and mitochondrial metabolism.FASEB J. 2022; https://doi.org/10.1096/fasebj.2022.36.S1.R5276Crossref Google Scholar, 15Das A. Nag S. Mason A.B. Barroso M.M. Endosome–mitochondria interactions are modulated by iron release from transferrin.J. Cell Biol. 2016; 214: 831-845Crossref PubMed Scopus (97) Google Scholar). As an example, recent studies demonstrated that iron turnover from ferritinophagy is critical to support mitochondrial iron sulfur cluster biogenesis and respiration in pancreatic cancer (16Ravichandran M. Hu J. Cai C. Ward N.P. Venida A. Foakes C. et al.Coordinated transcriptional and catabolic programs support iron dependent adaptation to RAS-MAPK pathway inhibition in pancreatic cancer.Cancer Discov. 2022; 12: 2198-2219Crossref PubMed Scopus (17) Google Scholar, 17Santana-Codina N. Del Rey M.Q. Kapner K.S. Zhang H. Gikandi A. Malcolm C. et al.NCOA4-mediated ferritinophagy is a pancreatic cancer dependency via maintenance of iron bioavailability for iron–sulfur cluster proteins.Cancer Discov. 2022; 12: 2180-2197Crossref PubMed Scopus (17) Google Scholar). To study the connection between mitophagy and iron balance, we generated a genetic, PINK1 loss-of-function model in colorectal cancer (CRC) cells. We identified PINK1-mediated mitophagy as a critical pathway for CRC cell proliferation and mitochondrial function. The loss of PINK1 led to increased sequestration of labile iron in FTN. The proliferative defects and cellular labile iron pool (LIP) in PINK1 knockdown (KD) was rescued by activating ferritinophagy via nuclear receptor coactivator 4 (NCOA4) overexpression. Overall, our study suggests that disruption of the canonical mitophagy pathway contributes to cytosolic iron imbalance, which can be rescued by activating ferritinophagy. We demonstrated that CRC cells utilize mitophagy to support metabolic rewiring under nutrient-deprived conditions (18Devenport S.N. Singhal R. Radyk M.D. Taranto J.G. Kerk S.A. Chen B. et al.Colorectal cancer cells utilize autophagy to maintain mitochondrial metabolism for cell proliferation under nutrient stress.Jci Insight. 2021; 6e138835Crossref PubMed Scopus (12) Google Scholar). We generated Kaplan–Meier plots from published transcriptomic data from colon cancer patients. We stratified gene expression data into high and low PINK1 expression based on median expression and compared regression-free survival over a span of 200 months. Log-ranked test indicated a significant p-value of 1.6e−5. Moreover, the hazard ratio analyses revealed improved survival of patients with low PINK1 expression level (Fig. 1A). In addition, mining the Human Protein Atlas, CRC cells robustly express PINK1 compared to several other cancers (Fig. 1B). We generated two independent doxycycline (DOX)-inducible shRNA targeting PINK1 (19Kane L.A. Lazarou M. Fogel A.I. Li Y. Yamano K. Sarraf S.A. et al.PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity.J. Cell Biol. 2014; 205: 143-153Crossref PubMed Scopus (867) Google Scholar). To test cell proliferation and viability, we counted cell number over time to account for proliferation and utilized colony formation assay to assess the ability of cells to form single unit colonies. When assessing growth differences between WT and PINK1 KD, we compared DOX-treated groups of shNT versus shPINK1 instead of between isogenic shPINK1 cells with and without DOX. In this study, we focused on two human CRC cell lines, HCT116 and SW480, which are widely used for colon cancer. The genomic profiles of these cell lines are well characterized, featuring activating KRAS mutations, which are representative of a significant proportion of mutations observed in CRC patients. Additionally, we generated shRNA constructs in a panel of other human and mouse CRC cell lines. Upon PINK1 KD, we observed proliferative defects and decreased colony-forming capacity in several CRC cell lines (HCT116, SW480, RKO, HT29, MC38, CT26) (Fig. 1, C and D). KD was validated by decreased PINK1 mRNA transcript (Fig. S1A). However, disruption of PINK1-independent mitophagy executors, parkin, BNIP3, NIX, FUNDC1, did not exhibit growth defects (Fig. S1, B and C). In addition, analysis of PINK1 KD cells in a subcutaneous xenograft model revealed decreased