CDC-48: Proteostasis control in tumor cell biology

Cancer Letters 337 (2013) 26–34

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Mini-review

P97/CDC-48: Proteostasis control in tumor cell biology Delphine Fessart a,c, Esther Marza b,c, Saïd Taouji b,c, Frédéric Delom a,c,⇑, Eric Chevet b,c,⇑ a

Inserm U1045, Bordeaux, France Inserm U1053, Bordeaux, France c Université Bordeaux Segalen, Bordeaux, France b

a r t i c l e

i n f o

Article history: Received 8 February 2013 Received in revised form 16 May 2013 Accepted 23 May 2013

Keywords: VCP p97 CDC-48 Proteostasis Cancer

a b s t r a c t P97/CDC-48 is a prominent member of a highly evolutionary conserved Walker cassette – containing AAA + ATPases. It has been involved in numerous cellular processes ranging from the control of protein homeostasis to membrane trafficking through the intervention of specific accessory proteins. Expression of p97/CDC-48 in cancers has been correlated with tumor aggressiveness and prognosis, however the precise underlying molecular mechanisms remain to be characterized. Moreover p97/CDC-48 inhibitors were developed and are currently under intense investigation as anticancer drugs. Herein, we discuss the role of p97/CDC-48 in cancer development and its therapeutic potential in tumor cell biology. Ó 2013 Elsevier Ireland Ltd. All rights reserved.

1. Introduction The ATPases associated with various cellular activities (AAA) family is a functionally diverse group of proteins that have a wide range of substrates. The AAA + family includes about 30,000 proteins that share common structural features including a highly conserved ATPase domain of about 200 amino acids, Walker A and B motifs and the second region of homology (SHR). These ATPases assemble into active oligomeric rings (usually hexamers) and form molecular motors that couple ATP hydrolysis to conformational changes generating mechanical forces on the bound substrate [27,55,85]. P97 (also named Valosin Containing Protein (VCP) in mammals, Cdc48 in yeast or Caenorhabditis elegans and TER94 in Drosophila melanogaster) is a prominent member of the magnesium-dependent Walker cassette AAA-ATPases. It is highly conserved throughout evolution (Fig. 1) and is very abundant in cells as it represents about 1% of all cytoplasmic proteins. It is mainly active as a homohexamer with each monomer containing two ATPase domains (called D1 and D2), the D2 domain being the most efficient. ATP hydrolysis generates energy to structurally remold or unfold proteins [17]. P97/CDC-48 was originally identified as a biochemical requirement for membrane fusion reactions

⇑ Corresponding authors. Addresses: Inserm U1045, Université Bordeaux Segalen, 146 rue Léo Saignat, 33000 Bordeaux, France. Tel.: +33 (0) 557575681; fax: +33 (0) 557571695 (F. Delom), Inserm U1053, Université Bordeaux Segalen, 146 rue Léo Saignat, 33000 Bordeaux, France. Tel.: +33 (0) 557579253 (E. Chevet). E-mail addresses: [email protected] (F. Delom), [email protected] (E. Chevet). 0304-3835/$ - see front matter Ó 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.canlet.2013.05.030

[22,59] but it rapidly turned out that this protein played biochemical and genetic roles in additional cellular pathways controlling protein homeostasis through its segregase activity [22]. Since the first discovery of CDC48 in Saccharomyces cerevisiae and its identification as p97/CDC-48 in mammals in the early 90’s [55], its involvement in diseases has been reported. This is particularly true for inclusion body myopathy associated with Paget disease of the bone and frontotemporal dementia (IBMPFD), which is an autosomal dominant disorder attributed to misense mutations in p97/ CDC-48 [99]. The central role of p97/CDC-48 in the control of protein homeostasis (proteostasis) through a direct action on protein substrates or through the regulation of membrane-based mechanisms [4,60] has raised the possibility that this protein could be involved in cancer. In this review, we describe p97/CDC-48 molecular and cellular functions and discuss its involvement in cancer development and propagation through the analysis of recent studies carried out in cell lines, animal models and descriptive clinicopathology. 2. Molecular functions of p97/CDC-48 2.1. P97/CDC-48 molecular functions – accessory and clients proteins P97/CDC-48 has many known protein substrates and is involved in diverse molecular functions. Specificity of the client protein partner and of the cell process is achieved by the interaction of p97/CDC-48 with specific accessory proteins or cofactors (i.e. reviewed by [50]) that can either bind the N- or the C-terminal end of p97/CDC-48 (Fig. 2A; [50]). One good example is the interaction

