Evidence of a role for antizyme and antizyme inhibitor as regulators of human cancer

Antizyme and its endogenous antizyme inhibitor have recently emerged as prominent regulators of cell growth, transformation, centrosome duplication, and tumorigenesis. Antizyme was originally isolated as a negative modulator of the enzyme ornithine decarboxylase (ODC), an essential component of the polyamine biosynthetic pathway. Antizyme binds ODC and facilitates proteasomal ODC degradation. Antizyme also facilitates degradation of a set of cell cycle regulatory proteins, including cyclin D1, Smad1, and Aurora A kinase, as well as Mps1, a protein that regulates centrosome duplication. Antizyme has been reported to function as a tumor suppressor and to negatively regulate tumor cell proliferation and transformation. Antizyme inhibitor binds to antizyme and suppresses its known functions, leading to increased polyamine synthesis, increased cell proliferation, and increased transformation and tumorigenesis. Gene array studies show antizyme inhibitor to be amplified in cancers of the ovary, breast, and prostate. In this review, we summarize the current literature on the role of antizyme and antizyme inhibitor in cancer, discuss how the ratio of antizyme to antizyme inhibitor can influence tumor growth, and suggest strategies to target this axis for tumor prevention and treatment.     

抗酶的研究进展

抗酶（antizyme）是当细胞内多胺水平升高时刺激机体合成的一种小分子量调节蛋白,能特异性地与鸟氨酸脱羧酶（omithine decarboxylase,ODC）结合,经泛素非依赖途径被26S蛋白酶体降解,从而使多胺合成减少;抗酶还可以调节多胺转运,以稳定细胞内多胺水平。近年来随着生物技术的不断发展,对抗酶的认识也逐步深入,本文综述了抗酶家族、合成、作用及定位等方面的研究进展。     

The antizyme family: polyamines and beyond

The family of antizymes functions as regulators of polyamine homeostasis. They are a class of small, inhibitory proteins, whose expression is regulated by a unique ribosomal frameshift mechanism. They have been shown to inhibit cell proliferation and possess anti-tumor activity. Antizymes bind ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. They inhibit its enzymatic activity and promote the ubiquitin-independent degradation of ODC by the 26S proteasome. In addition, they also negatively regulate polyamine transport. Antizyme-mediated, ubiquitin-independent degradation of ODC is conserved from yeast to man. But recent data suggest that this degradation pathway might not be restricted to ODC alone and could involve newly discovered antizyme binding partners. Interestingly, antizyme proteins have been strictly preserved over a vast evolutionary timeframe. Antizymes consequently represent an important class of proteins that regulate cell growth and metabolism by a diverse set of mechanisms that include protein degradation, inhibition of enzyme activity, small molecule transport and other, potentially not yet discovered properties.     

Antizyme targets cyclin D1 for degradation. A novel mechanism for cell growth repression

Overproduction of the ornithine decarboxylase (ODC) regulatory protein ODC-antizyme has been shown to correlate with cell growth inhibition in a variety of different cell types. Although the exact mechanism of this growth inhibition is not known, it has been attributed to the effect of antizyme on polyamine metabolism. Antizyme binds directly to ODC, targeting ODC for ubiquitin-independent degradation by the 26 S proteasome. We now show that antizyme induction also leads to degradation of the cell cycle regulatory protein cyclin D1. We demonstrate that antizyme is capable of specific, noncovalent association with cyclin D1 and that this interaction accelerates cyclin D1 degradation in vitro in the presence of only antizyme, cyclin D1, purified 26 S proteasomes, and ATP. In vivo, antizyme up-regulation induced either by the polyamine spermine or by antizyme overexpression causes reduction of intracellular cyclin D1 levels. The antizyme-mediated pathway for cyclin D1 degradation is independent of the previously characterized phosphorylation- and ubiquitination-dependent pathway, because antizyme up-regulation induces the degradation of a cyclin D1 mutant (T286A) that abrogates its ubiquitination. We propose that antizyme-mediated degradation of cyclin D1 by the proteasome may provide an explanation for the repression of cell growth following antizyme up-regulation.     

