Noxa: at the Tip of the Balance Between Life and Death

C Ploner1, R Koﬂer1,2 and A Villunger3

1Division of Molecular Pathophysiology, Biocenter, Innsbruck Medical University, Innsbruck, Austria; 2Tyrolean Cancer Research Institute, Innsbruck, Austria and 3Division of Developmental Immunology, Biocenter, Innsbruck Medical University, Innsbruck, Austria

Among all Bcl2 homology domain 3 (BH3)-only proteins putative homologue. Curiously, mouse and human known to date, APR/PMAIP1/Noxa, albeit showing Noxa differ significantly in size and the number of weak proapoptotic potential on its own, appears to be BH3 domains. Mouse Noxa, as well as the rat homo- crucial in fine-tuning cell death decisions by targeting the logue, contains two BH3 domains and both are about prosurvival molecule Mcl1 for proteasomal degradation. twice as long as the human isoform (Figure 1a). Because This event appears critical for cell death induction along no other mammalian sequence available in the database the mitochondrial Bcl2-regulated apoptosis pathway in to date, nor chicken or zebrafish variants of the response to factor deprivation or DNA damage, pre- gene, nor its putative common ancestor found in sumably by sensitizing the cell toward the action of Caenorhabditis elegans, CED-13, share this feature additional BH3-only protein family members. This review (Figure 1b), it has been suggested that this peculiarity aims to summarize the function of Noxa in normal is due a failed gene duplication/fusion event in physiology, stress-induced cell death and tumorigenesis. primordial ancestor of rats and mice (Coultas et al., Oncogene (2009) 27, S84–S92; doi:10.1038/onc.2009.46 2002). Noteworthy, like in mice and rats, the human gene contains three exons (Figure 1c) but the transcript Keywords: apoptosis; BH3-only proteins; p53 encoding the human protein (NM_21127) contains only sequences from exons 1 and 3, neglecting exon 2. This exon shows no similarity with mouse Noxa exon 2 and is only found in two splice variants, termed Noxa splicing Discovery, genes and transcripts variant 1 and 2, which encode predicted proteins of 136 and 70 aa in length, respectively (Wang and Sun, 2008). In 1990 Hijikata et al. isolated the first Noxa cDNA Both splice variants only share the first 19 aa clone from an adult T-cell leukemia (ATL) library in with Noxa (encoded by exon 1) but otherwise differ search for products involved in leukemogenesis. The vastly from the remaining Noxa protein sequence and transcript was rapidly induced by phorbol 12-myristate lack a BH3 domain. They are rapidly degraded through 13-acetate (PMA) treatment in human peripheral blood proteasome-dependent and -independent pathways mononuclear cells, Jurkat T acute lymphoblastic leuke- (Wang and Sun, 2008) and their function, if any, is mia (ALL) and human embryonic lung cells (Hijikata unknown. Thus, exon 2 of human Noxa is probably a et al., 1990) and was therefore named ATL-derived variant exon and functionally the human Noxa gene PMA-responsive gene (APR). Later on it was given the might therefore be considered a ‘two-exon gene’ like that HUGO designation PMA-induced protein 1 (PMAIP1). of cow, swine, chicken or zebrafish. However, the function of this protein remained unknown for another decade. APR/PMAIP1 was then rediscovered in a differential Regulation of Noxa expression display approach using mRNA from g-irradiated wild- type (wt) and IRF-1/p53 double-deficient mouse em- Initial observations indicated that Noxa is a primary bryonic fibroblasts (MEF). The isolated cDNA encoded p53-response gene. Noxa mRNA is rapidly induced after a 103 amino-acid (aa) protein, which the authors termed adenovirus-mediated introduction of p53 into MEF À/À Noxa (Latin for damage). Sequence analysis revealed derived from p53 or wt mice and in wt thymo- À/À that the Noxa protein contained no known structural cytes subjected to g-irradiation but in not p53 motif, with the exception of a Bcl2 homology (BH) controls. Promoter analysis of human Noxa revealed domain 3, putting it into the back and then steadily the presence of a bona fide p53 response element 195 bp growing BH3-only subgroup of proapoptotic Bcl2 upstream of the transcriptional start site (Figure 1c). family members (Oda et al., 2000). The search for the Northern blot analysis performed on mRNA isolated human counterpart revealed APR/PMAIP1 as the from various tissues of adult mice revealed constitutive low-level Noxa expression in brain, thymus, spleen, lung, kidney and testis as well as the intestinal tract (Oda Correspondence: Professor A Villunger, Division of Developmental Immunology, Biocenter, Innsbruck Medical University, A-6020 et al., 2000). Whole-body g-irradiation induced Noxa Innsbruck, Austria. mRNA in the thymus, spleen, jejunum and transverse E-mail: [email protected] colon (Oda et al., 2000; Fei et al., 2002). Interestingly, in NOXA in apoptotic cell death C Ploner et al S85

