Dysregulation of mitophagy in carcinogenesis and tumor progression

    Dysregulation of Mitophagy in Carcinogenesis and Tumor Progression Joon Young Chang, Hyon-Seung Yi, Hyeon-Woo Kim, Minho Shong PII: DOI: Reference:

S0005-2728(16)30691-0 doi:10.1016/j.bbabio.2016.12.008 BBABIO 47763

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BBA - Bioenergetics

Received date: Revised date: Accepted date:

14 October 2016 9 December 2016 21 December 2016

Please cite this article as: Joon Young Chang, Hyon-Seung Yi, Hyeon-Woo Kim, Minho Shong, Dysregulation of Mitophagy in Carcinogenesis and Tumor Progression, BBA Bioenergetics (2016), doi:10.1016/j.bbabio.2016.12.008

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Dysregulation of Mitophagy in Carcinogenesis and Tumor Progression

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Joon Young Chang1, 2, Hyon-Seung Yi1, Hyeon-Woo Kim1, 2, and Minho Shong1*

Research Center for Endocrine and Metabolic Diseases, Chungnam National University School

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of Medicine, Daejeon 301-721, Korea

Department of Medical Science, Chungnam National University School of Medicine, Daejeon

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301-721, Korea;

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Running title: Mitophagy and Cancer

*Corresponding author and person to whom reprint requests should be addressed: Minho Shong,

Research Center for Endocrine and Metabolic Diseases, Chungnam National University School of Medicine, Daejeon 301-721, Korea Phone: 82-42-280-6994 Fax: 82-42-280-7995 E-mail: MS ([email protected])

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Abstract The mitochondrial role in carcinogenesis and cancer progression is an area of active

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research, with many unresolved questions. Various aspects of altered mitochondrial function

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have been implicated in tumorigenesis and tumor progression, including mitochondrial

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dysfunction, a metabolic switch to aerobic glycolysis, and dysregulation of mitophagy. Mitophagy is a highly specific quality control process which eliminates dysfunctional

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mitochondria and promotes mitochondrial turnover, and is involved in the adaptation to nutrient

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stress by controlling mitochondrial mass. The dysregulation of mitochondrial turnover has both a positive and negative role in cancer. This review will begin with a basic overview of the

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molecular mechanisms of mitophagy, and highlight recent trends in mitophagy from cancer

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studies. We will conclude this review by discussing areas of research in normal mitophagy that have yet to be explored in the context of cancer such as mitochondrial proteases, the

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mitochondrial unfolded protein response, and mitokine action. Key Words: mitophagy, cancer, mitochondrial unfolded protein response, mitokine

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1. Introduction

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Mitochondria are essential double membrane-bound organelles with pleiotropic roles ranging from the production of cellular energy to the regulation of apoptosis and biosynthetic

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pathways. Mitochondria are highly dynamic and go through regular cycles of fusion and fission,

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as well as the maintenance of mitochondrial mass through mitobiogenesis and mitophagy. As the

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main organelle responsible for ATP production through oxidative phosphorylation, mitochondria are a regular source of cellular reactive oxidative species (ROS) from premature leakage of

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electrons from complexes I and III of the electron transport chain (ETC). Due to the endosymbiotic origin from proteobacteria, mitochondria contain a separate genome, or

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mitochondrial DNA (mtDNA) [1, 2]. The proximity of mtDNA to mitochondrial ROS, less

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robust repair mechanisms compared to nuclear DNA, and lack of histones make mtDNA highly

