when autophagy meets cancer through p62 sqstm1

Introduction The maintenance of cellular homeostasis de-pends on an exquisite balance between the biosynthesis and degradation (catabolism) of macromolecules. This balance is controlled by finely tuned mechanisms triggered by environ-mental signals. In eukaryotic cells, two main systems are responsible for protein degrada-tion. The first system is the ubiquitin-proteasome system that allows for the degrada-tion of proteins with a short half-life (generally less than 5 hours). Conversely, the second sys-tem, which is strictly dependent on the ly-sosome and represents approximately 99% of cellular proteins, is involved in the degradation of proteins with a half-life of up to 5 hours [1]. The latter system includes the targeting of pro-teins to the lysosomes and the further degrada-tion of these proteins by lysosomal hydrolases. The major process for the delivery of proteins to the lysosome is orchestrated by a specific and highly conserved mechanism called autophagy

or macro-autophagy [2]. Macro-autophagy, hereafter referred to simply as autophagy, is currently the most well studied of these degradation pathways, which is likely due to its important and paradoxical role in the control of cell death and survival. Active at the basal level in the resting cell, autophagy is in-duced under conditions of stress and thus be-haves as an adaptative survival mechanism, more particularly through amino acid recycling following the degradation of damaged organ-elles and macromolecules. In mammal cells, autophagy is a multistep process, including an initiation step, a nucleation step, an elongation step and, finally, a maturation step (Figure 1). The initiation and nucleation steps both con-verge to isolate de novo a portion of the intra-cellular membrane called a phagophore. The phagophore then invaginates, and its ends can fuse to generate a double-membraned vesicle referred to as the autophagosome. The auto-phagosome structure is delimited by several

Am J Cancer Res 2012;2(4):397-413 www.ajcr.us /ISSN:2156-6976/ajcr0000126

Review Article When autophagy meets cancer through p62/SQSTM1 Alexandre Puissant1*, Nina Fenouille2*, Patrick Auberger3-5

1Dana-Farber Cancer Institute, Pediatric-Oncology department, 450 Brookline Avenue, Boston, MA 02215, USA; 2Massachusetts Institute of Technology, 77 Massachusetts Avenue, Cambridge, MA 02139-4307, USA; 3INSERM U1065, Team Cell Death, Differentiation, Inflammation and Cancer, Nice, France; 4Equipe labellisée par la Ligue Nationale Contre le Cancer (2011-2013); 5University of Nice Sophia-Antipolis, Nice, France. *These authors contrib-uted equally to this work Received May 29, 2012; Accepted June 14, 2012; Epub June 28, 2012; Published July 15, 2012 Abstract: Although p62/SQSTM1 was initially identified as an essential mediator of NFκB signaling, several recent studies have also highlighted its important role at the crossroad between the mTOR or MAPK signaling pathways and selective autophagy. The p62 structure containing important interaction domains attests to the ability of this protein to regulate and modulate the activation of these signaling pathways during tumor formation and propagation. The second very important function of this protein is to act as a molecular adaptor between the autophagic machinery and its substrates. Consequently, p62 is degraded following an increase in autophagic flux for which this protein cur-rently serves as an indicator. However, the measurement of p62 expression strictly as a marker of autophagic flux is still controversial and can be misinterpreted mainly because this protein is subject to complex regulation at both the transcriptional and post-translational levels. Finally, because p62 is an autophagic substrate, it acts as a molecular link between cancer and autophagy by conferring a high level of selectivity through the degradation of important sig-naling molecules. Keywords: Paget’s disease, mTOR, NFκB, NRF2, MAPK, Atg, ROS, ubiquitin, protein aggregates, oxidative stress

p62/SQSTM1, a molecular link between selective autophagy and cancer

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lipidic layers that sequester the cytoplasmic content and/or organelles [2]. The strict origin of the autophagosome is still unknown, but it is thought that several cellular compartments, including the endoplasmic reticulum (ER), the Golgi/trans-Golgi apparatus and the plasma membrane, can participate in the genesis of the autophagosome. Finally, following an additional maturation step, the autophagosome becomes an amphisome after it fuses with multivesicular endosomes (Figure 1). During this step, the am-phisome is acidified via the activation of proton pumps contributed by the endosomes. Ulti-

mately, this amphisome will fuse with a ly-sosome to become an autolysosome in which the internal content is degraded by lysosomal enzymes (Figure 1). From yeast to mammals, all of the processes of autophagosome biogenesis are controlled by the specific interaction of several protein com-plexes composed of Atg proteins (autophagy-related genes). Of a total of 30 Atg proteins identified to date, approximately 50% play an essential role in the formation and elongation of the phagophore. With the exception of Atg9,

Figure 1. Schematic view of the autophagic process and its components. Macro-autophagy, or autophagy, is a multistep process including an initiation step, a nucleation step, an elongation step and, finally, a maturation step. In response to nutrient deprivation, the complex formed by ULK1, mAtg13, Atg101 and FIP200 is activated to initiate the de novo isolation of a portion of intracellular membrane referred to as a phagophore. Thenceforth, a complex formed by the Beclin 1/Vps34/Vps15 complex and two conjugation systems composed of Atg5-Atg12 and LC3-PE (LC3-II) play significant roles in the nucleation and elongation steps responsible for the invagination, elongation and closure of the phagophore to generate a structure called an autophagosome. This autophagosome undergoes two maturation steps: first, it fuses with multivesicular endosomes to generate an amphisome, which becomes acidified due to the presence of membrane proton pumps provided by endosomes; secondly, the amphisome fuses with ly-sosomes to generate autolysosomes, which specialize in the degradation of the material sequestered by the auto-phagosome. Membrane permeases then participate in the export and recycling of the products of this degradation.


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none of these proteins possesses a transmem-brane domain. Once recruited into the cyto-plasm, Atg proteins bind transiently to the mem-branes of the phagophore (or the pre-autophagosomal structure, PAS in yeast) and the autophagosome [3]. The Atg proteins can be classified into three functional groups according to the autophagy step in which they participate (Figure 1): 1) The complex composed of the serine/threonine kinase ULK1 (or its yeast homolog Atg1), mAtg13, FIP200 and Atg101 are involved in the initiation step of the phagophore. 2) The Atg6/Beclin 1-Atg14/Atg14L-Vps34-Vps15 and Beclin 1-UVRAG-Vps34-Vps-15 complexes are required for phagophore nucleation. 3) The two conjuga-tion systems composed of Atg5-Atg12 and Atg8/LC3-PE are essential for the elongation and closure of the autophagosome [2]. Notably, the mAtg9/Atg9 complex is important during the induction of autophagy, although its exact role in the autophagic process remains unknown. Atg9 does not belong to any of the above-mentioned functional groups, but it appears to be important during all steps of autophagosome biogenesis [4]. In contrast to what was initially thought, macro-autophagy is not only a non-selective mecha-nism for the degradation of cellular constitu-ents, but it is also now widely known that this form of autophagy is involved in the specific targeting of organelles within the cell. This tar-geting is called mitophagy in the case of mito-chondria, pexophagy for peroxisomes and ER-phagy for the endoplasmic reticulum. Finally, the mechanism of autophagy responsible for the degradation of unfolded or aggregated pro-teins is referred to as aggrephagy [5]. The selectivity of autophagy results in alterna-tive molecular mechanisms that connect with and engage the autophagic machinery to use this machinery to digest specific substrates. This process requires key scaffold proteins, which have only been partially identified, that may be associated with several autophagic components to influence the selective degrada-tion of some substrates. Importantly, several recent studies have highlighted the role of two of these adaptor proteins, p62 and NBR1, whose function is to specifically address ubiquit-inated and sometimes aggregated protein sub-strates for autophagic degradation [6]. Both of

these proteins exhibit a high level of homology, and, although the role of NRB1 is better under-stood than that of p62, both proteins appear to share common functions. The present review will first focus on the structure/function rela-tionship of p62 and then describe how p62 is regulated via several oncogenic or tumor-suppressor signaling pathways. As a molecular adaptor between the autophagic machinery and its substrates, p62 is degraded during this autophagic process. This property led some authors to use this protein as an index for the autophagic flux measurement. We will discuss more deeply the ambiguity that occurs when using this protein as a hallmark of auto-phagic flux. Finally, we will broaden our investigation into the role of autophagy in tumor development, and we will assess the impact of p62 as a mo-lecular link between autophagy and cancer. Structure and function relationship p62/SQSTM1 (Sequestosome 1, ZIP3) contains multiple major domains such as PB1 (aa. 21–103) and ZZ (aa. 128-163) domains that confer the ability to interact with key components in-volved in essential signaling pathways. Two other very important domains, the UBA (aa. 386-440) and LIR (aa. 321-345) domains, cause p62 to function as an adaptor between auto-phagy and ubiquitinated proteins (Figure 2). Interaction of p62 with major signaling path-ways via the Phox and Bpem 1 (PB1) and the central zinc finger (ZZ) domains P62 is a multifunctional protein characterized initially by its ability to bind atypical protein kinase C (PKC zeta and iota) via its N-terminal PB1 (Phox and Bpem1) domain (Figure 2). Het-erodimerization of p62 with atypical PKC is cru-cial for the regulation of the NFκB pathway. In-deed, following binding of the RANK ligand (RANK-L) to its cognate receptor RANK in osteo-clasts, the association of p62 with either atypi-cal PKC or with TRAF6 via its TRAF6-binding domain (TBD, aa. 225-250) contributes to the activation of IKK (Ik-B Kinase) and to the nu-clear translocation of the transcription factor NFκB [7]. It has been recently reported that the MAP kinase kinase 3 (MEKK3) can activate TRAF6 to promote nuclear relocalization of


