Cell cycle and apoptosis regulator 2 at the interface between DNA damage response and cell physiology

Accepted Manuscript Title: Cell cycle and apoptosis regulator 2 at the interface between DNA damage response and cell physiology Authors: Martina Magni, Giacomo Buscemi, Laura Zannini PII: DOI: Reference:

S1383-5742(18)30007-3 https://doi.org/10.1016/j.mrrev.2018.03.004 MUTREV 8238

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Please cite this article as: Martina Magni, Giacomo Buscemi, Laura Zannini, Cell cycle and apoptosis regulator 2 at the interface between DNA damage response and cell physiology, Mutation Research-Reviews in Mutation Research https://doi.org/10.1016/j.mrrev.2018.03.004 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Cell cycle and apoptosis regulator 2 at the interface between DNA damage response and cell physiology

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Martina Magni1, Giacomo Buscemi2 and Laura Zannini3,*

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Running Title: CCAR2 role in DDR and cell physiology.

Department of Medical Oncology and Hematology, Fondazione IRCCS Istituto Nazionale dei

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Tumori, Via Venezian 1, 20133 Milan, Italy. 2 Department of Biosciences, University of Milan, via

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Celoria 26, 20133 Milan, Italy. 3Istituto di Genetica Molecolare, Consiglio Nazionale delle Ricerche

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(IGM-CNR), via Abbiategrasso 207, 27100 Pavia, Italy.

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* To whom correspondence should be addressed. Tel: +390382546363; Fax: +390382422286;

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ABSTRACT

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Email: [email protected]

Cell cycle and apoptosis regulator 2 (CCAR2 or DBC1) is a human protein recently emerged as a novel and important player of the DNA damage response (DDR). Indeed, upon genotoxic stress,

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CCAR2, phosphorylated by the apical DDR kinases ATM and ATR, increases its binding to the NAD+-dependent histone deacetylase SIRT1 and inhibits its activity. This event promotes the acetylation and activation of p53, a SIRT1 target, and the subsequent induction of p53 dependent apoptosis. In addition, CCAR2 influences DNA repair pathway choice and promotes the chromatin relaxation necessary for the repair of heterochromatic DNA lesions. However, besides DDR, 1

CCAR2 is involved in several other cellular functions. Indeed, through the interaction with transcription factors, nuclear receptors, epigenetic modifiers and RNA polymerase II, CCAR2 regulates transcription and transcript elongation. Moreover, promoting Rev-erbα protein stability and repressing BMAL1 and CLOCK expression, it was reported to modulate the circadian rhythm. Through SIRT1 inhibition, CCAR2 is also involved in metabolism control and, suppressing RelB

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and p65 activities in the NFkB pathway, it restricts B cell proliferation and immunoglobulin production. Notably, CCAR2 expression is deregulated in several tumors and, compared to the non-

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neoplastic counterpart, it may be up- or down-regulated. Since its up-regulation in cancer patients is usually associated with poor prognosis and its depletion reduces cancer cell growth in vitro, CCAR2 was suggested to act as a tumor promoter. However, there is also evidence that CCAR2

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functions as a tumor suppressor and therefore its role in cancer formation and progression is still

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unclear. In this review we discuss CCAR2 functions in the DDR and its multiple biological

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activities in unstressed cells.

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Keywords: DNA damage, apoptosis, DNA repair, genomic stability.

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1. INTRODUCTION Tumorigenesis is a multistep process driven by genetic alterations in oncogenes and tumor suppressor genes that induce the progressive transformation of a normal cell into a malignant one [1]. These alterations may be the result of physical DNA damage caused by genotoxic agents (e.g. chemicals, radiations, cigarette smoking, food additives and toxins) or harmful metabolites (e.g.

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free oxygen radicals and lipid peroxidation products), or the consequence of mistakes made during DNA replication prior to cell division. Indeed every day, each cell of the human body is exposed to

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challenges that, with different mechanisms, generate from 50000 to 500000 DNA lesions per day [2]. If these lesions or errors are not repaired, they will become fixed in the genome, possibly leading to chromosomal instability and tumor formation.

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To prevent the replication of damaged DNA, all organisms have evolved an intricate signalling

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network that, in eukaryotes, is called the DNA damage response (DDR) [3]. DDR is a network of

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molecular pathways that detects DNA lesions and, depending on the severity, either repairs them or

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promotes premature and permanent cellular senescence or commits the cell to apoptosis (i.e.

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programmed cell death), thereby restricting the expansion of abnormal cells. Therefore the correct activation of the DDR is important for cancer prevention, as also underlined by the finding, in

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cancer patients, of mutations in genes encoding DDR proteins. However, although DDR defects favour tumor formation and progression, they also constitute a weakness that can be exploited by

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therapy [4], because cancer cells with a reduced ability to repair DNA lesions are more susceptible to DNA-damaging drugs than non-neoplastic cells that can tolerate the treatment.

