PINK1 (phosphatase and tensin homolog deleted on chromosome 10 (PTEN)-induced kinase 1), a Parkinson’s disease-associated gene, was identified originally because of its induction by the tumor-suppressor PTEN. PINK1 promotes cell survival and potentially metastatic functions and protects against cell stressors including chemotherapeutic agents. However, the mechanisms underlying PINK1 function in cancer cell biology are unclear. Here, using several model systems, we show that PINK1 deletion significantly reduced cancer-associated phenotypes including cell proliferation, colony formation and invasiveness, which were restored by human PINK1 overexpression. Results show that PINK1 deletion causes major defects in cell cycle progression in immortalized mouse embryonic fibroblasts (MEFs) from PINK1−/− mice, and in BE(2)-M17 cells stably transduced with short hairpin RNA against PINK1. Detailed cell cycle analyses of MEF cell lines from several PINK1−/− mice demonstrate an increased proportion of cells in G2/M and decreased number of cells in G1 following release from nocodazole block. This was concomitant with increased double and multi-nucleated cells, a reduced ability to undergo cytokinesis and to re-enter G1, and significant alterations in cell cycle markers, including failure to increase cyclin D1, all indicative of mitotic arrest. PINK1−/− cells also demonstrated ineffective cell cycle exit following serum deprivation. Cell cycle defects associated with PINK1 deficiency occur at points critical for cell division, growth and stress resistance in cancer cells were rescued by ectopic expression of human PINK1 and demonstrated PINK1 kinase dependence. The importance of PINK1 for cell cycle control is further supported by results showing that cell cycle deficits induced by PINK1 deletion were linked mechanistically to aberrant mitochondrial fission and its regulation by dynamin-related protein-1 (Drp1), known to be critical for progression of mitosis. Our data indicate that PINK1 has tumor-promoting properties and demonstrates a new function for PINK1 as a regulator of the cell cycle.
PINK1 (phosphatase and tensin homolog deleted on chromosome 10 ( PTEN)-induced kinase 1) was first identified in HeLa cells as a gene upregulated by overexpression of the central tumor suppressor, PTEN.1 Subsequently, loss of function mutations in PINK1 were discovered to cause autosomal recessive Parkinson’s disease,2 and a wealth of research on PINK1 function in neuronal and other cell systems emerged.3, 4, 5, 6, 7, 8 The PINK1 gene encodes a 581-amino-acid protein with a highly conserved serine/threonine kinase domain, a C-terminal auto-regulatory sequence and a mitochondrial-targeting signal.9,10 PINK1 is ubiquitously expressed,11 and is a major regulator of mitochondrial quality control, including bioenergetics and the triad of fission, fusion and mitophagy.12, 13, 14 PINK1 also demonstrates significant cytoprotective and anti-apoptotic functions,5,6,15,16 including via the phosphatidylinositol 3 kinase/Akt/mammalian target of rapamycin axis17,18 proteasomal19, 20, 21 and autophagic pathways.22,23
Many of the functions of PINK1 draw increasing attention to the importance of this kinase in regulation of cell survival systems outside those protecting from neurodegeneration in Parkinsonism. This is especially pertinent when considering cell survival in cancer, where a number of studies have indicated a potential role for PINK1 in tumorigenesis.17,24, 25, 26 However, the mechanisms underlying PINK1 function in cancer biology are not clear and have been the subject of only a few investigations, with conflicting evidence as to whether PINK1 has tumor-promoting or -suppressive activity. When considering tumor promotion, PINK1 is necessary for optimal activation of insulin-like growth factor-1 receptor–phosphatidylinositol 3 kinase/Akt signaling, a well-described oncogenic pathway.18 Moreover, PINK1-mediated activation of Akt via mammalian target of rapamycin complex 2 increases migration, a key feature of invasive cancer cells.17 High-throughput RNA interference screens have identified PINK1 deletion as a primary sensitizer of chemoresistant cancer cells to cell death following treatment with paclitaxel26 and as a target for treatment of malignancies with DNA mismatch repair deficiencies.25 In the context of tumor suppression, PINK1 is induced by PTEN1 and FOXO3a27 tumor suppressors, and associates with Beclin-1, another tumor suppressor.22 Parkin, an autosomal recessive Parkinson’s disease-causing gene, can function downstream of PINK1 (reviewed in Jin and Youle28and Wilhelmus and Nijland29) and is also a tumor suppressor.30, 31, 32 The PINK1 gene is located on chromosome 1p361, a region postulated to contain tumor-suppressive activity.33 Such opposing context-dependent pro- and antitumorigenic properties are emerging to be common for many genes with tumorigenic potential,34,35 and it has been postulated that PINK1 may have a dual role, being either anti-apoptotic or anti-growth depending on cellular context.24