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Fig. 1. Phylogenetic analysis of the Cdc48/TER94/VCP family in 14 species. The blue box indicates the tree generated in vertebrates whereas the yellow box indicates the one generated in invertebrates (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

Fig. 2. P97/CDC-48 adaptors/effectors in VCP cellular functions. (A) Four major molecular functions of p97/CDC-48 in regulating mitosis, membrane traffic, DNA repair, proteostasis. A fifth functional group that has to be further defined in the future is proposed (Other). (B) Molecular mechanism of p97/CDC-48 function in the ubiquitin system. An accessory protein (A, listed in 2A) allows for the recruitment of p97/CDC-48 to the polyubiquitinated substrate/client (S). ATP hydrolysis allows substrate segregation from its polyprotein complex and interaction with either E4 or Deubiquitinating enzymes (DUB) to control the fate of the segregated protein.

of p97/CDC-48 with ubiquitinated client proteins [38]. In that case, the accessory protein is not only required for this interaction to occur but it is also involved in the fate of the ubiquitinated client protein. These cofactors contain an ubiquitin-X domain (UBX) and form the largest family of p97/CDC-48 accessory proteins [21]. The UBX domain is an 80 aa domain that is mostly present at the carboxyl terminus of a variety of eukaryotic proteins and that binds the N-terminal part of p97/CDC-48 ([50], Fig. 2A). This family includes the Npl4/Ufd1 heterodimer and p47. Other domains present in different accessory proteins were identified to play critical roles in the interaction between p97/CDC-48 ubiquitinated client protein, such as the PUB domain (peptide N-glycosidase/ubiqui-

tin-associated) found on the PNGase and UBXD1, the PUL domain, which both bind the C-terminal tail of p97/CDC-48 [38] (see Fig. 3). 2.2. Segregase vs. degradation activity It is well established that the ubiquitin–proteasome system mediates degradation of misfolded or damaged proteins to maintain cellular protein homeostasis (proteostasis) [60], and selectively removes regulatory proteins in critical signaling pathways [22]. The role of p97/CDC-48 in this regard has been shown to be dual at least and to occur through a mechanism different than the one observed for classical ubiquitin-shuttling factors that deli-

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Fig. 3. P97/CDC-48 cellular functions. Seven major cellular functions of p97/CDC-48 are proposed (non exhaustively) to contribute to membrane traffic and fusion (yellow, orange, red) and to protein degradation mechanisms (green, light blue, dark blue, purple). All these cellular functions are linked to basic cellular mechanisms essential for tumor cell survival and proliferation (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.).

ver ubiquitin-conjugated substrates to the proteasome [6,21,99]. Indeed unlike other shuttle proteins, which simply bind to both ubiquitin chains and components of the proteasome, p97/CDC-48 uses the energy of ATP hydrolysis to structurally remodel or unfold its clients, and therefore helps extract them from cellular structures, segregate them from binding partners or generate initial unfolded stretches to facilitate degradation by the proteasome ([99]; Fig. 2B). This is best illustrated with the well-studied core complex p97/CDC-48–Ufd1–Npl4, which triggers extraction of client proteins from complexes or cellular surfaces to favor in most cases proteasome-mediated degradation [99]. In addition to substrate segregation, p97/CDC-48 has also been proposed to assist the proteasome function through the selective unfolding a subset of client proteins to enhance their degradation ([99]; Fig. 2B). Alternatively specific complexes associated with mono-ubiquitination and proteasome-independent membrane trafficking events (e.g. p97/ CDC-48–p47 and p97/CDC-48–UBXD1) are thought to promote membrane protein segregation rather than extraction/degradation [41]. Another role for p97/CDC-48 in this context is to actively participate in the coordination and modulation of ubiquitination [65,99]. Unlike the clearly established role of p97/CDC-48 in proteostasis control, the regulatory mechanisms for controlling the assembly of these specific complexes and the resulting cellular outcomes remain to be fully understood. 3. Cellular functions of p97/CDC-48 The diverse, yet proteostasis-oriented, molecular functions of p97/CDC-48 have been associated with a large variety of cellular processes ranging from transcription regulation to protein aggregates control (Fig. 3), which are described below. 3.1. Protein quality control 3.1.1. Organelle homeostasis 3.1.1.1. ER-associated degradation. The functional contribution of p97/CDC-48 and its accessory proteins has been best characterized in the Endoplasmic Reticulum Associated Degradation (ERAD)

