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A20 expressing TuMors And AnTicAncer drug resisTAnce

cleide Gonçalves da Silva,1 Darlan conterno Minussi,1 christiane ferran1 and Markus Bredel*,2

1Division of Vascular and Endovascular Surgery, Center for Vascular Biology Research and the Transplant Institute, Department of Surgery, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, Massachusetts, USA; 2Department of Radiation Oncology, Comprehensive Cancer Center, University of Alabama at Birmingham, Birmingham, Alabama, USA. Corresponding Author: Markus Bredel—Email: mbredel@uab.edu

Abstract: resistance to anticancer drugs is a major impediment to treating patients with cancer. the molecular mechanisms deciding whether a tumor cell commits to cell death or survives under chemotherapy are complex. Mounting evidence indicates a critical role of cell death and survival pathways in determining the response of human cancers to chemotherapy. Nuclear factor−kB (Nf-kB) is a eukaryotic transcription factor on the crossroad of a cell’s decision to live or die. under physiological conditions, Nf-kB is regulated by a complex network of endogenous pathway modulators. tumor necrosis factor a induced protein 3 (tnfaip3), a gene encoding the a20 protein, is one of the cell’s own inhibitory molecule, which regulates canonical Nf-kB activation by interacting with upstream signaling pathway components. Interestingly, a20 is also itself a Nf-kB dependent gene, that has been shown to also exert cell-type specific anti- or pro-apoptotic functions. recent reports suggest that a20 expression is increased in a number of solid human tumors. this likely contributes to both carcinogenesis and response to chemotherapy. these data uncover the complexities of the mechanisms involved in a20s impact on tumor development and response to treatment, highlighting tumor and drug-type specific outcomes. While a20-targeted therapies may certainly add to the chemotherapeutic armamentarium, better understanding of a20 regulation, molecular targets and function(s) in every single tumor and in response to any given drug is required prior to any clinical implementation. current renewed appreciation of the unique molecular signature of each tumor holds promise for personalized chemotherapeutic regimen hopefully comprising specific a20-targeting agents i.e., both inhibitors and enhancers.

The Multiple Therapeutic Targets of A20, edited by christiane ferran. ©2014 landes Bioscience and Springer Science+Business Media.

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inTroducTion

With the ever-growing number of chemotherapeutic molecules reaching the clinic in the past couple of decades, chemotherapy has assumed an important role in the management of human cancer. It is now common practice to have chemotherapy as part of a routine triad regimen including surgery and radiation therapy. chemotherapy significantly prolongs patients’ survival in various types of cancer, in particular metastatic cancers that otherwise cannot be eradicated. however, for other types of cancer, chemotherapy remains a last resort option rather than an established beneficial component of a multimodality treatment regimen. the cancer field still struggles with a substantial degree of variability in efficacy of some chemotherapeutic agents depending on cancer histological types, and even within the same tumor type on the different subtype. this has been ascribed to the unique molecular signature of the tumor and also to the uniqueness of the environment in which it develops i.e., host environment and genetic makeup.

accordingly, there is growing consensus in the field that chemotherapy failure in distinct cancer subgroups is mostly linked to genetic and epigenetic modifications prompting molecular resistance.1 over the past decades, analysis of drug resistance of human cancers has become a topic of intense research that yielded a better understanding of the molecular mechanisms utilized by cancer cells to escape treatment. this research enhanced chances for discovery of novel and targeted therapies2-4

oF solid TuMors And resisTAnce To cheMoTherApy

In the late 50s, pioneering studies aimed at understanding the mechanism(s) of action and the molecular basis for tumor resistance to methotrexate (MtX) set the stage for tailored chemotherapy regimen based on the molecular making of a given tumor. these studies were first to show that defects in metabolic pathways known to be targeted by MtX explained tumor resistance to this particular drug.5 few years later, in the 1960s, seminal experiments by frank Schabel and howard Skipper6 together with clinical studies performed by holland, frei and freireich7 demonstrated that combination of several drugs, each with a different site of action, was most effective in preventing development or selection of drug-resistant tumor cells. this, then novel, strategy fueled additional research aimed at uncovering the mechanism(s) accounting for tumor drug resistance. It is this understanding of cancers’ evasion mechanisms that prompted development of tailored multi-drug therapeutic strategies aimed at circumventing tumor’s non-responsiveness by simultaneously targeting alternate resistance pathways.