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NFκB by binding to the PB1 domain of p62 [8]. Conversely, p62 can recruit, via its PB1 domain, CYLD, a deubiquitin ligase that acts as an inhibi-tor of TRAF6 following NFκB activation [9]. This modulation of the NFκB pathway by p62 is es-sential for proper osteoclastogenesis. Finally, it has been reported that the PB1 domain of p62 can also interact with ERK1 to promote adipo-genesis [10]. Importantly, this PB1 domain is also required

for the homodimerization of the protein [11, 12] or its association with NBR1, which allows its oligomerization. For example, in ALK-positive large B cell lymphoma, which is characterized by p62-ALK translocation, homodimerization of p62 via its PB1 domain has been suggested to be decisive for the constitutive activity of the ALK kinase that is responsible for this hemato-poietic malignancy [13]. In addition to the PB1 domain, p62 possesses a central zing finger domain (ZZ domain) that interacts with RIP

Figure 2. Structure and interacting partners of p62/SQSTM1. The structure of p62/SQSTM1 is composed of domains required for its interaction with autophagic machinery and with signaling pathways involved in the antioxidant re-sponse, inflammation, metabolism and, ultimately, in cancer. The N-terminal Phox and Bpem1 (PB1, aa. 21-103) domain is responsible for the self-oligomerization of p62 or the heterodimerization with the p62 homolog protein NBR1. This PB1 domain is also involved in the binding of p62 to atypical PKC (aPKC) or to ERK1. Next, the central zing finger ZZ domain (aa. 128-163) and the TRAF6-binding domain (TBD, aa. 225-250) interact with RIP1 and TRAF6, respectively, to regulate the NFkB pathway. Between these two domains is the region that interacts with the Raptor protein, which is involved in the activation of the metabolic kinase mTOR. Through the C-terminal LC3-interacting region (LIR, aa. 321-345) and ubiquitin-associated domain (UBA, aa.386-440), p62 links the autophagic machinery to ubiquitinated protein substrates to promote the selective degradation of these substrates. The affinity of the UBA domain for ubiquitinated lysine residues is modulated by caseine kinase 2 (CK2)-mediated phosphoryla-tion of serine 403. Finally, the Keap-interacting region (KIR, aa. 346-359) binds Keap1 to induce the nuclear translo-cation of the NRF2 transcription factor engaged in the antioxidant response.


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(receptor-interacting protein) to modulate the NFκB pathway in conjunction with atypical PKC [14]. In conclusion, a recent report has identified a new region of p62 located between the ZZ and TBD domains that is responsible for the interac-tion of p62 with the mTOR regulator Raptor [15] (Figure 2). In mammalian cells, mTOR is part of two distinct complexes: mTORC1 (formed by the mTOR, Raptor and GβL proteins) and mTORC2 (formed by mTOR, Rictor, mSin1, GβL and Pro-tor) [16]. mTORC1 is highly sensitive to the nu-triment deprivation that leads to the inactivation of the serine/threonine kinase mTOR. Con-versely, mTOR is activated following normal nu-triment input. The interaction of p62 with Rap-tor and, subsequently, mTORC1 is dependent

on the uptake of amino ac-ids that participate in the lysosomal membrane local-ization of mTORC1 to pro-mote Rag GTPase-mediated mTOR activation [15]. p62/SQSTM1, an adaptor between autophagic ma-chinery and ubiquitinated proteins via its Ubiquitin-Binding (UBA) and LC3-Interacting (LIR) regions The ubiquitin-binding do-main (UBA): The C-terminal ubiquitin-binding domain (UBA) of p62 binds with a moderate affinity to the ubiquitin bound to the lysine residues in position 48 and with a strong affinity to the lysine in position 63 (Figures 2 and 3). The affinity of the UBA domain for ubiquiti-nated lysine residues is drastically increased follow-ing the caseine kinase 2-mediated phosphorylation of serine 403 [17] (Figure 3). The UBA domain of p62 is frequently mutated in Paget’s disease. This pathol-ogy is characterized by an increase in the number and the activity of osteoclasts,

leading to excessive bone resorption and anomalies of the bone architecture. Although the molecular mechanisms involved in this ex-cessive osteoclastogenesis are currently un-known, 14 mutations of the p62 gene have been reported to occur in this disease [18]. The first discovered point mutation leads to a recur-rent proline-to-leucine substitution (P392L) not only in familial forms of Paget’s disease but also in patients with no known familial history. Sub-sequently, stop (K378X, A390X, L394X, E396X) and non-sense (A381V, P387L, S399P, M404T, M404V, G411S, L413F, G425R) mutations have been identified in the UBA domain coding sequence of the p62 gene [18]. It is assumed that approximately 40% of familial forms and 8% of sporadic cases of patients suffering Paget’s disease harbor a mutation in the gene

Figure 3. Involvement of p62/SQSTM1 and NBR1 in the degradation of ubiquiti-nated and misfolded proteins through selective autophagy. The p62/SQSTM1 and NBR1 proteins contain a ubiquitin-binding domain (UBA domain), a LC3-interacting region (LIR domain) and two domains for oligomerization and polym-erization (CC and PB1 domains, respectively). Using the UBA domain to interact with misfolded and ubiquitinated proteins and the LIR domain to bind to the LC3 protein, these two proteins act as adaptors between the autophagic machinery and the ubiquitinated substrates, which will be selectively eliminated by aggre-phagy. This process is largely used to eliminate Tau protein aggregates that accumulate during Alzheimer’s disease. Casein kinase 2 (CK2) phosphorylates p62 to increase the affinity of the UBA-binding domain for ubiquitinated lysines.


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encoding p62. To date, no link has been estab-lished between the mutations found in the UBA domain of p62 and the etiology of Paget’s dis-ease [19]. The LC3-interacting region (LIR) domain: As mentioned above, the LC3 (Atg8) proteins, which can be divided into seven members, are key components of the autophagy machinery required for the elongation and closure of the autophagosome. To assure their proper func-tions, LC3 proteins are specifically cleaved at their C-termini by the Atg4 proteases to expose a C-terminal glycine residue, which is then con-jugated to a phosphatidylethanolamine (PE) group to form LC3-II [3]. This maturate form of LC3 is tightly bound to the autophagosomal membranes. The targeting of p62 via the auto-phagic machinery is dependent on the associa-tion of this protein with all isoforms of the LC3 protein via an 11 amino-acid portion of LIR lo-cated in the 332-343 region [12] (Figure 2). More recently, NBR1 has also been identified as an LC3 binding partner. The overall organization of the NBR1 protein is similar to that of p62, with the noticeable exception of two coiled-coil (CC1 and CC2) domains that allows for the oli-gomerization of NBR1 following its linkage to ubiquitinated proteins [6] (Figures 2 and 3). The LIR domain of the LC3 protein family can also interact with NDP52, BNIP3, NIX and TP53INP1 [20-23]. Whereas BNIP3 and NIX are key regula-tors of mitophagy, particularly during erythroid differentiation [20, 21], TP53INP1 regulates autophagy-dependent cell death (also called type II cell death) [22]. The interaction of p62 with LC3-II is required for the autophagy-mediated elimination of unfolded ubiquitinated long-half-life proteins (Figure 3). Thus, it has been shown that the knock-down of the LC3 protein triggers the intracytoplasmic accumulation of ubiquitinated protein aggre-gates bound to p62 and/or NBR1 proteins [6, 24]. In addition, another study demonstrated that the ubiquitination of soluble cytosolic pro-teins or organelles (especially proteins embed-ded in peroxysomal membranes) are sufficient to target these substrates for autophagic degra-dation [25]. These data clearly demonstrate that p62 is a receptor for the specific elimina-tion of ubiquitinated proteins via autophagy. Because it has only recently been identified, the biological function of NBR1 is not yet fully un-derstood. It is thought, however, that this pro-