pathway [87]. ERAD is a highly conserved process that is responsible for the clearance of misfolded proteins localized in the ER [87]. Terminally misfolded proteins in the ER retrotranslocate into the cytosol where they are degraded through a proteasomal dependent mechanism [87]. In this context, p97/CDC-48 plays a major role in first extracting the misfolded protein from the ER retro-translocon, followed by the de-glycosylation (if the protein is N-glycosylated (PGNase)) and then ubiquitination, before targeting it to the proteasome [20,74]. Although the information about number, types, and activities of p97/CDC-48 accessory proteins involved in ERAD is currently quite extensive, the central question of the precise role(s) of p97/CDC-48 and their regulation modes remain to be fully elucidated. 3.1.1.2. Mitochondrial quality control. Mitochondria have evolved a specific quality control machinery to eliminate improperly folded proteins [99]. Moreover proteins at the cytosolic side of the outer mitochondrial membrane are also subject to ubiquitination and proteasomal degradation [99]. Recent illustrations of the latter phenomenon have pointed towards a critical role for p97/CDC-48 in the degradation of two outer membrane proteins, Mcl1 and mitofusin-1 (Mfn1; [33]). Acting in a similar manner to ERAD, p97/CDC-48, which is found associated with the outer mitochondrial membrane, binds to the ubiquitinated proteins and targets them for proteasomal degradation. Moreover, p97/CDC-48-mediated ubiquitination and extraction of Mfn1 prevents fusion of damaged and intact mitochondria, thereby facilitating their disposal by autophagy ([54,72]; see below). 3.1.2. Protein aggregate control 3.1.2.1. Autophagy. p97/CDC-48 has also been involved in the degradation of cytosolic aggregates through an aggresome-autophagy (aggrephagy) dependent pathway [72,99]. Aggregates are first polyubiquitinated by the E3-ligase Parkin, thereby facilitating the cooperation between HDAC6, a dynein motor–complex retrograde transport to form aggresomes and allow the recruitment of the p62-NBR1-ALFY complex to form the autophagosome and then the autophagolysosome [31,41,73]. P97/CDC-48 has been shown

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to interact with HDAC6 and loss of p97/CDC-48 or HDAC6 leads to a defect in autophagosome–lysosome fusion [30]. Both expression of p97/CDC-48 R155H and A232E IBMPFD mutants as well as p97/ CDC-48 silencing result in the accumulation of autophagosomes, which are however, unable to mature into autophagolysosomes and degrade protein aggregates [73]. Although these observations robustly demonstrate the relationship between p97/CDC-48 and autophagosomes maturation, the mechanism(s) by which p97/ CDC-48 promotes degradation of protein aggregates still remains unclear. 3.1.2.2. Proteasome. It is well established that p97/CDC-48 facilitates the degradation of individual proteins in the nucleus and cytosol in regulatory processes [38]. Among the known mammalian substrates targeted for degradation through a p97/CDC-48dependent mechanism are IkBa [46], HIF1a ([1]; see below) and neurofibromin-1 [82] in human cells. Although these events are dependent on the accessory proteins Ufd1–Npl4 and the mechanism(s) by which p97/CDC-48 prompts the degradation of these substrates remains unclear. 3.2. Membrane traffic 3.2.1. Endoplasmic reticulum – golgi biogenesis P97/CDC-48 is known to mediate membrane fusion events and is required for reassembly of the Golgi apparatus, the ER and the nuclear membrane during mitosis [23,40,63,64,78]. In the p97/ CDC-48-mediated membrane fusion, both p47 and VCIP135 were identified as essential accessory proteins, to form the SNARE containing complex p97/CDC-48/p47/VCIP135/syntaxin5 [76]. In this model p47 would bind to monoubiquitinated proteins and VCIP135 could bring a deubiquitinating activity (at least in vitro) that could in turn provide a regulatory mechanism to the fusion system [76]. Moreover, phosphorylation events occurring on p97/ CDC-48 [43,48,103] and on p47 [77] were shown to regulate fusion events in the secretory pathway. As a consequence the mechanisms involving p97/CDC-48 in fusion events in the early secretory pathway are still to be fully characterized. However post-translational modifications by phosphorylation or mono-ubiquitination might represent novel aspects to include in the understanding of p97/CDC-48 function in the ER. 3.2.2. Endolysosomal sorting In 1993, the first identification of p97/CDC-48 in mammals showed a strong association of this protein with clathrin [57]. However, the functional relevance of this interaction for endocytic trafficking remained unclear until recently. Recent reports showed indeed the involvement of p97/CDC-48 in endocytic processes [7]. On the one hand, p97/CDC-48 binding to the early endosomal antigen-1 (EEA1) would disrupt EEA1 oligomers in an Ufd1–Npl4 independent fashion and regulate endosome fusion and dynamics [67]. On the other hand, the Meyer group reported an interaction of p97/ CDC-48 with Caveolin-1 along with UBXD1 but independently of Ufd1-Npl4 or p47 [35,68]. Even though these studies present different molecular mechanism, in both studies significant defects in endosomal trafficking of transferrin receptor [67] and of EGF receptor [68] are observed. 3.3. DNA damage and transcription Chromatin-associated functions of p97/CDC-48 are illustrated by its interaction with the DNA-repair factors BRCA1 [49], the Werner syndrome protein WRN [24], and DNA unwinding factor DUF [88]. Moreover p97/CDC-48 has been shown to be in a complex with ATM and the Bloom syndrome ReqQ helicase BLM ortholog in C. elegans [9]. Furthermore p97/CDC-48 is phosphorylated after

