Somatic and germline mutations in the tumor suppressor gene park2 impair pink1/parkin-mediated mitophagy in lung cancer cells

ABSTRACT _PARK2_, which encodes Parkin, is a disease-causing gene for both neurodegenerative disorders and cancer. Parkin can function as a neuroprotector that plays a crucial role in the

regulation of mitophagy, and germline mutations in _PARK2_ are associated with Parkinson’s disease (PD). Intriguingly, recent studies suggest that Parkin can also function as a tumor

suppressor and that somatic and germline mutations in _PARK2_ are associated with various human cancers, including lung cancer. However, it is presently unknown how the tumor suppressor

activity of Parkin is affected by these mutations and whether it is associated with mitophagy. Herein, we show that wild-type (WT) Parkin can rapidly translocate onto mitochondria following

mitochondrial damage and that Parkin promotes mitophagic clearance of mitochondria in lung cancer cells. However, lung cancer-linked mutations inhibit the mitochondrial translocation and

ubiquitin-associated activity of Parkin. Among all lung cancer-linked mutants that we tested, A46T Parkin failed to translocate onto mitochondria and could not recruit downstream mitophagic

regulators, including optineurin (OPTN) and TFEB, whereas N254S and R275W Parkin displayed slower mitochondrial translocation than WT Parkin. Moreover, we found that deferiprone (DFP), an

iron chelator that can induce mitophagy, greatly increased the death of A46T Parkin-expressing lung cancer cells. Taken together, our results reveal a novel mitophagic mechanism in lung

cancer, suggesting that lung cancer-linked mutations in _PARK_2 are associated with impaired mitophagy and identifying DFP as a novel therapeutic agent for _PARK2_-linked lung cancer and

possibly other types of cancers driven by mitophagic dysregulation. You have full access to this article via your institution. Download PDF SIMILAR CONTENT BEING VIEWED BY OTHERS PARKIN

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June 2023 INTRODUCTION Parkin is an important E3 ubiquitin ligase that is activated by phosphorylated ubiquitin and promotes the polyubiquitination of various substrates [1]. Under normal

conditions, Parkin localizes throughout the nucleus and cytosol, and it can translocate onto damaged mitochondria to promote the ubiquitination of mitochondrial proteins and trigger

mitophagy under stress conditions [2]. Loss-of-function mutations in _PARK2_, the gene encoding Parkin, cause Parkinson’s disease (PD) [3]. However, Parkin knockout mice did not display any

neurodegenerative phenotype but developed spontaneous hepatocellular carcinoma and tumor growth [4, 5], indicating the complexity of Parkin’s functions and associated disease mechanisms.

Moreover, the expression level of Parkin was found to be decreased in several human cancers, including lung cancer [6, 7], and somatic mutations in _PARK2_ were found in glioblastoma and

lung cancer [8, 9]. Importantly, overexpression of Parkin inhibited cell growth and proliferation in multiple cancer cell lines [10,11,12], indicating that Parkin can act as a tumor

suppressor and that its dysfunction may contribute to oncogenesis in somatic cells. Autophagy is an important degradation pathway responsible for cell homeostasis [13]. Although the role of

autophagy in cancer cell survival and death has been extensively explored, the relationship between cancer and the selective forms of autophagy, including mitophagy, is not yet well

understood [14]. Parkin acts as a crucial mediator in mitophagy regulation [2, 14]. Recent research advances in mitophagy have revealed that under normal conditions, Parkin displays an

autoinhibited state, since the active site of Parkin is sealed by the REP domain [15], whereas under disease conditions or mitochondrial stress, Parkin is phosphorylated by mitochondrial