tumor burden with decreased tumor volume, tumor weight, and proliferation and increased TUNEL staining following DOX treatment (Fig. 2, A–D). This data demonstrates that PINK1 has an essential role in mediating CRC cell growth.Figure 2PINK1 is required for colorectal cancer’s in vivo growth. A, HCT116 and SW480 shNT, shPINK1.2, and shPINK1.3 tumor were injected into the flanks of NOD/SCID mice to measure tumor volume over time with or without doxycycline (DOX) chow. B, endpoint tumor weight was measured. C, percent (%) TUNEL staining and (D) BrdU staining was quantified. ∗∗∗ indicates p < 0.0005, ∗∗∗∗ indicates p < 0.0001. (mean −/+ SEM, N = 6 or 8 per condition).View Large Image Figure ViewerDownload Hi-res image Download (PPT) Mitophagy is the process by which damaged mitochondria are degraded and the components are recycled to support cell growth. Upon loss of mitochondrial membrane potential, PINK1 accumulates on the outer mitochondrial membrane (OMM) and recruits E3 ligase Parkin to coordinate the decoration of OMM and OMM proteins with phospho-ubiquitin (pUb) chains (20Koyano F. Okatsu K. Kosako H. Tamura Y. Go E. Kimura M. et al.Ubiquitin is phosphorylated by PINK1 to activate parkin.Nature. 2014; 510: 162-166Crossref PubMed Scopus (1017) Google Scholar). pUb chains subsequently recruit autophagic adaptors such as p62 to deliver mitochondria to autophagosomes for degradation (11Allen G.F.G. Toth R. James J. Ganley I.G. Loss of iron triggers PINK1/Parkin-independent mitophagy.EMBO Rep. 2013; 14: 1127-1135Crossref PubMed Scopus (361) Google Scholar). PINK1 KD prevented accumulation of pUb in response to carbonyl cyanide m-chlorophenylhydrazone, a mitochondrial uncoupler (19Kane L.A. Lazarou M. Fogel A.I. Li Y. Yamano K. Sarraf S.A. et al.PINK1 phosphorylates ubiquitin to activate Parkin E3 ubiquitin ligase activity.J. Cell Biol. 2014; 205: 143-153Crossref PubMed Scopus (867) Google Scholar) (Fig. 3A). In addition, we sought to characterize mitophagy flux utilizing a mitochondrially targeted tandem Cox8-mCherry-GFP mitophagy reporter (21Rojansky R. Cha M.-Y. Chan D.C. Elimination of paternal mitochondria in mouse embryos occurs through autophagic degradation dependent on PARKIN and MUL1.Elife. 2016; 5e17896Crossref PubMed Scopus (210) Google Scholar) (Fig. 3B). GFP is a pH-sensitive fluorophore, while mCherry is pH-insensitive. Upon the internalization of mitochondria into the acidic autophagosome, the localized GFP signal is quenched, whereas mCherry will remain fluorescent. PINK1 KD effectively decreased mitophagy flux (Fig. 3C). Moreover, we observed an increase in mitochondrial DNA content, which indicated accumulation of mitochondria (Fig. 3D). Next, mitochondrial function was investigated following PINK1 KD. Seahorse analysis demonstrated lower basal oxygen consumption rate (OCR) and maximal respiration in PINK1 KD cells (Figs. 3E and S2, A and B). ETC is tightly coupled with mitochondrial membrane potential, and PINK1 KD cells had increased mitochondrial membrane potential, which is indicative of mitochondrial hyperpolarization. Previously, mitochondria hyperpolarization has been shown to occur under chronic inhibition of ETC CI inhibition and reduced CII, CIII, and CIV activity (22Forkink M. Manjeri G.R. Liemburg-Apers D.C. Nibbeling E. Blanchard M. Wojtala A. et al.Mitochondrial hyperpolarization during chronic complex I inhibition is sustained by low activity of complex II, III, IV and V.Biochim. Biophys. Acta. 2014; 1837: 1247-1256Crossref PubMed Scopus (72) Google Scholar) (Fig. 3F). Mitochondrial ETC complex I is also required to oxidize NADH to sustain NAD pools. We observed elevated levels of NADH/NAD ratio upon PINK1 KD (Fig. 3G). Mitochondria integrate the metabolism of nutrients such as glucose, glutamine, and fatty acids to coordinate energy production, the regulation of redox homeostasis, and other biosynthetic precursors. Liquid chromatography tandem mass spectrometry–based metabolomics was used to profile changes in central carbon metabolism upon PINK1 KD. Here, we observed a downregulation of the reduced glutathione (GSH) to oxidized glutathione (GSSG) ratio and nucleotides (i.e. ADP, AMP, UDP, IMP) in SW480 (Fig. 4, A and B, and Table