constitutive tumor resistance to chemotherapy can either result from genes normally expressed by the tissue of origin of the tumor or from “de novo” genetic alterations that occurred during carcinogenesis. regardless, such ‘intrinsic” resistance of tumors, based on their genetic make-up, is responsible for primary failure or inadequacy of initial chemotherapeutic regimen (fig. 1). additionally, although early drug treatment can achieve substantial cell killing in certain tumors, it may also select for a clonal variant of tumor cells that resists specific chemotherapy drugs. this process of drug selection could lead, via clonal expansion, to tumor repopulation and recurrence with acquired resistance to subsequent treatment. two principles guide “acquired” tumor resistance phenotype (fig. 1). first, inherent genetic instability of tumor cells can favor the expansion of a low number of resistant cell clones that were present before initiation of therapy. Second, specific drug mediated signals to cancer

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67A20 expressing TuMors And AnTicAncer drug resisTAnce

cells would cause their “de novo” acquisition of a resistance phenotype. Indeed, anticancer agents have proven mutagenic potential that could trigger mutations in genes within key molecular pathways involved in drug response (genetic level). anti-cancer agents can also, through epigenetic-dependent and independent mechanisms, induce the coordinated expression of protective stress response or drug resistance genes, or modify the cellular metabolism of tumor cells, in a way that amplifies tumor resistance to chemotherapy (fig. 1).1,3

Mounting evidence indicates that alteration in cell death and/or survival pathways is key to human cancers’ response to chemotherapy. the molecular mechanisms deciding whether a tumor cell commits to cell death or survives under therapy are both complex and under fine regulation. the transcription factor Nf-kB is one among other important regulators of cancers’ fate. Nf-kB controls the expression of a wide variety of genes that actively participate in controlling cell survival, cell proliferation, angiogenesis and metastasis. constitutive activation of Nf-kB is frequently observed in different types of cancer and has been correlated with tumor development, growth, progression and radio/chemoresistance.8 therefore, targeting Nf-kB signaling is considered a promising strategy to inhibit tumor

Figure 1. relationship between drug effect and drug resistance/sensitivity of human cancers (reprinted with permission from: Bredel M et al. lancet oncol 2004; 5:89–100).

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68 The MulTiple TherApeuTic TArgeTs oF A20

growth and metastasis and to increase efficiency of chemotherapy. In addition to constitutive expression of activated Nf-kB in some cancers, genotoxic stress i.e., DNa damage caused by chemotherapeutic agents, also leads to Nf-kB activation. however, whether activation of Nf-kB in this context results in protection from or sensitization to the genotoxic agent remains controversial and likely varies according to tumor cell type and/or the nature/dose of the chemotherapeutic agent.9 for instance, treatment of gliomas with o6-alkylating agents induces Nf-kB activation and subsequent overexpression of anti-apoptotic genes in tumor cells, promoting their resistance to chemotherapy.10 also, resistance of multiple myeloma to the cytotoxic effects of Melphalan and Doxorubicin is ascribed to these drugs’ ability to activate Nf-kB. consequently, addition of the Nf-kB inhibitor Bortezomib to such chemotherapeutic regimen overcomes tumor resistance.11 In contrast, activation of Nf-kB by the second mitochondria-derived activator of caspases (Smac) mimetic, BV6, is required to prime glioblastomas to temozolomide (tMZ) mediated apoptosis.12

In addition to Nf-kB activation, ubiquitination, a post-translational modification that adds one (mono) or several (poly) lysine-linked ubiquitin chains to proteins, has been implicated in both carcinogenesis and tumor resistance to chemotherapy.13 classically, ubiquitination, performed by the coordinated activation of ubiquitin-activating enzymes (E1), ubiquitin-conjugating enzymes (E2), and ubiquitin-ligases (E3), affects proteins sub-cellular localization and activation status (k6 and k63-linked) or targets them for lysosomal or proteasomal degradation (k11 and k48). this process is reversible as mono/polyubiquitin chains can be removed by a set of deubiquitinating enzymes (DuB).14 Specific modifications of tumors’ “ubiquitome” print have been implicated in the pathogenesis of malignancy per se. Indeed, mutation of some proto-oncogenes, in a way that inhibits their ubiquitination and subsequent degradation, causes them to accumulate in cells, promoting tumor development. as an example, a mutant variant of the guanine nucleotide exchange factor, DBl, lacking the N-terminus domain, no longer binds the E3-ligase chIP, hence escaping ubiquitin-dependent proteasomal degradation.15 consequently, this onco-DBl variant accumulates in cells, causing persistent activation of GtPases and cell transformation.16 alternatively, heightened expression of E3 ligases can cause ubiquitin-dependent degradation of important cell cycle regulators, relieving physiologic brakes and resulting in uncontrolled proliferation, a hallmark of tumorigenesis. for instance, overexpression of the E3 ligase Skp2 in a number of oral, epithelial, colo-rectal, and breast cancers causes excessive ubiquitin-dependent degradation of the cyclin Dependent kinase Inhibitor and potent cell cycle brake p27, fueling cell proliferation and tumor growth.13,17 Beyond its involvement in carcinogenesis, modifications in the ubiquitination status of several key proteins involved in initiation and progression of apoptotic signals within tumor cells, namely the kinase receptor- Interacting Protein 1 (rIP1) and caspase-8,18 have also been implicated in resistance to chemotherapy.