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DNA damage [24,53,81]. The replication-licensing factor Cdt1 was degraded by mechanisms depending on the p97/CDC-48Ufd1–Npl4 and CRL ligase complex CUL4–DDB1–CDT2 complexes and on the interaction with PCNA in cells and in vitro [66]. However, another study showed in that p97/CDC-48 helps to remove ubiquitinated Cdt1 from mitotic chromatin, which provides an explanation for the requirement of CDC-48 proteins and Ufd1– Npl4 for replication in C. elegans [19]. Finally p97/CDC-48 plays a role in DNA repair through its Ufd1–Npl4-dependent and RNF8/ 168 recruitment at double-strand breaks. P97/CDC-48 mediates removal of the lysine-48-conjugated substrates to allow proper assembly of downstream signaling factors, such as Rad51, BRCA1 and 53BP1 [65]. This reveals roles of p97/CDC-48 in chromatinassociated processes that warrant ad hoc transcription, replication and stability of the genome.

4. P97/CDC-48 in cancer Proteostasis control [60] in cancer development and its therapeutic targeting has gained a lot of interest over the past 10 years. This was first illustrated by the clinically relevant use of proteasome inhibitors (e.g. Bortezomib, [36]) or HSP90 inhibitors [25,26]. Recently, the concept has been further developed by De Raedt and colleagues who have shown that agents enhancing proteotoxic stress (such as HSP90 inhibitors), induce tumor regression in aggressive mouse models, but only when combined with rapamycin (immunosuppressant drug). They demonstrate that the toxicity of reactive oxygen species produced upon HSP90 inhibition is enhanced by the suppression of glutathione by rapamycin [16]. This opens new therapeutic avenues in which p97/CDC-48 could represent an extremely interesting target. In certain types of cancers, e.g., colorectal carcinomas, gastric carcinoma, pancreatic endocrine neoplasms, hepatocellular carcinoma [45,97], non-small cell lung carcinoma (NSCLC) [79], esophageal squamous cell carcinoma (ESCC) [89], gingival squamous cell carcinoma (GSCC) [95], prostate cancer [75] and follicular thyroid cancer [98], the expression level of p97/CDC-48 is markedly elevated and has been shown to be associated with poor prognosis or been associated with the presence of auto-antibodies in the serum [44], proposing p97/CDC-48 expression levels as a useful marker for the progression of these cancers [3,89,90,92–98,104]. Recently, global genomic analysis of pancreatic cancer has confirmed p97/CDC-48 overexpression by Serial analysis of gene expression (SAGE) [28]. This study identified p97/CDC-48 as one of the few known recurrent amplicons at the DNA level associated with tumor metastasis [28]. The potential anti-apoptotic role of p97/CDC-48, has been first described in 1991 by Shirogane et al. [71] and was further substantiated by the fact that increased levels of p97/CDC-48 strongly correlate with poor prognosis and metastasis of various human cancers, i.e., cells that are characterized by decreased ability to undergo apoptosis [3,75,89–98,104]. Serum p97/CDC-48 levels were also measured in ovarian carcinoma, nonHodgkin’s lymphoma and breast, colon, pancreatic, lung, and prostate cancer patients [42]. Increased serum p97/CDC-48 levels were observed in the majority of cancer cases, with the exception of patients with lung or prostate cancer. Moreover, serum p97/CDC-48 levels were increased in some ovarian carcinoma, breast cancer, and colon cancer patients who did not otherwise display increased levels of widely used serum tumor markers for their cancer. Finally, several somatic mutations were identified in p97/CDC-48 encoding gene in human cancers as presented in the COSMIC database (http://cancer.sanger.ac.uk/cosmic/gene/analysis?ln= VCP#histo). These mutations correspond to 19 missense substitutions, 2 nonsense substitutions, 6 synonymous substitutions and 1 frameshift deletion. Although these mutations were not