Many of the mitochondrial functions ascribed to PINK1 are centrally linked to regulation of cell death and survival in cancer cells. Mitochondrial fission is strongly associated with apoptosis36,37 and mitochondrial DNA mutations, altered metabolic pathways, increased mitochondrial reactive oxygen species, and deregulated mitochondrial dynamics contribute to the development of malignant cells.38, 39, 40, 41 It is well documented that regulation of the fission/fusion axis via the mitochondrial fission GTPase dynamin-related protein-1 (Drp1) is a major function of PINK1 in Drosophila.42, 43, 44 In dividing mammalian cells, PINK1 deletion causes excessive fission, and deletion of Drp1 can rescue the PINK1-null phenotype.13,45 Importantly, the mitochondrial fission/fusion and bioenergetic balance is critical for cell cycle progression, cell division and growth, with fission being essential for equal distribution of mitochondria during mitosis,46, 47, 48 whereas fused mitochondrial networks are important for progression from G1 to S phase49 and stress resistance during starvation.50,51
Much evidence thus supports a function for PINK1 in cancer cell biology, although the underlying mechanism(s) are unclear. Therefore, this study aimed to investigate PINK1 function in cancer cell phenotypes. Our results show, for the first time, that PINK1 deletion causes cell cycle defects associated with aberrant regulation of mitochondrial fission, and highlight PINK1 as a regulator of the cell cycle and as a candidate oncogene.
PINK1 deletion suppresses several cancer-associated phenotypes
Initial experiments aimed to determine the impact of PINK1 deletion on tumor-associated phenotypes including cell proliferation, colony formation, migration and invasiveness (Figure 1). Cells with PINK1 deletion including several immortalized mouse embryonic fibroblast (MEF) lines derived from n=3 PINK1−/− mice, HeLa cells with stable knockdown of PINK1 and MCF-7 cells with transient knockdown of PINK1, all demonstrated significantly reduced proliferation rates compared with controls (Figure 1a). The slower growth rate of PINK1−/− compared with PINK1+/+ MEFs, was rescued by overexpression of human wild-type PINK1 (hPINK1res), but not by the triple kinase dead52 PINK1 mutant (hPINK1K219A/D362A/D384A) (Figure 1a). Colony formation assays revealed a significant decrease in clonogenic potential in PINK1−/− compared with PINK1+/+ MEFs, which was restored in hPINK1res cells, but not by hPINK1K219A/D362A/D384A (Figure 1b). Reduced colony formation was also observed in HeLa cells with stable PINK1 knockdown (Figure 1c), although not in MCF-7 cells with transient PINK1 deletion via small interfering RNA (siRNA) (data not shown), most likely due to the transient nature of siRNA deletion. Though deletion of PINK1 did not alter cell migration in wound-healing assays of fibroblasts, overexpression of human PINK1 caused a significant increase in cell migration (Figure 1d), as was found also using transwell migration assays (data not shown). In addition, PINK1−/− cells were found to be less invasive than their PINK1+/+ counterparts (Figure 1e), which was rescued in hPINK1res MEFs. Taken together, these results indicate that PINK1 is required for key cell phenotypes associated with cancer progression.
PINK1 deficiency alters cell cycle profile and increases the frequency of multi-nucleated cells
We were next interested to examine the mechanism by which PINK1 deletion reduced tumorigenic phenotypes. Here, we focused first on analysis of immortalized MEF cell lines derived from three pairs of PINK1+/+ and PINK1−/− mice. Microscopic examination of these PINK1−/− MEFs revealed morphological differences compared with PINK1+/+ MEFs, whereby PINK1−/− cells appeared larger and flatter, a description commonly used for senescent cells.53 Use of a senescence assay to detect β-galactosidase activity (Figure 2a) and cell cycle analysis (Figure 2b) with late passage (p6) non-immortalized MEFs as a positive control showed only p6 MEFs were senescent, as shown by reduced cycling cells and increased cells in G0/G1.