among thousands of E3 ligases expressed in humans, the ubiquitin editing enzyme and potent Nf-kB inhibitory protein a20 recently gained accrued attention in the cancer field. Indeed, recent studies suggest that altered a20 expression contributes to both carcinogenesis and resistance of solid tumors to chemotherapy.19 this chapter will focus on the role of a20 in development of solid tumors and their response to chemotherapeutic agents.

The zinc Finger proTein A20

the zinc finger protein a20 is a key endogenous modulator of Nf-kB signaling. In humans, this 790-amino acid protein is encoded by tnfaip3 gene on chromosome 6q23.

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69A20 expressing TuMors And AnTicAncer drug resisTAnce

a20 was originally identified in endothelial cells as a Nf-kB dependent gene whose expression is rapidly induced by tNf.20 a20’s potent Nf-kB inhibitory activity was first observed in endothelial cells treated with tNf, lPS, the Pkc activator phorbol myristate acetate (PMa), or h2o2, and in Mcf7 breast carcinoma cells treated with tNf or interleukin-1.21,22 the molecular mechanisms supporting a20’s Nf-kB inhibitory function involves the cooperative activity of its two ubiquitin-editing domains, as reviewed in another chapter of this book.23 the N-terminal domain of a20 that harbors an ovarian tumor (otu)-like deubiquitinase motif, removes lysine-63 (k63)-linked ubiquitin chains from rIP. the c-terminal zinc finger domain, that comprises a E3 ubiquitin ligase motif, then polyubiquitinates rIP with k48-linked ubiquitin chains, thereby targeting it for proteasomal degradation.23 In addition to targeting rIP and hence tNf signaling, a20 also interrupts Nf-kB trans-activation signals by interacting with the tNfaIP3-induced protein 1 (tNIP1) or a20 binding inhibitor of Nf-kB (aBIN)-1 protein. recent evidence suggest a cooperative mechanism for tNIP1 and tNfaIP3 in inhibiting Nf-kB in that tNIP1 may physically and functionally link tNfaIP3 to the key regulator of the Ikk signalosome NEMo/Ikkg. this would facilitate tNfaIP3-mediated de-ubiquitination of NEMo/Ikkg, interrupting Nf-kB.24 alternatively, a20 has also been recently shown to inhibit NEMo/Ikkg through a non-catalytic mechanism involving ubiquitin-dependent recruitment of a20 to NEMo.25

In contrast to its anti-inflammatory function that has been ubiquitously documented, a20 has been ascribed anti- or pro-apoptotic functions, depending on cell type and/or apoptotic stimulus.26-32 Indeed, we have previously reported that a20 serves a potent cytoprotective function in endothelial cells as it protects these cells from death receptor (tNf and faS), and natural killer cells mediated apoptosis by inhibiting proteolytic cleavage of apical caspases-8 and -2, Bid cleavage and release of cytochrome c thus preserving mitochondrion integrity.26 this potent anti-apoptotic function of a20 was also documented in other cell types namely, hepatocytes and b cells.27,32 In contrast, a20 was rather pro-apoptotic in smooth muscle cells, as its overexpression sensitized them to cytokine and faS-mediated apoptosis.31

akin cell-type specific effects of a20 on apoptotic pathways, a20 also affected necrotic events in a cell type and stimulus dependent manner. Indeed, while a20 protected endothelial cells and hepatocytes from complement and h2o2-mediated necrosis, respectively, it promoted oxidative stress mediated necrosis of hela cells.26,33,34

to date, very little is known about the precise impact of a20 on apoptosis and necrosis pathways in tumors. however, based on the above mentioned data that were performed in non-cancer cells, whether primary or cell lines, we expect a similar complex picture in cancers, whereby the impact of a20 on apoptotic and/or necrotic events would be determined by the primary cell-type of the tumor, its unique molecular signature, and the chemotherapeutic agent applied.

Notably, a20 is also a key regulator of innate and adaptive immunity.35,36 Different from most cells in the body, which display very low basal a20 levels that are only upregulated in response to inflammatory stimuli, a20 is constitutively expressed at high levels in lymphoid organs. Indeed, immature and mature thymocyte subpopulations, resting peripheral t cells and B cells have significant a20 levels at baseline. In these cells a20 is downregulated upon t and B-cell activation, which allows control for cell proliferation and survival.37 Interestingly, in contrast to all other cells, tNf fails to induce a20 expression in activated t cells.37 a detailed description of the role of a20 in lymphoid organs will be reviewed in another chapter.