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apparently located at hot-spots, it appears that 7 mutations (including the frameshift deletion, 5 missense mutations and 1 synonymous mutation) concentrate in the WalkerB domain, thereby suggesting alterations in the catalytic domain of p97/CDC-48. The functional relevance of these mutations in cancer remains to be determined. 4.1. P97/CDC-48 and canonical cancer pathways 4.1.1. NFjB P97/CDC-48 and/or adaptors have been involved in the direct regulation of some key cancer-relevant proteins such as IjBa, an inhibitor of pro-survival function of Nuclear Factor kappa B (NFjB). Activation of the NFjB pathway is commonly observed during tumorigenesis [34]. NFjB is a transcription factor that upon stimulation translocates into the nucleus and triggers the expression of genes that promote cell proliferation thus protecting cells against apoptosis [5]. P97/CDC-48 has been proposed to be critically involved in the dissociation and proteasomal degradation of the inhibitor of NFjB: IjBa [2,15]. Therefore, increased levels of p97/ CDC-48 may result in decreased levels of IjBa and consequently in hyperactive NFjB signaling promoting cell proliferation. In fact, p97/CDC-48 has been identified to be specifically upregulated in the highly metastatic murine osteosarcoma cell line (LM8) [2] as compared to the control cell line [2]. Overexpressing p97/CDC-48 in control cells mimics the increased metastasis observed in LM8 cells [2]. P97/CDC-48 overexpression correlated with constantly activated NFjB, suggest that high levels of p97/CDC-48 may indeed promote cell proliferation and cell survival through hyperactive NFjB signaling [2]. The effects of p97/CDC-48 as a regulator of NFkB mediated tumor metastasis [2] has been demonstrated to be mediated by its ubiquitin–proteasome system (UPS) function. Recently, it has been demonstrated a role of p97/CDC-48 in controlling the protein levels of critical metastatic regulator-NFjB and tumor suppressor-p53 in NSCLC [79]. Valle and colleagues have suggested that p97/CDC-48 regulates NSCLC tumor- genesis and metastasis via NFjB and p53 by a UPS-mediated mechanism [79] that need to be further characterized. Moreover a link between p97/CDC-48 function and the alteration of the NFkB pathway coupled to an exacerbated inflammatory response has been reported by Custer and colleagues [14]. In this context, mice expressing mutants p97 (R155H or A232E), which develop a Paget disease of bone and fronto-temporal dementia (IBMPFD)-like pathology, display inappropriate activation of the NFkB signaling cascade. Based on this observation and on the report of somatic mutations found in the p97/CDC-48 gene in human cancers, one could postulate that similar alterations of the NFkB signaling cascade could occur in cancers and lead to inflammatory responses promoting cancer development. 4.1.2. Akt Klein and colleagues [37] have demonstrated that p97/CDC-48 is phosphorylated by the anti-apoptotic affinity-regulating kinase (Akt). Akt is another important mediator of cell survival and cell proliferation [47]. It promotes cell survival by indirectly activating NFjB signaling [34]. P97/CDC-48 has been shown to be a target of Akt signaling in MCF-7 breast cancer cells [80]. Indeed, p97/CDC48 interacts and co-localizes with Akt upon Akt activation [80]. Three serines in p97/CDC-48 were phosphorylated by Akt upon stimulation [80]. Replacement of these serines with alanines resulted in a p97/CDC-48 triple mutant (S351/745/747A) that upon expression in breast cancer cells markedly decreased the Akt-mediated pro-survival effect of fibroblast growth factor 2 (FGF2) and strikingly inhibited the activation of NFjB by FGF2 in breast cancer cells [80]. Therefore, increased levels of p97/CDC-48 promote cell