However, these results did reveal significant differences in the cell cycle profile of PINK1−/− compared with PINK1+/+ cells, whereby deletion of PINK1 caused a significant increase in the number of cells in G2/M phase, concomitant with a significant decrease in the percentage of cells in G0/G1 phases (Figure 2b). These changes were rescued by overexpression of human PINK1, although not rescued by the PINK1K219A/D362A/D384A mutant (Figure 2b). This observation was supported by morphological analysis of each of the three independently derived PINK1−/− MEF lines, which consistently revealed a highly significant increase in the frequency of double and multi-nucleated cells compared with PINK1+/+ MEFs (Figure 2c). This phenotype was rescued in hPINK1res MEFs and significantly less effectively in hPINK1K219A/D362A/D384A MEFs. (Figure 2c). Results further showed that, similar to MEFs lacking PINK1, BE(2)-M17, a dopaminergic neuroblastoma cell line, stably transduced with short hairpin RNA (shRNA) sequences directed against PINK113 demonstrated a significant increase in the number of cells in G2/M phase, concomitant with a significant decrease in the percentage of cells in G0/G1 phases, compared with cells with control shRNA and the parental BE(2)-M17 line (Supplementary Figure 1).
Taken together, these results indicate that PINK1 deletion alters cell cycle profile, increasing both the number of cells in G2/M and multi-nucleated cells, a phenotype that would impact on effective cell division and growth, which is rescued by overexpression of PINK1 and involves PINK1 kinase activity.
PINK1 deletion impairs cell division during G2/M
Next, we performed a more stringent analyses of the impact of PINK1 deletion at G2/M phase following synchronization with the mitotic spindle inhibitor nocodazole. The percentage of cells in G2/M did not reduce over time in PINK1−/− MEFs following release from nocodazole block, as seen in PINK1+/+ MEFs (Figure 3a, Supplementary Figure 2). Thus, PINK1−/− cells remained in G2/M phase up to 2 h post-nocodazole release (Figure 3a), which was rescued by overexpression of hPINK1. A high proportion of hPINK1K219A/D362A/D384A MEFs also remained in G2/M, but by 2 h, this population was reduced, although not to the same extent as PINK1+/+ or hPINK1res MEFs.
The inability of PINK1-deficient cells to exit G2/M phase was also observed using double immunofluorescence with 4,6-diamidino-2-phenylindole and α-tubulin, which showed that the majority of PINK1−/− cells had not divided 2 h post-release from nocodazole block (Figure 3b). PINK1-deficient cells underwent chromosome segregation and nuclear envelope reformation, indicating that this defect occurs at cytokinesis. In contrast at this time point, PINK1+/+, and hPINK1res cells had divided into two daughter cells associated only by the tubulin-rich midbody, indicative of the end stage of cytokinesis. hPINK1K219A/D362A/D384A MEFs showed both dividing cells and multi-nucleated cells, indicating only partial rescue of the cell division defect at this time point (Figure 3b). Together, these data indicate that the inability to progress from G2/M to G1 induced by PINK1 deficiency is rescued by human PINK1 and involves PINK1 kinase activity. Immunofluorescence microscopy revealed that human PINK1 localized to distinct poles at the periphery of the mitotic plane throughout mitosis and showed increased colocalization with the tubulin cytoskeleton at cytokinesis (Supplementary Figure 3).
Results further showed significant alterations in the profile of key cell cycle-associated proteins in PINK1−/− compared with PINK1+/+ fibroblasts up to 24 h post-release from nocodazole block (Figure 3c), consistent with the altered cell cycle profile. Phospho-histone H3, which is phosphorylated early in mitosis, is not phosphorylated at 6 h or even up to 24 h following nocodazole release in PINK1−/− cells, as occurs in PINK1+/+ MEFs, with partial rescue in hPINK1res cells, indicating that PINK1−/− cells do not undergo subsequent mitosis at the same rate as the control cells. Total histone H3 levels were not significantly different in PINK1−/−, PINK1+/+ or hPINK1res cells. Moreover, cyclin D1, whose increase in G1 phase is essential for cell cycle entry, is markedly reduced at all time points in PINK1−/− compared with PINK1+/+ cells, with cyclin D1 expression levels restored toward wild-type levels in hPINK1res cells.