S1). Together with the bioenergetic profiling data in Figure 2, this evidence indicates that losing the ability to recycle mitochondria via PINK1-dependent mitophagy inhibits mitochondrial respiration and contributes to cellular metabolic dysfunction. To reverse proliferation defects that resulted from PINK1 KD, based on our metabolomics data, we supplemented cells with the antioxidants N-acetylcysteine or glutathione ethyl ester, or the nucleosides (adenosine, thymidine, cytosine, and guanosine) (Fig. 4, C and D). However, these metabolites did not rescue the growth defects in PINK1 KD cells. To resolve elevated NADH/NAD ratio, we employed Lactobacillus brevis NADH oxidase (LbNOX) and mitochondrial targeted LbNOX (mtLbNOX), which oxidize NADH to water, in order to determine the impact of reductive stress on our PINK1 KD phenotype. Here too, we failed to rescue PINK1 KD (23Titov D.V. Cracan V. Goodman R.P. Peng J. Grabarek Z. Mootha V.K. Complementation of mitochondrial electron transport chain by manipulation of the NAD+/NADH ratio.Science. 2016; 352: 231-235Crossref PubMed Scopus (227) Google Scholar) (Fig. S2A). Additionally, the NADH buildup from defective ETC complex I can be rescued by expression of a yeast version of NADH oxidase, NDI1 (24Seo B.B. Kitajima-Ihara T. Chan E.K. Scheffler I.E. Matsuno-Yagi A. Yagi T. Molecular remedy of complex I defects: rotenone-insensitive internal NADH-quinone oxidoreductase of saccharomyces cerevisiae mitochondria restores the NADH oxidase activity of complex I-deficient mammalian cells.Proc. Natl. Acad Sci. U. S. A. 1998; 95: 9167-9171Crossref PubMed Scopus (145) Google Scholar). NDI1 rescued phenformin-dependent proliferative defect; it did not rescue proliferation in PINK1 KD cells. This demonstrates that increasing the oxidation of NADH is not sufficient to restore PINK1-dependent proliferative defects (Fig. S2E). Lastly, the pan-caspase/apoptosis inhibitor (z-VAD-FMK), ferroptosis inhibitor (Ferrostatin 1; Fer1), and necroptosis inhibitor (Necrostatin 1; Nec1) did not reverse growth defects in PINK1 KD cells (Fig. S2B). Collectively, these data suggested that metabolic dysregulation was secondary of the growth suppressive and mitochondrial dysfunction phenotypes following PINK1 KD. In addition to their above noted role in metabolism, mitochondria are also important hubs for cellular iron utilization. Iron sulfur clusters (Fe-S) and heme biosynthetic pathways initiate in the mitochondria. Iron and iron-containing cofactors are critical for electron transport and redox balance as iron is a redox active element that can shuttle electrons along the respiratory chain. Moreover, we have shown that nucleotide metabolism requires iron for pyrimidine/purine biosynthesis (25Schwartz A.J. Goyert J.W. Solanki S. Kerk S.A. Chen B. Castillo C. et al.Hepcidin sequesters iron to sustain nucleotide metabolism and mitochondrial function in colorectal cancer epithelial cells.Nat. Metab. 2021; 3: 969-982Crossref PubMed Scopus (38) Google Scholar). Acute depletion of mitochondrial iron via deferiprone induces mitophagy, thus linking mitochondrial turnover to cellular iron homeostasis (11Allen G.F.G. Toth R. James J. Ganley I.G. Loss of iron triggers PINK1/Parkin-independent mitophagy.EMBO Rep. 2013; 14: 1127-1135Crossref PubMed Scopus (361) Google Scholar). To measure the mitochondrial iron pool, we utilized a flow cytometry–compatible mitochondrial iron stain, Mito-FerroGreen. With this, we observed that PINK1 KD decreased mitochondrial iron levels (Fig. 5A). Mitochondrial iron is sensitive to cytosolic iron perturbation. For example, extracellular iron depletion induces hypoxia-inducible factor and thus drives the expression of mitochondrial iron transport SLC25A37 (26Li C. Zhang Y. Cheng X. Yuan H. Zhu S. Liu J. et al.PINK1 and PARK2 suppress pancreatic tumorigenesis through control of mitochondrial iron-mediated Immunometabolism.Dev. Cell. 2018; 46: 441-455.e8Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar, 27Cuadros A.M. Fernandez-Garcia J. Planque M. Altea-Manzano P. Schalley T. Vermeire I. et al.In vivo CRISPR screen defines Slc25a37 as an organ-specific regulator of antioxidant metabolism in metastasis.bioRxiv. 