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70 The MulTiple TherApeuTic TArgeTs oF A20

relevant to the immune regulatory functions of a20, it attenuates antigen presentation by dendritic cells.38-41 If this function is beneficial for the prevention of auto-immunity and to promote tolerance toward mismatched allografts, it may hamper the development of anti-tumor immune responses. accordingly, a20 silencing in dendritic cells may represent a promising strategy for successful implementation of anti-tumor vaccines.38

A20: The BeAuTy or The BeAsT?

two different a20 expression patterns have been linked to cancer; decreased or increased expression/function of a20. Most of the former cancers are represented by B-cell lymphomas where a20 was inactivated by deletion, promoter methylation, frame-shift mutations, and/or nonsense mutations that result in a20 truncations or point mutations.42-44 In these tumors, persistent Nf-kB activation secondary to loss of a20 function/expression is at the heart of the pathogenesis of B-cell lymphomas, fueling uncontrolled proliferation of these cells and their resistance to apoptosis.45 the implication of loss of a20 in the pathogenesis of lymphomas will be discussed in another chapter of this book, as this chapter focuses on a20 in solid cancers.

a20 is expressed at high levels in several solid tumors. Even though its high expression levels have not been formally linked to tumor pathogenesis, they have often been associated with advanced disease and/or poor prognosis. Numerous examples of high a20 expressing solid tumors with poor survival rate illustrate this association. Indeed, a20 is highly expressed in undifferentiated nasopharyngeal carcinoma, poorly differentiated head and neck squamous cell carcinomas of the skin, Er-negative breast cancers, hepatocellular carcinoma and glioblastomas.46-49 In glioblastomas, increased expression of a20 maps to the glioma stem cells-like compartment. these progenitor cells obviate a high self-renewal potential and as such are implicated in tumor growth and propagation. Expression of a20 in these cells protects them from apoptosis and promotes their proliferation, while conversely, a20 silencing limits their proliferation and increases their susceptibility to apoptosis-inducing agents.50

Even if association studies still fail to fully characterize the involvement of a20 in the pathogenesis of cancer development and progression, they prompted additional studies aimed at determining, in each cancer type, whether overexpression of an anti-inflammatory, and sometimes anti-apoptotic protein such as a20 has oncogenic potential, or at the contrary is expressed as part of the cell response to counteract the oncogenic process. In contrast to the knowledge gap that still persists in determining the role of a20 in carcinogenesis, its involvement in tumor resistance to chemotherapeutic agents is better documented. however, even in this field of drug resistance, a20 keeps assuming opposite roles depending on the cancer and/or the drug category under study.

role oF A20 in AnTicAncer drug resisTAnce

Within the last decade, it has become clear that a20 is a major player in anticancer drug resistance. Several reports indicate that a20 levels outside the physiologic range are implicated in the molecular mechanism(s) of cell evasion from drug-induced cytotoxicity. Interestingly, a20 modulates different signaling networks along pathways targeted by

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71A20 expressing TuMors And AnTicAncer drug resisTAnce

different chemotherapeutic agents. this results in either increasing or decreasing resistance of a given tumor to a specific drug. In this chapter, we will detail the role of a20 as a modulator of tumor response to traIl and o6-alkylating agents.

Heightened Expression of A20 Contributes to Tumor Resistance to TRAIL

tumor Necrosis factor-related apoptosis-Inducing ligand (traIl), a member of the tNf superfamily, was readily characterized as a selective apoptosis inducing agent in transformed cells,51 and a key physiologic player in tumor surveillance.52,53 as for tNf, active traIl is a homotrimeric molecule that binds five different receptors, two of which, death receptors Dr4 and Dr5, recruit fas-associated death domain (faDD) and the apical caspase-8 to form the Death Inducing Signaling complex (DISc).54,55 this causes caspase-8 dimerization and cleavage within the DISc, which activates downstream executioner caspases to promote apoptosis.55 Because initially thought to selectively target tumor, while preserving normal cells, traIl was hailed as a potentially “ideal” chemotherapeutic agent.56 however, the promise of traIl-based therapies in cancer was hampered by the emergence of traIl-resistant tumors.57 although, the molecular basis for tumor resistance to traIl is still not totally resolved, several mechanisms have been described in different tumor types. these include increased expression of the anti-apoptotic proteins c-flIP and Bcl-238,58 and relevant to this chapter increased expression of the ubiquitin-editing enzyme, a20.

