Noxa: at the tip of the balance between life and death

Keywords: apoptosis, BH3-only proteins, p53

Apoptosis induction and subcellular localization

Overexpression of mouse Noxa in HeLa cervical carcinoma and other cancer cells causes significant apoptosis, which is associated with its preferential localization to mitochondria (Oda et al., 2000). Apoptosis induced by Noxa requires a functional BH3 domain, and is accompanied by cytochrome c release and subsequent caspase activation. No binding of Noxa to proapoptotic Bax or Bak was reported so far, indicating that Noxa promotes Bax-mediated mitochondrial dysfunction indirectly by inhibiting antiapoptotic members of the Bcl2 family (Oda et al., 2000; Shibue et al., 2003).

Overexpressed Noxa protein localizes preferentially to mitochondria, as assessed by immunostaining (Oda et al., 2000). Interestingly, Noxa seems to require an intact BH3 domain not only for cell killing but also for its mitochondrial localization, suggesting that its association with this organelle may be secondary to its interaction with the prosurvival Bcl2-like molecules that it antagonizes. However, expression of a human GFP/Noxa fusion protein and truncation mutants thereof in HeLa cells revealed that aa 41–50 may constitute an bona fide mitochondrial targeting domain (MTD; Figure 1a; Seo et al., 2003). This sequence is conserved between human, mouse, rat and chicken Noxa, and a human Noxa construct lacking the MTD domain but still containing the BH3 domain was unable to induce cell death presumably due to mislocalization (Seo et al., 2003). However, because mitochondrial localization of mouse Noxa was also prevented by replacement mutations in the two BH3 domains (without altering MTD), both domains might be required for proper mitochondrial targeting. Alternatively, given the small size of human Noxa and the fact that this deletion starts immediately C-terminal to the BH3 domain, it may impair overall conformation and its ability to interact with Bcl2 and its homologues. This, rather than the destruction of a putative mitochondrial localization signal, might then cause inappropriate subcellular localization. In any rate, because cell biological data on endogenous Noxa protein expression are still lacking, its subcellular localization, before and after cell death induction, remains to be determined.

Molecular basis for the limited proapoptotic potential of Noxa

Although Noxa significantly contributes to apoptosis induced by p53 overexpression or DNA damage, in diverse cells such as Saos-2, BAF-3 pre-B cells or MEF, its apoptotic potential, when overexpressed on its own, appears to be quite variable (Oda et al., 2000; Schuler et al., 2003). Thus, Noxa overexpression per se entailed significant cell death in some systems (Oda et al., 2000), but in many other cell types, including MEF or human CEM T ALL cells, it proved poorly apoptotic (Chen et al., 2005; 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 Noxa shows the most restricted potential to neutralize prosurvival Bcl2 molecules. Although Bim and Puma can bind all survival Bcl2 proteins with comparable affinity, Noxa selectively binds to Mcl1 and, with lower affinity, Bfl1/A1 (Chen et al., 2005). The molecular basis for this selectivity depends on critical aa residues in the amphipathic α-helical BH3 domain contacting key residues in the hydrophobic groove of the antiapoptotic Bcl2-like prosurvival proteins, formed by parts of the BH1, BH2 and BH3 domains. As shown by high-resolution structure analyses of Bclxl/Bim complexes, binding of Bim to Bclxl depends on the interaction of hydrophobic pockets, located in the binding groove of Bclxl, with four conserved hydrophobic residues in the BH3 domain of Bim (Liu et al., 2003). Replacement of one or more of these critical residues interferes with the binding specificity and affinity of the whole BH3-only protein. This suggests that the binding properties of a given BH3 molecule depend only on the nature of its BH3 domain and hence exchange of the critical residues leads to altered binding specificity of the whole molecule. In fact, exchange of only two aa in the BH3 domain of human Noxa, that is, K35E and F32I (also called Noxa m3 mutant), increased its binding efficiency for Bclxl more than 100-fold (Chen et al., 2005). Moreover, exchange of the key contact residues of one BH3-only molecule into another transmits the binding characteristic of the donor BH3 molecule. When the Noxa BH3 domain was grafted into Bim (that usually shows high proapoptotic potential due to its capacity to bind all Bcl2 prosurvival molecules), the hybrid lost the cell death inducing potential of the BimS isoform that was used in this experiment. This correlated with the imposed binding capacity restricted to Mcl1. The same was true for BimS chimeras containing the BH3 domain of Bad, which bound Bclxl but no longer Mcl1 (Chen et al., 2005). Both mutants of BimS, however, can complement each other in promoting apoptosis. These experiments support the hypothesis that the nature of BH3 domains defines binding specificity and affinity of the whole BH3-only molecule and that mitochondrial cell death ensues when all types of Bcl2-like prosurvival molecules present in a given cell type are neutralized by BH3-only proteins. Alternative modes of BH3-only protein-mediated apoptosis are discussed in more detail by Letai and co-workers in this issue of Oncogene.