Figure 1 (a) Sequence comparison of the human, mouse and rat Noxa protein. The mitochondrial targeting sequence (MTD) and the Bcl2 homology domain 3 (BH3) domains (A- and B motif) are shown in bold, respectively. (b) Sequence alignment of BH3 domains from ancestral Egl-1, related CED-13 and Noxa from different species. (c) Noxa gene structure, transcription factor binding sites and reported mRNA transcripts. Untranslated regions and intronic sequences are shown in white, coding sequence in black. the latter two organs, induction of Noxa occurred speculative, this suggests a possible tumor suppressor efﬁciently also in the absence of p53. This indicated that, function for Noxa independent of the p53 status of a in certain tissues, Noxa induction in response to DNA cell, maybe as a downstream target of p73. Consistently, damage can occur in a p53-independent manner. Along the transactivating isoform of p73 was shown to be able the same line, Noxa induction was also observed in to induce transcription of this BH3-only protein and cell response to HDM2 inhibition in p53À/À HCT116 colon death (Flinterman et al., 2005). cancer cells (Lau et al., 2008) and in response to onco- Not surprisingly, p53 and its relatives are not the only genic stress, caused by E1a overexpression in p53-deﬁ- regulators of Noxa expression, and numerous other cient Saos-2 cells (Flinterman et al., 2005). Although regulators and control mechanisms have been described

Oncogene NOXA in apoptotic cell death C Ploner et al S86 p73 and/or by direct upregulation of several BH3-only proteins, including Noxa (Hershko and Ginsberg, 2004). In fact, the Noxa gene promoter contains a bona fide E2f1-binding site (Figure 1c) that allows transcription of this BH3-only protein in a p53-independent manner. Remembering that the human Noxa homologue, APR/PMAIP1, was initially discovered as a PMA- responsive gene in malignant T cells, Alves et al. (2006) studied regulation and function of Noxa in response to PMA and mitogenic stimulation with anti-CD3 and anti-CD28 crosslinking antibodies in mature human T cells. These treatments prompted proliferation and, somewhat counterintuitive, a strong upregulation of Noxa mRNA and protein. The response was independent of p53, but interfering with protein kinase C (PKC) signaling using pharmacological inhibitors abrogated Noxa induction, implicating novel PKC family members, presumably T-cell-selective PKC, in signal trans- duction downstream of the T-cell receptor in the regulation of Noxa expression (Alves et al., 2006). In this context it is worth mentioning that Noxa is induced by the common g-chain of the receptors for cytokines interleukin (IL)-7 and IL-15, although the underlying molecular mechanisms are not well understood (Alves et al., 2006). Although studying the molecular mechanism how immunological memory T cells might escape cell death after antigen clearance, Yamashita et al. (2008) observed Figure 2 Multiple signals can induce Noxa expression. Noxa that the polycomb group (PcG) gene Bmi1 represses transcription and protein expression can be activated by diverse Noxa gene expression. PcG genes control transcription apoptotic or mitogenic signals in a p53-dependent as well as by remodeling chromatin structures by constituting -independent manner. Noxa exerts its proapoptotic function polycomb repressive complexes enabling DNA methyla- mainly by neutralizing the prosurvival Bcl2 proteins Mcl1/A1, tion at CpG islands (Wang et al., 2004). Loss of Bmi1 facilitating activation of Bax and/or Bak proteins. did not interfere with memory T-cell proliferation but excess cell death was observed, which correlated with increased expression of several proapoptotic genes (Figure 2). Thus, like Puma, the second p53 target BH3- including Bax, Noxa and Puma, in line with the capacity only protein gene induced after DNA damage, Noxa of Bmi1 to repress the Ink4a/Arf gene locus and was also found induced under hypoxic conditions. subsequent p53 signaling. However, neither loss of the However, although hypoxia-induced expression of Ink4a/Arf locus nor loss of p53 rescued T helper 2 (Th2) Puma is mainly dependent on p53, Noxa was reported memory cell generation in the absence of one or both as a direct target of Hif1a, able to mediate hypoxic cell alleles of Bmi1, and Noxa expression was still elevated death in a p53-independent manner. A hypoxia-respon- in these compound mutant memory T cells. Subsequent sive element was identified in the Noxa promoter analysis demonstrated that Bmi1 was required for DNA (Figure 1c) and a general upregulation of Noxa during CpG methylation of the Noxa gene locus (Yamashita hypoxia was observed in various normal and malignant et al., 2008). Indeed, compared to wt Th2 cells, tissues (Kim et al., 2004). Potentially relevant for novel trimethylation of