PINK1 at the N-terminal ubiquitin-like (UBL) domain and interacts with phosphorylated ubiquitin on the mitochondrial outer membrane [16]. After a series of conformational changes, the RING

finger E3 catalytic center is exposed, and Parkin acts as an activated E3 ubiquitin ligase [2, 15]. According to recent studies, Parkin can facilitate the ubiquitination of outer

mitochondrial membrane (OMM) proteins and therefore enhance the recognition of polyubiquitinated mitochondria by mitophagic receptors, such as optineurin (OPTN) or NDP52 [17, 18]. These

receptors can bind to autophagosomal proteins, such as LC3 and target ubiquitinated mitochondria for lysosomal degradation [19]. Moreover, a recent report showed that TFEB, the master

transcription factor regulating the expression of autophagic and lysosomal genes, is a downstream regulator of mitophagy [20]. The above process, which is very powerful for clearing damaged

mitochondrial and is the most well-understood form of mitophagy, is called PINK1/Parkin-mediated mitophagy [2]. According to previous studies, somatic _PARK2_ mutations in lung cancer

include the A46T, N254S and H279P mutations [8], while germline mutations in _PARK2_ are found in familial lung cancer [9]. It is worth noting that all these lung cancer-linked mutations are

highly enriched in ubiquitin-associated domains of Parkin. In detail, the A46T mutation occurs in the UBL domain, whereas the N254S, R275W and H279P mutations occur in the RING1 domain of

Parkin [8, 9]. These findings suggest that Parkin-associated ubiquitin regulation may contribute to the disease pathogenesis underlying lung cancer. However, whether these mutations affect

Parkin-mediated mitophagy and the potential role of this alteration in lung cancer tumorigenesis remain unknown. In this study, we showed that these lung cancer-linked mutations abolish the

recognition of damaged mitochondria by autophagosomes due to the loss of mitochondrial ubiquitination driven by Parkin. Importantly, we identified that deferiprone (DFP), which is known as a

mitophagy enhancer [21], can induce cell death in lung cancer cells expressing mutant Parkin. MATERIALS AND METHODS PLASMID CONSTRUCTION AND SIRNAS The FLAG, FLAG-Parkin, HA-Ub, GFP-OPTN,

mCherry-C1 and GFP-TFEB plasmids were described before [22,23,24,25,26,27]. mCherry-Parkin was generously provided by Richard Youle (Addgene #23956); BFP-mito was generously provided by Gia

Voeltz (Addgene #49151). FLAG-tagged and mCherry-tagged A46T, N254S and R275W Parkin mutants were generated by site-directed mutagenesis with a MutanBEST kit (Takara, Shiga, Japan).

pcDNA3.1-mt-Keima was generated by inserting mKeima cDNA into the vector at the _Kpn_I and _EcoR_I sites. All constructs were verified by sequencing. Small interfering RNAs targeting human

OPTN and PINK1 were synthesized by Shanghai GenePharma. CELL CULTURE AND TREATMENTS Human alveolar epithelial cells (A549) were cultured in DMEM (Gibco, Grand Island, NY, USA) containing 10%

FBS (Gibco), 100 U/mL penicillin and 100 mg/mL streptomycin (Gibco). Cells were transfected with DNA plasmids using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) 24 h after splitting

and with siRNA using Lipofectamine RNAiMAX (Invitrogen) upon splitting. Propidium iodide (PI, Sigma, St. Louis, MO, USA; 0.5 mg/mL) staining was used to detect dead cells. Antimycin A1,

chloroquine (CQ), CCCP and Hoechst were purchased from Sigma. Oligomycin was purchased from Calbiochem. Bafilomycin A1 (Bafi A1) was obtained from Selleckchem, and MitoTracker Red CMXRos was

purchased from Thermo Fisher Scientific (Waltham, MA, USA). IMMUNOBLOT ANALYSIS AND IMMUNOFLUORESCENCE For immunoblot analysis, cells were lysed with cell lysis buffer [22, 25, 27].