Based on recent literature, abnormally high levels of a20 have been implicated in conferring traIl resistant phenotype to several types of tumors. the ability of a20 to bind and post-translationally modify expression or activation of apical caspase-8 or rIP1 has been identified as key to this process.46,48 however, and even if caspase-8 is seemingly the ultimate target of a20 in most tumors tested, tumor type and cell line specific mechanisms causing a20 to decrease caspase-8 expression or activity have been identified.

one of these mechanisms was described in glioblastoma tissues.46 In an elegant study, Bellail et al. demonstrate increased levels of a20 in most glioblastomas as compared with normal cerebral tissue. furthermore, these authors identify a direct correlation between high a20 levels and in vitro resistance to traIl. Mechanistically, heightened a20 protein levels in glioblastoma cells leads to the formation of a pre-ligand assembly complex (Plac) comprising a20, rIP1, traf2 and Dr5 at the cellular membrane. upon traIl stimulation, faDD and caspase-8 are recruited to Plac to form the death inducing signaling complex (DISc) but fail to further engage apoptotic signals because of the inability of caspase-8 to undergo autocatalytic proteolysis. a series of knockdown experiments proved that both a20 and rIP1 are necessary to inhibit caspase-8 cleavage within the DISc. Indeed, in the proposed sequence of events, a20 binds rIP1 and causes, through its E3 ubiquitin ligase activity, k-63 linked polyubiquitination of rIP1. In turn, k63-linked polyubiquitinated rIP1 binds the protease domain of pro-caspase-8, which inhibits its dimerization and cleavage, preventing activation of the caspase cascade and execution of the apoptotic program, in other words conferring resistance to traIl-induced apoptosis (fig. 2a).46 In traIl-sensitive glioblastoma cell lines a20 is barely expressed, and neither a20 nor rIP1 are detected in Plac. the link between heightened a20 levels and resistance to traIl was also demonstrated in hepatocelullar carcinoma cell lines. here again, a mechanism involving polyubiquitination of rIP1 by the E3 ligase domain of a20 and secondary inhibition of caspase-8 activation is proposed, although the exact molecular basis linking these two observations is still not clear.48

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72 The MulTiple TherApeuTic TArgeTs oF A20

In unpublished work, we have identified a second mechanism by which a20 interrupts traIl-mediated apoptosis, in the prostate cancer cell line, Pc3 at the level of caspase-8 (Benhaga et al., Ms. In preparation). In these cells, overexpression of a20 promotes its binding to pro-caspase-8, causing its ubiquitination and subsequent degradation in the proteasome. further studies are ongoing to verify that pro-caspase-8 is indeed a novel target for the E3 ubiquitin ligase activity of a20, and that this results in k-48 linked polyubiquitination of pro-caspase-8. It is well documented in the literature that k-48 linked polyubiquitination of proteins is the most common post-translational modification (PtM) that targets them to proteasomal degradation.59,60 this would agree with the well-described a20-dependent k48-linked polyubiquitination of rIP1 followed by its proteasomal degradation, reported as key for inhibition of Nf-kB in response to tNf.23,61

another mechanism of a20-mediated inhibition of pro-caspase-8 activity was described in non-small cell lung cancer cell lines. In these cells, traIl binding to its ligand causes the recruitment of E3 ubiquitin ligase cullin 3 to the DISc. In turn, DISc associated cullin 3 causes both k48- and k63-linked polyubiquitination of pro-caspase-8, which facilitates its aggregation, autocatalytic cleavage and activation.62 In a20 overexpressing

Figure 2. Mechanisms of a20-mediated tumor resistance to traIl. homotrimeric traIl bind death receptors (Dr) that recruit fas-associated death domain (faDD) and the apical caspase-8 to the Death Inducing Signaling complex (DISc). this causes caspase-8 dimerization and cleavage within the DISc, which activates downstream executioner caspases to promote cell apoptosis. a) In a20 overexpressing, traIl resistant glioblastomas heightened a20 protein levels lead to the formation at the cellular membrane of a pre-ligand assembly complex (Plac) that associates to Dr5, comprising a20, receptor Interacting Protein 1(rIP1) and tNf receptor associated factor 2 (traf2). traIl binding to Dr5, initiates a20-induced k63-linked polyubiquitination of rIP1, which promotes its interaction with pro-caspase-8 to inhibit caspase-8 dimerization and autocatalytic activation. B) alternatively, in small cell lung cancer cell lines, traIl binding to Dr allows recruitment of the E3 ubiquitin ligase, cullin 3, to the DISc. DISc associated cullin 3 causes both k48- and k63-linked polyubiquitination of pro-caspase-8, which facilitates its aggregation, autocatalytic cleavage and activation. however, in a20 overexpressing, traIl resistant cells, a20 gets also recruited to the DISc, where through its otu domain, it deubiquitinates pro-caspase-8 and inhibits its aggregation, therefore annulling the effect of the pro-apoptotic facilitator, cullin 3. Green (gray) arrows depict traIl-apoptotic pathways, whereas red (dark gray) arrows signify a20-mediated blockade of this pathway.

