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

Oncogene (2009) 27, S84–S92 & 2009 Macmillan Publishers Limited All rights reserved 0950-9232/09 $32.00 www.nature.com/onc REVIEW 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.

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