The mitotic kinase (MBP, mitotic-promoting factor) cyclin B1/Cdk1 is essential for initiation of the mitotic programme with reduction of cyclin B1 levels being necessary for mitotic exit.46 Cyclin B1 and Cdk1 expression profiles are altered in PINK1−/− compared with PINK1+/+ MEFs, with cyclin B1 failing to increase at later time points and increased Cdk1 expression at all time points in PINK1−/− MEFs (Figure 3c). Unlike cyclin D1 expression and histone H3 phosphorylation, these changes were not rescued in hPINK1res MEFs. p53 levels are similar up to 24 h post-release from nocodazole block in all cells. These results were also reflected in BE(2)-M17 cells transduced with the PINK1 shRNA, which showed significantly reduced cyclin D1 expression, and significantly increased phospho-histone H3 levels and Cdk1 expression compared with cells with control shRNA (Supplementary Figure 4a). Together, these data indicate that PINK1 promotes progression through mitosis and cytokinesis to G1.
Multi-nucleation in PINK1-deficient cells is associated with increased mitochondrial fission
PINK1 function is inextricably linked with the maintenance of effective mitochondrial dynamics. Mitochondrial fission/fusion transitions are critical for cell cycle progression especially through G2/M to G147, although this has never been investigated in the context of PINK1. Mitochondrial fission during mitosis is essential for the equal distribution of mitochondria to daughter cells,46 whereas inhibition of fission and transition to a fused state permits progression through cytokinesis to G147. We thus hypothesized that the regulation of mitochondrial fission/fusion by PINK1 is important mechanistically in the transition to G1, and that defects in this regulation are an underlying mechanism in the failure of PINK1−/− cells to complete mitosis, leading to multi-nucleation.
Confocal microscopy revealed a small but significant decrease in mitochondrial interconnectivity (Figure 4a) but not elongation (Figure 4a), both measures of mitochondrial fusion,54 in single nucleated PINK1−/− MEFs compared with PINK1+/+ cells. Importantly, however, PINK1−/− cells that exhibited multi-nucleation were found to have a highly significant decrease in both mitochondrial interconnectivity and elongation, indicating major mitochondrial fragmentation and fission in cells which fail to divide (Figure 4a). Exogenous expression of hPINK1 rescues this fission and hPINK1res cells demonstrated significantly increased levels of mitochondrial interconnectivity and elongation compared with PINK1−/− multi-nucleated cells (Figure 4a). Similar results were observed when cells were fixed and stained with the nuclear stain 4,6-diamidino-2-phenylindole to identify multiple nuclei (Figure 4b).
PINK1-deficiency induced G2/M defects associate with impaired Drp1 regulation
The GTPase Drp1 is the major mediator of mitochondrial fission, and Drp1 function has been linked to PINK1 previously,13,45 albeit never in the context of cell cycle progression where Drp1 function is critical.46,47,55 During mitosis, Drp1 expression, mitochondrial recruitment and activity increase inducing mitochondrial fission. Mitochondria are distributed to daughter cells in metaphase, and Drp1 expression and activity decreases subsequently in anaphase and cytokinesis, through degradation via the anaphase-promoting complex (APC/Ccdh1),48 as mitochondria return to a more fused morphology.46,55,56
Therefore, we investigated whether impaired regulation of Drp1 is associated with the cell cycle defects caused by PINK1 deficiency. Immunoblot analysis showed Drp1 expression was significantly higher in PINK1−/− (Figure 5a) compared with PINK1+/+ MEFs, which was restored by overexpression of human PINK1, but less effectively in cells expressing the kinase dead hPINK1K219A/D362A/D384A or partial kinase dead hPINK1K219A mutants.52 Double immunofluorescence of Drp1 and MitoTracker Green (MTG) showed Drp1 colocalized more strongly with mitochondria in PINK1−/− compared with PINK1+/+ fibroblasts, which was most evident in cells that fail to divide and exhibit multi-nucleation (Figure 5b). Similarly, PINK1−/− cells overexpressing hPINK1K219A/D362A/D384A demonstrated increased Drp1/MitoTracker Green colocalization only in their multi-nucleated cells. Overexpression of hPINK1 rescued Drp1 cytoplasmic localization (Figure 5b).
