肿瘤中甲硫氨酸代谢及其相关基因的表达调控

甲硫氨酸(methionine)作为人体必需氨基酸,生理功能多样,在肿瘤代谢重编程过程中具有重要意义。研究发现,多种肿瘤细胞对外源性甲硫氨酸存在依赖性,该效应被称为Hoffman效应。在人体内,甲硫氨酸经甲硫氨酸循环代谢,参与一碳单位代谢、叶酸循环,以及多胺、谷胱甘肽、半胱氨酸和核苷酸等多种物质的合成。肿瘤中常出现甲硫氨酸代谢的改变,并伴随甲硫氨酸代谢相关酶基因表达的异常,其中以甲硫氨酸腺苷转移酶(methionine adenosyltransferase, MAT)相关基因表达改变及甲硫腺苷磷酸化酶(methylthioadenosine phosphorylase,MTAP)基因的缺失最为常见,二者可分别引起甲硫氨酸循环及甲硫氨酸补救合成途径的异常,进而导致甲基供体S-腺苷甲硫氨酸(S-adenosylmethionine, SAM)的生成减少和甲硫腺苷(methylthioadenosine, MTA)的堆积,其与肿瘤的发生、发展和转移等活动密切相关。由甲硫氨酸的代谢改变和代谢酶的基因表达异常,分别衍生出2种不同的治疗策略,即甲硫氨酸限制疗法和靶向治疗。本文将从甲硫氨酸代谢出发,阐述肿瘤中甲硫氨酸依懒性、肿瘤细胞MAT和MTAP相关基因的表达调控,并概述甲硫氨酸相关肿瘤治疗方案的新进展与新问题,为肿瘤治疗方案的进一步探索提供新思路。     

Functional Identification of APIP as Human mtnB,a Key Enzyme in the Methionine Salvage Pathway

The methionine salvage pathway is widely distributed among some eubacteria, yeast, plants and animals and recycles the sulfur-containing metabolite 5-methylthioadenosine (MTA) to methionine. In eukaryotic cells, the methionine salvage pathway takes place in the cytosol and usually involves six enzymatic activities: MTA phosphorylase (MTAP, EC 2.4.2.28), 5′-methylthioribose-1-phosphate isomerase (mtnA, EC 5.3.1.23), 5′-methylthioribulose-1-phosphate dehydratase (mtnB, EC: 4.2.1.109), 2,3-dioxomethiopentane-1-phosphate enolase/phosphatase (mtnC, EC 3.1.3.77), aci-reductone dioxygenase (mtnD, EC 1.13.11.54) and 4-methylthio-2-oxo-butanoate (MTOB) transaminase (EC 2.6.1.-). The aim of this study was to complete the available information on the methionine salvage pathway in human by identifying the enzyme responsible for the dehydratase step. Using a bioinformatics approach, we propose that a protein called APIP could perform this role. The involvement of this protein in the methionine salvage pathway was investigated directly in HeLa cells by transient and stable short hairpin RNA interference. We show that APIP depletion specifically impaired the capacity of cells to grow in media where methionine is replaced by MTA. Using a Shigella mutant auxotroph for methionine, we confirm that the knockdown of APIP specifically affects the recycling of methionine. We also show that mutation of three potential phosphorylation sites does not affect APIP activity whereas mutation of the potential zinc binding site completely abrogates it. Finally, we show that the N-terminal region of APIP that is missing in the short isoform is required for activity. Together, these results confirm the involvement of APIP in the methionine salvage pathway, which plays a key role in many biological functions like cancer, apoptosis, microbial proliferation and inflammation.     

The structure of human 5'-deoxy-5'-methylthioadenosine phosphorylase at 1.7 A resolution provides insights into substrate binding and catalysis.