Investigations into the contribution of Noxa and Puma to p53-dependent apoptosis uncovered that NIH3T3 or MEF, transformed with adenovirus E1a, died in the presence of transgenic Noxa or Puma, whereas wt cells were protected against Noxa- but not Puma-induced cell death (Shibue et al., 2006). In E1a-transformed cells Noxa enhanced activation and oligomerization of Bax but not of Bak, whereas overexpression of Puma activated both (Shibue et al., 2006). This observation is supported by studies analysing mitochondrial targeting of Noxa in isolated mitochondria of mouse and rat liver cells where Noxa-mediated cytochrome c release was reported to occur independent from Bak oligomerization (Seo et al., 2003). Taken together, these observations suggest that Noxa kills cells in a mainly Bax-dependent but Bak-independent manner. However, this is somewhat surprising, given that Noxa has been repeatedly implicated in the disruption of Mcl1/Bak complexes, triggering Bak oligomerization during apoptosis induction in cells as diverse as MEF, HEK-293T, multiple myeloma and B-cell lymphomas (Willis et al., 2005; Inoue et al., 2007; Morales et al., 2008). This apparent discrepancy might be explained by the fact that oncogenic stress may not only impact expression of prosurvival molecules but also their subcellular localization as repeatedly observed for Bax, which is usually found in the cytoplasm of primary cells, but redistributes to mitochondria after transformation. Once at the mitochondria, Bax may be restrained efficiently by Mcl1, and Mcl1/Bax complexes may then be targeted by Noxa. However, it remains unclear why Noxa should selectively trigger oligomerization of Bax under these conditions, unless Mcl1 binds much more avidly to Bax than Bak, which may then be restrained by other prosurvival molecules such as Bclxl (Willis et al., 2005). Whatever the molecular mechanism may be, it is interesting to note that although primary MEF engage both Noxa and Puma for cell killing in response to UV irradiation, in E1a/ras-transformed cells Puma becomes dispensable for this process and Noxa accounts for all p53-induced death observed (Naik et al., 2007).

In summary, these data indicate that Noxa can engage Bax or Bak for apoptosis induction, but the engagement of these proteins depends on the cell type, cellular state and apoptotic stimulus applied. Furthermore, Noxa-induced cell death may become more relevant for apoptosis induction in malignant or highly proliferating versus primary or differentiated cells, suggesting its importance as tumor suppressor with therapeutic potential (see below).