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The cellular response to DNA damage is orchestrated by several proteins and the major coordinators of these molecular mechanisms are the ATM-CHK2 and ATR-CHK1 signalling. While the ATM-CHK2 pathway is mainly implicated in the response to double strand breaks (DSBs), the ATR-CHK1 signalling cascade is activated by replication stress [5,6]. However, since single-strand DNA (ssDNA) is, in some cases, generated during the first steps of DNA repair, the ATR-Chk1 pathway is indirectly activated also in response to DSBs [7]. In addition, DSBs can also 3

be introduced during the replication of DNA sequences harboring lesions; this event is primarily observed when the leading-strand DNA polymerases come across single-strand nicks or abasic sites [5]. Therefore, cells can frequently activate both the ATM–Chk2 and ATR–Chk1 pathways. Upon DNA damage, ataxia telangiectasia mutated (ATM) and ataxia telangiectasia and Rad3 related (ATR), cooperating with mediator proteins and the transducer kinases, phosphorylate a

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multitude of substrates to promote the proper cellular response to lesions, which can be DNA repair (eventually coupled with cell cycle arrest at checkpoints), senescence, or apoptosis induction [8]. In

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particular, the mediators accumulate at sites of DNA damage to promote ATM and ATR activation [9,10] and recruit proteins to facilitate the repair of the break [11]. Instead, the transducer kinases, checkpoint kinase 2 (CHK2) for ATM [12] and checkpoint kinase 1 (CHK1) for ATR [13],

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propagate the DNA damage signal and intensify or switch the ATM-ATR response by

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phosphorylating effector proteins.

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Importantly, the DDR is also implicated in other physiological cellular functions aimed at the

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preservation, replication and division of the whole genome or at DNA rearrangements, such as

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telomere length maintenance [14], mitochondrial DNA repair [15], viral DNA processing [16], cell cycle regulation [17], circadian clock control [18] stem cell genome stability maintenance [19],

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mitosis and meiosis regulation [17,20] and antibodies production (reviewed in [21]). Given the complexity of these signalling mechanisms, new proteins and molecules regulating the

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DDR at different levels are continuously discovered. Here, we review the DDR functions of cell cycle and apoptosis regulator 2 (CCAR2), a recently identified player of these pathways. Indeed,

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CCAR2 was found to be involved in the DDR by regulating apoptosis induction, repair of DSBs and senescence. Moreover emerging roles for this protein in other crucial cellular events that require the maintenance or restoration of DNA integrity, such as the response to mitochondrial stress, have been described. However, CCAR2 is also involved in other various biological processes, as reported below, and therefore it constitutes an interesting topic for scientist working in different fields of biology. 4

2. CCAR2 DISCOVERY AND STRUCTURE In 2002, Hamaguchi and colleagues [22] discovered a region of chromosome 8 frequently deleted in breast cancer patients. This locus contains four known genes and two previously uncharacterized genes that they named deleted in breast cancer 1 (DBC1) and deleted in breast cancer 2 (DBC2). However, that same study showed that, differently from DBC2, most breast tumors retained DBC1

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expression and further research confirmed its expression in different types of cancers. Therefore, this protein, in consideration of its discovered functions and homologies, has recently been

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officially renamed cell cycle and apoptosis regulator 2 or CCAR2.

Human CCAR2 is a nuclear protein highly conserved in eukaryotes. A paralog of CCAR2, named cell division cycle and apoptosis regulator protein 1 (CCAR1 or CARP-1) is present in vertebrates

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[23] and was described as a pro-apoptotic protein and ATM substrate [24,25]

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CCAR2 is a single polypepotide of 923 a.a. organized in several functional domains (Figure 1); at

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the N-terminal region there are a S1-like RNA-binding domain (a.a. 55-112), important for the

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interaction with RNA molecules, a nuclear localization sequence (a.a. 202-219), which determines

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its subcellular compartmentalization, and a Leucine Zipper domain (a.a. 243-264), involved in the interaction with other proteins. Furthemore, a catalytically inactive Nudix hydrolase domain,

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involved in NAD binding, was identified among a.a. 339-462. At the C-terminus, an inactive EFhand domain (a.a. 704-748), usually implicated in Ca2+ binding, and a coiled-coil domain (a.a. 794-

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918), with a role in protein-protein interactions and apoptosis regulation were also reported [23]. Through these functional domains, CCAR2 participates to many cellular processes such as

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apoptosis, DNA repair, senescence, transcription, metabolism, circadian cycle, epigenetic regulation and B cell development (Figure 2).