tein can interact directly with p62 within the same ubiquitinated protein aggregates [6, 26, 27]. Conversely, p62 is not only required for the proper elimination of ubiquitinated proteins but also participates in the autophagic clearance of non-ubiquitinated substrates. This fact is em-phasized in a recent publication by Watanabe et al. in which the authors assessed the impact of the overexpression of an isoform of STAT5A, STAT5A_ΔE18, which has the ability to form non-ubiquitinated aggregates [28]. Despite the ab-sence of ubiquitinated residues in STAT5A_ΔE18 aggregates, p62 is able to bind to these aggregates via its PB1 domain to medi-ate the elimination of these aggregates by auto-phagy [28]. Influence of the PB1 and UBA domains on the autophagic clearance of p62/SQSTM1 targets: the case of neurodegenerative diseases The PB1 and UBA domains are both required for the proper localization of p62 within cytoplas-mic aggregates (either ubiquitinated or not) that are referred to as p62-bodies. Thus, the inhibi-tion of autophagy leads to both an intracellular accumulation of p62 and an increase in the number of p62-bodies. In a clinical context, the dissection of the mechanisms sustaining the autophagic elimination of p62-bodies is a very important challenge in the development of new treatments to circumvent neurodegenerative disorders, such as Huntington’s chorea. Huntington’s chorea is characterized by strong neurologic deficits that affect muscle coordina-tion and lead to cognitive decline, psychiatric problems and, ultimately, to the death of the patients. Huntington’s disease is caused by a mutation in the gene encoding huntingtin. Mu-tant huntingtin accumulates in the cytoplasm where it forms inclusions that are deleterious to neuronal cells. It has been shown that p62 can surround and associate with such inclusions and interact with the LC3-II protein to promote the degradation of these inclusions via an auto-phagic process [12, 29]. In agreement with this observation, deletion of the UBA domain abro-gates the protective effect of p62 and increases the amount of cell death induced by the mutant huntingtin [29]. The accumulation of p62 into cytoplasmic inclu-


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sion bodies has also been described in other neuropathies such as Alzheimer’s disease, Parkinson’s disease and amyotrophic lateral sclerosis, which is also known as Charcot dis-ease. The evolution of Alzheimer’s disease is strikingly associated with the pathological accu-mulation of filamentous structures called PHFs (paired helical filaments) in the cytoplasm of neuronal cells. These structures are essentially formed by the Tau protein, which belongs to the microtubule-associated protein (MAP) family and plays an important role in microtubule sta-bilization. The aberrant accumulation of Tau in neuronal cells leads to the apoptotic cell death that is associated with neurofibrillar degenera-tion. It has been reported that p62 colocalizes with these Tau-positive inclusions in neuronal cells [30, 31]. Similarly, p62 also colocalizes with α-synuclein, whose accumulation is associ-ated with Parkinson’s disease. The functional link between Tau and p62 has been highlighted in p62-deficient mice that exhibit a pathological accumulation of Tau associated with a massive neuronal degeneration [32]. These findings, therefore, clearly reflect the preventive role of p62 in the emergence of neurodegenerative disorders. Very interestingly, the inhibition of autophagic degradation pathways has also been associated with the genesis of Alzheimer’s disease [33, 34]. Indeed, the autophagosome-lysosome sys-tem participates to the elimination of Tau pro-tein aggregates [35, 36]. Although this hypothe-sis still lacks formal evidence, it is likely that p62 is the molecular clutch necessary for the autophagic clearance of Tau or α-synuclein pro-teins. Thus, the inhibition of autophagy or the knockdown of p62 would lead to a toxic intracel-lular accumulation of these proteins. A recent study by Du et al. highlighted an interesting mechanism responsible for blocking p62 ex-pression, which may explain the onset of Alz-heimer’s disease. Staining of brain slides from p62-/- mice with 8-OHdG, an oxidative stress sensor, revealed an increased level of reactive oxygen species (ROS) production compared with that of the control mice [37]. One consequence of this exacerbated ROS production is the al-teration of the p62 promoter as mice get older. This deterioration of the p62 promoter results in a blockade of p62 expression that impairs the autophagic elimination of Tau aggregates that are the cause of the neuropathy [37]. The au-thors next reported a negative feedback loop in

which increased ROS production, resulting from the occurrence of Tau protein clusters, blocks the expression of p62 due to the alteration of its promoter. Finally, this blocked expression con-tributes to a further increase in the accumula-tion of Tau aggregates that, in turn, exacerbates intracellular ROS production [37]. Regulation of the p62/SQSTM1 intracellular level As previously mentioned, the expression of p62 is highly regulated at the transcriptional level. However, we will also describe a second auto-phagy-dependent mechanism involved in the control of intracellular levels of p62 (Figure 4). Transcriptional regulation of p62/SQSTM1 Transcriptional regulation dependent on the transcription factor NRF2: One of the main modes of transcriptional regulation of p62 is dependent on the NRF2 transcription factor, which belongs to the basic leucine zipper (bZIP) family of transcription factors. In response to an oxidative stress such as H2O2, NRF2 specifically binds to the antioxidant-responsive element (ARE motif) located in the p62 promoter to pro-mote the expression of p62 mRNA [38]. In the absence of oxidative stress, NRF2 is maintained as an inactive protein in the cytoplasm through its interaction with the E3 ubiquitin ligase KEAP1 (Kelch-like ECH-associated protein 1) [39]. In addition to its specific regulation by NRF2, p62 can interact directly with KEAP1 via its KIR domain that is juxtaposed to the LC3-interacting (LIR) domain. Hence, p62 is able to dissociate NRF2 from KEAP1, thus promoting the activation and nuclear translocation of NRF2 [40]. This process contributes to the in-duction of a positive feedback loop in which p62 activates NRF2 to drive its own transcrip-tion [38]. This mechanism provides a pertinent explanation for the increased ROS production observed in Tau-positive neuronal cells of p62-/- mice [37]. Although p62 deficiency is sufficient to impair the autophagic degradation pathway and result in an oxidative burst, the sustained inactivation of NRF2 by KEAP1 in p62-/- mice could also participate to amplify this deleterious process. In contrast, given that p62 is degraded during the autophagic process [12], one can assume that a defect in autophagy may cause an in-


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crease in NRF2 transcriptional activity. This non-canonical mechanism for NRF2 activation would strictly rely on p62 degradation during auto-phagy [41]. These findings are corroborated in vivo by the specific invalidation of Atg7 in mouse livers in which the inhibition of auto-phagy triggers the formation of adenomas con-taining p62-positive aggregates, which are also found in 25% of hepatocellular carcinoma cases [42]. In correlation with the presence of p62-positive aggregates, NRF2 activation is also detected in these hepatocellular carcinoma samples. Based on these reports, we conclude that there may be an intriguing link between the variation in the activity of the autophagic ma-chinery and the p62-dependent oxidative stress response during carcinogenesis. This striking interrelation between NRF2 and p62 is also involved in the regulation of other signaling pathways. Thus, following the non-canonical p62-mediated activation of the NRF2 pathway, NQO1, the transcriptional target of NRF2, would stabilize the tumor suppressor p53 [43]. The p62/NRF2/NQO1 complex also plays a role in the maintenance of mitochondrial sta-bility during cell aging. The ROS accumulation linked to aging alters mitochondrial functions. Hence, whereas the tissues of p62-deficient mice display an accelerated aging phenotype,

the overexpression of NQO1 in this p62-/- con-text maintains the integrity of the mitochondrial membrane potential [44]. Another study demon-strated that the NRF2/p62 pathway is neces-sary for signaling through the TLR4/Myd88 re-ceptor [45, 46]. Following the engagement of the TLR4 receptor by LPS or E Coli, the p38MAPK pathway is activated, leading to the nuclear translocation of NRF2 and the in-creased expression of p62. Transcriptional regulation independent of the transcription factor NRF2: p62 expression is not only dependent on NRF2 activity. Indeed, other stimuli including phorbol 12-myristate 13-acetate (PMA), calcium and IL-3, which act inde-pendently of NRF2, drastically increase the ex-pression of p62 mRNA in a very short time (from 30 minutes to 2 hours) [47]. In agreement, an analysis of the 5’-flanking region of the p62 pro-moter revealed the presence of binding sites for several transcription factors, such as SP-1, AP-1, NF-κB and Ets-1 [48]. Importantly, Duran et al. show that Ras-transformed fibroblasts ex-hibit a high level of p62 mRNA, which is not ob-served after invalidation of the AP-1 binding site located upstream on the p62 promoter [49]. Thus, these findings strongly suggest that the constitutive activity of the Ras/MEK/ERK1/2 pathway regulates p62 transcription via the AP-

Figure 4. The intracellular level of p62/SQSTM1 is regulated by a fine balance between transcriptional regulation and post-translational autophagic degradation. The intracellular level of p62/SQSTM1 is dependent on the combination of incoming and outgoing flux. The incoming flux is powered by the transcriptional regulation of p62 and the neosyn-thesis boost of p62 in response to various stimuli, whereas the outgoing flux is influenced by autophagic activity. Because some inducers of autophagy (i.e., Resveratrol) exert their effect at the levels of both the synthesis and deg-radation of p62, it is sometimes difficult to interpret the mechanisms underlying this fluctuation. The only way to avoid any misinterpretation is to use pharmacological agents that are able to block either the incoming (i.e Actinomy-cin D) or outgoing (i.e Bafilomycin A1, Pan-cathepsin inhibitors) flux.