Proteins were then separated by SDS–PAGE and transferred onto PVDF membranes (Millipore, Billerica, MA, USA). The primary antibodies anti-GAPDH and anti-Hsp60 were purchased from

Proteintech. Horseradish peroxidase-conjugated sheep anti-mouse antibody (Jackson ImmunoResearch Laboratories, PA, USA) was used as the secondary antibody. Proteins were detected using an

ECL detection kit (Thermo Fisher Scientific). Immunoblot analysis was performed with a standard protocol as previously reported [28,29,30,31,32,33]. For immunofluorescence, cells were fixed

with 4% paraformaldehyde, permeabilized with 0.1% Triton X-100 and blocked with 0.1% FBS. Then, cells were incubated sequentially with primary antibodies and secondary antibodies. Anti-HA

(Santa Cruz, CA, USA), anti-FLAG (Sigma) and anti-TOM20 (Proteintech, Rosemont, IL, USA) antibodies were used as primary antibodies. Alexa Fluor 405-conjugated goat anti-rabbit IgG (H+L)

(Invitrogen) and Alexa Fluor 594-conjugated goat anti-mouse IgG (Proteintech) were used as secondary antibodies. Eventually, cells were visualized using a Nikon [34] or Zeiss LSM710 confocal

microscope [35, 36]. Briefly, the imaging analysis was performed by researchers who were blinded to the study, and fluorescence was quantified using ImageJ software. For live cell imaging,

cells were stained with MitoTracker Red CMXRos and Hoechst as described in Fig. S4. STATISTICAL ANALYSIS Parkin and OPTN mitochondrial localization was determined by visual inspection in 10 

min intervals. PI-positive cells were considered nonviable. Data are presented as the means ± SDs. Comparisons were performed using Prism 6.0 (GraphPad Software). _P_ < 0.05 was

considered statistically significant. RESULTS BOTH SOMATIC AND GERMLINE MUTATIONS OF PARKIN IN LUNG CANCER IMPAIR PARKIN-MEDIATED MITOPHAGY Parkin, the E3 ubiquitin ligase, has been

identified as a tumor suppressor in several cancers, including lung cancer. We were interested in several _PARK2_ lung cancer mutations, including the somatic mutations A46T and N254S, and a

recurrent germline mutation, R275W (Fig. 1a) [8, 9]. To elucidate the effect of these Parkin mutations on mitophagy, we investigated the mitochondrial translocation of Parkin and

mitochondrial clearance in mutant Parkin-overexpressing lung cancer (A549) cells. As shown in Fig. 1, wild-type (WT) Parkin translocated onto damaged mitochondria, followed by mitochondrial

recruitment of the mitophagy receptor OPTN following A/O treatment, a standard method to induce cellular mitophagy [17]. However, Parkin with the lung cancer-related mutations—A46T, N254S

and R275W—showed delayed mitochondrial translocation to different degrees (Fig. 1b, d). Long-term mitophagy induction resulted in complete clearance of mitochondria in cancer cells with WT

Parkin. In contrast, mutant Parkin-expressing cells showed impaired mitochondrial clearance (Fig. 1c, e). PINK1/PARKIN-MEDIATED MITOPHAGY IS BLOCKED BY PARKIN MUTATIONS IN LUNG CANCER CELLS

Although PINK1/Parkin-mediated mitophagy has been extensively investigated, the relationship between mitophagy and lung tumorigenesis remains elusive. To understand the role of

PINK1/Parkin-mediated mitophagy in lung cancer cells, we used a long-term mitophagic degradation assay and investigated the turnover of mitochondrial proteins. Immunoblot analysis showed

that the level of the mitochondrial matrix protein Hsp60 was decreased dramatically in cells expressing WT Parkin (Fig. 2a). Depletion of the mitophagic receptor OPTN caused a reduction in

mitochondrial degradation, even in the presence of WT Parkin, which suggested that OPTN is a downstream mitophagic regulator of Parkin in lung cancer cells. Consistent with the fluorescence

results shown in Fig. 1, A46T and N254S mutant Parkin had no significant effect on mitochondrial degradation, regardless of whether OPTN was knocked down (Figs. 2a and S1a). To verify the

effect of PINK1 on Parkin-associated mitophagy in lung cancer cells, we knocked down the crucial mitochondrial initiator PINK1, which is reported to phosphorylate Parkin and ubiquitin on

mitochondria [2]. Compared to cells with endogenous PINK1 expression, PINK1-deficient lung cancer cells did not exhibit mitochondrial clearance upon A/O or CCCP treatment (Figs. 2b and S1b).

To further test whether the degradation of the mitochondrial protein Hsp60 occurs through autophagy but not other degradation systems, we blocked the autophagy pathway with Bafi A1 or CQ.