proliferation in providing a switch between the pro-survival pathways mediated by Akt and NFjB. 4.1.3. HIF1a One another promising adaptor, based on recent functional studies, UBXD7, should be looked at for potential altered expression in malignant cells. UBXD7 facilitates the degradation of HIF1a, an important promoter of angiogenesis, tumor growth, and metastases [1]. Also, HIF1a overexpression is also a frequent occurrence in many different solid tumor types [70]. Alexandru and colleagues have shown that HIF1a is a substrate of p97/CDC-48 and that UBXD7 mediates its interaction with VCP. 4.1.4. DNA damage BRCA1, a breast/ovarian cancer susceptibility gene, undergoes mutations in as many as 50% of familial breast tumors and is involved in DNA damage repair. P97/CDC-48 physically associates with the BRCA1 protein [102]. In vitro studies revealed that p97/ CDC-48, via its N-terminal region, binds to amino acid residues 303–625 in the BRCA1 protein [102]. These results suggested that p97/CDC-48, by binding to BRCA1, participates in the DNA damage-repair function as an ATP transporter, possibly facilitating the transcription-coupled repair. 4.2. Control of p97/CDC-48 expression in cancer and prognosis How p97/CDC-48 expression is regulated in cancer cell lines is still an open question. Analysis for transcriptional regulation of p97/CDC-48 gene in mammary carcinoma cell line (MCF7) revealed that two transcription factors bound to the 50 region of the p97/CDC-48 gene for p97/CDC-48 transactivation [62,101]: the pre B-cell transcription factor 1 (PBX1) [62] and the E74-like factor 2 (ELF2) [101]. The knocked-down expression of either PBX1 or ELF2 by small interfering RNA decreased the level of p97/CDC-48 gene expression. These findings indicate that both PBX1 and ELF2 are necessary for p97/CDC-48 transcription. Chromatin immunoprecipitation (ChIP) assay revealed that PBX1 binds to the two motifs located in a highly conserved region of the p97/ CDC-48 promoter. The mutation at these motifs significantly decreased the promoter activity, indicating that PBX1 transactivates p97/CDC-48 promoter through these two motifs [62]. In the highly conserved region, one ELF2 binding motif was present. The binding of ELF2 to this motif has been shown by ChIP assay [101]. Since knock-down of these factors reduced both p97/CDC-48 levels and cell viability thereby increasing the susceptibility of the cells to undergo apoptosis, these factors may play an important role in the regulation of p97/CDC-48-mediated cell survival [62,101]. PBX2 has been identified as a highly related homolog to PBX1 [51]. PBX1 and PBX2 are important members of the PBX family, which function as a transcription factor in cooperation with Homeobox (Hox) proteins to regulate proliferation and differentiation of normal and cancer cells [32,56]. The roles of PBX1 and PBX2, on expression levels of p97/CDC-48 have been examined in NSCLC cell lines [61]. The PBX2 knocked down, but not PBX1, in NSCLC cell lines, decreased P97/CDC-48 mRNA. Thus, the correlation of PBX2 expression with p97/CDC-48 level indicates that PBX2 works as a transcription factor enhancing p97/CDC-48 expression [61]. From clinical studies on immune histochemical samples, only few reports show a link between PBX and p97/CDC-48. Nevertheless a correlation of expression level between PBX1, ELF2 and p97/CDC48 has been reported in breast cancer [62]. In another study by Qiu and colleagues [61], it has also been shown that high levels of PBX2 expression correlate with prognosis of NSCLC, therefore PBX2 could be a target molecule for treatment of NSCLC. Recently, the expression levels of p97/CDC-48 were also regulated by a microRNA (miR-129-5p), whose expression was negatively correlated