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1 binding domain present in its promoter. Our own results have implicated the JNK/c-Jun pathway in the regulation of p62 synthesis. In chronic myelogenous leukemia (CML) cells, the phytoalexin Resveratrol increases the expres-sion of p62 via the activation of the JNK path-way [50, 51]. In this context, treatment of CML cells with SP600125, a JNK inhibitor, abrogates the effect of Resveratrol on p62 expression. Similarly, hydroxytyrosol a natural compound found in olive oil, also activates the JNK/p62 pathway [52]. Collectively, these findings are in good agreement with the observation that Pur-kinje cells deficient in JNK1/2/3 have undetect-able levels of p62 relative to their control coun-terparts [53]. It remains, however, to decipher the exact molecular mechanisms by which JNK increases p62 expression. A recent study also reported an indirect transcriptional control of p62 via AP1 activation. KrasG12D-induced AP1 activation stimulates the transcription of IL-1α, which, in turn, stimulates the synthesis of p62 through NFκB activation [54]. Finally, a positive feedback loop in which newly synthesized p62 stimulates IKK2/β results in a constitutively activated NFκB pathway. Post-translational regulation of p62/SQSTM1 As mentioned above, p62 is also regulated at the protein level by the autophagic machinery. Following the fusion of lysosomes with auto-phagosomes, most of the Atg proteins are recy-cled into the cytosol, but some proteins present in the lumen of the autophagosomes that are necessary for the autophagic process are de-graded by lysosomal proteases, including cathepsins. For example, the LC3-phosphatidylethanolamine fraction (LC3-II) is insensitive to Atg4 deconjugation due to its membrane topology and therefore remains in the autophagosomal membrane where it is de-graded. Due to its association with LC3-II, p62 is also a substrate for lysosomal proteases. Hence, several pro-autophagic stimuli such as hypoxia, amino acid deprivation or treatment with autophagy inducers have been shown to induce p62 degradation and, subsequently, decrease its intracellular levels [55]. In contrast, the blockade of autophagy is linked to p62 ac-cumulation. Several pharmacological ap-proaches have been used to prevent p62 degra-dation during autophagy. The first approach consists of using Bafilomycin A1 to block the

membrane-bound lysosomal vacuolar ATPase (V-ATPase) to prevent the lysosomal lumen acidifi-cation responsible for cathepsin activation. The second approach consists of the use of a cock-tail of protease inhibitors (mainly Pepstatin A and E-64d) to inhibit the activation of lysosomal proteases [55] (Figure 4). Intracellular level of p62/SQSTM1 as a marker of autophagic flux Considering the fact that p62 is rapidly de-graded during autophagy, the analysis of its intracellular level by western blotting is used routinely to measure the autophagic flux in re-sponse to pro-autophagic stimuli. In a recent report, Larsen et al. described a reporter cell system assay based on the tetracyclin-inducible expression of GFP-p62 [56]. In this assay, cells transduced with this vector are treated for 24 h with tetracyclin to highly express the GFP-p62 fusion protein. Autophagy is then induced in these cells, and the decrease in fluorescence, which reflects the degradation of p62 following the activation of autophagic flux, is analyzed using flow cytometry [56]. According to these results, the GFP fluorescence emitted by the cells treated with the V-ATPase inhibitor Bafilo-mycin A1 does not change as a function of time because p62 is not degraded via the autophagic machinery [56]. In the future, this assay could be a tremendous asset for the large-scale screening of compounds or stimuli susceptible to the modulation of the autophagic flux. The fact that the intracellular expression of p62 is regulated at both the transcriptional and post-translational levels is a source of concern for the use of p62 as a general index to monitor autophagic flux. Indeed, if the autophagy in-ducer activates p62 transcription, the increased autophagic flux may not be sufficient enough to fully clear intracellular level of p62. Several re-cent studies using three compounds that are able to trigger autophagy, i.e., Resveratrol, PMA and AICAR, nicely illustrate this notion [51, 57, 58]. Whereas each of these three compounds increased the autophagic flux in CML cells, the intracellular level of p62 remained unchanged. In the case of Resveratrol, activation of the JNK/c-Jun pathway drastically increased p62 expression, an event crucial for triggering Res-veratrol-dependent autophagy. In this context, the intracellular level of p62 remained stable mainly due to the increase of its synthesis,


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which compensates for its autophagic degrada-tion (Figure 4). The full validation of p62 as a hallmark of autophagic flux would therefore necessitate measuring the flux by combining the pro-autophagic stimuli with a transcriptional inhibitor such as Actinomycin D to ensure that a potential increase in p62 transcription would not counteract the degradation of p62 via auto-phagy. An emerging role of p62/SQSTM1 in cancer As previously mentioned, p62 is at the cross-roads of several signaling pathways including Ras/Raf/MAPK and NFκB, whose functions are often modified during tumoral transformation to promote the proliferation, migration and inva-sion of malignant cells. Importantly, p62 also plays a crucial role in the regulation of mTOR activity [15] and in the regulation of autophagy. The implication of p62 in carcinogenesis is still controversial and depends on whether the pro-tein plays a role in pro- or anti-tumoral auto-

phagy or in the oncogenic signaling pathways. Modulation of autophagy during tumoral pro-gression During transformation, cells need to reprogram their metabolic machinery to escape cell death, to become autonomous and to acquire new in-vasive properties. It has been well established that autophagy is deregulated during carcino-genesis [59, 60]. However, in cancer cells, the molecular mechanisms underlying the tumor-promoting or, conversely, the tumor-suppressing roles of autophagy are still poorly understood. Some findings are in agreement with the re-quirement of autophagy during the early stages of oncogenesis, particularly at the stage of tu-mor dormancy (Figure 5). This clinically unde-tectable phase, which precedes the period of tumor growth, is characterized by a quiescent state. In the context of residual metabolic activ-ity, autophagy helps cells produce enough en-ergy to address stressful conditions, such as

Figure 5. Involvement of autophagy in carcinogenesis. During tumor growth, the autophagic process helps cancer cells overcome metabolic stress, particularly stresses linked to the absence of vascularization. Autophagy thus ap-pears, in this early phase, as a mechanism of adaptation to a metabolic stress. However, during the tumoral progres-sion phase, only autophagy-deficient cells may acquire mutations favoring their metastatic dissemination. Autophagy blockade enables the metastasis of cancer cells by promoting the accumulation of damaged organelles and intracel-lular ROS, both of which are responsible for an increased rate of DNA mutations. Moreover, this autophagy inhibition promotes the accumulation of high intracellular levels of p62 (that is no longer degraded), which contributes to the amplification of the oxidative stress linked to ROS production and leads to NFκB activation, two events involved in tumoral transformation. It is currently unknown how autophagic knockdown mechanisms occur to trigger the tumor dissemination step. Dr. White’s team used an allograft model based on cells that were monoallelically depleted of Beclin 1 (becn1+/-) to demonstrate this two-tiered tumor growth mechanism.


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during a treatment with chemotherapeutic agents, until the environment becomes more favorable [61]. It thus appears that in the initial phases of malignant transformation, autophagy is beneficial for the adaptation of tumoral cells to their microenvironment (Figure 5). The supply of nutrients and oxygen in the tumoral microen-vironment is relatively poor, and in this context, autophagy would be beneficial for tumor cell development [62]. Similarly, lysosomes that are involved in autophagic degradation also play a very important role during tumorigenesis, nota-bly through the activation of cathepsins [63]. An allograft model using apoptotic and/or auto-phagy-deficient mammary cells generated by overexpressing Bcl-2 (an anti-apoptotic protein) in cells monoallelically depleted of Beclin 1 (beclin 1+/- cells) greatly contributed to the un-derstanding of the role of autophagy in the steps following this dormancy, especially during the dissemination of cancer cells [64, 65]. In this model, the autophagy process reduces the genomic instability of cancer cells. This genomic instability promotes the acquisition of the sec-ondary mutations necessary for the growth and dissemination of these cells [66]. Consequently, these Beclin 1+/- cancer cells accumulate dam-aged organelles and intracellular ROS, which both contribute to increase the frequency of DNA mutations. These autophagy-deficient cells then accumulate high intracellular levels of p62 (that is not degraded), which contributes to the amplification of oxidative stress linked to ROS production and leads to NFκB activation, two events involved in tumoral transformation [67]. Other evidence argues that autophagy is capa-ble of reducing the rate of tumor growth. Thus, the inactivation of the pro-autophagic genes TSC1/2, Atg4c, LKB1 or Beclin 1 in mice favors tumor progression [61]. In addition, in numer-ous types of cancer cells, the pro-autophagic genes (LKB1, PTEN, p53, TSC1, TCS2, Beclin 1, UVRAG) are often inactivated, whereas the anti-autophagic genes (PI3K, Akt, Ras, Bcl-2) are overactivated, suggesting that autophagy repre-sents a barrier for cancer development [62, 68]. However, it should also be noted that the phe-notypes associated with the mutation of all or some of these genes could be independent of the pro-autophagic activity of the genes. Finally, only cells that are deficient in the auto-phagic pathways are able to acquire the muta-