Regulation of Mcl1 stability by Noxa

Given the fact that Noxa exclusively binds to Mcl1 and Bfl1/A1, cellular levels of these proteins determine susceptibility to Noxa-induced cell death of a given cell type. Inhibition or elimination of Mcl1 in response to cytotoxic signals is considered critical in cell death in a number of normal as well as malignant cells (Craig, 2002; Willis et al., 2005). However, targeting Mcl1 by transgenic overexpression of Noxa failed to induce significant cell death, for example, in immortalized MEF or NIH3T3 fibroblasts (Shibue et al., 2006), where survival depends on two or more Bcl2-like proteins such as Mcl1 and Bclxl (Willis et al., 2005). Consistently, Noxa needs to be functionally complemented by Bad, which targets Bcl2, Bclxl and Bclw, and overexpression of both BH3-only proteins induced cell death as efficiently as transgenic expression of Bim or Puma, able to neutralize all prosurvival Bcl2-like molecules (Chen et al., 2005).

An intriguing feature of Noxa is that it targets Mcl1 for proteasomal degradation. This event is considered to be a prerequisite for cell death in response to UV irradiation (Nijhawan et al., 2003), cytokine deprivation (Opferman et al., 2003), treatment with anticancer agents such as arsenic trioxide (Morales et al., 2008) or histone deacetylase inhibitors (Inoue et al., 2007). Although basal levels of Mcl1 appear to be regulated by the HECT- and BH3-domain-containing E3-ligase Mule/ARF-BP1 (Zhong et al., 2005), it is still unclear what regulates its degradation when complexed with Noxa, because the binding pocket is occupied with its BH3 domain. Interestingly, degradation of Mcl1 is not, as initially assumed, a prerequisite for apoptosis to occur. Interaction with Bim, for example, in response to GC treatment of acute lymphoblastic juvenile leukemia cells (Ploner et al., 2008), Puma overexpression (Mei et al., 2005) or arsenic trioxide treatment of myeloma cells (Morales et al., 2008), actually leads to its stabilization, presumably due to exclusion of Mule/ARF-BP1. The signature for degradation of the Mcl1/Noxa complex appears to be encoded in the C-terminal portion of the BH3 domain of Noxa (FRQKLL in human Noxa), as replacement of these residues with aa residues found in the same position in Bim (AYYARR) led to Mcl1 stabilization (Czabotar et al., 2007). Interestingly, although the Noxa BH3 domain in the context of Bim protein caused Mcl1 degradation, it failed to do so in the context of Bad, suggesting that residues outside the BH3 domain, functionally conserved in Noxa and Bim, contribute to this process (Czabotar et al., 2007).

Together all these studies reveal that Mcl1 degradation during cell death is uniquely associated with the formation of the Mcl1/Noxa complex whereas Mcl1/Bim or Mcl1/Puma interaction leads to its stabilization. This is of relevance considering the design of BH3 mimetics that may be able to bind to Mcl1, but may not necessarily promote its degradation. The selective (dis)advantage of Mcl1 degradation over stabilization during apoptosis induction, however, remains unclear at present. Identification of an Mcl1/Noxa-specific E3-ligase may allow some insight and rational explanation.

Although protein stability of the prosurvival Bcl2 homologue Bfl1/A1 also appears to be regulated by the proteasome and stability can be increased by Bim binding (Morales et al., 2008), no data are available addressing the possibility that A1/Noxa interactions may promote its degradation during apoptosis induction.

Noxa and the DNA-damage response

Two laboratories generated Noxa^−/− mice to study its function in vivo (Shibue et al., 2003; Villunger et al., 2003). Mice lacking Noxa show no overt phenotype, but more detailed analysis of stress responses confirmed a function for Noxa in p53-dependent, but also p53-independent cell death after DNA damage, albeit in a cell type and stimulus-dependent manner. Although hemopoietic cells from Puma^−/− mice resisted cell death induced by DNA damage or factor deprivation, cells from Noxa^−/− mice were normally sensitive to cell death induction. However, loss of either protein protected MEF cells from DNA-damage-induced apoptosis caused by etoposide treatment, subsequently also confirmed in Noxa^−/−Puma^−/− MEF that showed significantly greater resistance as cells from single knockout mice. Analysis of Noxa^−/−Puma^−/− mice revealed a major nonredundant function for Puma in apoptosis induced by γ-irradiation in MEF and most lymphocyte subsets, besides CD4⁺8⁺ thymocytes, where Puma and Noxa account for all cell death induced by p53. Similar observations were made in etoposide-treated MEF transformed with E1a (Michalak et al., 2008). Interestingly, as mentioned before, Noxa and Puma contribute equally to apoptosis induced by UV irradiation in primary MEF, but Noxa becomes the dominant cell death inducer in response to this stimulus after oncogenic transformation (Naik et al., 2007). Noteworthy, this pathway to apoptosis depends on c-Jun N-terminal kinase activation (Tournier et al., 2000) and Mcl1 degradation (Nijhawan et al., 2003) that is triggered selectively by binding of Noxa to Mcl1.