3. MULTIPLE FUNCTIONS OF CCAR2 IN APOPTOSIS REGULATION The major role of CCAR2 in the DDR seems to exactly be the regulation of apoptosis, a mechanism of programmed cell death characterized by blebbing, cell shrinkage, chromatin condensation, global 5

mRNA decay and nuclear and chromosomal DNA fragmentation, that cells activate when DNA lesions are irreparable [26]. Essentially, two apoptotic pathways exist, called extrinsic and intrinsic, and both of them can be activated in response to DNA damage [27] and regulated by CCAR2.

3.1 Apoptotic pathways

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In the extrinsic apoptotic pathway, extracellular ligands, such as tumor necrosis factor α (TNFα) and FAS ligand (FASL) associate with death receptors of TNF and FAS families, which then

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trimerize and recruit adapter molecules, such as FADD (FAS-associated death domain protein) and TRADD (TNFR1-associated death domain protein). This interaction allows the recruitment of procaspase 8 and leads to the formation of the death-inducing signalling complex (DISC) and to the

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autocatalytic activation of caspase 8 [26]. Caspase 8 is the initiator of a cascade of caspases that

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promotes the cleavage of several proteins in the cell, followed by cell disassembly, cell death, and,

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ultimately, phagocytosis and removal of the cell debris [28]. Activation of this pathway in response

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to DNA lesions is mainly due to c-Jun N-terminal kinase (JNK) and the transcription factor p53,

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which, respectively, induce the activation of FAS ligand (FASL) and FAS receptor (FASR) [27]. Differently, the intrinsic apoptotic pathway is especially activated by DNA damage, but also by

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other cell stresses like viral infection or cytokines deprivation. These stimuli produce intracellular signals that propagate trough mitochondria. Particularly, in response to a diffuse DNA damage,

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ATM and ATR promote p53 activation upon DNA lesions and active p53 then favours the transcription of PUMA, NOXA and BAX, which are pro-apoptotic proteins of the Bcl-2 family

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[27]. These proteins, interacting with the external membrane of the mitochondria, induce the release of cytochrome c from mitochondria and the activation of APAF1 (apoptotic protease activating factor 1). Activated APAF1 then oligomerizes into the apoptosome, a protein complex which interacts with and activates the initiator pro-caspases 9 that starts the caspases cascade and the apoptotic programme [26].

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3.2 The tumor suppressor protein p53 The tumor suppressor p53 is a transcription factor that plays important roles in both the intrinsic and extrinsic apoptotic pathways. This protein is mainly involved in the maintenance of genomic stability and its loss or mutation is responsible for tumor susceptibility [29]. In unstressed cells, the E3 ubiquitin ligase MDM2 maintains p53 at low levels promoting p53 poly-ubiquitination and

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proteasomal degradation [30]. Upon DNA damage, p53 is phosphorylated by the kinases ATM, ATR, CHK1 and CHK2 on several serine and threonine residues, thereby disrupting p53-MDM2

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interaction and stabilizing p53 [31]. Phosphorylated p53 then stimulates the recruitment of specific acetyltransferases, that acetylate p53 on multiple and specific residues finally leading to its transcriptional activation [31]. These acetylated residues are also commonly targeted for

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ubiquitination; therefore, since acetylation and ubiquitination on the same residue are mutually

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exclusive, this modification contributes also to p53 stabilization. Once fully activated, p53, driven

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by post-traslational modifications which direct it to determined promoters, induces the transcription

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of specific genes, thus leading to the proper cellular outcomes, like cell cycle arrest, senescence or

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apoptosis [27].

Given its importance in p53 activation, the acetylation of p53 is a finely tuned process, negatively

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regulated by histone deacetylases (HDAC) complexes of class I and III. The first p53-deacetylase identified is the class III NAD-dependent deacetylase sirtuin-1 (SIRT1), [32] which specifically

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deacetylates p53-K382, resulting in the reduction of apoptosis after DNA damage [33][34]; for this, SIRT1 activity towards p53 needs to be strictly controlled and, as better detailed below, the major

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biological inhibitor of SIRT1 discovered up to now is CCAR2 [33-36].

3.3 CCAR2 role in apoptotic pathways Initially, it was demonstrated that CCAR2 plays a pro-apoptotic function during TNFα-mediated apoptosis; in particular caspases process CCAR2 to generate p120 CCAR2 and p66 CCAR2 both containing the c-terminal region and the coiled-coil domain. The truncated forms of this protein are 7

then relocalized to the cytoplasm and their ectopic expression was found to induce mitochondrial clustering and mitochondrial matrix condensation, therefore sensitizing cells to TNFα-mediated apoptosis (Figure 3A) [37]. In 2008, two independent groups discovered that CCAR2 is also involved in the intrinsic apoptotic pathway. In particular they demonstrated that, upon genotoxic stress, CCAR2 increases its

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association with the deacetylase SIRT1 and this event leads to inhibition of SIRT1 activity, finally resulting in p53 acetylation, activation and induction of p53-dependent apoptosis (Figure 3B)

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[33,34].