tions required for their growth and metastatic dissemination [61, 62, 66] (Figure 5). There-fore, a failure to eliminate p62 represents an explanation of how the inhibition of autophagy can promote tumoral development. This inhibi-tion would favor the accumulation of pro-oncogenic proteins such as p62 that would pro-mote the development and propagation of tu-mors (Figure 5). If this hypothesis is correct, the use of autophagy modulators would represent a promising therapeutic strategy that would de-pend on the progress phase of the disease. Influence of autophagy on p62 tumoral func-tions Several other studies have associated the accu-mulation of p62 with tumoral transformation and/or progression. As previously mentioned, the defective clearance of p62 in Atg7-/- murine hepatocytes leads to the constitutive activation of the NRF2 transcription factor that is responsi-ble for hepatoma genesis [42]. Similarly, a re-cent report demonstrates an increased expres-sion of NRF2 and p62 in 34 and 37% of non-small-cell lung cancer (NSCLC) cases, respec-tively [69]. NSCLC patients exhibiting high p62 levels have a significantly worse prognosis than patients exhibiting a low level of p62 do [69]. Similarly, p62 is highly accumulated in breast cancer disease [70], the evolution of which (grade and formation of distant metastases) is significantly correlated with the degree of p62 expression [71]. The accumulation of p62 concomitant with autophagy blockade does not only result in the amplification of pro-tumoral signaling. Indeed, another study shows that the Wnt pathway can be targeted by p62-dependent autophagy [72]. Wnt signaling, which is essential during embry-onic development and whose deregulation is involved in numerous cancers, is transduced via a key protein, Dishevelled (Dsh, Dvl), that aggre-gates in cytoplasmic puncta to relay the signal from the Wnt receptor to multiple downstream effectors [73] (Figure 6). During starvation, Dsh is ubiquitinated and associates with p62 before being delivered to autophagosomes to be de-graded by the autophagic machinery. As a con-sequence, the Wnt pathway is inactivated [72, 73] (Figure 6). Although autophagy is thought to be a non-selective degradation process, this example illustrates a certain degree of plasticity and specificity of this process, which, through


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actors such as p62 protein, can target major oncogenic pathways to reprogram the tumor during its development. This selectivity, which is dependent on p62, is essential for autophagy, may affect tumor development and could repre-sent a tremendous asset for elaborating new anti-cancer strategies. The proof-of-principle of this type of therapy was provided by studying the effect of ATRA on acute promyelocytic leukemia (APL), a common vari-ant of acute myeloid leukemia (AML). This he-matopoietic malignancy is caused by the PML-RARα nuclear translocation, which fuses the genes encoding for promyelocytic leukemia (PML) and for the retinoic acid receptor alpha (RARα). ATRA is currently the leading treatment for this pathology and has recently been shown to induce PML-RARα degradation [74]. The au-thors showed that this degradation proceeds by autophagy following the binding of p62 to PML-

RARα [75]. By itself, the interaction between p62 and PML-RARα has no deleterious; how-ever, only because ATRA results in an increased autophagic flux, p62 is converted to a deadly adaptor that eliminates the oncogene responsi-ble for the disease. p62/SQSTM1, a protein working either as an essential participant in oncogenic signaling or as an inhibitor of tumor proliferation p62 is a scaffold protein, which, through its own degradation, selectively directs its targets to the autophagic machinery. However, this protein is not only a substrate allowing the autophagy to arbitrate tumor cells fate. This protein also acts intrinsically as a major pro-oncogenic regulator of several important signaling pathways. There-fore, it is possible that autophagy only inter-venes as a facilitator or an inhibitor of the pro-oncogenic action of p62. Thus, the ectopic over-

Figure 6. p62/SQSTM1 acts either as a pro-oncogene or as a tumor suppressor protein. Based on the presence of multiple interacting domains in p62/SQSTM1, this protein is able to positively modulate either the NFκB pathway by binding to the IKK activator TRAF6 or the mTOR pathway by interacting with Raptor. The interaction of p62 with Rap-tor and, subsequently, mTORC1 involves the localization of mTORC1 to the lysosomal membrane to promote Rag GTPase-mediated mTOR activation. During tumor development, this intricate link between p62 and the NFκB and mTOR pathways appears to be indispensable because the knockdown of p62 reduces the tumorigenicity related to hyperactivation. In contrast to its pro-oncogenic activity, p62 can also exert tumor a suppressive activity by promoting inactivation of the Wnt pathway via an autophagic process. Indeed, the Dsh protein that relays the signal of the acti-vated Wnt pathway is ubiquitinated during starvation and associates with p62 before its degradation by the autopha-gic machinery. In this particular context, p62 acts as a tumor suppressive autophagic adaptor.


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expression of p62 in apoptosis- and autophagy-deficient cells is sufficient to drive tumor forma-tion [62]. As previously mentioned, through its interaction with TRAF6, p62 plays an essential role in the regulation of the NFκB pathway (Figure 6). The p62-TRAF6 complex integrates and transduces signals in response to the activation of IL-1 [14], the nerve growth factor and the RANK-L recep-tors [76, 77]. In ductal pancreatic adenocarci-noma cells, the constitutive activation of Ras activates the NFκB pathway to trigger increased p62 synthesis, and this newly synthesized p62, in turn, interacts with TRAF6 to amplify Ras sig-naling in a positive feedback loop [15, 54]. The same observations arise from studies on lung cancer in which Ras activation stimulates the TRAF6-p62-NFκB complex to trigger an antioxi-dant response that is necessary to inhibit dele-terious ROS production [49]. Finally, p62 is strongly overexpressed in the subfraction of highly aggressive glioblastoma harboring a mes-enchymal oncogenic signature, and this overex-pression is concomitant with MAPK activation [78]. In this particular context, the inhibition of p62 drastically reduces the motility and inva-siveness of the glioblastoma cells. p62 also participates in the tumorigenic signal-ing mediated by the mTOR pathway (Figure 6). mTOR can form two distinct complexes: mTORC1 and mTORC2. The mTORC2 complex is relatively insensitive to nutrient deprivation and to rapamycin and is involved in the regulation of cell survival and actin cytoskeleton remodeling via Akt phosphorylation. Conversely, the mTORC1 complex, which is highly sensitive to nutrient starvation and to rapamycin stimula-tion, modulates autophagy [16, 79, 80] and plays an essential role in the regulation of cell growth and protein synthesis. mTORC1 is inhib-ited by the TSC complex (tuberous sclerosis complex), which is composed of the TSC1 and TSC2 proteins. TSC2, also called tuberine, con-tains a GAP (GTPase-activating protein) domain in its C-terminal region, which is involved in the inactivation of the mTORC1-activating small G protein (Ras-like GTPase) Rheb. Therefore, the TSC complex is a tumor suppressor, and its in-validation in mice results in the hyperactivation of the mTOR pathway and the occurrence of harmless tumors in several organs. A recent study established that p62 is necessary for the proper development of the tumors associated

with TSC2 invalidation [81]. Indeed, the survival of mice injected with TSC2-/- mouse embryonic fibroblasts is significantly increased when p62 expression is knocked down. According to these results, a link between TSC2 and p62 has been reported by Duran et al., who described an inter-action of p62 with mTOR and Raptor that was required for the signaling pathway that is de-pendent on the mTORC1 complex [15] (Figure 6). Subsequently, the same authors showed that the knockdown of p62 by shRNA inhibits the growth of mTOR hyperactive-driven tumor cells. In contrast, several other studies argue for a tumor-suppressive function of p62, especially during mitosis. In mammalian cells, the main protein kinases required for progression through the cell cycle belong to the cyclin-dependent kinase (CDK) family. CDK1 controls the checkpoint between the S/G2 and early mitotic phases. Importantly, p62 is phosphory-lated by CDK1 on threonine 269 and serine 272 during the early mitotic phase, allowing the cells to properly continue through mitosis [82]. More-over, these authors describe cancer cells ex-pressing a non-phosphorylatable mutant of p62 that exhibit a better tumoral phenotype in vitro and in vivo than cells expressing the wild-type form of p62. Therefore, it appears that the CDK1-dependent phosphorylation of p62 repre-sents a constraint for tumoral transformation, again highlighting the dual function of p62, which can act either as an oncogene or as a tumor suppressor depending on the cellular circumstances. In conclusion, p62 is a scaffold protein at the crossroads between pro-oncogenic signaling pathways (i.e., NFκB, MAPK, mTOR) and auto-phagic degradative pathways. Although p62 is by itself a significant participant in cellular transformation and tumor propagation, it is also regulated by autophagy, which can target some pathways or key proteins involved in tumoral development via its intermediate form. As a future clinical prospective, new anti-cancer strategies should benefit from the pivotal func-tion of p62 between tumorigenesis and auto-phagy to circumvent tumoral progression. Acknowledgements This work was supported by INSERM, the Ligue Nationale Contre le Cancer (LNCC), équipe la-


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bellisée 2008-2010, INCA (PL2010-0149) ,the Association pour la Rechercher contre le Cancer and the Fondation de France (FDF). P.A. is the recipient of a Contrat Hospitalier de Recherche Translationnel (CHRT 2010-2013) from the CHU of Nice, France. A.P. is the recipient of a fellow-ship from the Association pour la Recherche sur le Cancer (ARC). N.F. is the recipient of a fellow-ship from The Ludwig Fund for Cancer Re-search. Address correspondence to: Dr. Patrick Auberger, DR1 INSERM, Directeur INSERM U1065, Centre Méditerranéen de Médecine Moléculaire, Bâtiment ARCHIMED, 151 Route de Saint-Antoine de Gines-tière, BP 2 3194, 06204 Nice Cedex 3. Tel: 00 33 4 89 06 43 11; Fax: 00 33 4 89 06 42 21; E-mail : [email protected] References [1] Ohsumi Y. Molecular dissection of autophagy:

two ubiquitin-like systems. Nat Rev Mol Cell Biol 2001; 2: 211-216.