Phosphorylation of Drp1 at Ser585 rat sequence (Ser616 human Drp1 variant 1) by cyclin B1/Cdk1 is essential for mitotic entry and for increased Drp1-mediated mitochondrial fission during metaphase neccessary for mitochondrial distribution to daughter cells at cytokinesis.46,47 Thereafter, APC/Ccdh1-induced degradation of cyclin B1 coincides with reduced phospho-Drp1Ser585 levels, and increased Drp1 degradation essential for mitotic exit and increased mitochondrial fusion necessary for entry to G1.46,47 We found that this phosphorylation-induced regulation of Drp1 during mitosis is impaired in PINK1−/− compared with PINK1+/+ cells. Upon release from nocodazole block, Drp1 and phospho-Drp1Ser585 levels reduced slightly at 3 h in PINK1+/+ cells during G1 phase. Thereafter, both Drp1 expression and phospho-Drp1Ser585 levels increased at 6 h, as the cells initiate the next round of mitosis and phospho-Drp1Ser585 levels migrate predominantly as a distinctive higher molecular weight band (Figure 5c). In stark contrast, Drp1 and phospho-Drp1Ser585 levels were significantly higher at time zero and all subsequent time points following release from nocodazole block in PINK1-deficient cells, where phospho-Drp1Ser585 is only evident as the higher molecular weight phosphorylated band at all time points (Figure 5c). This is indicative of sustained phosphorylation at Drp1Ser585 concomitant with prolonged residence in the mitotic pre-cytokinesis phase in PINK1−/− cells. Together, these results indicate that the multi-nucleation and failure to exit mitosis caused by PINK1 deficiency is linked to excessive mitochondrial fission, increased Drp1 expression, increased and sustained phosphorylation of Drp1Ser585, and increased Drp1 mitochondrial localization. As Drp1Ser585 is phosphorylated by cyclin B1/Cdk1, we sought to determine whether cyclin B1/Cdk1 complexes were increased in PINK1−/− compared with PINK1+/+ fibroblasts. Cyclin B1 levels that co-immunoprecipitated with Cdk1 in PINK1−/− cells were marginally higher than in PINK1+/+ cells (Supplementary Figure 4b). Together with the higher Cdk1 levels in PINK1−/− cells, this could tentatively indicate increased activity of the complex.
Knockdown of Drp1 reduces multi-nucleation caused by PINK1 deletion
In order to further investigate the effect of mitochondrial fission on cell cycle defects in PINK1-deficient cells, we used an RNA interference approach to suppress Drp1 expression in PINK1−/− MEFs. Transfection with Drp1 siRNA resulted in a decrease of ~50% of Drp1 protein expression in PINK1−/− MEFs (Figure 6a, lane 3) reducing Drp1 levels to the level found in PINK1+/+ cells (Figure 6a, final lane). This resulted in reduced levels of fragmented mitochondria with increased levels of tubulated, interconnected mitochondria in PINK1−/− MEFs (Figure 6b), the characteristic morphology described previously following Drp1 knockdown.46 Morphological analysis further showed that Drp1 knockdown significantly reduced the frequency of multi-nucleated cells in PINK1−/− MEFs (Figure 6c). Thus, inhibition of mitochondrial fission, via reduction of Drp1, rescues multi-nucleation caused by PINK1 deficiency.
G0/G1 cell cycle exit impairment in PINK1-deficient cells is also associated with defective mitochondrial dynamics
A significant decrease in the number of cells in G0/G1 phases was detected in PINK1-deficient MEFs (Figure 2b). To investigate this more stringently, cells were synchronized at G0/G1 by removal of growth factors for 24 h, with subsequent release into serum and cell cycle analysis (Figure 7a, Supplementary Figure 5). Serum deprivation causes cells to exit the cell cycle at G0/G1 and to stop dividing, as occurred in PINK1+/+ fibroblasts (Figure 7a). In contrast, significantly fewer PINK1−/− cells arrested in G0/G1, with 10% of cells remaining in G2/M. This was partially rescued in hPINK1res, but not in hPINK1K219A MEFs, indicating PINK1 kinase dependence, for effective cell cycle exit. Upon restoration of serum, all cells re-entered the cell cycle, returning to their normal profile after 24 h (Supplementary Figure 5).