histone H3-K27 and methylation of treatment regimens of ischemia, elevated Noxa levels CpG islands of the Noxa gene promoter were reduced in were also detected after transient ischemia experiments Bmi1-deficient Th2 memory cells. Interestingly, binding in vivo and liposomal delivery of Noxa antisense oligo- of the Bmi1/PcG complex to the Noxa gene locus itself nucleotides significantly reduced infarct volumes of rat depended on the methylation status of the promoter brains (Kim et al., 2004). because knockdown of the methyltransferase Dnmt1 Another sequence-specific transcription factor impli- caused decreased binding of Bmi1 to the Noxa promoter cated in regulating Noxa expression is E2f1. Binding and increased mRNA expression (Yamashita et al., of viral oncoproteins such as human papillomavirus 2008). protein E7 or adenoviral E1a to the retinoblastoma gene Another interesting observation concerning Noxa product Rb causes deregulation of its activity entailing regulation was recently made in human ALL. We defective cell-cycle progression and apoptosis. Ectopic discovered that Noxa mRNA expression, as detected expression of E2f1 can trigger cell death not only in by whole-genome expression profiling, was repressed in a p53-dependent manner (through Arf) but also in a children with ALL undergoing systemic glucocorticoid p53-independent manner. The latter may occur through (GC) monotherapy (Schmidt et al., 2006; Ploner et al.,

Oncogene NOXA in apoptotic cell death C Ploner et al S87 2008). Subsequent analyses revealed that GC represses appears to be quite variable (Oda et al., 2000; Schuler not only Noxa mRNA but also its protein in a et al., 2003). Thus, Noxa overexpression per se entailed proteasome-dependent manner. This phenomenon may significant cell death in some systems (Oda et al., 2000), have clinical relevance because functional analyses but in many other cell types, including MEF or human suggested that Noxa repression by GC paradoxically CEM T ALL cells, it proved poorly apoptotic (Chen interferes with extent and kinetics of GC-induced et al., 2005; Ploner et al., submitted for publication). apoptosis (Ploner et al., submitted for publication). Several possibilities may explain these observations (see also review by DCS Huang and P Bouillet, in this issue on page S128). Compared to other BH3-only proteins Molecular mechanisms of Noxa-induced cell death Noxa shows the most restricted potential to neutralize prosurvival Bcl2 molecules. Although Bim and Puma Apoptosis induction and subcellular localization can bind all survival Bcl2 proteins with comparable Overexpression of mouse Noxa in HeLa cervical carci- affinity, Noxa selectively binds to Mcl1 and, with lower noma and other cancer cells causes significant apoptosis, affinity, Bfl1/A1 (Chen et al., 2005). The molecular basis which is associated with its preferential localization to for this selectivity depends on critical aa residues in the mitochondria (Oda et al., 2000). Apoptosis induced by amphipathic a-helical BH3 domain contacting key Noxa requires a functional BH3 domain, and is residues in the hydrophobic groove of the antiapoptotic accompanied by cytochrome c release and subsequent Bcl2-like prosurvival proteins, formed by parts of the caspase activation. No binding of Noxa to proapoptotic BH1, BH2 and BH3 domains. As shown by high- Bax or Bak was reported so far, indicating that Noxa resolution structure analyses of Bclxl/Bim complexes, promotes Bax-mediated mitochondrial dysfunction in- binding of Bim to Bclxl depends on the interaction of directly by inhibiting antiapoptotic members of the Bcl2 hydrophobic pockets, located in the binding groove of family (Oda et al., 2000; Shibue et al., 2003). Bclxl, with four conserved hydrophobic residues in the Overexpressed Noxa protein localizes preferentially BH3 domain of Bim (Liu et al., 2003). Replacement of to mitochondria, as assessed by immunostaining (Oda one or more of these critical residues interferes with the et al., 2000). Interestingly, Noxa seems to require an binding specificity and affinity of the whole BH3-only intact BH3 domain not only for cell killing but also for protein. This suggests that the binding properties of a its mitochondrial localization, suggesting that its asso- given BH3 molecule depend only on the nature of its ciation with this organelle may be secondary to its BH3 domain and hence exchange of the critical residues interaction with the prosurvival Bcl2-like molecules that leads to altered binding specificity of the whole it antagonizes. However, expression of a human GFP/ molecule. In fact, exchange of only two aa in the BH3 Noxa fusion protein and truncation mutants thereof in domain of human Noxa, that is, K35E and F32I (also HeLa cells revealed that aa 41–50 may constitute an called Noxa m3 mutant), increased its binding efficiency bona fide mitochondrial targeting