The mechanism involved in SIRT1 inhibition by CCAR2 has been extensively studied; it was reported that CCAR2 directly binds the deacetylase domain of SIRT1, disrupting its association

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with substrates [33,34]. Moreover it was also demonstrated that the C-terminus ESA (which means

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essential for SIRT1 activity) region of SIRT1, interacting with the deacetylase core, switches-on the

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catalytic activity of SIRT1. Thus, CCAR2, competing with the ESA domain, indirectly prevents

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SIRT1-dependent deacetylation of p53 [38]. The catalytic amino acid His363 and the NAD-binding

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residue Asn346 of SIRT1 have been also reported to be essential for SIRT1 binding to CCAR2 [39]. Moreover, CCAR2 sequence displays a catalytically inactive Nudix hydrolase domain, known

co-factor [23].

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to bind NAD+; therefore, CCAR2 may also modulate SIRT1 activity by sequestering its essential

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Some years after the discovery of CCAR2 as the main biological inhibitor of SIRT1, we and others reported that upon DNA damage CCAR2 receives from the apical kinases ATM and ATR the signal

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for the inhibition of SIRT1, via the phosphorylation of Thr454 [35,36]. This residue is then dephosphorylated by the phosphatase PP4, an essential event to fine tune the DNA damage signal [40]. Moreover, kinase suppressor of ras-1 (KSR1), a scaffolding protein in the mitogen activated protein kinase (MAPK) pathway, was found to indirectly reduce Thr454 phosphorylation upon etoposide exposure and to affect CCAR2-SIRT1 association (Figure 3B) [41].

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Subsequent studies by other groups further confirmed that the association between CCAR2 and SIRT1 is a strictly regulated event. In fact, alongside phosphorylation, other post-translational modifications have been found to finely regulate CCAR2 function. For instance, CCAR2 is acetylated by the histone acetyltransferase hMOF on Lys112 and Lys215; this event prevents CCAR2-SIRT1 interaction (Figure 3B). For this, hMOF activity towards CCAR2 is inhibited upon

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DNA damage in an ATM-dependent manner, to favour p53 activation [42]. Other acetylation sites on the N-terminal domain of CCAR2 and important for SIRT1 binding have been reported [39].

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Interestingly, these proteins seem to be involved in a feedback loop since at least three lysines of CCAR2 (Lys138, Lys162 and Lys215) are deacetylated by SIRT1 [39]. In addition CCAR2 sumoylation by SUMO 2/3 upon DNA damage promotes CCAR2 binding to SIRT1 and p53

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activation (Figure 3B) [43].

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Besides post translational modifications, also the association of CCAR2 with other proteins or

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cellular factors was demonstrated to be important for SIRT1 activity regulation.

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In fact, in addition to ATM and ATR, also the transducer kinase CHK2 is required for the CCAR2-

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dependent inhibition of SIRT1; indeed CHK2, phosphorylating the proteasome subunit REGγ promotes CCAR2-REGγ association which in turn induces CCAR2 binding to SIRT1 (Figure 3B)

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[44]. Conversely, CCAR2-SIRT1 complex formation is negatively regulated by breast cancer metastasis suppressor 1 (BRMS1) protein and by the long non coding RNA metastasis-associated

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lung adenocarcinoma transcript 1 (MALAT1) (Figure 3B). In unstressed cells, BRMS1 is bound to CCAR2 and interferes with its association to SIRT1. Upon etoposide treatment CCAR2 is released

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from BRMS1 and inhibits SIRT1 preventing p53 deacetylation [45]. Similarly, MALAT1, by directly interacting with the N-terminal region of CCAR2, restricts CCAR2-SIRT1 association and stimulates SIRT1-dependent p53 deacetylation and apoptosis inhibition [46]. However, CCAR2 can also regulate cell death through SIRT1-independent mechanisms. In fact, it has recently been reported that CCAR2 participates to p53 stabilization also through the competition with MDM2. Basically, CCAR2 prevents the proteasome mediated degradation of p53 9

by competing with MDM2 for the direct binding to the N-terminus and the DNA binding domain of p53 (Figure 3C) [47]. However, while CCAR2 mainly promotes apoptosis in response to different cellular stresses, unexpectedly, some evidence seems to suggest that in specific conditions this protein can also negatively regulate cell suicide. Indeed, in UV treated breast cancer cells, where the association

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between CCAR2 and SIRT1 is barely detectable, CCAR2 deficiency resulted in impaired activation of cell cycle checkpoints and in apoptosis induction [48]. Similarly it was also demonstrated that, in

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non small cell lung cancer cell lines, CCAR2 protects cells from genotoxic stress upon melatonin treatment in a SIRT1-independent manner [49].