Reduced mitochondrial fission and increased mitochondrial membrane potential (Δψm) have a protective role during serum deprivation-induced cell cycle exit.50,51 Flow cytometry analysis with the potentiometric dye tetramethylrhodamine, ethyl ester showed that Δψm was significantly reduced in PINK1−/− compared with PINK1+/+ fibroblasts (Figure 7b), as previously reported.14 Moreover, we found that PINK1−/− MEFs did not undergo an increase in Δψm following serum deprivation as observed in PINK1+/+ cells (Figure 7b). Serum deprivation caused no change to mitochondrial morphology in PINK1+/+ MEFs, with a mix of both interconnected mitochondrial networks and punctate, circular mitochondria observed (Figure 7c). In contrast, removal of serum in PINK1−/− MEFs caused a striking induction of punctate, fragmented mitochondria, which was in part prevented by overexpression of hPINK1 (Figure 7c).
Double immunofluorescence microscopy of Drp1 with pDsRed2-Mito corroborated these findings and showed increased mitochondrially localized Drp1 upon serum deprivation in PINK1−/− MEFs compared with PINK1+/+ cells (Figure 7d). A small percentage of mitotic cells were observed in PINK1−/− MEFs following 24-h incubation without growth factors supporting the cell cycle analysis (Figure 7d, bottom panel, 4,6-diamidino-2-phenylindole staining). These findings indicate that ineffective cell cycle exit caused by the absence of PINK1 is associated with increased Drp1-mediated mitochondrial fission and an inability to increase Δψm following serum deprivation. Taken together, our data indicate PINK1 regulates the cell cycle at stages critical for division, growth and stress resistance via regulation of mitochondrial function.
The past decade has seen substantial research into PINK1 function since its discovery as an autosomal recessive Parkinson’s disease-causing gene. Recently, increasing attention has implicated PINK1 in a number of processes linked to cancer,1,17,18,24, 25, 26 although limited information is available regarding fundamental mechanisms for PINK1 in cancer. This study demonstrates a new role for PINK1 as a cell cycle regulator at key points necessary for division, growth and stress resistance in cancer, and reveals that deletion of PINK1 inhibits several cancer-causing phenotypes including proliferation, colony formation and migration. Results show PINK1 kinase activity is necessary for proper cell cycle progression at G2/M specifically during cytokinesis, and during cell cycle exit at G0/G1. Although several reports have described varied roles for PINK1 in processes associated with cell cycle regulation, such as mitochondrial dynamics,13,42,45 calcium flux,13,57, 58, 59 phosphatidylinositol 3 kinase/Akt signaling17,18 and autophagy,28 this is the first study to directly implicate PINK1 in cell cycle control. These findings provide a potential mechanism, which could underlie several phenotypes previously described for PINK1 deficiency, and in the context of this study are of critical importance for cancer cell biology.
We also show that the regulation of Drp1-mediated mitochondrial fission is important in the control of cell cycle progression by PINK1 to transition from G2/M to G1 and to exit the cell cycle following serum deprivation. It is well documented that PINK1 regulates mitochondrial fission/fusion balance via Drp113, 42, 43, 44, 45, although this was not previously considered in the context of the cell cycle. Previous results, as also demonstrated here, showed that deletion of PINK1 in dividing cells causes excessive fission, rescued by Drp1 deletion.13,45 In direct contrast, in terminally differentiated tissues in Drosophila, Drp1 overexpression rescues PINK1-null phenotypes.43,44,60 This conundrum could be explained by our findings when considering the context-dependent regulation of Drp1 at different cell cycle stages in dividing cells and in terminally differentiated post-mitotic cells. Mitochondrial fission/fusion and bioenergetics are emerging to be critical for effective cell cycle progression.46,47,49,55,56 Our findings now draw attention to the importance of further understanding the mitochondrial functions of PINK1 in cell cycle regulation in the context of both cancer and neurodegeneration.