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susceptible to genetic mutations [3, 4]. However, recent evidence also suggests that mtDNA repair mechanisms are more developed than initially perceived [5]. Nevertheless, normal mitochondrial quality control is imperative for cellular homeostasis. Mitochondrial quality control is achieved through the process of mitochondrial dynamics, mitochondria-derived vesicles, and mitophagy [6]. Mitophagy is a type of selective macroautophagy, and degrades dysfunctional or excessive mitochondria through the process of the autophagosome-lysosome system. One regulator of mitophagy is the PTEN-induced putative kinase 1(PINK1)/Parkin pathway, which is triggered by membrane depolarization [7]. An alternate pathway exists where mitophagy is activated via Hif1α and is independent of PINK1/Parkin. The adaptor molecules in this case are BNIP3, NIX, and FUNDC1, which interact with LC3 at the outer mitochondrial membrane [7]. 3

Dysregulation of mitophagy

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contributes to the pathogenesis of various diseases including neurodegeneration, type II diabetes, and cancer [8-10].

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Cancer is a complex disease characterized by uncontrolled cellular proliferation, aerobic

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glycolysis, or the Warburg Effect, and aberrant mitochondrial quality control [11, 12]. Recent

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advances in mitophagy research in the context of cancer and non-cancer have provided significant insight in our understanding of mitochondrial quality control, and how its

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dysregulation leads to the onset of cancer. This review will begin with a brief overview of the

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molecular mechanisms of mitophagy, followed by recent studies in cancer mitophagy, and conclude with a discussion on areas of mitophagy research that have yet to be explored in the

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context of cancer.

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2. Molecular mechanisms of mitophagy

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Macroautophagy (hereafter autophagy) is an adaptive response which recycles cellular components by enclosing lipids, proteins, and organelles within an autophagasome, which is then fused with a lysosome to form an autolysosome to be degraded through lysosomal hydrolases. The hVps34-Beclin1 complex is initially formed, which is integral in the formation of the phagophore membrane. The autophagy-related (Atg) family of proteins coordinate the subsequent stages of autophagy. Atg7 is involved in the formation of a ubiquitin-like conjugation system which catalyzes the covalent linkage of Atg5 to Atg12 and Atg16L1. Atg8/LC3 lipidation of Atg4 leads to the processing of pro-LC3 to its active form LC3-I. LC3-I in turn is activated by Atg7 and transferred to Atg3. Finally, LC3-Atg3 is conjugated to phosphatidylethanolamine to form LC3-PE or LC3-II, which is recruited to autophagosomal membranes. A more detailed explanation is reviewed elegantly in another article [13]. 4

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Mitophagy is a highly specific form of macroautophagy which targets dysfunctional mitochondria and is also involved in the adaptation to nutrient stress. Mitochondrial membrane

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depolarization has been shown to induce mitophagy through the stabilization of PINK1 at the

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outer mitochondrial membrane [14, 15]. PINK1 in turn recruits the E3 ubiquitin ligase Parkin to

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the outer membrane, which leads to polyubiquitination of damaged mitochondria to be cleared by lysosomal degradation. Other adaptor proteins are known to regulate mitophagy such as

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BNIP3, NIX, and FUNDC1. The following sections will cover the adaptor proteins in more

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detail and is summarized in Figure 1. 2.1 Parkin-dependent mitophagy

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The PINK1/Parkin pathway has been well-characterized in mammalian cell models and is considered to be a major pathway for mitophagy [16]. PINK1 is a serine/threonine kinase which

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contains a predicted mitochondrial targeting sequence (MTS) in its N-terminus [17, 18]. Under normal conditions, PINK1 is translocated into the mitochondrial inner membrane where it is cleaved into a 52 kDa form by the mitochondrial protease PARL, and is further degraded by proteases within the mitochondria [19]. In depolarized mitochondria, PINK1 can no longer translocate into the mitochondria due to a decreased mitochondrial membrane potential. Instead PINK1 accumulates at the outer membrane of mitochondria and associates with the TOM20 complex. Parkin is recruited by PINK1 and is phosphorylated at serine 65 of its ubiquitin-like domain. This promotes the E3 ligase activity of Parkin, and further polyubiquitination is followed by the recruitment of the p62/SQSTM1 adaptor proteins and interaction with LC3 [20]. Finally, this complex is degraded by the autophagic machinery. However, it should be noted that not all cell lines express Parkin, and alternate mitophagy pathways exist. 5