2022; https://doi.org/10.1101/2022.09.03.506468Crossref Scopus (0) Google Scholar). Cells have an extensive regulatory network that monitors iron homeostasis (28Wang J. Pantopoulos K. Regulation of cellular iron metabolism.Biochem. J. 2011; 434: 365-381Crossref PubMed Scopus (710) Google Scholar). Excess cytosolic LIP can lead to oxidative damage (29Reif D.W. Ferritin as a source of iron for oxidative damage.Free Radic. Biol. Med. 1992; 12: 417-427Crossref PubMed Scopus (327) Google Scholar, 30Stäubli A. Boelsterli U.A. The labile iron pool in hepatocytes: prooxidant-induced increase in free iron precedes oxidative cell injury.Am. J. Physiol. 1998; 274: G1031-G1037PubMed Google Scholar). Thus, iron is tightly regulated through many overlapping and distinct pathways. FerroOrange measures total LIP, which was robustly decreased upon PINK1 KD (Fig. 5B). In contrast, total cellular iron was not changed, as assessed by inductively-coupled plasma mass spectrometry (ICP-MS), as well as other trace metal elements (Fig. 5C). High levels of iron lead to the upregulation of the iron storage protein FTN expression and stabilization, which is composed of FTN heavy chain (FTH1) and FTN light chain (FTL). FTN sequesters excessive iron, preventing cellular oxidative damage from excessive LIP. In PINK1 KD cells, increased FTH1 and FTL was observed (Fig. 5D). FTN accumulation observed in PINK1 KD cells is consistent in our subcutaneous xenograft tumors (Fig. 5E). The regulation of cellular iron homeostasis involves transferrin receptor–mediated iron uptake, endolysosomal trafficking and release, and posttranscriptional regulation of iron-related mRNA transcripts by iron response protein 2 (31Shah Y.M. Xie L. Hypoxia-inducible factors link iron homeostasis and erythropoiesis.Gastroenterology. 2014; 146: 630-642Abstract Full Text Full Text PDF PubMed Scopus (108) Google Scholar). We observed an increase in transferrin receptor expression in PINK1 KD HCT116, but there was no change in SW480. Iron response protein 2 level was also not altered upon PINK1 KD in both cell lines. To determine if cellular iron uptake is increased in PINK1 KD cells, the cells were treated with a heavy iron-57Fe isotope. Cellular iron-57Fe was measured by ICP-MS, and no change in iron uptake was observed (Figs. 5F and S3A). These data together suggest that canonical iron starvation response is not involved in PINK1-dependent FTN regulation. To understand the role of iron in the growth defects following PINK1 KD, we supplemented with ferric ammonium citrate (FAC) to rescue LIP and mitochondrial iron. Interestingly, growth was not rescued in the PINK1 KD cells (Fig. 6A). An increase in FTN levels upon FAC treatment was observed, but no increase in LIP or mitochondrial iron, indicating that introduction of exogenous iron was primarily integrated to FTN complexes (Fig. 6, B and C). Indeed, iron chelation with deferoxamine (DFO) decreased LIP (Fig. 6D). Cells compensate for the loss of cellular LIP via a cargo-specific autophagic degradation of FTN by ferritinophagy (32Kakhlon O. Cabantchik Z.I. The labile iron pool: characterization, measurement, and participation in cellular processes(1).Free Radic. Biol. Med. 2002; 33: 1037-1046Crossref PubMed Scopus (640) Google Scholar, 33Mancias J.D. Wang X. Gygi S.P. Harper J.W. Kimmelman A.C. Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy.Nature. 2014; 509: 105-109Crossref PubMed Scopus (964) Google Scholar). We show that DFO depleted FTN levels in WT cells, but PINK1 KD maintained higher FTN levels (Fig. 5D). This data suggested that there are defects in ferritinophagy that decrease LIP in the PINK1 KD cells. Ferritinophagy is coordinated by the cargo receptor NCOA4, which binds to both FTL and FTH1 and delivers FTN to autophagosomes for degradation and release of iron (33Mancias J.D. Wang X. Gygi S.P. Harper J.W. Kimmelman A.C. Quantitative proteomics identifies NCOA4 as the cargo receptor mediating ferritinophagy.Nature. 