domain (MTD; for Bclxl more than 100-fold (Chen et al., 2005). Figure 1a; Seo et al., 2003). This sequence is conserved Moreover, exchange of the key contact residues of one between human, mouse, rat and chicken Noxa, and a BH3-only molecule into another transmits the binding human Noxa construct lacking the MTD domain but characteristic of the donor BH3 molecule. When the still containing the BH3 domain was unable to induce Noxa BH3 domain was grafted into Bim (that usually cell death presumably due to mislocalization (Seo et al., shows high proapoptotic potential due to its capacity to 2003). However, because mitochondrial localization of bind all Bcl2 prosurvival molecules), the hybrid lost the mouse Noxa was also prevented by replacement cell death inducing potential of the BimS isoform that mutations in the two BH3 domains (without altering was used in this experiment. This correlated with the MTD), both domains might be required for proper imposed binding capacity restricted to Mcl1. The same mitochondrial targeting. Alternatively, given the small was true for BimS chimeras containing the BH3 domain size of human Noxa and the fact that this deletion starts of Bad, which bound Bclxl but no longer Mcl1 (Chen immediately C-terminal to the BH3 domain, it may et al., 2005). Both mutants of BimS, however, can impair overall conformation and its ability to interact complement each other in promoting apoptosis. These with Bcl2 and its homologues. This, rather than the experiments support the hypothesis that the nature of destruction of a putative mitochondrial localization BH3 domains defines binding specificity and affinity of signal, might then cause inappropriate subcellular the whole BH3-only molecule and that mitochondrial localization. In any rate, because cell biological data cell death ensues when all types of Bcl2-like prosurvival on endogenous Noxa protein expression are still lacking, molecules present in a given cell type are neutralized by its subcellular localization, before and after cell death BH3-only proteins. Alternative modes of BH3-only induction, remains to be determined. protein-mediated apoptosis are discussed in more detail by Letai and co-workers in this issue of Oncogene. Molecular basis for the limited proapoptotic potential Investigations into the contribution of Noxa and of Noxa Puma to p53-dependent apoptosis uncovered that Although Noxa significantly contributes to apoptosis NIH3T3 or MEF, transformed with adenovirus E1a, induced by p53 overexpression or DNA damage, in died in the presence of transgenic Noxa or Puma, diverse cells such as Saos-2, BAF-3 pre-B cells or MEF, whereas wt cells were protected against Noxa- but its apoptotic potential, when overexpressed on its own, not Puma-induced cell death (Shibue et al., 2006).

Oncogene NOXA in apoptotic cell death C Ploner et al S88 In E1a-transformed cells Noxa enhanced activation and of both BH3-only proteins induced cell death as oligomerization of Bax but not of Bak, whereas over- efficiently as transgenic expression of Bim or Puma, expression of Puma activated both (Shibue et al., 2006). able to neutralize all prosurvival Bcl2-like molecules This observation is supported by studies analysing (Chen et al., 2005). mitochondrial targeting of Noxa in isolated An intriguing feature of Noxa is that it targets Mcl1 mitochondria of mouse and rat liver cells where for proteasomal degradation. This event is considered to Noxa-mediated cytochrome c release was reported to be a prerequisite for cell death in response to UV occur independent from Bak oligomerization (Seo et al., irradiation (Nijhawan et al., 2003), cytokine deprivation 2003). Taken together, these observations suggest that (Opferman et al., 2003), treatment with anticancer Noxa kills cells in a mainly Bax-dependent but agents such as arsenic trioxide (Morales et al., 2008) Bak-independent manner. However, this is somewhat or histone deacetylase inhibitors (Inoue et al., 2007). surprising, given that Noxa has been repeatedly Although basal levels of Mcl1 appear to be regulated by implicated in the disruption of Mcl1/Bak complexes, the HECT- and BH3-domain-containing E3-ligase triggering Bak oligomerization during apoptosis induc- Mule/ARF-BP1 (Zhong et al., 2005), it is still unclear tion in cells as diverse as MEF, HEK-293T, multiple what regulates its degradation when complexed with myeloma and B-cell lymphomas (Willis et al., 2005; Noxa, because the binding pocket is occupied with its Inoue et al., 2007; Morales et al., 2008). This apparent BH3 domain. Interestingly, degradation of Mcl1 is not, discrepancy might be explained by the fact that as initially assumed, a prerequisite for apoptosis to oncogenic stress may not only impact expression of occur. Interaction with Bim, for example, in response to prosurvival molecules but also their subcellular localiza- GC treatment of acute