BACKGROUND: 5'-Deoxy-5'-methylthioadenosine phosphorylase (MTAP) catalyzes the reversible phosphorolysis of 5'-deoxy-5'-methylthioadenosine (MTA) to adenine and 5-methylthio-D-ribose-1-phosphate. MTA is a by-product of polyamine biosynthesis, which is essential for cell growth and proliferation. This salvage reaction is the principle source of free adenine in human cells. Because of its importance in coupling the purine salvage pathway to polyamine biosynthesis MTAP is a potential chemotherapeutic target. RESULTS: We have determined the crystal structure of MTAP at 1.7 A resolution using multiwavelength anomalous diffraction phasing techniques. MTAP is a trimer comprised of three identical subunits. Each subunit consists of a single alpha/beta domain containing a central eight-stranded mixed beta sheet, a smaller five-stranded mixed beta sheet and six alpha helices. The native structure revealed the presence of an adenine molecule in the purine-binding site. The structure of MTAP with methylthioadenosine and sulfate ion soaked into the active site was also determined using diffraction data to 1.7 A resolution. CONCLUSIONS: The overall quaternary structure and subunit topology of MTAP are similar to mammalian purine nucleoside phosphorylase (PNP). The structures of the MTAP-ligand complexes provide a map of the active site and suggest possible roles for specific residues in substrate binding and catalysis. Residues accounting for the differences in substrate specificity between MTAP and PNP are also identified. Detailed information about the structure and chemical nature of the MTAP active site will aid in the rational design of inhibitors of this potential chemotherapeutic target. The MTAP structure represents the first structure of a mammalian PNP that is specific for 6-aminopurines.     

Methylthioadenosine phosphorylase gene expression is impaired in human liver cirrhosis and hepatocarcinoma

Methylthioadenosine phosphorylase (MTAP) is a key enzyme in the methionine and adenine salvage pathways. In mammals, the liver plays a central role in methionine metabolism, and this essential function is lost in the progression from liver cirrhosis to hepatocarcinoma. Deficient MTAP gene expression has been recognized in many transformed cell lines and tissues. In the present work, we have studied the expression of MTAP in human and experimental liver cirrhosis and hepatocarcinoma. We observe that MTAP gene expression is significantly reduced in human hepatocarcinoma tissues and cell lines. Interestingly, MTAP gene expression was also impaired in the liver of CCl4-cirrhotic rats and cirrhotic patients. We provide evidence indicating that epigenetic mechanisms, involving DNA methylation and histone deacetylation, may play a role in the silencing of MTAP gene expression in hepatocarcinoma. Given the recently proposed tumor suppressor activity of MTAP, our observations can be relevant to the elucidation of the molecular mechanisms of multistep hepatocarcinogenesis.     

Polyamine depletion by ODC-AdoMetDC antisense adenovirus impairs human colorectal cancer growth and invasion in vitro and in vivo

BACKGROUND: Polyamine biosynthesis is controlled primarily by ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC). Polyamine concentrations are elevated in colorectal cancer. Depletion of polyamine content in colorectal cancer by chemotherapy is related to tumor regression and impaired tumorigenicity. The current study evaluates the therapeutic effects of antisense ODC and AdoMetDC sequences on colorectal cancer in vitro and in vivo. METHODS: Antisense ODC and AdoMetDC sequences were cloned into an adenoviral vector (Ad-ODC-AdoMetDCas). The human colon cancer cell lines, HT-29 and Caco-2, were infected with Ad-ODC-AdoMetDCas as well as with control vector. Viable cell counting, determination of polyamine concentrations, cell cycle analysis, and Matrigel invasion assays were performed in order to assess properties of tumor growth and invasiveness. Furthermore, the antitumor effects of Ad-ODC-AdoMetDCas were also evaluated in vivo in a nude mouse tumor model. RESULTS: Our study demonstrated that adenovirus-mediated ODC and AdoMetDC antisense expression inhibits tumor cell growth through a blockade of the polyamine synthesis pathway. This inhibitory effect cannot be reversed by the administration of putrescine. Tumor cells were arrested at the G1 phase of the cell cycle after gene transfer and had reduced invasiveness. The adenovirus also induced tumor regression in established tumors in nude mice. CONCLUSIONS: Our study suggests that Ad-ODC-AdoMetDCas has antitumor activity and therapeutic potential for the treatment of colorectal cancer.     