Shibue et al. examined the involvement of Noxa in the DNA damage response in vivo by analysing apoptosis of epithelial cells in the stem cell region of small intestinal crypts and cells in lamina propria after whole-body γ-irradiation. Noxa deficiency protected both cell types 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 apoptosis by loss of Noxa also led to a delayed onset of γ-irradiation disease-associated mortality. Noxa^−/− mice survived significantly longer than wt or p53^−/− animals. Taken together these results suggest that Noxa can be activated in response to γ-irradiation in a p53-dependent as well as -independent manner and may be prominent in the regulation of stem cell homeostasis, at least after DNA damage, as recently suggested also for Puma (see Review by Yu and Zhang, in this issue on page S71).

Possible functions for Noxa in the immune system

Although initial analysis of Noxa^−/− mice did not reveal any abnormalities in the hematopoietic system, Noxa appears to control maintenance of memory CD4⁺ T Th1/Th2 cell homeostasis. As discussed in Chapter 2, Yamashita et al. (2008) reported that the excessive Th2 memory T-cell death observed in mice defective in the PcG gene Bmi1 was at least in part due to lacking repression of Noxa gene expression by Bmi1. In support, ablation of Noxa expression on a Bmi1 hemizygous background rescued the observed defects in memory cell generation, consistent with a key role for Noxa in the maintenance of Th2 memory T-cell homeostasis.

Interestingly, in contrast to memory Th2 cells, induction of Noxa per se was inefficient in triggering significant cell death in proliferating T cells, presumably due to concomitant induction of prosurvival Bcl2-like molecules. It may, however, prime proliferating T cells for death when conditions no longer favor clonal expansion, for example, when growth factors or cytokines become limiting at the end of an immune response. This idea is also supported by the observations that Noxa is induced by the common γ-chain of the receptors for cytokines IL-7 and IL-15, able to promote cell division in the absence of antigenic stimulation in CD8⁺ T cells. However, loss of Noxa mildly interferes with IL-2-deprivation-induced death of activated T cells, albeit not as efficiently as loss of Bim or Puma (Bauer et al., 2006), as well as IL-15-deprivation-induced death of NK-cells, together with Bim (Huntington et al., 2007). Consistent with its role as a sentinel in proliferating cells, Jurkat T-cell clones carrying shRNAs-targeting Noxa showed better survival under conditions of metabolic stress due to glucose limitation. Subsequent biochemical analysis indicated that the high Noxa levels induced during the proliferative response contributed to cell death by neutralizing the prosurvival effects of Mcl1 and presumably also Bfl1/A1 (Alves et al., 2006). Therefore, we speculate that upregulation of Noxa in proliferating T cells might counterbalance enhanced expression of prosurvival molecules, thereby helping to restrain oncogenic transformation and/or autoimmunity. However, because Noxa-deficient mice develop neither of these pathologies, Noxa may perform these functions only in conjunction with other cell death regulators and/or control mechanisms.