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2.2 PINK1/Parkin-independent mitophagy

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In addition to mitochondrial depolarization, hypoxia can also induce mitophagy through two mitochondrial outer membrane proteins BNIP3 and NIX [21-23]. BNIP3 is highly expressed

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in the heart, liver, and muscle, while NIX is expressed in maturing reticulocytes [24-26]. Both

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NIX and BNIP3 are activated directly by hypoxia inducible factors (HIF) and contain a BH3

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domain which interacts with BCL-2 proteins [21]. The presence of an n-terminal LIR promotes the recruitment of the LC3/GABARAP complex [27]. In Hela cells, which do not contain

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endogenous Parkin, NIX was found to mediate mitophagy by binding to LC3 upon treatment with CCCP [27, 28]. Recently, a study found that the FUN14 domain-containing protein 1

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(FUNDC1) was a mitophagy receptor which was regulated by ULK1 phosphorylation [29, 30].

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FUNDC1 was found to increase in response to hypoxia via HIF1α, and binds to LC3 via its LIF

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motif to facilitate mitophagy through the autophagosome [29]. In addition, Mulan (Mul1) is also reported to mediate mitophagy in response to nutrient stress, and is induced by FOXO3 [31]. It remains to be seen how these varying mitophagic pathways are regulated in different organs and tissue, and whether there is any interaction or dominant activation of each effector in response to various physiological stressors. 3. Mitophagy and cancer Increasing evidence from various studies support the notion that dysregulation of mitophagy is an etiological factor in tumorigenesis (Figure 2). Loss-of-function mutations in Parkin lead to inhibition of mitophagy, and the accumulation of dysfunctional mitochondria resulting from increased ROS [32]. Mutations in the PARK2 gene (Parkin) are commonly found in various tumors as it is located at a fragile site in chromosome 6 (FRA6E, 6q26) [33-35]. 6

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Whereas tumorigenesis relies on inhibition of mitophagy, tumor progression likely relies on the presence of functional mitophagy. The following sections will discuss recent advances in our

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understanding of mitophagy in cancer and is summarized in Figure 2.

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3.1 Mutations in Park2 (Parkin)

Parkin is a E3 ubiquitin ligase responsible for the polyubiquitination of damaged

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mitochondria. Homozygous mutations in PARK2 or PARK6 have been linked to autosomal recessive juvenile Parkinsonism [36, 37]. The PARK2 gene is located at chromosome 6 (FRA6E,

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6q26), which is a fragile site and highly susceptible to genetic mutations [37]. Parkin knockout mice are prone to spontaneous hepatic tumors, while various cell models show increased

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tumorigenesis [38, 39]. PARK2 mutations are prevalent in many tumors which includes lung, melanoma, gliomas, and colon cancers, to name a few (Table 1) [40-46]. This suggests that

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PARK2 may be a tumor suppressive gene [40]. Recently, our group detected a homozygous point mutation which substituted valine with leucine (V380L) in the PARK2 gene of Hürthle cell tumor tissues and XTC.UC1 cells, resulting in dysfunctional autoubiquitination and decreased E3 ligase activity of Parkin [47]. The introduction of wild type Parkin sensitized XTC.UC1 to membrane depolarization-mediated cell death. Thus, loss-of-function mutations in the PARK2 gene generally result in increased tumorigenesis. 3.2 Mitophagy mediated by HIF1α - BNIP3, NIX, and FUNDC1 3.2.1 HIF1α and the tumor microenvironment The tumor microenvironment of solid tumors is highly hypoxic, and leads to the activation of HIF1α, a master transcriptional regulator of the hypoxic response. The hypoxic 7

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environment maintains a favorable microenvironment for cancer stem cells and increases cancer progression and metastatic potential [48, 49]. Whereas mitophagy is inhibited in tumorigenesis

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through PARK2 mutations, mitophagy is actually increased during cancer progression and

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metastasis via abnormal regulation of BNIP3. This dichotomy may be explained by the fact that

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excessive mitochondrial damage accrued during the initial stages of tumorigenesis would lead to decreased fitness of cancer cells. In contrast, NIX and FUNDC1 have not been studied

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extensively in the context of tumor progression, and require further investigation. Thus,

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increased mitophagy during tumor progression may be an adaptive response by tumors to increase survival.