lymphoblastic juvenile leukemia tion as repeatedly observed for Bax, which is usually cells (Ploner et al., 2008), Puma overexpression (Mei found in the cytoplasm of primary cells, but redis- et al., 2005) or arsenic trioxide treatment of myeloma tributes to mitochondria after transformation. Once at cells (Morales et al., 2008), actually leads to its the mitochondria, Bax may be restrained efficiently by stabilization, presumably due to exclusion of Mule/ Mcl1, and Mcl1/Bax complexes may then be targeted by ARF-BP1. The signature for degradation of the Mcl1/ Noxa. However, it remains unclear why Noxa should Noxa complex appears to be encoded in the C-terminal selectively trigger oligomerization of Bax under these portion of the BH3 domain of Noxa (FRQKLL in conditions, unless Mcl1 binds much more avidly to human Noxa), as replacement of these residues with aa Bax than Bak, which may then be restrained by other residues found in the same position in Bim (AYYARR) prosurvival molecules such as Bclxl (Willis et al., 2005). led to Mcl1 stabilization (Czabotar et al., 2007). Whatever the molecular mechanism may be, it is Interestingly, although the Noxa BH3 domain in the interesting to note that although primary MEF engage context of Bim protein caused Mcl1 degradation, it both Noxa and Puma for cell killing in response to UV failed to do so in the context of Bad, suggesting that irradiation, in E1a/ras-transformed cells Puma becomes residues outside the BH3 domain, functionally con- dispensable for this process and Noxa accounts for all served in Noxa and Bim, contribute to this process p53-induced death observed (Naik et al., 2007). (Czabotar et al., 2007). In summary, these data indicate that Noxa can engage Together all these studies reveal that Mcl1 degrada- Bax or Bak for apoptosis induction, but the engagement of tion during cell death is uniquely associated with the these proteins depends on the cell type, cellular state and formation of the Mcl1/Noxa complex whereas Mcl1/ apoptotic stimulus applied. Furthermore, Noxa-induced Bim or Mcl1/Puma interaction leads to its stabilization. cell death may become more relevant for apoptosis This is of relevance considering the design of BH3 induction in malignant or highly proliferating versus mimetics that may be able to bind to Mcl1, but may not primary or differentiated cells, suggesting its importance necessarily promote its degradation. The selective as tumor suppressor with therapeutic potential (see below). (dis)advantage of Mcl1 degradation over stabilization during apoptosis induction, however, remains unclear at present. Identification of an Mcl1/Noxa-specific E3- Regulation of Mcl1 stability by Noxa ligase may allow some insight and rational explanation. Given the fact that Noxa exclusively binds to Mcl1 and Although protein stability of the prosurvival Bcl2 Bfl1/A1, cellular levels of these proteins determine homologue Bfl1/A1 also appears to be regulated by the susceptibility to Noxa-induced cell death of a given cell proteasome and stability can be increased by Bim type. Inhibition or elimination of Mcl1 in response to binding (Morales et al., 2008), no data are available cytotoxic signals is considered critical in cell death in a addressing the possibility that A1/Noxa interactions may number of normal as well as malignant cells (Craig, promote its degradation during apoptosis induction. 2002; Willis et al., 2005). However, targeting Mcl1 by transgenic overexpression of Noxa failed to induce significant cell death, for example, in immortalized MEF Possible physiological functions for Noxa or NIH3T3 fibroblasts (Shibue et al., 2006), where survival depends on two or more Bcl2-like proteins such Noxa and the DNA-damage response as Mcl1 and Bclxl (Willis et al., 2005). Consistently, Two laboratories generated NoxaÀ/À mice to study its Noxa needs to be functionally complemented by Bad, function in vivo (Shibue et al., 2003; Villunger et al., which targets Bcl2, Bclxl and Bclw, and overexpression 2003). Mice lacking Noxa show no overt phenotype, but

Oncogene NOXA in apoptotic cell death C Ploner et al S89 more detailed analysis of stress responses confirmed a Interestingly, in contrast to memory Th2 cells, function for Noxa in p53-dependent, but also p53- induction of Noxa per se was inefficient in triggering independent cell death after DNA damage, albeit in a significant cell death in proliferating T cells, presumably cell type and stimulus-dependent manner. Although due to concomitant induction of prosurvival Bcl2-like hemopoietic cells from PumaÀ/À mice resisted cell death molecules. It may, however, prime proliferating T cells induced by DNA damage or factor deprivation, cells for death when conditions no longer favor clonal from NoxaÀ/À mice were normally sensitive to cell death expansion, for example, when growth factors or induction. However, loss of either