Degrons of yeast and mammalian ornithine decarboxylase enzymes make potent combination for regulated targeted protein degradation

Elevation of polyamine levels in eukaryotes leads to rapid degradation of ornithine decarboxylase (ODC), the first enzyme of polyamine biosynthesis pathway. ODC in yeast (yODC) has two domains, the Nα/β domain consisting of α/β barrel domain (α/β) preceded by an overhang of 50 residues at its N-terminus (N50) and β sheet domain at its C-terminus. Two degradation determinant signals or degrons in yODC sequence, namely the N50 and the antizyme-binding element (AzBE) housed in the α/β domain, are responsible for its degradation by proteasomes. Antizyme (Az) induced under polyamine excess binds to AzBE and delivers ODC to proteasome, while the N50 threads the protein into proteasome. It was previously reported by us that the peptide Nα/β of yODC acts as an independent transplantable degron, whose action can be modulated with the help of antizyme by varying polyamine levels. Mammalian ODC (mODC), in spite of its 40% sequence homology with yODC, is devoid of N50 of yODC and instead sports a C-terminal tail of 37 residues (CmODC). CmODC acts as an independent transplantable degron with no equivalent in yODC. The present study investigates the merits of employing the two degrons Nα/β and CmODC together for targeted protein degradation by expressing them in a chimeric fusion with green fluorescent protein (GFP). Our results establish that under the regulation of antizyme, the signals Nα/β and CmODC acting together enhance degradation better than either degron in isolation. The combination of Nα/β and CmODC can be employed to study the function of novel proteins through their rapid removal.

     

Recurrent Emergence of Catalytically Inactive Ornithine Decarboxylase Homologous Forms That Likely Have Regulatory Function

Ornithine decarboxylase (ODC) catalyzes the first and rate limiting step in the biosynthesis of polyamines in most eukaryotes. Because polyamines have pleiotropic and often dramatic effects on cellular processes at both high and low concentrations, ODC expression is tightly controlled. ODC is regulated by a family of polyamine-induced proteins, antizymes, which bind to, and inactivate it. In mammals, and apparently most vertebrates, antizymes are in turn antagonized by proteins called antizyme inhibitors. Antizyme inhibitors are homologs of ODC that have lost their decarboxylation activity but have retained their ability to bind antizyme, in most cases even more tightly than ODC. We present a phylogenetic analysis of over 200 eukaryotic homologs of ODC and evaluate their potential to be either true ODCs or catalytically inactive proteins that might be analogs of the previously identified antizyme inhibitors. This analysis yielded several orthologous groups of putative novel antizyme inhibitors each apparently arising independently. In the process we also identify previously unrecognized ODC paralogs in several evolutionary branches, including a previously unrecognized ODC paralog in mammals, and we evaluate their biochemical potential based on their pattern of amino acid conservation.     

Ornithine decarboxylase-antizyme is rapidly degraded through a mechanism that requires functional ubiquitin-dependent proteolytic activity.

Antizyme is a polyamine-induced cellular protein that binds to ornithine decarboxylase (ODC), and targets it to rapid ubiquitin-independent degradation by the 26S proteasome. However, the metabolic fate of antizyme is not clear. We have tested the stability of antizyme in mammalian cells. In contrast with previous studies demonstrating stability in vitro in a reticulocyte lysate-based degradation system, in cells antizyme is rapidly degraded and this degradation is inhibited by specific proteasome inhibitors. While the degradation of ODC is stimulated by the presence of cotransfected antizyme, degradation of antizyme seems to be independent of ODC, suggesting that antizyme degradation does not occur while presenting ODC to the 26S proteasome. Interestingly, both species of antizyme, which represent initiation at two in-frame initiation codons, are rapidly degraded. The degradation of both antizyme proteins is inhibited in ts20 cells containing a thermosensitive ubiquitin-activating enzyme, E1. Therefore we conclude that in contrast with ubiquitin-independent degradation of ODC, degradation of antizyme requires a functional ubiquitin system.     