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3.2.2 BNIP3 and NIX in cancer mitophagy

Both BNIP3 and NIX promote mitophagy in hypoxic conditions and are induced by

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HIF1α. The BNIP3 contains a LIR region which can interact with Atg8 to induce mitophagy [50]. There are conflicting reports as to the role of BNIP3 in tumorigenesis and tumor progression. In a melanoma cell line, shRNA lentiviral knockdown of BNIP3 reduced cell migration and vascular mimicry by remodeling the cytoskeletal actin configuration, which suggests a protumorigenic role [51]. Additionally, in hepatocellular carcinoma cells, the upregulation of BNIP3 was induced by ERK/HIF1α while suppressing the mTOR/S6K1 pathway to activate autophagy and increase the resistance to anoikis [52]. Furthermore, in adenoid cystic carcinoma cells, hypoxia-induced upregulation of BNIP3 was observed, while inhibition of autophagy led to decreased tumor invasion [53]. However, other studies show that BNIP3 has anti-tumorigenic properties and suggest it has a role as a tumor suppressor gene [54]. This is supported by a study showing that mitophagy 8

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defects arising from BNIP3 loss led to increased metastasis in a mouse model of mammary tumorigenesis [55]. In addition, epigenetic silencing of BNIP3 was shown to increase the

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aggressiveness of pancreatic cell lines [56]. This was supported by immunohistochemical

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analyses of pancreatic specimens from cancer patients [57]. 59% of patient samples had

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undetectable levels of BNIP3, which generally correlated with reduced survival. One possible explanation for this dichotomy is that BNIP3 can be alternatively spliced via deletion of exon 3,

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resulting in a truncated splice variant with pro-survival properties which is dependent on PKM2

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[58]. It is likely that BNIP3 functions as a tumor suppressor gene, and its alternative splicing or variable transcriptional regulation via Sp3 may contribute to its pro-tumorigenic role [59]. In

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contrast to BNIP3, the role of NIX in tumor progression remains relatively obscure, and requires

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further investigation.

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3.2.3 FUNDC1 in cancer mitophagy

The role of FUNDC1 in cancer mitophagy is not as concrete as its role in hypoxiamediated mitophagy was only discovered in 2012. However, further inspection of its mechanism may reveal novel targets of cancer mitophagy research. FUNDC1 is a novel outer mitochondrial membrane receptor, and is involved in hypoxia-induced mitophagy by binding to LC3 via its LIR motif [29]. ULK1, a critical mediator of autophagy, is upregulated and translocates to fragmented mitochondria in response to hypoxia and mitochondrial uncouplers. ULK1 was found to phosphorylate FUNDC1 at serine 17 and facilitate its binding to LC3 [60]. Further studies revealed that the BCL2L1-PGAM5-FUNDC1 axis regulated hypoxia-induced mitophagy [30]. A recent study provides evidence that the Tyr18 phosphorylation status in the LIR acts as a molecular switch that regulates mitophagic function of FUNDC1 [61]. The molecular 9

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mechanisms of FUNDC1 have only recently begun to be unraveled. It remains to be seen

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whether FUNDC1 also plays an important role in cancer mitophagy.