Lessons from mice and humans

Mice lacking both or even only a single allele of p53 develop tumors with high incidence. In contrast, no spontaneous tumor development was observed in Noxa^−/− and even Noxa^−/−Puma^−/− double-deficient animals, up to an observation period of more than 1 year (Shibue et al., 2003; Michalak et al., 2008). This 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 cell-cycle arrest and apoptosis, have to be impaired simultaneously. Interestingly, studies with knock-in mice that express an apoptosis-defective, but cell-cycle inhibitory competent mutant of p53 (R172P) indicate an equally prominent role for these two p53 functions in the suppression of thymic lymphoma development (Liu et al., 2004). Whether loss of Noxa may impact tumor formation caused by DNA damage remains to be tested and it will be interesting to see if loss of Noxa on a p21-deficient background increases the rate of spontaneous tumorigenesis in animal lacking this CDK inhibitor.

So far, the connection between Noxa and tumor suppression is mainly circumstantial. In mice bearing a liver-specific deletion of c-Jun, the development of chemically induced hepatocellular carcinoma (HCC) is significantly delayed, revealing the oncogenic potential of c-Jun in an in vivo model (Eferl et al., 2003). c-Jun, a central component of the transcription factor Ap1, is a major regulator of proliferation and apoptosis in liver cells and loss of c-Jun in HCC leads to an increased number of apoptotic cells that display increased mRNA levels of p53 and Noxa (Eferl et al., 2003). Induction of Noxa in the absence of c-Jun seems to be specific, because the expression of other proapoptotic p53 target genes, such as Bax, Puma or Apaf-1, was unaffected. However, it was not formally demonstrated that the observed upregulation of Noxa mRNA was the consequence of p53 activation, leaving the possibility that Noxa and p53 may be independently activated by loss of c-Jun. Taken together, these observations indicate that c-Jun promotes tumor cell survival by inhibiting the proapoptotic action of p53, maybe by inhibiting expression of Noxa.

Most recently, the impact of loss of Noxa on c-Myc-driven B-cell lymphomagenesis has been studied. Aberrant expression of c-Myc leads to p53 activation and subsequent induction of apoptosis that may engage Noxa and/or Puma to restrain tumor formation. Eμ-Myc transgenic mice lacking Puma develop lymphomas significantly earlier than Eμ-Myc mice, consistent with the initial hypothesis (Hemann et al., 2004; Garrison et al., 2008). Loss of Noxa, however, did not accelerate tumor formation, but only did so on a sensitized background, (haplo)insufficient for Puma (Michalak et al., 2009). This observation suggests a modifier, rather than a tumor suppressor function for Noxa, at least in this model system. Even more puzzling, loss of Noxa clearly delayed the onset of c-Myc-driven immature preB cell, but had no impact on the development of mature IgM⁺ B-cell tumors. Although it is unclear whether Noxa is induced on c-Myc-driven oncogenic stress in IgM⁺ B cells, one may speculate that in its 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 explanation remains to be formerly tested, the finding itself suggests that under certain conditions BH3-only proteins possess oncogenic potential (Michalak et al., 2008).

Following up on the idea that tumors with functional p53 may have lost Noxa function, Lee et al. (2003) investigated 78 colon adenocarcinomas, 53 advanced gastric adenocarcinomas, 83 non-small-cell lung cancers, 76 breast carcinomas, 33 urinary bladder transitional cell carcinomas and 90 HCCs for their Noxa status. However, apart from one transitional cell carcinoma of the urinary bladder, which carried a somatic mutation in the Noxa gene, all tumor samples contained wt Noxa sequences. Moreover, when analysed in vitro the observed mutation had no impact on the proapoptotic activity of Noxa (Lee et al., 2003). Comparison of Noxa mRNA expression levels in 94 colorectal adenocarcinoma and adjacent normal mucosa samples by quantitative RT-PCR failed to reveal any pathology-associated differences, nor were mutations found in the Noxa coding sequence (Jansson et al., 2003). Interestingly, though, integrative genomic analysis of small-cell lung carcinoma revealed a correlation between amplification on 18q and sensitivity to the BH3-mimetic ABT-737 (Olejniczak et al., 2007). Resistance of cancer cells to ABT-737 is strongly associated with the relative expression of Mcl1, which it cannot antagonize (see below and review by Labi et al., 2008). The region of gain contains Bcl2 that can be potently blocked by ABT-737, but also Noxa that effectively neutralizes Mcl1 (van Delft et al., 2006), suggesting that 18q21–23, or maybe even Noxa expression alone, may be a clinically relevant predictor for responsiveness to Bcl2 family inhibitors (Olejniczak et al., 2007). Along that line, in B-CLL cells expression of Noxa protein was found frequently increased, when compared with tonsillar B cells (Mackus et al., 2005) and may be causal for the high sensitivity of these tumors to ABT-737 treatment (Oltersdorf et al., 2005).