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73A20 expressing TuMors And AnTicAncer drug resisTAnce

non-small lung cancer cell lines, a20 gets also recruited to the DISc, where through its otu domain, it deubiquitinates pro-caspase-8 and inhibits its aggregation, therefore annulling the effect of the pro-apoptotic facilitator, cullin 3 (fig. 2B).62

Decreased A20 Levels Promotes Resistance to O6‑Alkylating Agents

In contrast with heightened a20 supporting tumor resistance to traIl, decreased a20 levels is linked to resistance of certain tumors, namely glioblastoma multiforme, to alkylating agents.19 In this form of tumor, even after aggressive therapeutic intervention, a significant number of residual and highly metastatic glioblastoma cells remain viable and proliferative, causing high recurrence rate and limiting patients’ survival.63 With this grim prognosis, alkylating drugs remain the most effective chemotherapeutic molecules used to treat these patients. alkylating agents exert their cytotoxic effects by forming DNa alkylating adducts, which cause DNa damage and mutation and inhibit DNa replication. Monofunctional methylating agents such as temozolomide (tMZ), bifunctional alkylating agents such as nitrogen mustards, and chloroethylating agents such as 1,3-bis(2-chloroethyl)-1-nitrosourea (BcNu) are the most commonly used alkylating agents.64 tMZ forms o6-methylated adducts on guanine base (o6MeG). o6MeG:t mispairs activate mismatch repair systems (MMr) which inevitably fail, leading to DNa fragmentation, cell cycle arrest and eventually cell death.65 Similarly, BcNu causes primary chloroethyl adducts at o6G inducing complex DNa damage.64 ultimately, cell death induced by alkylating agents results from p53-dependent activation of the intrinsic apoptosis pathway i.e., implicating non-receptor mediated signals causing mitochondrial depolarization, release of cytochrome c release, and activation of initiator pro-caspase 9 followed by that of executioner caspase 366,67 In contrast to traIl-mediated apoptosis, this process is mostly caspase-8 independent.

upon profiling glioblastoma multiforme cells for gene expression signatures associated with in vitro and/or in vivo resistance to the DNa alkylating drugs tMZ and BcNu, we found significantly lower a20 transcripts in glioblastoma cell lines that were resistant to such chemotherapeutic agents.19

It is well documented that efficiency of alkylating drugs is limited by cellular resistance mechanisms that develop during tumor progression. upregulation of the o6-alkylating-methylguanine-DNa methyltransferase (MGMt), an enzyme that specifically removes o6MeG from DNa, is the most frequent resistance mechanism of brain tumors to alkylating agents.68 however, addition of MGMt inhibitors to chemotherapeutic regimen using DNa alkylating agents is not always successful in stopping progression of these tumors, suggesting the presence of other molecular mechanism(s) implicated in tumor resistance to alkylating agents. In glioblastomas, p53 mutations that hamper its nuclear translocation and ability to transcribe cDkI p21, have been reported as one additional mechanism counteracting cell cycle inhibition caused by alkylating drugs.69 In human melanomas, colorectal carcinomas, and melanoma cell lines, tMZ induced akt-dependent activation of both canonical and non-canonical Nf-kB pathways has also been reported as a molecular mechanism of resistance.70 In fact, in melanoma cell lines, sirNa-mediated knockdown of the rela/p65 Nf-kB member, or addition of the Nf-kB inhibitor, Ikkg NEMo Binding Domain (NBD) peptide, overcame this resistance and allowed for tMZ-induced cell death to proceed. this agrees with data reporting that placenta growth factor (PGf)-mediated activation of Nf-kB in melanoma cell lines also amplifies their resistance to tMZ, a process again reversed by addition of the Nf-kB

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74 The MulTiple TherApeuTic TArgeTs oF A20

inhibitor, Dehydroxymethylepoxyquinomicin (DhMEQ).71 additional supportive evidence include data showing that the Mcf-7 derived kD12 human breast carcinoma cell line that express a dominant negative form of akt and fail to activate Nf-kB in response to tMZ, is approximately 6-fold more sensitive to tMZ than Mcf-7 cell lines that do not express dominant negative akt.72 relevant to our work in glioblastomas, we and others have demonstrated that tMZ causes Nf-kB activation in these tumors19,73 which together with lower levels of the Nf-kB brake, a20, would keep the Nf-kB pro-survival pathway unchecked, and hence contribute to tumor resistance to alkylating agents (fig. 3).19