[2] Puissant A, Robert G and Auberger P. Targeting autophagy to fight hematopoietic malignancies. Cell Cycle 9: 3470-3478.

[3] Mehrpour M, Esclatine A, Beau I and Codogno P. Overview of macroautophagy regulation in mammalian cells. Cell Res 2010; 20: 748-762.

[4] Tanida I. Autophagy basics. Microbiol Immunol 2011; 55: 1-11.

[5] Codogno P, Mehrpour M and Proikas-Cezanne T. Canonical and non-canonical autophagy: variations on a common theme of self-eating? Nat Rev Mol Cell Biol 2011; 13: 7-12.

[6] Kirkin V, McEwan DG, Novak I and Dikic I. A role for ubiquitin in selective autophagy. Mol Cell 2009; 34: 259-269.

[7] Duran A, Serrano M, Leitges M, Flores JM, Picard S, Brown JP, Moscat J and Diaz-Meco MT. The atypical PKC-interacting protein p62 is an important mediator of RANK-activated os-teoclastogenesis. Dev Cell 2004; 6: 303-309.

[8] Nakamura K, Kimple AJ, Siderovski DP and Johnson GL. PB1 domain interaction of p62/sequestosome 1 and MEKK3 regulates NF-kappaB activation. J Biol Chem 2010; 285: 2077-2089.

[9] Jin W, Chang M, Paul EM, Babu G, Lee AJ, Reiley W, Wright A, Zhang M, You J and Sun SC. Deubiquitinating enzyme CYLD negatively regu-lates RANK signaling and osteoclastogenesis in mice. J Clin Invest 2008; 118: 1858-1866.

[10] Lee SJ, Pfluger PT, Kim JY, Nogueiras R, Duran A, Pages G, Pouyssegur J, Tschop MH, Diaz-Meco MT and Moscat J. A functional role for the p62-ERK1 axis in the control of energy homeo-stasis and adipogenesis. EMBO Rep 2010; 11: 226-232.

[11] Ichimura Y, Kumanomidou T, Sou YS, Mizu-shima T, Ezaki J, Ueno T, Kominami E, Yamane T, Tanaka K and Komatsu M. Structural basis for sorting mechanism of p62 in selective auto-phagy. J Biol Chem 2008; 283: 22847-22857.

[12] Pankiv S, Clausen TH, Lamark T, Brech A, Bruun JA, Outzen H, Overvatn A, Bjorkoy G and Johansen T. p62/SQSTM1 binds directly to Atg8/LC3 to facilitate degradation of ubiquiti-nated protein aggregates by autophagy. J Biol Chem 2007; 282: 24131-24145.

[13] Takeuchi K, Soda M, Togashi Y, Ota Y, Sekigu-chi Y, Hatano S, Asaka R, Noguchi M and Mano H. Identification of a novel fusion, SQSTM1-ALK, in ALK-positive large B-cell lymphoma. Haematologica 2011; 96: 464-467.

[14] Sanz L, Diaz-Meco MT, Nakano H and Moscat J. The atypical PKC-interacting protein p62 chan-nels NF-kappaB activation by the IL-1-TRAF6 pathway. EMBO J 2000; 19: 1576-1586.

[15] Duran A, Amanchy R, Linares JF, Joshi J, Abu-Baker S, Porollo A, Hansen M, Moscat J and Diaz-Meco MT. p62 is a key regulator of nutri-ent sensing in the mTORC1 pathway. Mol Cell 2011; 44: 134-146.

[16] Wullschleger S, Loewith R and Hall MN. TOR signaling in growth and metabolism. Cell 2006; 124: 471-484.

[17] Matsumoto G, Wada K, Okuno M, Kurosawa M and Nukina N. Serine 403 phosphorylation of p62/SQSTM1 regulates selective autophagic clearance of ubiquitinated proteins. Mol Cell 2011; 44: 279-289.

[18] Morissette J, Laurin N and Brown JP. Sequesto-some 1: mutation frequencies, haplotypes, and phenotypes in familial Paget's disease of bone. J Bone Miner Res 2006; 21 Suppl 2: P38-44.

[19] Michou L, Collet C, Morissette J, Audran M, Thomas T, Gagnon E, Launay JM, Laplanche JL, Brown JP and Orcel P. Epidemiogenetic study of French families with Paget's disease of bone. Joint Bone Spine 2011.

[20] Hanna RA, Quinsay MN, Orogo AM, Giang K, Rikka S and Gustafsson AB. Microtubule-Associated Protein 1 Light Chain 3 (LC3) Inter-acts with Bnip3 to Selectively Remove Endo-plasmic Reticulum and Mitochondria via Auto-phagy. J Biol Chem 2012.

[21] Novak I, Kirkin V, McEwan DG, Zhang J, Wild P, Rozenknop A, Rogov V, Lohr F, Popovic D, Oc-chipinti A, Reichert AS, Terzic J, Dotsch V, Ney PA and Dikic I. Nix is a selective autophagy receptor for mitochondrial clearance. EMBO Rep 2009; 11: 45-51.

[22] Seillier M, Peuget S, Gayet O, Gauthier C, N'Guessan P, Monte M, Carrier A, Iovanna JL and Dusetti NJ. TP53INP1, a tumor suppressor, interacts with LC3 and ATG8-family proteins through the LC3-interacting region (LIR) and promotes autophagy-dependent cell death. Cell Death Differ 2012.

[23] von Muhlinen N, Thurston T, Ryzhakov G, Bloor


411 Am J Cancer Res 2012;2(4):397-413

S and Randow F. NDP52, a novel autophagy receptor for ubiquitin-decorated cytosolic bacte-ria. Autophagy 2010; 6: 288-289.

[24] Shvets E, Fass E, Scherz-Shouval R and Elazar Z. The N-terminus and Phe52 residue of LC3 recruit p62/SQSTM1 into autophagosomes. J Cell Sci 2008; 121: 2685-2695.

[25] Kim PK, Hailey DW, Mullen RT and Lippincott-Schwartz J. Ubiquitin signals autophagic degra-dation of cytosolic proteins and peroxisomes. Proc Natl Acad Sci USA 2008; 105: 20567-20574.

[26] Lamark T, Kirkin V, Dikic I and Johansen T. NBR1 and p62 as cargo receptors for selective autophagy of ubiquitinated targets. Cell Cycle 2009; 8: 1986-1990.

[27] Lamark T, Perander M, Outzen H, Kristiansen K, Overvatn A, Michaelsen E, Bjorkoy G and Johansen T. Interaction codes within the family of mammalian Phox and Bem1p domain-containing proteins. J Biol Chem 2003; 278: 34568-34581.

[28] Watanabe Y and Tanaka M. p62/SQSTM1 in autophagic clearance of a non-ubiquitylated substrate. J Cell Sci 2011; 124: 2692-2701.

[29] Bjorkoy G, Lamark T, Brech A, Outzen H, Per-ander M, Overvatn A, Stenmark H and Johansen T. p62/SQSTM1 forms protein aggre-gates degraded by autophagy and has a protec-tive effect on huntingtin-induced cell death. J Cell Biol 2005; 171: 603-614.

[30] Kuusisto E, Salminen A and Alafuzoff I. Ubiq-uitin-binding protein p62 is present in neuronal and glial inclusions in human tauopathies and synucleinopathies. Neuroreport 2001; 12: 2085-2090.

[31] Zatloukal K, Stumptner C, Fuchsbichler A, Heid H, Schnoelzer M, Kenner L, Kleinert R, Prinz M, Aguzzi A and Denk H. p62 Is a common compo-nent of cytoplasmic inclusions in protein aggre-gation diseases. Am J Pathol 2002; 160: 255-263.