In addition it was recently discovered that a fraction of CCAR2 interacts with the mitochondrial

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chaperone heat shock protein 60 (Hsp60). This complex resides in the mitochondria [50] where it

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protects cells from apoptosis induced by treatment with rotenone, an inhibitor of mitochondrial

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respiratory chain complex I which causes oxidative stress and damage to mitochondrial DNA [50].

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Finally, it was also demonstrated that CCAR2 is able to suppress anoikis, a particular form of

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apoptosis characterized by cell detachment from the extracellular matrix, promoting the

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transcription of anti-apoptotic NF-κB target genes [51,52].

4. DNA REPAIR REGULATION BY CCAR2

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Among DNA lesions, DSBs are the most dangerous, because they affect both the DNA strands and, if not rapidly repaired they can lead to aneuploidy, genome rearrangements and cell death. For this

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reason, eukaryotic cells have evolved a variety of molecular mechanisms to repair DNA DSBs. These mechanisms can be essentially divided into two categories: non homologous end joining (NHEJ) and homologous recombination (HR) [8]. Basically, NHEJ, after an endonucleases mediated processing of the broken DNA, promotes the direct ligation of ligatable DNA ends; contrarily, HR needs a homologous DNA strand for the synthesis of the nucleotides that are lost during the initial resection of broken DNA ends, performed 10

by CtIP. During this step, ssDNA ends 3’overhang, important for the subsequent search and invasion of the homologous double helix, are generated. These ssDNA ends are then extended by a DNA polymerase which copies the homologous sequence. Since NHEJ does not require a homologous template, it is active throughout all the cell cycle, while HR is more specific for S and G2 phases, when a replicated sister chromatid is available [53]. HR is considered an error free

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mechanism of DNA repair, since it "copies" DNA information from homologous sequences; in contrast, NHEJ, allowing small insertions or deletions, is an error prone mechanism [53].

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Up to now, the role of CCAR2 in DNA repair has not been extensively studied; a first paper reported that silencing of CCAR2 affects the normal efficiency of HR, suggesting that it is required for this repair pathway [54]; however, more recently another group reported that CCAR2 promotes

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NHEJ over HR. In particular they show that CCAR2 is recruited at DNA damage sites and interacts

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with CtIP, antagonizing its activity and limiting DNA ends resection [55].

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It is now well established that also the chromatin environment in the surroundings of a DSB

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significantly influences DNA repair [56]; in particular, DNA lesions that occur in heterochromatin

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are repaired with a slower kinetics compared to those occurring in active euchromatin [57], because their repair is more complex and requires additional steps of chromatin remodelling and relaxation

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[57,58]. To date, several proteins have been described to play a role in the regulation of chromatin dynamics in response to DNA lesions. Among them, the transcriptional corepressor KRAB domain-

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associated protein 1 (KAP1) allows the formation of transcriptionally inactive heterochromatin through the recruitment of nucleosome remodelers, histone deacetylases and methyltransferases.

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However, in response to DNA lesions, KAP1 is phosphorylated by ATM on Ser824 [59] and by CHK2 on Ser473 [60,61], by that favouring chromatin relaxation and heterochromatic DNA repair. Recently, CCAR2 was found to be involved in these pathways. Indeed, in response to DNA lesions, CCAR2, promoting the full activation of CHK2 and the subsequent phosphorylation of KAP1, favours heterochromatin relaxation and the repair of heterochromatic DSBs [62].

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Finally another role for CCAR2 in DNA repair was recently proposed [63]. In fact, it was demonstrated that, besides SIRT1, CCAR2 also binds to and inhibits poly (ADP) ribose polymerase (PARP1) [63], a protein with a critical role in various DNA repair pathways [64]. This complex formation is negatively regulated by NAD+, an essential co-factor for the activities of PARP1 and sirtuins, which binds the Nudix hydrolase domain present in the central region of CCAR2.

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Therefore, as the cellular concentration of NAD+ declines with aging, the association of CCAR2 with PARP1 increases, lastly leading to defective DNA repair and accumulation of DNA damage

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[63].

5. CCAR2 AND CELLULAR SENESCENCE

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Another outcome of DDR is the induction of cellular senescence, which is characterized by the

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promotion of a permanent cell cycle arrest and by the cellular acquisition of senescence features,

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like flattening and vacuolization [65]. This phenomenon is involved in the creation of a barrier

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against the replication of cells with DNA damage or activated oncogenes, but also in organism

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aging. Recently, also obesity was associated with the increase of cellular senescence which finally leads to inflammation through the induction of senescent associated secretory phenotype (SASP)

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[66]. Given CCAR2 role in the regulation of SIRT1 and HDAC3 (see below), both involved in the regulation of these pathways, it is not surprising that this protein could have a role also in the