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3.3 KRAS mutations and cancer mitophagy

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The KRAS is a proto-oncogene which is commonly mutated in the form of a single nucleotide substitution, and results in its hyperactivation [62]. This leads to constitutive

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activation of a proliferative pathway involving PI3K, and upregulation of GLUT1, which contributes to a metabolic switch toward aerobic glycolysis [63-65]. KRAS mutations are

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commonly found in lung, colorectal, and pancreatic cancers, to name a few [66-68].

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In the context of cancer mitophagy, several studies provide consistent evidence that in

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tumors with oncogenic transformation of KRAS, autophagy leads to increased malignancy of tumors. In Rat2 cells with oncogenic KRASV12 transformation, inhibition of autophagy resulted

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in restoration of normal mitochondrial respiratory function. These results were supported by correlative data from hepatocellular carcinoma tissue, where increased LC3-II was noticed only in cells with oncogenic KRASV12 transformation and low glucose levels [69]. Subsequent experiments in knockout mice provide further support for the role of autophagy in tumor malignancy. In a mouse model of non-small-cell lung cancer with K-rasG12D activation, the loss of Atg7 altered the tumor fate from adenoma and carcinoma to the more benign, but rare oncocytoma with increased number of dysfunctional mitochondria [70]. Atg5 mutant mice with KRas(G12D) activation show impaired progression of lung cancer [71]. Taken together, in tumors with KRAS mutations, mitophagy may actually increase the malignancy of tumors, which differs from other models of cancer where mitophagy during tumorigenesis inhibits malignancy. 10

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4. Unexplored areas of cancer mitophagy

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Various molecules and cellular events have been shown to affect mitophagy. Mitochondrial proteases such as USP30, PARL, and Htra2 interact with Parkin and PINK1, and

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may possibly modulate mitophagic responses during tumorigenesis or cancer progression. The

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mitochondrial unfolded protein response (UPRmt) is a retrograde response activated by

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proteotoxic stress. As mitochondrial dysfunction is an integral component during the initial stages of tumorigenesis, mechanistic insight on the interaction between these two pathways may

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lead to novel therapeutic approaches. This is relevant especially in light of Sirt3, which is closely involved in both mitophagy and the UPRmt [72]. Finally, cancer cachexia is a common affliction

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seen in cancer patients [73]. A recent article shows that skeletal muscle autophagy is increased in

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cancer patients [74]. This suggests that a signaling factor may mediate cell non-autonomous

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autophagic responses in cancer patients. Several secretory molecules have been reported to be secreted from tissues or organs with mitochondrial dysfunction [75, 76] . Putative mitokine factors such as Fgf21 and Gdf15 are elevated in the serum of cancer patients [77, 78]. Gdf15 in particular has been reported to be correlated with cachexia in cancer patients [79]. Therefore, the final section will discuss possible interactions between the various stress mediators and mitophagy or autophagy, and discuss these observations in relation to cancer (Figure 3). 4.1 Mitochondrial proteases and mitophagy The mitochondria protein environment is regulated by both the nuclear and mitochondrial genome. Of the approximately 1200 known mitochondrial proteins in humans, 99% are encoded by the nuclear DNA [80]. The mitochondrion consists of an outer and inner membrane, and two enclosed compartments: the intermembrane space and the mitochondrial matrix. Mitochondrial 11

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proteins usually have a mitochondrial targeting sequence in their N-terminus, and are dependent on the mitochondrial membrane potential for import into the specific mitochondrial

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compartments [81]. Most proteins are imported in an unfolded state, and are reliant on the

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various chaperones within the mitochondrial compartments for proper folding and function. In

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order to regulate this dynamic environment, mitochondria contain various proteases and

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chaperones to maintain protein homeostasis, or proteostasis [82]. Mitochondrial proteases are classified based on their localization. Thus mitochondrial

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proteases can be defined as outer membrane, intermembrane space, inner membrane, and matrix proteases. The inner membrane proteases are further divided into m-AAA and i-AAA proteases

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depending on the orientation of the catalytic site. A detailed list and function of the various