[32] Babu JR, Geetha T and Wooten MW. Sequesto-some 1/p62 shuttles polyubiquitinated tau for proteasomal degradation. J Neurochem 2005; 94: 192-203.

[33] Nixon RA, Wegiel J, Kumar A, Yu WH, Peterhoff C, Cataldo A and Cuervo AM. Extensive involve-ment of autophagy in Alzheimer disease: an immuno-electron microscopy study. J Neuropa-thol Exp Neurol 2005; 64: 113-122.

[34] Nixon RA and Yang DS. Autophagy failure in Alzheimer's disease--locating the primary de-fect. Neurobiol Dis 2011; 43: 38-45.

[35] Wang Y, Martinez-Vicente M, Kruger U, Kaushik S, Wong E, Mandelkow EM, Cuervo AM and Mandelkow E. Synergy and antagonism of macroautophagy and chaperone-mediated autophagy in a cell model of pathological tau aggregation. Autophagy 2010; 6: 182-183.

[36] Wang Y, Martinez-Vicente M, Kruger U, Kaushik S, Wong E, Mandelkow EM, Cuervo AM and

Mandelkow E. Tau fragmentation, aggregation and clearance: the dual role of lysosomal proc-essing. Hum Mol Genet 2009; 18: 4153-4170.

[37] Du Y, Wooten MC and Wooten MW. Oxidative damage to the promoter region of SQSTM1/p62 is common to neurodegenerative disease. Neurobiol Dis 2009; 35: 302-310.

[38] Jain A, Lamark T, Sjottem E, Larsen KB, Awuh JA, Overvatn A, McMahon M, Hayes JD and Johansen T. p62/SQSTM1 is a target gene for transcription factor NRF2 and creates a posi-tive feedback loop by inducing antioxidant re-sponse element-driven gene transcription. J Biol Chem 2010; 285: 22576-22591.

[39] Itoh K, Wakabayashi N, Katoh Y, Ishii T, Igarashi K, Engel JD and Yamamoto M. Keap1 re-presses nuclear activation of antioxidant re-sponsive elements by Nrf2 through binding to the amino-terminal Neh2 domain. Genes Dev 1999; 13: 76-86.

[40] Komatsu M, Kurokawa H, Waguri S, Taguchi K, Kobayashi A, Ichimura Y, Sou YS, Ueno I, Saka-moto A, Tong KI, Kim M, Nishito Y, Iemura S, Natsume T, Ueno T, Kominami E, Motohashi H, Tanaka K and Yamamoto M. The selective auto-phagy substrate p62 activates the stress re-sponsive transcription factor Nrf2 through inac-tivation of Keap1. Nat Cell Biol 2010; 12: 213-223.

[41] Lau A, Wang XJ, Zhao F, Villeneuve NF, Wu T, Jiang T, Sun Z, White E and Zhang DD. A non-canonical mechanism of Nrf2 activation by autophagy deficiency: direct interaction be-tween Keap1 and p62. Mol Cell Biol 2010; 30: 3275-3285.

[42] Inami Y, Waguri S, Sakamoto A, Kouno T, Na-kada K, Hino O, Watanabe S, Ando J, Iwadate M, Yamamoto M, Lee MS, Tanaka K and Koma-tsu M. Persistent activation of Nrf2 through p62 in hepatocellular carcinoma cells. J Cell Biol 2011; 193: 275-284.

[43] Bui CB and Shin J. Persistent expression of Nqo1 by p62-mediated Nrf2 activation facili-tates p53-dependent mitotic catastrophe. Bio-chem Biophys Res Commun 2011; 412: 347-352.

[44] Kwon J, Han E, Bui CB, Shin W, Lee J, Lee S, Choi YB, Lee AH, Lee KH, Park C, Obin MS, Park SK, Seo YJ, Oh GT, Lee HW and Shin J. Assur-ance of mitochondrial integrity and mammalian longevity by the p62-Keap1-Nrf2-Nqo1 cas-cade. EMBO Rep 2012; 13: 150-156.

[45] Fujita K, Maeda D, Xiao Q and Srinivasula SM. Nrf2-mediated induction of p62 controls Toll-like receptor-4-driven aggresome-like induced structure formation and autophagic degrada-tion. Proc Natl Acad Sci USA 108: 2011; 1427-1432.

[46] Fujita K and Srinivasula SM. TLR4-mediated autophagy in macrophages is a p62-dependent type of selective autophagy of aggresome-like induced structures (ALIS). Autophagy 2011; 7:


412 Am J Cancer Res 2012;2(4):397-413

552-554. [47] Lee YH, Ko J, Joung I, Kim JH and Shin J. Imme-

diate early response of the p62 gene encoding a non-proteasomal multiubiquitin chain binding protein. FEBS Lett 1998; 438: 297-300.

[48] Vadlamudi RK and Shin J. Genomic structure and promoter analysis of the p62 gene encod-ing a non-proteasomal multiubiquitin chain binding protein. FEBS Lett 1998; 435: 138-142.

[49] Duran A, Linares JF, Galvez AS, Wikenheiser K, Flores JM, Diaz-Meco MT and Moscat J. The signaling adaptor p62 is an important NF-kappaB mediator in tumorigenesis. Cancer Cell 2008; 13: 343-354.

[50] Puissant A and Auberger P. AMPK- and p62/SQSTM1-dependent autophagy mediate Res-veratrol-induced cell death in chronic myeloge-nous leukemia. Autophagy 2010.

[51] Puissant A, Robert G, Fenouille N, Luciano F, Cassuto JP, Raynaud S and Auberger P. Res-veratrol promotes autophagic cell death in chronic myelogenous leukemia cells via JNK-mediated p62/SQSTM1 expression and AMPK activation. Cancer Res 2010; 70: 1042-1052.

[52] Zou X, Feng Z, Li Y, Wang Y, Wertz K, Weber P, Fu Y and Liu J. Stimulation of GSH synthesis to prevent oxidative stress-induced apoptosis by hydroxytyrosol in human retinal pigment epithe-lial cells: activation of Nrf2 and JNK-p62/SQSTM1 pathways. J Nutr Biochem 2011.

[53] Xu P, Das M, Reilly J and Davis RJ. JNK regu-lates FoxO-dependent autophagy in neurons. Genes Dev 2011; 25: 310-322.

[54] Ling J, Kang Y, Zhao R, Xia Q, Lee DF, Chang Z, Li J, Peng B, Fleming JB, Wang H, Liu J, Lemis-chka IR, Hung MC and Chiao PJ. KrasG12D-induced IKK2/beta/NF-kappaB activation by IL-1alpha and p62 feedforward loops is required for development of pancreatic ductal adenocar-cinoma. Cancer Cell 2012; 21: 105-120.

[55] Klionsky DJ, Abeliovich H, Agostinis P, Agrawal DK, Aliev G, Askew DS, Baba M, Baehrecke EH, Bahr BA, Ballabio A, Bamber BA, Bassham DC, Bergamini E, Bi X, Biard-Piechaczyk M, Blum JS, Bredesen DE, Brodsky JL, Brumell JH, Brunk UT, Bursch W, Camougrand N, Cebollero E, Cecconi F, Chen Y, Chin LS, Choi A, Chu CT, Chung J, Clarke PG, Clark RS, Clarke SG, Clave C, Cleve-land JL, Codogno P, Colombo MI, Coto-Montes A, Cregg JM, Cuervo AM, Debnath J, Demarchi F, Dennis PB, Dennis PA, Deretic V, Devenish RJ, Di Sano F, Dice JF, Difiglia M, Dinesh-Kumar S, Distelhorst CW, Djavaheri-Mergny M, Dorsey FC, Droge W, Dron M, Dunn WA, Jr., Duszenko M, Eissa NT, Elazar Z, Esclatine A, Eskelinen EL, Fesus L, Finley KD, Fuentes JM, Fueyo J, Fuji-saki K, Galliot B, Gao FB, Gewirtz DA, Gibson SB, Gohla A, Goldberg AL, Gonzalez R, Gonzalez-Estevez C, Gorski S, Gottlieb RA, Haussinger D, He YW, Heidenreich K, Hill JA, Hoyer-Hansen M, Hu X, Huang WP, Iwasaki A, Jaattela M, Jackson