Ornithine decarboxylase antizyme inhibitor 2 regulates intracellular vesicle trafficking

Antizyme inhibitor 1 (AZIN1) and 2 (AZIN2) are proteins that activate ornithine decarboxylase (ODC), the key enzyme of polyamine biosynthesis. Both AZINs release ODC from its inactive complex with antizyme (AZ), leading to formation of the catalytically active ODC. The ubiquitously expressed AZIN1 is involved in cell proliferation and transformation whereas the role of the recently found AZIN2 in cellular functions is unknown. Here we report the intracellular localization of AZIN2 and present novel evidence indicating that it acts as a regulator of vesicle trafficking. We used immunostaining to demonstrate that both endogenous and FLAG-tagged AZIN2 localize to post-Golgi vesicles of the secretory pathway. Immuno-electron microscopy revealed that the vesicles associate mainly with the trans-Golgi network (TGN). RNAi-mediated knockdown of AZIN2 or depletion of cellular polyamines caused selective fragmentation of the TGN and retarded the exocytotic release of vesicular stomatitis virus glycoprotein. Exogenous addition of polyamines normalized the morphological changes and reversed the inhibition of protein secretion. Our findings demonstrate that AZIN2 regulates the transport of secretory vesicles by locally activating ODC and polyamine biosynthesis.     

Functional Roles of the Dimer-Interface Residues in Human Ornithine Decarboxylase

Ornithine decarboxylase (ODC) catalyzes the decarboxylation of ornithine to putrescine and is the rate-limiting enzyme in the polyamine biosynthesis pathway. ODC is a dimeric enzyme, and the active sites of this enzyme reside at the dimer interface. Once the enzyme dissociates, the enzyme activity is lost. In this paper, we investigated the roles of amino acid residues at the dimer interface regarding the dimerization, protein stability and/or enzyme activity of ODC. A multiple sequence alignment of ODC and its homologous protein antizyme inhibitor revealed that 5 of 9 residues (residues 165, 277, 331, 332 and 389) are divergent, whereas 4 (134, 169, 294 and 322) are conserved. Analytical ultracentrifugation analysis suggested that some dimer-interface amino acid residues contribute to formation of the dimer of ODC and that this dimerization results from the cooperativity of these interface residues. The quaternary structure of the sextuple mutant Y331S/Y389D/R277S/D332E/V322D/D134A was changed to a monomer rather than a dimer, and the K _d value of the mutant was 52.8 µM, which is over 500-fold greater than that of the wild-type ODC (ODC_WT). In addition, most interface mutants showed low but detectable or negligible enzyme activity. Therefore, the protein stability of these interface mutants was measured by differential scanning calorimetry. These results indicate that these dimer-interface residues are important for dimer formation and, as a consequence, are critical for enzyme catalysis.     

Ornithine decarboxylase antizyme in kidneys of male and female mice.

Antizyme, a protein inhibitor of ornithine decarboxylase (ODC), was shown to be induced in mouse kidney by repeated injection of putrescine. Antizyme was also present as a complex with ODC in the kidney of untreated mouse. The amount of the renal ODC-antizyme complex was 3-fold higher in male mice than in female mice. On the contrary, the proportion of ODC present as a complex with antizyme was 24-fold higher in females than in males, and the decay of renal ODC activity after cycloheximide treatment was about 5-fold more rapid in females than in males. Administration of testosterone to female mice, a procedure known to prolong the half-life of renal ODC, increased both ODC activity and the content of ODC-antizyme complex, but decreased the antizyme/ODC ratio in the kidney. These results are consistent with the previous observation in HTC cells that the decay rate of ODC activity in the presence of cycloheximide correlated well with the proportion of ODC present as a complex with antizyme, suggesting the ubiquitous role of antizyme in ODC degradation.     

Regulated degradation of ornithine decarboxylase requires interaction with the polyamine-inducible protein antizyme.

Intracellular degradation of vertebrate ornithine decarboxylase (ODC) is accelerated by polyamines, the products of the pathway controlled by ODC. Antizyme, a reversible, tightly binding protein inhibitor of ODC activity, is believed to be involved in this process. Mouse and Trypanosoma brucei ODCs are structurally similar, but the trypanosome enzyme, unlike that of the mouse, is not regulated by intracellular polyamines when expressed in hamster cells (L. Ghoda, D. Sidney, M. Macrae, and P. Coffino, Mol. Cell. Biol. 12:2178-2185, 1992). We found that mouse ODC interacts with antizyme in vitro but trypanosome ODC does not. To localize the region necessary for binding, we made a series of enzymatically active chimeric mouse-trypanosome ODCs and tested them for antizyme interaction. Replacing residues 117 to 140 within the 461-amino-acid mouse ODC sequence with the equivalent region of trypanosome ODC disrupted both antizyme binding and in vivo regulation. Formation of an antizyme-ODC complex is therefore required for regulated degradation.     