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proteases have been reviewed by Quiros and colleagues [83]. Several mitochondrial proteases

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have been reported to interact and influence the activity of known regulators of mitophagy. The USP30 is a mitochondrial protease tethered to the outer mitochondrial membrane. USP30 is a deubiquitinase, and opposes the actions of Parkin, thereby antagonizing mitophagy. Addition of recombinant USP30 in whole cell lysates treated with CCCP leads to deubiquitination and cleavage of the Lys 6 and Lys 11 dimers of polyubiquitin [84]. USP30 was further shown to regulate apoptosis. Depletion of USP30 enhanced depolarization-induced cell death in Parkin-overexpressing cells [85]. Thus USP30 is a vital regulator of PINK1/Parkinmediated mitophagy. It remains to be seen whether USP30 function is upregulated or downregulated during tumorigenesis and tumor progression, respectively. PARL is a rhomboid intramembrane serine protease localized at the inner mitochondrial membrane, and is involved in the cleavage of 60kDa PINK1 in healthy mitochondria [19, 86]. 12

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One study found that deficiency of PARL led to decreased recruitment of Parkin, suggesting that PINK1 processing by PARL is an important event in functional PINK1 activity and recruitment

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of Parkin [87]. A more recent study supports the previous results as PARL abrogation led to a

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retrograde translocation of an intermembrane space-bridging PINK1 import intermediate [88].

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However, in this article the PINK1 intermediate was translocated to the outer membrane where it recruited Parkin and phenocopied depolarization-induced mitophagy. Therefore, it is uncertain

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whether PARL is involved in tumorigenesis or tumor progression, and requires further

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investigation.

HtrA2 is a mitochondrial intermembrane space serine protease. Cellular stress results in

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the release of HtrA2 into the cytosol, where it co-localizes with Parkin [89]. Cytosolic HtrA2

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was found to cleave Parkin and irreversibly inhibit its E3 ligase activity. Inactivation of HtrA2

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protease activity in mice exposed to H2O2 resulted in increased expression of Mulan, a mitochondrial E3 ubiquitin ligase, and resulted in increased mitophagy [90]. Thus HtrA2 is a negative regulator of mitophagy. Further studies are required to determine whether HtrA2 regulation is involved in tumorigenesis. 4.2 Mitochondrial unfolded protein response and mitophagy As mentioned in the previous section, mitochondrial proteostasis is tightly regulated by the various chaperones and proteases. Perturbations in mitochondrial function lead to a disruption in the protein folding environment. This leads to increased unfolded proteins and proteotoxicity, thereby activating a retrograde stress response defined as the UPRmt, and is characterized by an upregulation of chaperones such as Hsp60, and the matrix protease Clpp [91].

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The UPRmt was originally identified in COS7 cells transfected with mutated ornithine transcarbamylase (∆OTC) which led to the compartment-specific upregulation of the Hsp60 and

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Clpp in the absence of endoplasmic reticulum (ER) stress [92]. Subsequent studies in C. elegans

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showed that the UPRmt increased worm lifespan and is an area of active research in the field of

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aging [93, 94]. One elegant study showed that the master regulator of the UPRmt in worms is the activating transcription factor related to stress (ATFS1) [95]. In mammalian models, mitonuclear

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protein imbalance was shown to activate the UPRmt [96]. It is uncertain at this point as to how the

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UPRmt is regulated in mammals, as the mammalian homolog or ortholog of ATFS1 has yet to be discovered.