WT, Jiang X, Jin S, Johansen T, Jung JU, Kadowaki M, Kang C, Kelekar A, Kessel DH, Kiel JA, Kim HP, Kimchi A, Kinsella TJ, Kiselyov K, Kitamoto K, Knecht E, Komatsu M, Komi-nami E, Kondo S, Kovacs AL, Kroemer G, Kuan CY, Kumar R, Kundu M, Landry J, Laporte M, Le W, Lei HY, Lenardo MJ, Levine B, Lieberman A, Lim KL, Lin FC, Liou W, Liu LF, Lopez-Berestein G, Lopez-Otin C, Lu B, Macleod KF, Malorni W, Martinet W, Matsuoka K, Mautner J, Meijer AJ, Melendez A, Michels P, Miotto G, Mistiaen WP, Mizushima N, Mograbi B, Monastyrska I, Moore MN, Moreira PI, Moriyasu Y, Motyl T, Munz C, Murphy LO, Naqvi NI, Neufeld TP, Nishino I, Nixon RA, Noda T, Nurnberg B, Ogawa M, Oleinick NL, Olsen LJ, Ozpolat B, Paglin S, Palmer GE, Papassideri I, Parkes M, Perlmutter DH, Perry G, Piacentini M, Pinkas-Kramarski R, Prescott M, Proikas-Cezanne T, Raben N, Rami A, Reggiori F, Rohrer B, Rubinsztein DC, Ryan KM, Sadoshima J, Sakagami H, Sakai Y, Sandri M, Sasakawa C, Sass M, Schneider C, Seglen PO, Seleverstov O, Settleman J, Shacka JJ, Shapiro IM, Sibirny A, Silva-Zacarin EC, Simon HU, Simone C, Simonsen A, Smith MA, Spanel-Borowski K, Srinivas V, Steeves M, Stenmark H, Stromhaug PE, Subauste CS, Sugimoto S, Sul-zer D, Suzuki T, Swanson MS, Tabas I, Take-shita F, Talbot NJ, Talloczy Z, Tanaka K, Tanida I, Taylor GS, Taylor JP, Terman A, Tettamanti G, Thompson CB, Thumm M, Tolkovsky AM, Tooze SA, Truant R, Tumanovska LV, Uchiyama Y, Ueno T, Uzcategui NL, van der Klei I, Vaquero EC, Vellai T, Vogel MW, Wang HG, Webster P, Wiley JW, Xi Z, Xiao G, Yahalom J, Yang JM, Yap G, Yin XM, Yoshimori T, Yu L, Yue Z, Yuzaki M, Zabirnyk O, Zheng X, Zhu X and Deter RL. Guidelines for the use and interpretation of assays for monitoring autophagy in higher eu-karyotes. Autophagy 2008; 4: 151-175.

[56] Larsen KB, Lamark T, Overvatn A, Harneshaug I, Johansen T and Bjorkoy G. A reporter cell system to monitor autophagy based on p62/SQSTM1. Autophagy 2010; 6: 784-793.

[57] Colosetti P, Puissant A, Robert G, Luciano F, Jacquel A, Gounon P, Cassuto JP and Auberger P. Autophagy is an important event for mega-karyocytic differentiation of the chronic mye-logenous leukemia K562 cell line. Autophagy 2009; 5: 1092-1098.

[58] Robert G, Ben Sahra I, Puissant A, Colosetti P, Belhacene N, Gounon P, Hofman P, Bost F, Cassuto JP and Auberger P. Acadesine kills chronic myelogenous leukemia (CML) cells through PKC-dependent induction of autopha-gic cell death. PLoS One 2009; 4: e7889.

[59] Hoyer-Hansen M and Jaattela M. Autophagy: an emerging target for cancer therapy. Autophagy 2008; 4: 574-580.

[60] Maiuri MC, Tasdemir E, Criollo A, Morselli E, Vicencio JM, Carnuccio R and Kroemer G. Con-trol of autophagy by oncogenes and tumor sup-


413 Am J Cancer Res 2012;2(4):397-413

pressor genes. Cell Death Differ 2009; 16: 87-93.

[61] Corcelle EA, Puustinen P and Jaattela M. Apop-tosis and autophagy: Targeting autophagy sig-nalling in cancer cells -'trick or treats'? FEBS J 2009; 276: 6084-6096.

[62] Mathew R, Karantza-Wadsworth V and White E. Role of autophagy in cancer. Nat Rev Cancer 2007; 7: 961-967.

[63] Kroemer G and Jaattela M. Lysosomes and autophagy in cell death control. Nat Rev Cancer 2005; 5: 886-897.

[64] Karantza-Wadsworth V, Patel S, Kravchuk O, Chen G, Mathew R, Jin S and White E. Auto-phagy mitigates metabolic stress and genome damage in mammary tumorigenesis. Genes Dev 2007; 21: 1621-1635.

[65] Karantza-Wadsworth V and White E. Role of autophagy in breast cancer. Autophagy 2007; 3: 610-613.

[66] Mathew R, Kongara S, Beaudoin B, Karp CM, Bray K, Degenhardt K, Chen G, Jin S and White E. Autophagy suppresses tumor progression by limiting chromosomal instability. Genes Dev 2007; 21: 1367-1381.

[67] Mathew R, Karp CM, Beaudoin B, Vuong N, Chen G, Chen HY, Bray K, Reddy A, Bhanot G, Gelinas C, Dipaola RS, Karantza-Wadsworth V and White E. Autophagy suppresses tumori-genesis through elimination of p62. Cell 2009; 137: 1062-1075.

[68] Gozuacik D and Kimchi A. Autophagy as a cell death and tumor suppressor mechanism. Onco-gene 2004; 23: 2891-2906.

[69] Inoue D, Suzuki T, Mitsuishi Y, Miki Y, Suzuki S, Sugawara S, Watanabe M, Sakurada A, Endo C, Uruno A, Sasano H, Nakagawa T, Satoh K, Ta-naka N, Kubo H, Motohashi H and Yamamoto M. Accumulation of p62/SQSTM1 is associated with poor prognosis in patients with lung ade-nocarcinoma. Cancer Sci 2012; 103: 760-766.

[70] Thompson HG, Harris JW, Wold BJ, Lin F and Brody JP. p62 overexpression in breast tumors and regulation by prostate-derived Ets factor in breast cancer cells. Oncogene 2003; 22: 2322-2333.

[71] Rolland P, Madjd Z, Durrant L, Ellis IO, Layfield R and Spendlove I. The ubiquitin-binding pro-tein p62 is expressed in breast cancers show-ing features of aggressive disease. Endocr Re-lat Cancer 2007; 14: 73-80.

[72] Gao C, Cao W, Bao L, Zuo W, Xie G, Cai T, Fu W, Zhang J, Wu W, Zhang X and Chen YG. Auto-phagy negatively regulates Wnt signalling by promoting Dishevelled degradation. Nat Cell Biol 2010; 12: 781-790.

[73] Gao C and Chen YG. Selective removal of di-shevelled by autophagy: a role of p62. Auto-phagy 2011; 7: 334-335.

[74] Ozpolat B, Lopez-Berestein G and Mehta K. ATRA(ouble) in the treatment of acute promye-locytic leukemia. J Biol Regul Homeost Agents 2001; 15: 107-122.

[75] Wang Z, Cao L, Kang R, Yang M, Liu L, Zhao Y, Yu Y, Xie M, Yin X, Livesey KM and Tang D. Autophagy regulates myeloid cell differentiation by p62/SQSTM1-mediated degradation of PML-RARalpha oncoprotein. Autophagy 2011; 7: 401-411.

[76] Laurin N, Brown JP, Morissette J and Raymond V. Recurrent mutation of the gene encoding sequestosome 1 (SQSTM1/p62) in Paget dis-ease of bone. Am J Hum Genet 2002; 70: 1582-1588.

[77] Wooten MW, Geetha T, Seibenhener ML, Babu JR, Diaz-Meco MT and Moscat J. The p62 scaf-fold regulates nerve growth factor-induced NF-kappaB activation by influencing TRAF6 polyubiquitination. J Biol Chem 2005; 280: 35625-35629.

[78] Galavotti S, Bartesaghi S, Faccenda D, Shaked-Rabi M, Sanzone S, McEvoy A, Dinsdale D, Con-dorelli F, Brandner S, Campanella M, Grose R, Jones C and Salomoni P. The autophagy-associated factors DRAM1 and p62 regulate cell migration and invasion in glioblastoma stem cells. Oncogene 2012.

[79] Guertin DA and Sabatini DM. Defining the role of mTOR in cancer. Cancer Cell 2007; 12: 9-22.

[80] Hosokawa N, Hara T, Kaizuka T, Kishi C, Taka-mura A, Miura Y, Iemura S, Natsume T, Take-hana K, Yamada N, Guan JL, Oshiro N and Mi-zushima N. Nutrient-dependent mTORC1 asso-ciation with the ULK1-Atg13-FIP200 complex required for autophagy. Mol Biol Cell 2009; 20: 1981-1991.

[81] Parkhitko A, Myachina F, Morrison TA, Hindi KM, Auricchio N, Karbowniczek M, Wu JJ, Finkel T, Kwiatkowski DJ, Yu JJ and Henske EP. Tu-morigenesis in tuberous sclerosis complex is autophagy and p62/sequestosome 1 (SQSTM1)-dependent. Proc Natl Acad Sci USA 2011; 108: 12455-12460.

[82] Linares JF, Amanchy R, Greis K, Diaz-Meco MT and Moscat J. Phosphorylation of p62 by cdk1 controls the timely transit of cells through mito-sis and tumor cell proliferation. Mol Cell Biol 2011; 31: 105-117.