In support of this hypothesis, we found an inverse correlation between a20 levels and tumor resistance to o6-alkylating agents both in glioblastomas derived from patients that had been treated with BcNu, and in vitro studies performed on glioblastoma cell lines under

Figure 3. Mechanisms of tumor resistance to alkylating agents in low a20 expressing gliobastomas. alkylating agent temozolomide (tMZ) forms o6-methylated adducts on guanine bases (o6MeG), causing DNa fragmentation and subsequent apoptosis. however, simultaneous tMZ-induced activation of Nf-kB increases expression levels of Nf-kB dependent anti-apoptotic genes, contributing to secondary resistance to tMZ. this mechanism is contained in tumors expressing adequate levels of the Nf-kB inhibitory protein, a20, but is amplified in tumors expressing low levels of a20. Green arrows depict expected tMZ-induced genotoxic pathways. Purple arrows depict potential Nf-kB dependent resistance to tMZ. red (dark gray) arrows depict amplified Nf-kB activation in low a20 expressing tumors.

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resistance selection, i.e., when a more or less sensitive parental tumor cell line is exposed to (sub)lethal drug concentrations that would specifically select for resistant cells. resistance selection correlates with cells expressing less a20 transcripts and corresponding protein product,19 suggesting regulation of mrNa level as the primary mechanism controlling a20 protein abundance during resistance development.74 lower a20 expression correlated with heightened Nf-kB activation and, most likely, downstream anti-apoptotic Nf-kB dependent genes in these cells.19 It is still unclear whether selection for low a20-expressing and hence resistant tumor cells results from random emergence of mutations, causing a20 knockdown or from treatment-induced downregulation of a20. In the former scenario, random mutations, a phenomenon caused by inherent genetic instability of many human cancers, allows for high degree of focal genetic microheterogeneity within any given tumor. consequently, a20 low, Nf-kB high, resistant clone would be selected and would preferentially expand under chemotherapy, leading to secondary treatment failure. In the latter scenario, a20 knockdown may result from treatment-dependent repression of a20, as a consequence of mutagenesis or epigenetic pressure. Both non-mutually exclusive scenarios may explain the decrease of a Nf-kB dependent gene, like a20, in cells that demonstrate increased Nf-kB activation.Epigenetic mechanisms have been shown to regulate both tMZ resistance in glioblastomas and a20 mrNa levels in B-cell lymphomas. hypermethylation of the MGMt promoter, which decreases its transcription, is a key predictor of outcome in glioblastoma patients treated with BcNu or tMZ.75,76 constitutive activation of Nf-kB in diffuse large cell B-cell lymphomas is attributed to a20 knockdown by increased expression of mir-125a and mir-125b that regulate a20mrNa.77 alternatively, transcription of a20 could be independent from Nf-kB in these cells, as shown in other cell types.78

Downregulation of a20 in tumor cells that acquire a phenotype resistant to o6-alkylating agents coincides with paralleling changes in the abundance of other Nf-kB pathway members, including IkB proteins IkBa and IkBe, and a20-interacting proteins rIP and a20 binding inhibitor of Nf-kB (aBIN)-119 rIP, that is part of the Nf-kB trans-activation signal and a known target for the dual ubiquiting editing function of a2023 is significantly upregulated in tMZ resistant glioblastomas19 (fig. 4). a cooperative mechanism implicating imbalanced expression of a20 and rIP determines glioblastomas resistance to o6-alkylating agents, even if its exact mode of action remains ambiguous. What is known is that rIP-mediated Nf-kB activation by drug-induced DNa damage is not mediated by tNfr1 signaling.79 rather, upon receiving the nuclear signal reporting DNa damage, rIP is upregulated and directly initiates signaling pathways activating Nf-kB.79 Decreased levels of a20 in resistant tumor cells would relieve rIP regulation by a20-dependent degradation, therefore amplifying rIP initiated Nf-kB activation and expression of downstream anti-apoptotic genes. otherwise, in cells expressing sufficient amount of a20, it would bind rIP and cause its sequential de-ubiquitination (k-63-polyubiquitin chain)s, followed by k-48 poly-ubiquitination and targeting for proteasomal degradation, as detailed in chapter one of this book.