protein protected cytokines become limiting at the end of an immune MEF cells from DNA-damage-induced apoptosis response. This idea is also supported by the observations caused by etoposide treatment, subsequently also con- that Noxa is induced by the common g-chain of the firmed in NoxaÀ/ÀPumaÀ/À MEF that showed signifi- receptors for cytokines IL-7 and IL-15, able to promote cantly greater resistance as cells from single knockout cell division in the absence of antigenic stimulation in mice. Analysis of NoxaÀ/ÀPumaÀ/À mice revealed a major CD8 þ T cells. However, loss of Noxa mildly interferes nonredundant function for Puma in apoptosis induced with IL-2-deprivation-induced death of activated T cells, by g-irradiation in MEF and most lymphocyte subsets, albeit not as efficiently as loss of Bim or Puma (Bauer besides CD4 þ 8 þ thymocytes, where Puma and Noxa et al., 2006), as well as IL-15-deprivation-induced death account for all cell death induced by p53. Similar of NK-cells, together with Bim (Huntington et al., observations were made in etoposide-treated MEF 2007). Consistent with its role as a sentinel in proli- transformed with E1a (Michalak et al., 2008). Interest- ferating cells, Jurkat T-cell clones carrying shRNAs- ingly, as mentioned before, Noxa and Puma contribute targeting Noxa showed better survival under conditions equally to apoptosis induced by UV irradiation in of metabolic stress due to glucose limitation. Subsequent primary MEF, but Noxa becomes the dominant cell biochemical analysis indicated that the high Noxa levels death inducer in response to this stimulus after induced during the proliferative response contributed oncogenic transformation (Naik et al., 2007). Note- to cell death by neutralizing the prosurvival effects of worthy, this pathway to apoptosis depends on c-Jun Mcl1 and presumably also Bfl1/A1 (Alves et al., 2006). N-terminal kinase activation (Tournier et al., 2000) and Therefore, we speculate that upregulation of Noxa in Mcl1 degradation (Nijhawan et al., 2003) that is proliferating T cells might counterbalance enhanced triggered selectively by binding of Noxa to Mcl1. expression of prosurvival molecules, thereby helping to Shibue et al. examined the involvement of Noxa in the restrain oncogenic transformation and/or autoimmu- DNA damage response in vivo by analysing apoptosis of nity. However, because Noxa-deficient mice develop epithelial cells in the stem cell region of small intestinal neither of these pathologies, Noxa may perform these crypts and cells in lamina propria after whole-body functions only in conjunction with other cell death g-irradiation. Noxa deficiency protected both cell types regulators and/or control mechanisms. from cell death. This is of particular interest because cells in lamina propria still die in the absence of p53 (Shibue et al., 2003). Protection from gastrointestinal Noxa in tumor development apoptosis by loss of Noxa also led to a delayed onset of g-irradiation disease-associated mortality. NoxaÀ/À mice Lessons from mice and humans survived significantly longer than wt or p53À/À animals. Mice lacking both or even only a single allele of Taken together these results suggest that Noxa can be p53 develop tumors with high incidence. In contrast, activated in response to g-irradiation in a p53-dependent no spontaneous tumor development was observed in as well as -independent manner and may be prominent NoxaÀ/À and even NoxaÀ/ÀPumaÀ/À double-deficient in the regulation of stem cell homeostasis, at least after animals, up to an observation period of more than 1 DNA damage, as recently suggested also for Puma (see year (Shibue et al., 2003; Michalak et al., 2008). This Review by Yu and Zhang, in this issue on page S71). confirms that inactivation of the proapoptotic function of p53 is insufficient to promote tumor development but that two or more of its activities, such as induction of Possible functions for Noxa in the immune system cell-cycle arrest and apoptosis, have to be impaired Although initial analysis of NoxaÀ/À mice did not reveal simultaneously. Interestingly, studies with knock-in any abnormalities in the hematopoietic system, Noxa mice that express an apoptosis-defective, but cell-cycle appears to control maintenance of memory CD4 þ inhibitory competent mutant of p53 (R172P) indicate an T Th1/Th2 cell homeostasis. As discussed in Chapter equally prominent role for these two p53 functions in 2, Yamashita et al. (2008) reported that the excessive the suppression of thymic lymphoma development (Liu Th2 memory T-cell death observed in mice defective et al., 2004). Whether loss of Noxa may impact tumor in the PcG gene Bmi1 was at least in part due to formation caused by DNA damage remains to be tested lacking repression of Noxa gene expression by Bmi1. In and it will be interesting to see if loss of Noxa on a p21- support, ablation of Noxa expression on a Bmi1 deficient background increases the rate of spontaneous hemizygous background rescued the observed defects tumorigenesis in animal lacking this CDK inhibitor. in memory cell generation, consistent with a key role for So far, the connection between Noxa and tumor Noxa in the maintenance of Th2 memory T-cell homeo- suppression is mainly circumstantial. In mice bearing stasis. a liver-specific deletion of c-Jun, the development of