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One putative regulator of the UPRmt in mammals is the NAD-dependent protein

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deacetylase Sirt3. A recent study utilized Endo G, a mutated form of mitochondrial endonuclease

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G, to induce proteotoxic stress within the mitochondria. Concomitant upregulation of Hsp60 with Sirt3 was confirmed, and found to be reliant on ROS, as the inclusion of N-acetylcysteine (NAC), a mitochondrial antioxidant, abrogated Sirt3 induction. It was further confirmed that siRNA of Sirt3 led to decreased autophagy [72]. It should be noted that one weakness of this study is that the relationship between Sirt3 and the UPRmt is merely correlative, and requires further investigation. In contrast, the regulation of mitophagy by Sirt3 was confirmed in a separate study, where siRNA of Sirt3 in human glioma cells blunted the hypoxia-dependent localization of LC3 to the mitochondria [97]. The relevance of these findings to tumorigenesis and tumor progression need further confirmation in follow-up studies. 4.3 Mitokines and autophagy

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A mitochondrial cytokine, or mitokine, is defined as a secretory factor released from cells with mitochondrial perturbations. The term was first used in a study in C. elegans, which

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uncovered a cell non-autonomous induction of the UPRmt in the gut of worms when cco-1, the

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mammalian ortholog of the nuclear-encoded cytochrome c oxidase-1 subunit Vb/COX4, was

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knocked down in the brain [93]. The authors hypothesized that the cell non-autonomous induction of the UPRmt was mediated by an unknown signaling molecule, which they defined as

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a “mitokine”. The initial use of “mitokine” in a mammalian model was in the study of skeletal

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muscle-specific knockout of Atg7 in mice, which resulted in mitochondrial dysfunction and improved metabolic parameters. The authors identified fibroblast growth factor 21 (Fgf21) as a

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mitokine factor which improved systemic metabolism [75]. Thus a “mitokine” in the mammalian

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sense can be considered as a secretory factor released from an organ or tissue with mitochondrial

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dysfunction, which can modulate and re-establish systemic metabolic homeostasis. Several groups have found that growth differentiation factor 15 (Gdf15) is elevated in patients with mitochondrial disease [76, 98]. Our group has also confirmed Gdf15 secretion in mice with a skeletal muscle-specific knockout of the mitoribosomal factor Crif1 (unpublished data). Similar to Fgf21, Gdf15 treatment in genetically obese mice conferred a beneficial metabolic phenotype. This favorable metabolic phenotype is supported by studies in Gdf15 transgenic mice which show improved metabolic parameters and lower levels of systemic inflammation [99, 100]. Thus Gdf15 is a likely candidate along with Fgf21 as a putative mitokine factor. Both Fgf21 and Gdf15 have been reported to be elevated in cancer patients [77, 78]. In fact, Gdf15 has been reported to be a driver of cachexia in mice [79]. Cachexia is seen in about 15

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45% of cancer patients and is characterized by systemic inflammation, negative energy balance,

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and loss of lean body mass, with or without wasting of adipose tissue [73]. A recent study in cancer patients showed that autophagy was increased in the skeletal muscle of cachexic cancer

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patients as assessed by protein levels of LC3B-II [74]. The intermediary signaling factors were

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not mentioned. It is possible that autophagy may have been utilized to mobilize macromolecule

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stores. Whether mitokine factors were involved in this cell non-autonomous response requires further study.

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Presently, the relationship between putative mitokine factors and distal autophagy is correlative at best. It is also unclear whether these putative mitokine factors are increased in

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cancer patients due to mitochondrial dysfunction or some form of tissue damage. In mouse

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models of tissue-specific mitochondrial dysfunction, mitokines generally confer positive

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metabolic effects in the form of improved glucose tolerance, insulin sensitivity, and white adipose tissue browning [75]. However, it is possible that in cancer cachexia, mitokines may contribute to the constant catabolic state, of which autophagy may be one mechanism to mobilize macromolecules from distal tissues to fuel primary tumor growth. Further investigation in this area may yield mechanistic insight on the role of mitochondrial dysfunction and mitokines in cancer, and may also lead to novel therapeutic targets. 5. Concluding remarks Recent advances in mitophagy research in the context of cancer and non-cancer studies have revealed significant insight on the molecular mechanisms of mitochondrial quality control. The PINK1/Parkin regulated mitophagy has been studied widely and loss-of-function mutations in the PARK2 gene are commonly found in various tumors. Mitophagy can be induced by an 16

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alternate pathway involving HIF1α-mediated activity of BNIP3, NIX, and FUNDC1. BNIP3 has been studied extensively in the context of cancer, and shown to have a dichotomous role in

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tumorigenesis, likely due to an alternatively spliced isoform and variable transcriptional

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regulation via Sp3. However, studies on the role of NIX in cancer mitophagy is relatively lacking.