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with that of p97/CDC-48 in HCC samples [45]. Experiments carried out in cell lines revealed that down-regulation of miR-129-5p led to increase in p97/CDC-48 expression [45]. 4.3. P97/CDC-48 as an anticancer target? Given the essential nature of p97/CDC-48, altered expression or mutation of p97/CDC-48 is expected to have pathological consequences. Supporting this expectation, an elevated level of p97/ CDC-48 is detected in many cancer cells and is associated with a poor prognosis in carcinoma. Overall, these observations suggest that p97/CDC-48 may be a potential molecular target for the treatment of cancer. Disruption of p97/CDC-48 function, via RNA interference or over expression of ATPase deficient protein in tumor cell lines, has been shown to cause cell death [39,86]. Thus, a p97/CDC-48 inhibitor has the potential as a novel small molecule cancer therapeutic. If a specific p97/CDC-48 inhibitor is available, it will be also useful as another tool for the detailed analysis of p97/CDC-48 function both in vitro and in vivo. Seven p97/CDC-48 inhibitors have been reported so far: (1) Eeyarestatin I [82], (2) 2-Anilino-4-aryl-1,3-thiazole [8], (3) the Syk inhibitor III [11], (4) N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ) [11,13], (5) Sorafenib [100], (6) alkylsulfanyl-1,2,4-triazoles [58] and (7) Xanthohumol [69]. 4.3.1. The ERAD inhibitor Eeyarestatin I Recently, it has been shown that p97/CDC-48 inhibition controls NSCLC proliferation and progression by regulating tumor cell growth, migration, and apoptosis [79]. Valle and colleagues demonstrated the therapeutic potential of targeting p97/CDC-48 by a small-molecule functional inhibitor (EerI, Eeyarestatin I). In both in vitro and in vivo models, p97/CDC-48 inhibition by EerI significantly reduced NSCLC tumor growth [79]. Recent studies have revealed that EerI binds to both p97/CDC-48 and the ER membrane and inhibits p97/CDC-48-dependent protein degradation as well as p97/CDC-48-associated deubiquitinating enzymes [83,84], but not p97/CDC-48 ATPase activity. It has been revealed that p97/ CDC-48-associated deubiquitination is involved in ERAD. Thus, EerI or another potent p97/CDC-48 inhibitor may have added potential as a novel cancer therapeutic. 4.3.2. The 2-anilino-4-aryl-1,3-thiazole compound A high throughput screening (HTS) assay looking for p97/CDC48 inhibitors has shown that 2-anilino-4-aryl-1,3-thiazoles are potent drug-like inhibitors of p97/CDC-48 [8]. The identified compounds show low nanomolar p97/CDC-48 potency, a structure– activity relationships trend, and an activity in cellular assay. It inhibited both p97/CDC-48 ATPase activity and p97/CDC-48-associated protein degradation. This class of compounds represented the first disclosure of small, drug-like p97/CDC-48 inhibitors with potential utility as a cancer therapeutic [8]. 4.3.3. The Syk inhibitor III Syk inhibitor III irreversibly inhibits p97/CDC-48 ATPase activity through its interaction with the Cystein 522 within the D2 ATPase domain of p97/CDC-48 and the ubiquitinated client protein [12]. 4.3.4. The N2,N4-dibenzylquinazoline-2,4-diamine (DBeQ) DBeQ was identified as a selective, potent, reversible, and ATP-competitive p97/CDC-48 inhibitor by screening a library of chemical compounds [11]. DBeQ inhibited ERAD and autophagy, and induced apoptosis by activating several caspases [11]. DBeQ also showed antiproliferative activity on human cancer cells [11]. It has been demonstrated that inhibition of the ubiquitin–protea-