Induction and completion of mitosis is essential for cancer cell proliferation, and much of the cell cycle machinery is increased in cancer cells.61 Indeed, cyclin D1, found here to be reduced in PINK1-deficient cells, is a major proto-oncogene and driver of cell cycle progression.62 PINK1 inhibition was shown previously to sensitize breast cancer cells to chemotherapeutic treatment with paclitaxel,26 implicating PINK1 in chemoresistance. Paclitaxel inhibits spindle disassembly, causing cell death in dividing cells.63 Here we show that deletion of PINK1 alone can prevent cell division, indicating a potential mechanism through which PINK1 inhibition and treatment with paclitaxel synergistically kills cancer cells, and lending further credence to PINK1 as a potential target in chemoresistant tumors. Impaired mitosis in PINK1-deficient cells will constrain proliferation and likely result in death, however, failure to properly complete cell division and aneuploidy are feature of cancer cells.64 Thus, in certain contexts, PINK1 deletion may facilitate gain of chromosomes, increasing the frequency of chromosomal aberrations and explain the opposing functions described for PINK1 in cancer.24
Cell cycle aberrations have primary functions in cancer but are also implicated in neurodegenerative disorders, where abortive cell cycle re-entry is mechanistically linked to cell death of post-mitotic neurons.65, 66, 67, 68 Thus, these results have clear importance for understanding PINK1 with respect to cell cycle regulation in cancer, but also when considering the protective function of PINK1 against neurodegeneration in Parkinson’s disease.
Materials and methods
Generation of PINK1−/− mice and derived MEF cell lines
PINK1−/− knockout and PINK1+/− heterozygous knockout mice were generated by Wolfgang Wurst and Daniela Vogt-Weisenhorn (Helmholtz Center, Munich, Germany) and immortalized MEFs were generated as previously described.14 Immortalized MEFs from three pairs of PINK1−/− and matched PINK1+/+ mice were generated. In brief, PINK1+/− mice were interbred to generate mutant mice and wild-type littermate controls. Embryonic day 13 embryos were dissected, heads and red organs removed and used for genotyping. The rest of the bodies were chopped up in cell culture dishes containing Dulbecco’s modified Eagle's medium supplied with 50% fetal bovine serum and 1% penicillin/streptomycin. Cultures were expanded and serum decreased to 10% fetal bovine serum after the attainment of consistent growth. Afterward cultures were immortalized by transfection with simian virus 40 (SV40) large T-antigen. PINK1−/− MEFs were stably transfected with a plasmid containing human PINK1 (hPINK1 construct Origene (Rockville, MD, USA)), the partial kinase dead hPINK1K219A and triple kinase dead hPINK1K219A/D362A/D384A mutants using site-directed mutagenesis (Stratagene, Santa Clara, CA, USA). hPINK1 expression was confirmed using RNA extraction and analysis. As indicated, experiments were performed using MEF lines from three pairs of PINK1+/+ and PINK1−/− mice.
Cell culture and cell synchronization
MEFs, MCF-7 and HeLa cells were cultured in dulbecco’s modified eagle's medium supplemented with 10% fetal bovine serum and 2 mM L-glutamine in a humidified atmosphere containing 5% CO2 at 37 °C. BE(2)-M17 cells transduced with PINK1 shRNA or with control shRNA were kindly provided by Mark Cookson and Alexandra Beilina NIA, Bethesda, MD, USA and were transduced and cultured as previously described.13 G2/M synchronization was achieved by treatment with 40 ng/ml nocodazole in complete media for 16 h. Cells were synchronized at G0/G1 phase by incubation with serum-free dulbecco’s modified eagle's medium for 24 h.
Plasmids, siRNA and shRNA transfection
MEFs were transfected with pDsMitoRed2-Mito (Clontech, Palo Alto, CA, USA) using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s instructions and incubated with DNA/Lipofectamine transfection mix for at least 12 h before being imaged on a Zeiss LSM 510 META Confocal microscope (Jena, Germany) or being fixed for immunofluorescence. For siRNA knockdown, MCF-7 cells or PINK1−/− MEFs were transfected for 24–48 h with 100 nM negative control siRNA (Ambion, Austin, TX, USA), PINK1-targeting siRNA (Qiagen, Germantown, MD, USA) or Drp1-targeting siRNA (Sigma, St Louis, MO, USA) using HiPerfect (Qiagen) according to the manufacturer’s instructions. Stable PINK1 knockdown was achieved in HeLa cells using a 29mer shRNA construct against human PINK1 cloned into a retroviral untagged vector (Origene). The shRNA sequence was 5′-IndexTermCCAGAACCTGGAGGTGACAAAGAGCACCG-3′. Cells were transfected with Lipofectamine 2000 and selected with 5 μg/ml puromycin.