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control of senescence. Indeed it was demonstrated that during cellular stress induced by obesity CCAR2 plays an important role in the onset of cellular senescence and inflammation. Accordingly,

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CCAR2-KO mice, fed with high fat diet, display less features of cellular senescence than controls in their adipose tissue, consistently with the finding of reduced levels of senescent markers in preadipocytes [67] and reduced expression of inflammatory genes in the early stages of adipogenesis [68]. Moreover, that same study, demonstrated that CCAR2 is involved also in DNA damage induced cellular senescence. In fact H2O2 treatment of 3T3-L1 preadipocytes led to the rapid increase of CCAR2 binding to and inhibition of HDAC3, which finally result in the induction 12

of p16ink4a and p21waf1 expression, two essential upstream elements of the senescence programme [67].

6. CCAR2 FUNCTIONS BEYOND THE DDR Besides DDR, CCAR2 is involved in the regulation of a variety of other cellular processes; these

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CCAR2 functions may be or not be mediated by its negative regulation of SIRT1 and confirm for this protein a multifunctional role in cellular physiology. The main CCAR2 functions beyond DDR

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are briefly summarized below.

6.1 Chromatin remodelling and epigenetic modifications

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As previously described, CCAR2 inhibits the activity of SIRT1, an enzyme that deacetylates both

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histone and non histone proteins [32,69]. Moreover, CCAR2 binds and inhibits the enzymatic

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activity of the histone methyltransferase SUV39H1 and the histone deacetylase HDAC3 [70,71];

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interestingly the product of KLLN gene, killin, interacts with CCAR2 and prevents the inhibition of

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SUV39H1 leading to the increase of histone H3-K9 methylation [72]. Thus, via the modulation of these chromatin remodelers, CCAR2 has been proposed to influence chromatin status and

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epigenetic modifications.

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6.2 Transcription regulation and modulation of nuclear receptors' functions CCAR2 was described to form a complex with the RNA polymerase II and the protein ZNF326.

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The generated complex, named DBIRD, regulates transcript elongation rates and alternative splicing of a set of exons located in A-T rich DNA [73]. However, it is not yet clear whether the RNA-binding domain of CCAR2 plays a role in this function. In addition CCAR2 regulates the activity of several transcription factors, such as TFII-I, PEA3, PROX1 and NFkB, and was demonstrated to have a role in transcription complex assembly and RNA polymerase II recruitment to target enhancers [54,74-76]. 13

CCAR2 also interacts with and regulates the stability and function of several nuclear receptors. Indeed it was found to enhance the transcriptional activity of androgen receptor (AR) and androgen receptor-variant 7 (AR-V7) [77,78] and to promote suppression of apoptosis through the ligandindependent interaction with estrogen receptor α (ERα, the predominant isoform of the receptor expressed in breast cancer cells) [79]. Moreover, CCAR2 is required for transcriptional functions of

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retinoic acids receptor (RAR, [80]) and it binds and inhibits estrogen receptor β (ERβ), the receptor that protects from the proliferative effects of estrogen in breast tissue, with a ligand-independent

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interaction, [81]. Finally, CCAR2 associates with and inhibits, in a ligand-independent manner, the liver X receptor α (LXRα), which is involved in lipid metabolism [82].

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6.3 Circadian clock modulation

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Recent data suggest that CCAR2 may be a modulator of circadian rhythm, an intracellular

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oscillatory system which programs daily rhythms and regulates metabolic pathways. Indeed it was

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found to interact with and promote the protein stability and repressive function of the nuclear

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receptor Rev-erbα, which is a major coordinator of circadian clock, capable to also integrate circadian rhythm and metabolism [83]. Through Rev-erbα, CCAR2 also represses the expression of

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the transcriptional activator BMAL1 [83]. Moreover, proteomic studies in T cells further confirmed a regulatory role for CCAR2 in the circadian clock, through its interaction with several proteins of

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the PERIOD (PER) complex which represses the expression of circadian gene targets [84]. These studies also reveal that CCAR2 protein abundance is important to temporally control the expression

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of CLOCK and BMAL1 transcription factors [84]. Indeed ectopic expression of CCAR2 caused respectively the loss and the slowdown of CLOCK and BMAL1 oscillations. These results suggest that the CCAR2 mediated regulation of circadian rhythm may be exerted at different levels, also in consideration of its interaction with the DBIRD complex which also comprises PER proteins.