Oncogene NOXA in apoptotic cell death C Ploner et al S90 chemically induced hepatocellular carcinoma (HCC) is Noxa coding sequence (Jansson et al., 2003). Interest- significantly delayed, revealing the oncogenic potential ingly, though, integrative genomic analysis of small-cell of c-Jun in an in vivo model (Eferl et al., 2003). c-Jun, a lung carcinoma revealed a correlation between amplifi- central component of the transcription factor Ap1, is a cation on 18q and sensitivity to the BH3-mimetic ABT- major regulator of proliferation and apoptosis in liver 737 (Olejniczak et al., 2007). Resistance of cancer cells to cells and loss of c-Jun in HCC leads to an increased ABT-737 is strongly associated with the relative expres- number of apoptotic cells that display increased mRNA sion of Mcl1, which it cannot antagonize (see below and levels of p53 and Noxa (Eferl et al., 2003). Induction of review by Labi et al., 2008). The region of gain contains Noxa in the absence of c-Jun seems to be specific, Bcl2 that can be potently blocked by ABT-737, but also because the expression of other proapoptotic p53 target Noxa that effectively neutralizes Mcl1 (van Delft et al., genes, such as Bax, Puma or Apaf-1, was unaffected. 2006), suggesting that 18q21–23, or maybe even Noxa However, it was not formally demonstrated that the expression alone, may be a clinically relevant predictor observed upregulation of Noxa mRNA was the con- for responsiveness to Bcl2 family inhibitors (Olejniczak sequence of p53 activation, leaving the possibility that et al., 2007). Along that line, in B-CLL cells expression Noxa and p53 may be independently activated by loss of of Noxa protein was found frequently increased, when c-Jun. Taken together, these observations indicate that compared with tonsillar B cells (Mackus et al., 2005) c-Jun promotes tumor cell survival by inhibiting the and may be causal for the high sensitivity of these proapoptotic action of p53, maybe by inhibiting tumors to ABT-737 treatment (Oltersdorf et al., 2005). expression of Noxa. Noxa expression levels, however, failed to correlate Most recently, the impact of loss of Noxa on c-Myc- with overall or disease-free survival in human colon driven B-cell lymphomagenesis has been studied. Aber- carcinoma, whereas relative expression levels of Bim or rant expression of c-Myc leads to p53 activation and Puma did correlate well with these parameters (Sinic- subsequent induction of apoptosis that may engage rope et al., 2008). Finally, Noxa was also found deleted Noxa and/or Puma to restrain tumor formation. Em- in the Burkitt-derived cell line Elijah and loss of one Myc transgenic mice lacking Puma develop lymphomas allele was observed in the related Karpas 106, UPN1 significantly earlier than Em-Myc mice, consistent with and Namalwa cell lines (Mestre-Escorihuela et al., the initial hypothesis (Hemann et al., 2004; Garrison 2007). Two of these cell lines showed inactivating et al., 2008). Loss of Noxa, however, did not accelerate mutations in the remaining allele. Mutational screening tumor formation, but only did so on a sensitized in 15 primary B-NHL biopsies, however, failed to reveal background, (haplo)insufficient for Puma (Michalak inactivating mutations but identified polymorphisms in et al., 2009). This observation suggests a modifier, 3 out of 15 promoter regions analysed. Analysis of rather than a tumor suppressor function for Noxa,at protein expression by immunohistochemistry on biopsy least in this model system. Even more puzzling, loss of samples from 367 B-NHL patients failed to reveal Noxa clearly delayed the onset of c-Myc-driven im- significant disease-associated changes, but resembled the mature preB cell, but had no impact on the development expression found in the nonmalignant counterpart of of mature IgM þ B-cell tumors. Although it is unclear these tumor cells (Mestre-Escorihuela et al., 2007), whether Noxa is induced on c-Myc-driven oncogenic suggesting that deregulation of Noxa expression is not stress in IgM þ B cells, one may speculate that in its significant in the pathogenesis of B-NHL. absence compensatory activation of other BH3-only proteins such as Puma or Bim may be more effective in removing (pre)malignant preB cells. Although this Noxa and the cellular response to anticancer agents explanation remains to be formerly tested, the finding Although a possible involvement of Noxa in cancer- itself suggests that