Proliferation and colony formation assays
Cells were seeded at 4 × 104 cells per well in a 24-well plate. To monitor cell growth at intervals, attached cells were removed from quadruplicate wells using trypsin-ethylenediaminetetra acetic acid and viable cells counted by Trypan blue exclusion. Anchorage-independent cell growth was determined by assaying colony formation in soft agarose as described previously.69 In all, 1 × 103 cells were resuspended in 0.33% low-melting point agarose (Sigma) in dulbecco’s modified eagle's medium/10% fetal bovine serum and plated in triplicate onto 35-mm dishes containing a 2-ml base agarose layer (0.6%). After 14 days, colonies were stained with 0.01% crystal violet in 20% ethanol and counted.
Migration and invasion assays
Cells were plated in triplicate in six-well plates. Confluent monolayers were scratched using a sterile tip (time 0 h) and cells were allowed to migrate into the wound for 8 h. Wound closure was determined comparing the same field at 0 and 8 h using T-Scratch software.70 For invasion assays, 1 × 105 cells were allowed to migrate into a 5 μM pore Transwell, pre-coated with 7 mg/ml Matrigel (BD Biosciences, Heidelberg, Germany). After 24 h, cells were fixed with 100% methanol, stained with 0.1% crystal violet and counted using a light microscope.
Cell cycle analysis and flow cytometry
For cell cycle analysis, cells were resuspended in ice-cold phosphate-buffered saline. Before flow cytometry, NP-40 and propidium iodide (Sigma) were added at a final concentration of 0.1% and 50 μg/ml, respectively. DNA content was measured in the FL2 channel using CellQuest software (Becton Dickinson, Oxford, UK). Δψm was measured by incubation with 50 nM tetramethylrhodamine, ethyl ester (Molecular Probes, Eugene, OR, USA) before being harvested for flow cytometry, with Δψm measured in the FL2 channel.
Confocal microscopy and morphological analysis
Fluorescence images were acquired Zeiss LSM 510 META confocal microscope fitted with a 63 × /1.4 plan apochromat lens (Jena, Germany). For details of confocal microscopy and morphological analysis, see Supplementary Materials and methods.
Western blot analysis
Proteins were extracted for sodium dodecyl sulfate–polyacrylamide gel electrophoresis and western immunoblot analysis was performed as described in Supplementary Materials and methods.
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We are grateful to Mark Cookson and Alexandra Beilina, National Institute on Aging, Bethesda, MD, USA for providing us with BE(2)-M17 cells transduced with control and PINK1 shRNA. We thank Rosemary O’Connor, School of Biochemistry and Cell Biology, University College Cork (UCC) for many helpful discussions, Sandra Yeomans UCC for technical assistance and Daniela Vogt-Weisenhorn, Helmholtz Centrum München, for helpful input. This work was funded by the Health Research Board of Ireland, PhD Scholars’ Programme in Cancer Biology. Support from Science Foundation Ireland (SFI) (RFP), the FWO Foundation for Scientific Research Belgium, a Methusalem grant of the Flemish Government and the KU Leuven, and the Helmholtz Alliance for Mental Health in an Ageing Society is also gratefully acknowledged. BDS is the Arthur Bax and Anna Vanluffelen Chair for Alzheimer’s disease. The Molecular Cell Biology group, UCC, provided access to a Zeiss 510 Confocal microscope, funded by an SFI Programme Grant to Mary W McCaffrey.
The authors declare no conflict of interest.
Supplementary Information accompanies this paper on the Oncogene website
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O'Flanagan, C., Morais, V., Wurst, W. et al. The Parkinson’s gene PINK1 regulates cell cycle progression and promotes cancer-associated phenotypes. Oncogene 34, 1363–1374 (2015). https://doi.org/10.1038/onc.2014.81
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