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some system and ER homeostasis may offer attractive therapeutic possibilities for certain types of human cancer. 4.3.5. Sorafenib This multikinase inhibitor is used for the treatment of hepatocellular carcinoma (HCC), Sorafenib treatment prevents p97/CDC48 tyrosine phosphorylation, and that this has been correlated with enhanced membrane association, thereby promoting an unbalance of p97/CDC-48 subcellular distribution. Interestingly, DBeQ, enhances sorafenib-mediated toxicity in cultured cells. Our study showed a novel mechanism for sorafenib-mediated cell death in HCC, which depends on the integrity of the secretory pathway; and we have identified p97/CDC-48 phosphorylation as a potential target for improved sorafenib treatment efficacy in patients [100]. 4.3.6. The alkylsulfanyl-1,2,4-triazoles This compound represents a new class of p97/CDC-48 inhibitors, which was identified through biochemical screening of a collection of more than one million compounds. It inhibits the enzyme through an allosteric mechanism not previously described [58]. Further work will be necessary to evaluate its in vivo efficacy. 4.3.7. Xanthohumol Xanthohumol, a prenylated chalcone present in hops (Humulus lupulus L.) and beer, has been found to modulate autophagy [69]. By using Xanthohumol immobilized beads, p97/CDC-48 was identified as a Xanthohumol-binding protein. This indicated that Xanthohumol inhibited the function of p97/CDC-48, thereby allowing the impairment of autophagosome maturation. These findings not only reveal the molecular mechanism of Xanthohumol-modulated autophagy but may also explain how Xanthohumol exhibits various biological activities. In conclusion, based on the dual therapeutic potential of p97/ CDC-48 inhibition as well as its ability to regulate critical oncogene and tumor suppressor protein levels, it is conceivable that specific inhibitors or stimulators for the chaperone activity of p97/CDC-48 will prove to be chemopreventive and chemotherapeutic agents for cancer diseases. 5. Conclusion and perspectives From the diversity of cellular functions at the level of membrane fusion and traffic as well as in the control of protein homeostasis that can be achieved by p97/CDC-48, it is difficult to anticipate the exact contribution(s) of this protein in oncogenesis. Most of the functions in which p97/CDC-48 has been involved are relevant to cancer development with for instance the control of cell-cycle control, autophagy, proteostasis, endocytic processes or DNA-damage mechanisms. In this context the characterization of p97/CDC-48 activity, the identification of p97/CDC-48 regulatory pathways in select cancers, the characterization of p97/CDC-48 preferential adapters in cancer conditions and the subsequent consequences on p97/CDC-48 functions should provide some indications on the contribution of this protein to specific cancer phenotypes. At first, the characterization of the impact of p97/CDC-48 natural mutations found in cancer on the activity of the protein could represent an interesting approach to not only evaluate the impact of structure modulation on p97/CDC-48 function but also to functionally evaluate the impact that these changes could have on cancer cells phenotypes (such as migration, invasion, proliferation and metastasis). Approaches combining traditional molecular cell biology and more recent technologies such as genome editing [29] could represent powerful tools to address such problematic. The

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second point of interest to better understand the roles of p97/CDC48 in cancer (and perhaps more generally in physiology and pathophysiology) would be to systematically map p97/CDC-48 post-translational modifications. Already, significant information has been gathered in various experimental systems reporting that p97/CDC-48 was subjected to phosphorylation and acetylation and that this regulated its functions [18,52], thus evaluating p97/CDC48 status in cancer relevant models represent an interesting option that could document p97/CDC-48 localization and activity. Moreover experimental approaches that could quantitatively determine the nature of p97/CDC-48 associated adapter (i.e. DUB) proteins in cellular models and in pathological tissues could provide some information on the main role played by this protein in the corresponding pathology (for instance UBDX7 and the regulation of the hypoxia-inducible factor pathway [1]). This could be combined with the evaluation of p97/CDC-48 in pathological tissues (e.g. tumors) using immunological methods and with clinical data for determining p97/CDC-48 functions that could be associated with poor patient outcome. Based on the above mentioned information and the relevance of p97/CDC-48 as a therapeutic target, intensive efforts have been deployed and led to identify specific inhibitors of p97/CDC-48, which have now to be further investigated as anticancer agents with encouraging results in model systems [8,10,100]. The extent of p97/CDC-48 functions will presumably still grow in the coming years, and it is therefore conceivable that the use of p97/CDC-48 inhibitors might also be applied to other pathologies. 6. Conflict of interest

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The authors declare no conflicts of interest. Acknowledgements We apologize to all colleagues whose work could not be cited owing to space limitations. This work was funded by grants from Institut National du Cancer (INCa) to EC and Ligue Nationale Contre le Cancer (Comités de Dordogne et de Gironde) to EC and FD. DF was funded by a fellowship from La Ligue Contre le Cancer (Comité Dordogne) and EM was funded by a post-doctoral fellowship from La fondation ARC pour la recherche contre le cancer.

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