under certain conditions BH3-only ogenesis is still under investigation, its importance as a proteins possess oncogenic potential (Michalak et al., drug target became evident when analysing the efficiency 2008). of the BH3-mimetic ABT-737. Using a nutation nuclear Following up on the idea that tumors with functional magnetic resonance structure-based approach to target p53 may have lost Noxa function, Lee et al. (2003) the BH3-binding groove of Bclxl, Abbott Laboratories investigated 78 colon adenocarcinomas, 53 advanced designed a cell permeating BH3 mimetic (Oltersdorf gastric adenocarcinomas, 83 non-small-cell lung can- et al., 2005) that efficiently antagonized Bcl2, Bclxl and cers, 76 breast carcinomas, 33 urinary bladder transi- Bclw, but was ineffective in killing tumor cells expres- tional cell carcinomas and 90 HCCs for their Noxa sing high levels of Mcl1 (van Delft et al., 2006; Tahir status. However, apart from one transitional cell carci- et al., 2007; Konopleva et al., 2008). Knockdown of noma of the urinary bladder, which carried a somatic Mcl1 helped to restore sensitivity to ABT-737 treatment mutation in the Noxa gene, all tumor samples contained in per se resistant colon and bladder cell lines (Lin et al., wt Noxa sequences. Moreover, when analysed in vitro 2007). So, to overcome ABT resistance development of a the observed mutation had no impact on the proapop- Noxa-like BH3 mimetic or a mimetic that also targets totic activity of Noxa (Lee et al., 2003). Comparison of Mcl1 is highly desirable (reviewed in more detail by Noxa mRNA expression levels in 94 colorectal adeno- Letai and Ni Chonghaile, in this issue on page S149). carcinoma and adjacent normal mucosa samples by As indicated Noxa is not only clearly relevant for p53- quantitative RT-PCR failed to reveal any pathology- induced apoptosis triggered by classical anticancer associated differences, nor were mutations found in the agents such as etoposide or g-irradiation but also for

Oncogene NOXA in apoptotic cell death C Ploner et al S91 tumor cell death triggered by the cyclin-dependent found in humans, activation of this BH3-only protein kinase inhibitor R-roscovitine, actually leading to appears critical for the cellular response to anticancer reduced Mcl1 protein levels (Hallaert et al., 2007), treatment regimens, such as g-irradiation, the applica- inhibition of histone deacetylases, triggering de novo tion of chemotherapeutic drugs, and also novel com- synthesis of Noxa (Inoue et al., 2007) or, somewhat pounds such as the BH3-mimetic ABT-737. Restoring counterintuitive, proteasome inhibition. In malignant or enhancing the tumor cell ability to increase Noxa for melanoma, but not primary melanocytes, as well as targeting Mcl1 and/or Bfl1/A1 expression or mimicking multiple myeloma, but not peripheral blood lympho- this function by small molecular compounds should cytes, nonselective inhibition of the proteasome by significantly increase treatment efficiency of current and Bortezomib/Velcade causes an increase in the expression novel treatment strategies against cancer. However, a of Noxa preceding tumor cell apoptosis. In contrast, more detailed knowledge on Noxa biology is required, Bim is induced by this treatment in normal as well because aberrant activation of this BH3-only protein in cancer cells, but the former are highly resistant to may well compromise the efficiency of the immune this drug (Fernandez et al., 2005; Qin et al., 2005). system, for example, by prompting memory T-cell Noteworthy, induction of Noxa is not simply a death, or by limiting the clonal expansion of antigen- consequence of impaired proteasomal degradation of activated T cells. Noxa/Mcl1 complexes but occurs also at the transcriptional level in a c-Myc-dependent manner and independent from the p53 status of the tumor cell (Nikiforov Conflict of interest et al., 2007). For a more detailed review on this topic see the chapter by M Soengas (in this issue). Taken The authors declare no conflict of interest. together, a correlation between proliferation potential of the cell and sensitivity toward Noxa becomes again apparent, suggesting a possible link between the Acknowledgements formation of Mcl1/Noxa complexes, cell-cycle progression and apoptosis induction. The work in our laboratories was supported by fellowships and grants from the Austrian Science Fund (FWF): Y212-B13 START, the Doctoral College MCBO, the SFB021, projects P18747 and P18571, the Association for International Cancer Conclusions Research (AICR), EU-FP7 (ApopTrain) and the Tyrolean Science Fund (TWF). We apologize to the many scientists in Although so far only a limited correlation between the this field whose excellent research was not cited but was only expression of Noxa and tumor development has been referred to indirectly through reviews.

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