Stable siRNA-mediated silencing of antizyme inhibitor: regulation of ornithine decarboxylase activity

Ornithine decarboxylase (ODC) is the rate-limiting enzyme involved in the biosynthesis of polyamines essential for cell growth and differentiation. Aberrant upregulation of ODC, however, is widely believed to be a contributing factor in tumorigenesis. Antizyme is a major regulator of ODC, inhibiting ODC activity through the formation of complexes and facilitating degradation of ODC by the 26S proteasome. Moreover, the antizyme inhibitor (AZI) serves as another factor in regulating ODC, by binding to antizyme and releasing ODC from ODC-antizyme complexes. In our previous report, we observed elevated AZI expression in tumor specimens. Therefore, to evaluate the role of AZI in regulating ODC activity in tumors, we successfully down-regulated AZI expression using RNA interference technology in A549 lung cancer cells expressing high levels of AZI. Two AZI siRNAs, which were capable to generate a hairpin dsRNA loop targeting AZI, could successively decrease the expression of AZI. Using biological assays, antizyme activity increased in AZI-siRNA-transfected cells, and ODC levels and activity were reduced as well. Moreover, silencing AZI expression decreased intracellular polyamine levels, reduced cell proliferation, and prolonged population doubling time. Our results directly demonstrate that downregulation of AZI regulates ODC activity, intracellular polyamine levels, and cell growth through regulating antizyme activity. This study also suggests that highly expressed AZI may be partly responsible for increased ODC activity and cellular transformation.     

Properties and fluctuations in vivo of rat liver antizyme inhibitor.

Antizyme inhibitor was highly purified from rat liver by using affinity chromatography. It has some structural resemblance to ornithine decarboxylase (ODC), as judged from Mr, immunoreactivity and reversible binding with antizyme. However, unlike hepatic amounts of ODC and ODC-antizyme complex, that of antizyme inhibitor did not show much fluctuation upon putrescine treatment, whereas it decreased as rapidly as ODC decay in the presence of cycloheximide. These results suggested that antizyme inhibitor is an independent regulatory protein rather than a derivative of ODC. Changes in hepatic amounts of antizyme inhibitor, antizyme and ODC upon feeding suggested that antizyme inhibitor may play a role in ODC regulation by trapping antizyme and thereby suppressing ODC degradation. A monoclonal antibody to rat liver antizyme inhibitor was obtained. This antibody was shown to be utilizable for a simple assay of antizyme-inhibitor activity in tissue extracts.     

Transgenic mouse models for studies of the role of polyamines in normal,hypertrophic and neoplastic growth

Transgenic mice expressing proteins altering polyamine levels in a tissue-specific manner have considerable promise for evaluation of the roles of polyamines in normal, hypertrophic and neoplastic growth. This short review summarizes the available transgenic models. Mice with large increases in ornithine decarboxylase (ODC), S-adenosylmethionine decarboxylase or antizyme, a protein regulating polyamine synthesis by reducing polyamine transport and ODC in the heart, have been produced using constructs in which the protein is expressed from the alpha -myosin heavy-chain promoter. These mice are useful in studies of the role of polyamines in hypertrophic growth. Expression from keratin promoters has been used to target increased synthesis of ODC, spermidine/spermine-N(1)-acetyltransferase (SSAT) and antizyme in the skin. Such expression of ODC leads to an increased sensitivity to chemical and UV carcinogenesis. Expression of antizyme inhibits carcinogenesis in skin and forestomach. Expression of SSAT increases the incidence of skin papillomas and their progression to carcinomas in response to a two-stage carcinogenesis protocol. These results establish the importance of polyamines in carcinogenesis and neoplastic growth and these transgenic mice will be valuable experimental tools to evaluate the importance of polyamines in mediating responses to oncogenes and studies of cancer chemoprevention.     