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The application of unexplored areas of mitophagy research to cancer studies is promising, and increased knowledge of mitophagy adaptor protein interactions with mitochondrial proteases, the

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UPRmt, and mitokine action may potentially lead to novel therapeutic approaches to treat cancer.

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Conflict of interest

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The authors declare no conflict of interest.

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Acknowledgements

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We apologize to the authors whose work was not included. This work was supported by the Basic Science Research Program through the NRF funded by the Ministry of Science, ICT, and Future Planning, Korea (NRF- 2014M3A9D8034464). References

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Figure 1. Schematic diagram for mitophagy pathways. Top, HIF1α-mediated induction of BNIP3, NIX, and FUNDC1 lead to mitophagy. BNIP3 and NIX have been reported to be activated in the pancreas, liver, and lungs, while FUNDC1 has been mentioned in in vitro studies. Hypoxia leads to the recruitment of the adaptor molecules to the outer mitochondrial membrane, thereby activating mitophagy through the autophagosome. Bottom, PINK1/Parkin-mediated mitophagy. Mitochondrial damage leads to accumulation of PINK1 at the outer mitochondrial membrane and recruitment of Parkin, further leading to mitochondrial degradation via the autophagosome machinery.

Figure 2. Schematic diagram of cancer mitophagy. Top, Parkin mutations result in abnormal mitophagy and leads to tumorigenesis. Bottom, in established tumors, hypoxia leads to HIF1αmediated induction of BNIP3, NIX, or FUNDC1, leading to mitophagy and increased tumor survival. BNIP3 plays a dichotomous role in tumor progression of various organs including liver, pancreas, and lungs. Possible activation or inactivating mutations can both increase tumor survival. In contrast, the role of NIX and FUNDC1 remains obscure, and is depicted with a question mark.

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Table 1. Park2 mutations in various tumors.

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Figure 3. Schematic diagram of outstanding areas of mitophagy research. Top left, mitochondrial proteases USP30 and HtrA2 inhibit Parkin, thus blocking mitophagy and possibly leading to tumorigenesis. Top right, Sirt3 possibly regulates both the UPRmt and mitophagy during tumorigenesis and tumor progression, respectively. Bottom right, mitokines are secreted from primary tumors and target skeletal muscle, where they facilitate cell non-autonomous autophagy to mobilize macromolecules to be utilized by primary tumors.

Mutation type

Notes

DNA copy number loss

Glioma

Intragenic deletions Splice variants Splice variants Single nucleotide polymorphisms Germline mutations V380L amino acid substitution

PARK2 deletions in APC mutant mice Inactivating mutations human tissue H20,H1, H5 isoforms Human lung adenocarcinomas Human lung cancer and COPD tissue Sporadic tumors and cell lines Abnormal autoubiquination and decreased E3 ligase activity

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Melanoma Thyroid Hürthle cell

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Lung

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Tumor type Colorectal

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Figure 1

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Figure 2

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Figure 3

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Highlights

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 Mitophagy is a key regulatory process for mitochondrial quality control

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 Mitophagy is mediated by Pink1/Parkin and Hif1α-mediated adaptors BNIP3, NIX, and FUNDC1.

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 Mitophagy is inhibited during tumorigenesis via mutations in Parkin  Mitophagy is dysregulated during tumor progression via mutations in BNIP3

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 Further studies in mitochondrial proteases, UPRmt, and mitokines may lead to novel therapeutics

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