Noxa expression levels, however, failed to correlate with overall or disease-free survival in human colon carcinoma, whereas relative expression levels of Bim or Puma did correlate well with these parameters (Sinicrope et al., 2008). Finally, Noxa was also found deleted in the Burkitt-derived cell line Elijah and loss of one allele was observed in the related Karpas 106, UPN1 and Namalwa cell lines (Mestre-Escorihuela et al., 2007). Two of these cell lines showed inactivating mutations in the remaining allele. Mutational screening in 15 primary B-NHL biopsies, however, failed to reveal inactivating mutations but identified polymorphisms in 3 out of 15 promoter regions analysed. Analysis of protein expression by immunohistochemistry on biopsy samples from 367 B-NHL patients failed to reveal significant disease-associated changes, but resembled the expression found in the nonmalignant counterpart of these tumor cells (Mestre-Escorihuela et al., 2007), suggesting that deregulation of Noxa expression is not significant in the pathogenesis of B-NHL.

Noxa and the cellular response to anticancer agents

Although a possible involvement of Noxa in cancerogenesis is still under investigation, its importance as a drug target became evident when analysing the efficiency of the BH3-mimetic ABT-737. Using a nutation nuclear magnetic resonance structure-based approach to target the BH3-binding groove of Bclxl, Abbott Laboratories designed a cell permeating BH3 mimetic (Oltersdorf et al., 2005) that efficiently antagonized Bcl2, Bclxl and Bclw, but was ineffective in killing tumor cells expressing high levels of Mcl1 (van Delft et al., 2006; Tahir et al., 2007; Konopleva et al., 2008). Knockdown of Mcl1 helped to restore sensitivity to ABT-737 treatment in per se resistant colon and bladder cell lines (Lin et al., 2007). So, to overcome ABT resistance development of a Noxa-like BH3 mimetic or a mimetic that also targets Mcl1 is highly desirable (reviewed in more detail by Letai and Ni Chonghaile, in this issue on page S149).

As indicated Noxa is not only clearly relevant for p53-induced apoptosis triggered by classical anticancer agents such as etoposide or γ-irradiation but also for tumor cell death triggered by the cyclin-dependent kinase inhibitor R-roscovitine, actually leading to reduced Mcl1 protein levels (Hallaert et al., 2007), inhibition of histone deacetylases, triggering de novo synthesis of Noxa (Inoue et al., 2007) or, somewhat counterintuitive, proteasome inhibition. In malignant melanoma, but not primary melanocytes, as well as multiple myeloma, but not peripheral blood lymphocytes, nonselective inhibition of the proteasome by Bortezomib/Velcade causes an increase in the expression of Noxa preceding tumor cell apoptosis. In contrast, Bim is induced by this treatment in normal as well in cancer cells, but the former are highly resistant to this drug (Fernandez et al., 2005; Qin et al., 2005). Noteworthy, induction of Noxa is not simply a consequence of impaired proteasomal degradation of 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 et al., 2007). For a more detailed review on this topic see the chapter by M Soengas (in this issue). Taken together, a correlation between proliferation potential of the cell and sensitivity toward Noxa becomes again apparent, suggesting a possible link between the formation of Mcl1/Noxa complexes, cell-cycle progression and apoptosis induction.

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