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6.4 Control of cellular metabolism SIRT1 plays important roles in cellular metabolism, like the regulation of lipids, glucose and insulin metabolism [85]. Thus, repressing SIRT1, also CCAR2 is involved in metabolic mechanisms. For instance, in mice CCAR2-SIRT1 interaction is strongly increased during high-fat diet (HFD) and almost absent during starvation; this fact well explains the changes of SIRT1 activity observed

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during different metabolic conditions [86]. In addition, CCAR2-/- mice have high levels of hepatic SIRT1 and are protected against the development of liver steatosis and inflammation [86]. More

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recently, it has been reported that CCAR2 is involved in the development of fat tissue dysfunctions and metabolic diseases during caloric surplus; in particular, during HFD CCAR2-/- mice become more obese than WT mice but are characterized by insulin sensitivity, develop atherosclerosis less

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frequently and live longer [87]. In addition CCAR2 depletion was associated with enhanced

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adipocyte differentiation and increased intracellular accumulation of lipids [68]. Moreover CCAR2

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promotes brown remodelling of white adipose tissue through the SIRT1 dependent deacetylation of

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peroxisome proliferator activator receptor (PPAR) γ [88]. Interestingly, metabolic abnormalities of

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CCAR2-/- mice seem to be due to the SIRT1-dependent hyperactivation of stearoyl-coenzyme A desaturase 1 (SCD1), which finally leads to accumulation of unsaturated fatty acids in both mice

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plasma and tissues [89].

Of note, CCAR2 is also implicated in the regulation of glucose metabolism. Indeed by modulating

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Rev-erbα and SIRT1 activities, it regulates the expression of phosphoenolpyruvate carboxykinase

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(PEPCK) which has a critical role in gluoconeogenesis control [90].

6.5 Regulation of B cell development

To guarantee an optimal immune response against pathogens, the activation of B cells and immunoglobulin (Ig) production are required. Deregulation of these processes may lead to increased Ig accumulation and augmented susceptibility to autoimmune diseases [91]. An essential signalling mechanism implicated in B cell activation is the alternative NFkB pathway which uses 15

RelB and p65 dimers as transcription factor. Recently CCAR2 was reported to play a role also in this signalling mechanism. Indeed CCAR2 interacts with and suppresses RelB and p65 activities therefore restricting B cell proliferation and Ig production [76,92]. In addition it was demonstrated that CCAR2 is phosphorylated at the C-terminus by the NFkB regulator IKKα and that this modification promotes its binding to and inhibition of RelB [92]. In accordance with these findings,

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diseases, such as experimental induced myasthenia gravis [76,92].

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CCAR2 -/- mice show increased antibodies production and increased susceptibility to autoimmune

7. CCAR2 AND CANCER

The name initially assigned to CCAR2 [22] generated a lot of confusion about its role in cancer.

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Indeed studies following its discovery clarified that CCAR2 is not deleted in breast cancer; on the

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contrary it is frequently reported to be up-regulated in this type of tumor [74,93,94]. Up to now,

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several studies highlighted a deregulation of CCAR2 expression in cancer tissues (Table 1), but,

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since it can be both up-regulated or down-regulated, the role of this protein in tumor formation and

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cancer progression is still controversial. Usually upregulation of CCAR2 expression in cancer patients correlates with poor prognosis and distant metastatic relapse [93,95]; nonetheless it was

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demonstrated by several groups that loss of CCAR2 specifically prevents the growth of cancer cells [75,96] and this phenomenon seems to be mediated by the inactivation of AKT pathway caused by

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CCAR2 absence [97]. However, also a role for CCAR2 in the regulation of Wnt/β-catenin pathway [75,98], which promotes colorectal cancer progression, and in the stabilization of androgen receptor

A

[96], which is involved in osteosarcoma development, has been described. These studies therefore indicated that CCAR2 is a tumor promoter and this hypothesis is further corroborated by the finding that CCAR2 inhibits the transcriptional function of the tumor suppressor breast cancer associated 1 (BRCA1) [99]. However in other cases high CCAR2 expression correlates with a more favourable outcome [100] and, in a recent study performed in mice, CCAR2 deletion was reported to be tumor

16

prone in a non oncogenic background [47]. These data, although disputed [89], suggest that CCAR2 is a tumor suppressor. Moreover, also the role of CCAR2 in the regulation of the DDR and DNA repair seems to support a tumor suppressor function for this protein. However, as reported above, CCAR2, on one hand,

hand, is involved in anoikis resistance, a hallmark of cancer [51,52,101].

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promotes p53 dependent apoptosis of cancer cells via SIRT1 inhibition [33,34], while, on the other

Most importantly, also its relation with SIRT1 is multifaceted and complicates the comprehension

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of CCAR2 role in cancer progression. Indeed the function of SIRT1 in tumorigenesis is still elusive, since this protein acts as a tumor promoter or suppressor in a cell-specific manner [102]. However, it was demonstrated that the correlation between CCAR2 and SIRT1 expression is lost in breast

U

cancer specimens [103], that their interaction is disrupted in breast cancer cells [104] and that

N

CCAR2 does not inhibit SIRT1 function in liver cancer [105].