Interestingly, tNIP1/aBIN-1 is concomitantly downregulated with a20 in glioblastoma cells resistant to o6-alkylating agents.19 aBIN-1 interacts with a20 and cooperates with it in a negative feedback regulation of Nf-kB.24 as reviewed in chapter 2 of this book, aBIN-1 physically and functionally links a20 to NEMo/Ikkg and facilitates a20-induced de-ubiquitination of NEMo/Ikkg, causing Nf-kB inhibition. aBIN-1 silencing abrogates a20-dependent de-ubiquitination of NEMo/Ikkg and, in turn, a20 silencing impairs aBIN-1’s ability to inhibit Nf-kB activation.24 accordingly, concurrent downregulation of a20 and aBIN-1 maintains the phenotype of resistant cells by forestalling upregulation of other regulatory proteins that can countervail excessive Nf-kB activity.

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the implication of heightened Nf-kB activation in promoting tMZ-resistance of the glioblastoma cell line t98G lends support to our data. In this low a20 expressing glioblastoma cell line, authors demonstrate that addition of resveratrol reverts cell resistance to tMZ by specifically blocking tMZ-induced NfkB activation.73 Interestingly, while the t98G glioblastoma cell line resists the cytotoxic effect of tMZ, it is rather sensitive to traIl.46 this brings our story full circle as it exemplifies finely regulated specificity of a20 as a modulator of tumor resistance to anti-cancer drugs, not only on basis of cell type but also of chemotherapeutic agent.

from a clinical standpoint, a critical step in allocating a significant role for a20 in modulating cancer resistance to chemotherapeutic drugs is to define the best way to translate in vitro read-outs to predictive parameters of patients’ response to chemotherapy. using glioblastomas as a case study, resistance of human glioblastomas to o6-alkylating agents and patients’ prognosis under such chemotherapy could be deduced from the state of expression of a20 as part of a multi (four)-gene predictor, including syndecan 1 (SDc1), human lymphocyte homing receptor cD44, f Box Protein 32 (fBXo32) in addition to a20/tNfaIP3 (fig. 5). While, mere a20 levels were able to predict response, predictor models that include multiple endogenous modulators outperform the predictive power

Figure 4. a20 and rIP transcript abundance in vitro and in vivo in resistant glioblastoma cell lines. Gene expression by microarray and real-time reverse-transcription polymerase chain reaction (qrt-Pcr) of tNfaIP3 and rIP. Bar graphs indicate the microarray-assessed gene expression in resistant cells relative to the corresponding parental cells, the upper panel (downregulation of tNfaIP3 in gray/green) and lower panel (upregulation of rIP in gray/red) heat maps correlate the parentally normalized expression between microarray (array-EXP) and qrt-Pcr and reports corresponding parentally transformed gene copy numbers (microarray-based comparative genomic hybridization [array-cGh]). heat maps have been masked (black squares) to only show fluorescent ratios indicating at least ± 2-fold changes in resistant cells vs. sensitive cells (reprinted with permission from: Bredel M et al. j clin oncol 2006; 24:274–287). a color version of this figure is available online at www.landesbioscience.com/curie.

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of each individual molecule.19 Work is devoted to extend multi-gene predictor panels in management of cancer therapies, and we believe that these panels will often include a20.

conclusion

Mounting evidences linking a20 to development of solid tumors and response to chemotherapy ultimately highlights its promise as a novel therapeutic target in cancer. however, development of therapeutic anti-cancer strategies targeting a20 requires a refined understanding of the molecular mechanism(s) underlying a20s role in carcinogenesis and drug resistance. In this chapter, we attempted to illustrate the intricacies of these mechanisms by showing opposite associations between low a20 levels in a given cancer type (glioblastomas) and sensitivity to traIl vs. resistance to o6-alkylating agents, while a mirror image is expected for high a20 expressing tumors. this calls for thorough and careful tailoring of any given a20-targeting approach based on tumor type and/or anticancer agent used. With the expansion of cutting edge technologies allowing rapid and thorough determination of the molecular signature of each tumor, one may hope for personalized chemotherapeutic regimen that would include a20-targeting agents.

Figure 5. a20-including four-gene outcome prediction model in 31 glioblastomas. results of unsupervised hierarchical cluster analysis based on the weighted expression of 4 resistance-associated transcripts. according to cox proportional hazards regression analysis, p = 0.028 for a20 and p = 0.022 for the model as a continuous variable, and log-rank p = 0.007 for the model as a class based on the two major subgroups defined by unsupervised hierarchical clustering. (reprinted with permission from: Bredel M et al. j clin oncol 2006; 24:274–287). a color version of this figure is available online at www.landesbioscience.com/curie.

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AcknoWledgMenTs

this work was supported supported by r01 hl080130, r01 Dk063275, r01 hl021796, jDrf 1–2007–567, r21 Dk091822 to cf and DcM was supported by a fellowship from National council for Scientific and technological Development, cNPq, universidade federal de ciências da Saúde de Porto alegre, Brasil.

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