The results showed that these autophagy inhibitors abolished Parkin-mediated mitophagy (Fig. 2c). LUNG CANCER-LINKED MUTATIONS ABOLISH MITOCHONDRIAL RECRUITMENT OF PARKIN AND UBIQUITIN AND

DIMINISH NUCLEAR TRANSLOCATION OF TFEB DURING MITOPHAGY To further gain mechanistic insights into the failure of mitophagy driven by these Parkin mutations, we next performed a time-course

experiment to detect the speed of translocation of Parkin and OPTN onto mitochondria during mitophagy. As shown in Fig. 3, WT Parkin rapidly translocated to mitochondria in ~2 h, while N254S

Parkin finished this process at 3 h. Strikingly, A46T Parkin did not show strong mitochondrial localization within similar times (Fig. 3a, b). Therefore, we conclude that lung cancer-linked

UBL mutations in Parkin lead to a significant failure of mitochondrial Parkin and receptor recruitment, whereas RING 1 mutations delay recruitment. We next examined the translocation of

TFEB, which reflects TFEB activity, during Parkin-mediated mitophagy, since it has been shown that TFEB functions downstream of Parkin during mitophagy [20]. We found that translocation of

Parkin onto mitochondria was accompanied by TFEB nuclear translocation (Fig. 4a). However, the lung cancer-related Parkin mutations showed different effects. TFEB was expressed completely in

the cytoplasm in cells with A46T Parkin expression and partially in the nucleus in cells with N254S Parkin expression (Fig. 4a). As an E3 ubiquitin ligase, Parkin is known to ubiquitinate

damaged mitochondria and promote their clearance via autophagy. To determine the ubiquitin ligase activity of mutant Parkin, we tested the ubiquitination of mitochondria. The lung

cancer-related Parkin mutants failed to ubiquitinate damaged mitochondria as WT Parkin did (Figs. 4b and S2). Our observation indicates that mutation of Parkin impaired its E3 ligase

function in mitochondria. LUNG CANCER MUTANT PARKIN ATTENUATES MITOPHAGIC FLUX Based on the observation that mutated Parkin exhibits greatly delayed mitochondrial translocation and defective

clearance of Hsp60, we next visualized mitophagic flux using a fluorescence-sensitive probe, mt-Keima. According to previous studies, mt-Keima is a mitochondria-located fluorescent protein

that is pH-stable. Keima emits different signals with changes in pH. The signal peaks at 586 nm in acidic conditions, such as in the lysosomal lumen, or at 440 nm in neutral pH conditions.

Therefore, acidic autolysosomes containing mitochondria, which reflects mitophagy, can be detected as “561 nm” signals. Meanwhile, nonlysosomal mitochondria, which reflect the pool of

mitochondria that are not targeted by mitophagy, can be detected as “458 nm” signals [37, 38]. We found that 561 nm signals existed when mitochondria were damaged for a short time (Fig. 5,

left region, and Fig. S3), indicating that a portion of mitochondria were delivered to acidic lysosomes. Twenty-four hours of mitochondrial damage generated an overwhelming ratio of 561/458 

nm signals in Parkin-expressing cells, indicating that most mitochondria were targeted by mitophagy (Fig. 5b, right region). Regarding the lung cancer-related Parkin mutations, the R275W and

N254S mutations produced partial effects compared with those of WT Parkin, while the A46T mutation blocked most of the mitophagic flux. TREATMENT WITH DFP PREVENTS THE SURVIVAL OF MUTANT