2014; 509: 105-109Crossref PubMed Scopus (964) Google Scholar). In addition to liberating FTN-bound iron for other iron-dependent processes, NCOA4 has been reported to be important for mitochondrial iron balance and respiration (34Fujimaki M. Furuya N. Saiki S. Amo T. Imamichi Y. Hattori N. Iron supply via NCOA4-mediated ferritin degradation maintains mitochondrial functions.Mol. Cell. Biol. 2019; 39e00010–e00019Crossref PubMed Scopus (37) Google Scholar). To rescue FTN accumulation in PINK1 KD cells, NCOA4 was over expressed (Fig. S3B). NCOA4 expression was sufficient to rescue proliferative defects of PINK1 KD and restore LIP in these cells (Fig. 6, E and F). Overall, here we demonstrated that PINK1 loss leads to mitochondrial dysfunction and iron accumulation in FTN. Liberating the sequestered iron from the FTN complex by inducing ferritinophagy was able to compensate for PINK1 KD. This data suggests an essential role of mitophagy in regulating the LIP via ferritinophagy. Mitochondria are important biosynthetic and metabolic hubs in tumorigenesis. Functional mitochondria are critical to support cancer cell growth (35Weinberg S.E. Chandel N.S. Targeting mitochondria metabolism for cancer therapy.Nat. Chem. Biol. 2015; 11: 9-15Crossref PubMed Scopus (992) Google Scholar). Stressors in the tumor microenvironment such as hypoxia and nutrient dysregulation and intrinsic factors driving mitochondrial DNA heteroplasmy require adaptations to mitochondrial dynamics, turnover, and programs for cancer cell survival (36Hertweck K.L. Dasgupta S. The landscape of mtDNA modifications in cancer: a tale of two cities.Front. Oncol. 2017; 7: 262Crossref PubMed Scopus (56) Google Scholar, 37Petros J.A. Baumann A.K. Ruiz-Pesini E. Amin M.B. Sun C.Q. Hall J. et al.mtDNA mutations increase tumorigenicity in prostate cancer.Proc. Natl. Acad Sci. U. S. A. 2005; 102: 719-724Crossref PubMed Scopus (695) Google Scholar). The induction of mitophagy requires exogenous expression of parkin and mitochondrial damage to remove the mitochondrial pool (38Narendra D. Tanaka A. Suen D.-F. Youle R.J. Parkin is recruited selectively to impaired mitochondria and promotes their autophagy.J. Cell Biol. 2008; 183: 795-803Crossref PubMed Scopus (2973) Google Scholar). Mitophagy is essential in colon cancer growth, however, the underlying mechanisms are unclear. The present work demonstrates that inhibition of PINK1-mediated mitophagy decreases colon cancer growth in a panel of colon cancer-derived cell lines. CRC cells have high basal mitophagy (18Devenport S.N. Singhal R. Radyk M.D. Taranto J.G. Kerk S.A. Chen B. et al.Colorectal cancer cells utilize autophagy to maintain mitochondrial metabolism for cell proliferation under nutrient stress.Jci Insight. 2021; 6e138835Crossref PubMed Scopus (12) Google Scholar), and failure to execute mitophagy leads to proliferative defects. Mechanistically, we show that colon cancer cells are dependent on PINK1 to maintain mitochondrial respiration. Although we did observe metabolite changes such as decreased reduced glutathione and several nucleotide species, rescue with these metabolites was not sufficient to restore PINK1-dependent proliferative defects. Rather, we demonstrate that PINK1 KD decreases the LIP, and restoring ferritinophagy was sufficient to rescue cell proliferation (Fig. 7). The critical role of mitochondria in iron homeostasis is well appreciated. Mitochondrial uptake of iron via the mitoferrins (SLC25A28/37) is essential for the biosynthesis of iron sulfur clusters proteins and heme (39Lill R. Hoffmann B. Molik S. Pierik A.J. Rietzschel N. Stehling O. et al.The role of mitochondria in cellular iron–sulfur protein biogenesis and iron metabolism.Biochim. Biophys. Acta. 2012; 1823: 1491-1508Crossref PubMed Scopus (390) Google Scholar). Iron sulfur clusters and heme are both important cofactors that support ETC, nucleotide biosynthesis, and DNA replication (40Ye H. Rouault T.A. Human iron−sulfur cluster assembly, cellular iron homeostasis, and disease.Biochemistry. 2010; 49: 4945-4956Crossref PubMed Scopus (209) Google Scholar). Although iron is a critical micronutrient essential for many biological processes, excessive free iron can be cytotoxic and exert oxidative stress (41Yang W.S. Stockwell B.R. Ferroptosis: death by lipid peroxidation.Trends Cell Biol. 2016; 26: 165-176Abstract Full Text Full Text PDF PubMed Scopus (1466) Google Scholar). Therefore, the balance between iron storage in multimeric FTN complexes and FTN turno