Subcellular localization of antizyme inhibitor 2 in mammalian cells: Influence of intrinsic sequences and interaction with antizymes

Ornithine decarboxylase (ODC) and the antizyme inhibitors (AZIN1 and AZIN2), regulatory proteins of polyamine levels, are antizyme‐binding proteins. Although it is widely recognized that ODC is mainly a cytosolic enzyme, less is known about the subcellular distribution of AZIN1 and AZIN2. We found that these proteins, which share a high degree of homology in their amino acid sequences, presented differences in their subcellular location in transfected mammalian cells. Whereas ODC was mainly present in the cytosol, and AZIN1 was found predominantly in the nucleus, interestingly, AZIN2 was located in the ER‐Golgi intermediate compartment (ERGIC) and in the cis‐Golgi network, apparently not related to any known cell‐sorting sequence. Our results rather suggest that the N‐terminal region may be responsible for this particular location, since its deletion abrogated the incorporation of the mutated AZIN2 to the ERGIC complex and, on the other hand, the substitution of this sequence for the corresponding sequence in ODC, translocated ODC from cytosol to the ERGIC compartment. Furthermore, the coexpression of AZIN2 with any members of the antizyme family induced a shift of AZIN2 from the ERGIC to the cytosol. These findings underline the complexity of the AZs/AZINs regulatory system, supporting early evidence that relates these proteins with additional functions other than regulating polyamine homeostasis. J. Cell. Biochem. 107: 732–740, 2009. © 2009 Wiley‐Liss, Inc.     

Antizyme to ornithine decarboxylase is present in the liver of starved rats.

Antizyme to ornithine decarboxylase (ODC) and ODC-antizyme complex were both present in liver cytosols of starved rats. The antizyme was identified by its molecular weight, kinetic properties, formation of a complex with ODC, and reversal of its inhibition by antizyme inhibitor. The average amount of antizyme in liver cytosols of starved rats was 0.1 unit/mg of protein, roughly corresponding to basal hepatic ODC activity in rats fed ad libitum. The presence of ODC-antizyme complex was detected by using antizyme inhibitor. These results indicate that antizyme participates in the regulation of ODC activity in vivo under physiological conditions.     

Existence of antizyme and ornithine decarboxylase-antizyme complex in RK13 kidney cells

It has been reported that 'antizyme', a protein inhibitor of ornithine decarboxylase (ODC) induced by its product, is not found in rat or mouse kidney. We determined whether antizyme was present in rabbit kidney cells (RK13) in culture. Antizyme could be induced in these cells by putrescine treatment, a substantial portion being in the particulate fraction in contrast with hepatic antizyme. Furthermore, ODC-antizyme complex was present even in untreated cells. Pretreatment of cells with putrescine increased the relative amount of ODC-antizyme complex and accelerated decay of ODC. These results support the ubiquitous existence of antizyme and its role in ODC degradation.     

Inducible expression of antizyme 1 in prostate cancer cell lines after lentivirus mediated gene transfer

The prostate has the highest level of polyamines among all tissues, and it is the only tissue in which polyamines are purposely synthesized for export. It has been suggested that the high local polyamine concentrations suppress cell growth of primary prostatic carcinomas and that this growth control is lost when cancer cells metastasize. It has also been shown that the sensitivity to polyamine-induced growth arrest correlates with antizyme induction in prostate carcinoma cell lines. In this study, we evaluated the sensitivity of poorly metastatic (LNCaP) and highly metastatic (DU145) prostate cancer cell lines to conditional antizyme 1 over-expression. Antizyme 1 induction resulted in a marked loss of ODC activity and polyamine uptake in both cell lines. However, the proliferation of LNCaP cells was repressed by antizyme 1 induction, whereas the proliferation of DU 145 cells was not affected. Neither cell line showed any reduction in polyamine pools after manipulation nor did polyamine addition into the medium save the LNCaP cells from the growth retardation. The growth inhibition of LNCaP cells was accompanied by accumulation of cells in the G1 phase and depletion of cyclin E1 protein. These results confirm that different prostate cancer cell lines show diverse sensitivities to antizyme 1 which may not be directly polyamine related. The high gene transfer capacity of the used lentiviral vector makes the present approach a useful tool to study the therapeutic potential of antizyme 1 both in cell cultures and experimental animals.