PARKIN-EXPRESSING LUNG CANCER CELLS According to previous reports, strong induction of mitophagy can trigger cancer cell death [39]. We found that long-term Parkin-mediated mitophagy

significantly induced cell death in lung cancer cells (Fig. 6a). However, expression of lung cancer-related Parkin mutants, especially the A46T mutant, resulted in decreased cell death

compared to that seen with WT Parkin (Fig. 6a). Interestingly and importantly, we found that DFP, an iron chelator and mitophagy inducer available for clinical use [21], significantly

increased the death of A46T Parkin-expressing cancer cells (Fig. 6b). Moreover, we found that in contrast to A/O treatment, DFP treatment did not affect mitochondrial dynamics and membrane

potential (Fig. S4), indicating that DFP was not toxic to mitochondrial function and that it can induce cancer cell death by increasing mitophagic signaling. Taken together, these data

suggest that induction of mitophagy may be a potential treatment for lung cancer caused by Parkin loss-of-function mutations. DISCUSSION Autophagy plays a fundamental role in regulating the

intracellular clearance of organelles, proteins and pathogens. Previous studies have shown that autophagy is tightly associated with a number of human diseases, including neurodegenerative

diseases, inflammatory diseases and cancer [14]. It is worth noting that studies of PINK1/Parkin-mediated mitophagy have mainly focused on neurodegenerative diseases, such as PD, whereas the

pathogenic relationship between PINK1/Parkin-mediated mitophagy and cancer development remains elusive. The current concept of mitophagy is that cells may use mitophagy to remove damaged

mitochondria to protect themselves against toxicity generated by damaged mitochondria, such as reactive oxygen species (ROS). However, emerging evidence indicates that long-term induction of

mitophagy in Parkin-expressing cells would lead to cell death through mechanisms currently unknown but possibly associated with apoptosis. For example, a previous report showed that

ceramide, a known tumor suppressor widely used in preclinical and clinical studies in cancer research, could trigger cancer cell death and tumor suppression through lethal mitophagy [39].

Consistent with this finding, we found that DFP treatment significantly decreased the survival of lung cancer cell lines expressing pathogenic Parkin mutants (Fig. 6b). One issue in the

field of autophagy is the lack of chemicals that can directly enhance downstream signaling in the pathway, such as inducers of autophagosome–lysosome fusion or autophagosome–substrate

recognition. Given that DFP treatment increased the number of autolysosome-targeted mitochondria and benefits of the research progress made in the pharmacological regulation of mitophagy

[40], our study not only is helpful for understanding the contribution of PINK1/Parkin-mediated mitophagy to the pathogenesis of lung cancer development but also offers experimental evidence

for the effective treatment of lung cancer and possibly other types of human cancers. Mechanistically, the A46T mutation may affect the conformation of the UBL domain of Parkin, thereby

either inhibiting the phosphorylation of UBL or blocking UBL release, which, in turn, stops the subsequent activation of the Parkin E3 catalytic center. In addition, the N254S and R275W

mutations are in the RING1 domain, which may inhibit the interaction between phosphorylated ubiquitin and the RING1 domain, thereby decreasing the E3 activity of Parkin. To date, it is not

understood why both PD and cancer, two entirely different diseases, can be caused by inactivating mutations in Parkin. Additionally, it is intriguing that the single mutation R275W in

_PARK2_ can either cause PD or cause cancer. There are several possibilities. First, non-cell-autonomous toxicity may also contribute to dopaminergic neuron degeneration in PD, since

germline mutation in _PARK2_ will result in loss of Parkin function across all cell types, including glial cells such as microglia, astrocytes and oligodendrocytes, in the central nervous

system. Second, various types of cellular stress in affected neurons or tumor cells may lead to different expression levels of mitophagy-related proteins. The combination of this variety and

Parkin dysfunction may cause different biological consequences; therefore, the R275W germline mutation, which has already been shown to impair mitophagy [41], is associated with both PD and

cancer. The last but perhaps the most important consideration is that dopaminergic neurons are more vulnerable when mitochondrial functions decline during aging. In support of this idea,

Parkin-null mice did not develop a neurodegenerative phenotype under normal conditions, whereas they exhibited dopaminergic neuron-specific degeneration when exposed to mitochondrial stress

[42]. Given that PD is a late-onset, age-related disease with increasing mitochondrial damage during disease progression, it is reasonable to postulate that Parkin-inactivating mutations

would accelerate the accumulation of damaged mitochondria accompanied by high levels of toxic species, such as ROS, eventually resulting in the death of dopaminergic neurons and the

development of PD during the aging process. Taken together, our findings suggest that both PD-linked and cancer-linked mutations in _PARK2_ can inhibit mitophagy in different ways, and