A

Moreover, CCAR2 functions in the activation of the pro-tumorigenic ERα [79,106], AR [96] and

M

AR-V7 [78] and in the inhibition of anti-tumorigenic ERβ [81] are other contradictory evidences

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about its role in cancer.

Finally, since obesity is associated with cancer [107], the role of CCAR2 in fatty acids

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accumulation could also contribute to explain its function in tumorigenesis, further providing a

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potential mechanism for the increase of cancer incidence in metabolic diseases [89].

8. CONCLUSIONS AND PERSPECTIVES

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More than 15 years after the discovery of CCAR2, many efforts have been made with the purpose to understand the role of this protein in normal cell physiology and cancer progression. It is now well established that CCAR2 is a new player of DDR pathways with multiple functions in these signalling mechanisms. On the contrary its role in tumorigenesis and cancer progression is still controversial. However, although much has been understood about CCAR2 function, much remains

17

to be disclosed with the purpose to reconcile its different activities and to finally understand if it can be considered a tumor suppressor or promoter. In the next years, new molecular approaches and clinical analyses will provide the necessary tools for the comprehension of the global and cell specific CCAR2 functions and to determine if CCAR2 can be used as target for cancer therapy or as prognostic factor for cancer patients. In case of

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positive results, these studies will hopefully allow the design of the best strategies to use CCAR2 activity modulators for clinical purposes and the development of compounds modulating CCAR2

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interactions that could be relevant for cancer therapy. Moreover, in consideration of recent advances, we think that these results will be useful also to extend our knowledge of metabolic

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also be a target for the therapy of these kind of pathologies.

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diseases and of the role played by CCAR2 in this context, therefore establishing if this protein could

M

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CONFLICT OF INTEREST

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ACKNOWLEDGMENTS

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The authors declare no conflict of interests

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M. Magni is a recipient of Umberto Veronesi Foundation fellowship.

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FIGURE LEGENDS Figure 1: CCAR2 PROTEIN PRIMARY STRUCTURE. CCAR2 protein domains and their functions are illustrated in the picture. NLS, nuclear localization signal; Leu Zipper, leucine zipper; NUDIX, nucleoside diphosphate linked to X; EF hand, E and F alpha elices of parvalbumin.

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Figure 2: THE MANY CELLULAR FUNCTIONS OF CCAR2 PROTEIN. As better detailed in the text, CCAR2 is involved in many cellular functions. Indeed CCAR2 can positively and

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negatively regulate apoptosis, DNA repair and transcription; it promotes chromatin remodelling and senescence, represses B cell development and circadian clock induced oscillations and regulates

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metabolism.

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Figure 3: CCAR2 ROLES IN CELL DEATH REGULATION. A) In response to TNFα,

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CCAR2 is cleaved by caspases in p120 CCAR2 and p66 CCAR2. Truncated proteins relocalized to the cytoplasm where they promote mitochondrial clustering and apoptotic cell death. B) Upon DNA

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damage CCAR2 inhibits SIRT1, promoting p53 acetylation and apoptosis induction. These events

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are positively regulated by the ATM and ATR-dependent phosphorylation of CCAR2-Thr454, by the sumoylation of CCAR2-Lys 591 and by the association of CCAR2 with the proteasome subunit

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REGγ phosphorylated by CHK2. Conversely, SIRT1 inhibition by CCAR2 is counteracted by the phosphatase PP4 and the kinase KSR1, that reduce CCAR2-Thr454 phosphorylation, by the hMOFdependent acetylation of CCAR2 Lys112 and Lys215 and by BRMS1 and the long non coding

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RNA MALAT1 which interfere with SIRT1-CCAR2 association. C) CCAR2, competing with the Mdm2 mediated ubiquitination and degradation of p53, favours p53 stabilization.

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TABLE Table 1. Changes in CCAR2 expression detected in various types of cancer.

[96] [52,108-110]

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[111] [74,93,94] [112] [113] [114]

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References

[115] [75] [105] [116] [116] [117] [95]

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Up-regulated Up-regulated Down-regulated Up-regulated Down-regulated Down-regulated Up-regulated

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Ovarian cancer Breast cancer Soft tissue sarcoma Clear cell renal cell carcinoma Laryngeal and hypopharyngeal carcinoma Diffuse large B cell lymphoma Colorectal cancer Liver cancer Lung adenocarcinoma Lung squamous cell carcinoma Pancreatic cancer Esophageal squamous cell carcinoma Gallbladder cancer Hepatocellular carcinoma Squamous cell carcinoma

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Osteosarcoma Gastric cancer

DBC1 expression (Relative to nonneoplastic tissue) Up-regulated Up-regulated Down-regulated Up-regulated Up-regulated Up-regulated Up-regulated Down-regulated

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Cancer Type

[100] [118,119] [120]

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Up-regulated Up-regulated Up-regulated

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