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2015;87:371–81. CAS  PubMed  PubMed Central  Google Scholar  Download references ACKNOWLEDGEMENTS This work was supported by the National Natural Science Foundation of China (Nos. 31771117,

31701222 and 31571053), the National Key Plan for Scientific Research and Development of China (No. 2017YFC0909100), a Project Funded by Jiangsu Key Laboratory of Neuropsychiatric Diseases

(BM2013003) and a Project Funded by the Priority Academic Program Development of the Jiangsu Higher Education Institutes (PAPD). AUTHOR INFORMATION Author notes * These authors contributed

equally: Zeng-li Zhang, Na-na Wang AUTHORS AND AFFILIATIONS * Department of Respiration, The Second Affiliated Hospital of Soochow University, Suzhou, 215004, China Zeng-li Zhang & 

Min-hua Shi * Jiangsu Key Laboratory of Neuropsychiatric Diseases and College of Pharmaceutical Sciences, Soochow University, Suzhou, 215123, China Na-na Wang, Qi-lian Ma, Yang Chen, Li Yao,

 Hong-feng Wang & Zheng Ying * School of Pharmacy, Key Laboratory of Molecular Pharmacology and Drug Evaluation (Yantai University), Ministry of Education, Yantai University, Yantai,

264005, China Zheng Ying * Jiangsu Key Laboratory of Preventive and Translational Medicine for Geriatric Diseases, College of Pharmaceutical Sciences, Soochow University, Suzhou, 215021,

China Zheng Ying * Key Laboratory of Nuclear Medicine, Ministry of Health, Jiangsu Key Laboratory of Molecular Nuclear Medicine, Jiangsu Institute of Nuclear Medicine, Wuxi, 214063, China Li

Zhang * National University of Singapore (Suzhou) Research Institute, Suzhou, 215123, China Qiu-shi Li Authors * Zeng-li Zhang View author publications You can also search for this author

inPubMed Google Scholar * Na-na Wang View author publications You can also search for this author inPubMed Google Scholar * Qi-lian Ma View author publications You can also search for this

author inPubMed Google Scholar * Yang Chen View author publications You can also search for this author inPubMed Google Scholar * Li Yao View author publications You can also search for this

author inPubMed Google Scholar * Li Zhang View author publications You can also search for this author inPubMed Google Scholar * Qiu-shi Li View author publications You can also search for

this author inPubMed Google Scholar * Min-hua Shi View author publications You can also search for this author inPubMed Google Scholar * Hong-feng Wang View author publications You can also

search for this author inPubMed Google Scholar * Zheng Ying View author publications You can also search for this author inPubMed Google Scholar CONTRIBUTIONS ZY, HFW, MHS and QSL designed

the experiments and drafted the manuscript; ZLZ, NNW, QLM, YC, LY and LZ performed the experiments. CORRESPONDING AUTHORS Correspondence to Hong-feng Wang or Zheng Ying. ETHICS DECLARATIONS

COMPETING INTERESTS The authors declare no competing interests. SUPPLEMENTARY INFORMATION SUPPLEMENTARY INFORMATION RIGHTS AND PERMISSIONS Reprints and permissions ABOUT THIS ARTICLE CITE

THIS ARTICLE Zhang, Zl., Wang, Nn., Ma, Ql. _et al._ Somatic and germline mutations in the tumor suppressor gene _PARK2_ impair PINK1/Parkin-mediated mitophagy in lung cancer cells. _Acta

Pharmacol Sin_ 41, 93–100 (2020). https://doi.org/10.1038/s41401-019-0260-6 Download citation * Received: 22 March 2019 * Accepted: 21 May 2019 * Published: 08 July 2019 * Issue Date:

January 2020 * DOI: https://doi.org/10.1038/s41401-019-0260-6 SHARE THIS ARTICLE Anyone you share the following link with will be able to read this content: Get shareable link Sorry, a

shareable link is not currently available for this article. Copy to clipboard Provided by the Springer Nature SharedIt content-sharing initiative KEYWORDS * autophagy * mitophagy * Parkin *

ubiquitin * cancer