Methylthioadenosine and polyamine biosynthesis in a Saccharomyces cerevisiae meu1delta mutant

As part of our studies on polyamine biosynthesis in yeast, the metabolism of methylthioadenosine was studied in a mutant that lacks methylthioadenosine phosphorylase (meu1delta). The nucleoside accumulates in this mutant and is mainly excreted into the culture medium. Intracellular accumulation of the nucleoside is enough to account for the inhibition of spermidine synthase and thus to indirectly regulate the polyamine content of the meu1delta cells. By comparing the results with this mutant with a meu1delta spe2delta mutant that cannot synthesize spermidine or spermine, we showed that >98% of methylthioadenosine is produced as a byproduct of polyamine synthesis (i.e., from decarboxylated S-adenosylmethionine). In contrast, in MEU1+ SPE2+ cells methylthioadenosine does not accumulate and is metabolized through the methionine salvage pathway. Using a met15delta mutant we show that this pathway (i.e., involving polyamine biosynthesis and methylthioadenosine metabolism) is a significant factor in the metabolism of methionine, accounting for 15% of the added methionine.     

Expression and Function of Methylthioadenosine Phosphorylase in Chronic Liver Disease

To study expression and function of methylthioadenosine phosphorylase (MTAP), the rate-limiting enzyme in the methionine and adenine salvage pathway, in chronic liver disease.

Design

MTAP expression was analyzed by qRT-PCR, Western blot and immunohistochemical analysis. Levels of MTA were determined by liquid chromatography-tandem mass spectrometry.

Results

MTAP was downregulated in hepatocytes in murine fibrosis models and in patients with chronic liver disease, leading to a concomitant increase in MTA levels. In contrast, activated hepatic stellate cells (HSCs) showed strong MTAP expression in cirrhotic livers. However, also MTA levels in activated HSCs were significantly higher than in hepatocytes, and there was a significant correlation between MTA levels and collagen expression in diseased human liver tissue indicating that activated HSCs significantly contribute to elevated MTA in diseased livers. MTAP suppression by siRNA resulted in increased MTA levels, NFκB activation and apoptosis resistance, while overexpression of MTAP caused the opposite effects in HSCs. The anti-apoptotic effect of low MTAP expression and high MTA levels, respectively, was mediated by induced expression of survivin, while inhibition of survivin abolished the anti-apoptotic effect of MTA on HSCs. Treatment with a DNA demethylating agent induced MTAP and reduced survivin expression, while oxidative stress reduced MTAP levels but enhanced survivin expression in HSCs.

Conclusion

MTAP mediated regulation of MTA links polyamine metabolism with NFκB activation and apoptosis in HSCs. MTAP and MTAP modulating mechanisms appear as promising prognostic markers and therapeutic targets for hepatic fibrosis.     

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.     

Mechanisms of degradation of the fungal ornithine decarboxylase

Ornithine decarboxylase (ODC) is the first enzyme in polyamine biosynthesis in numerous living organisms, from bacteria to mammalian cells. Its control is under negative feedback regulation by the end products of the pathway. In dimorphic fungi, ODC activity and therefore polyamine concentrations are related to the morphogenetic process. From the fission yeast Schizosaccharomyces pombe to human, polyamines induce antizyme synthesis which in turn inactivates ODC. This is hydrolyzed by the 26S proteasome without ubiquitination. The regulatory mechanism of antizyme on polyamines is conserved, although to date no antizyme homology has been identified in some fungal species. The components that are responsible for regulating polyamine levels in cells and the current knowledge of ODC regulation in dimorphic fungi are presented in this review. ODC degradation is of particular interest because inhibitors of this pathway may lead to the discovery of novel antifungal drugs.     

Chemical genetic screening for compounds that preferentially inhibit growth of methylthioadenosine phosphorylase (MTAP)-deficient Saccharomyces cerevisiae

Methylthioadenosine phosphorylase (MTAP), a key enzyme in the methionine salvage pathway, is inactivated in a variety of human cancers. Since all human tissues express MTAP, it would be of potential interest to identify compounds that selectively inhibit the growth of MTAP-deficient cells. To determine if MTAP inactivation could be targeted, the authors have performed a differential chemical genetic screen in isogenic MTAP(+) and MTAP(-) Saccharomyces cerevisiae. A low molecular weight compound library containing 30,080 unique compounds was screened for those that selectively inhibit growth of MTAP(-) yeast using a differential growth assay. One compound, containing a 1,3,4-thiadiazine ring, repeatedly showed a differential dose response, with MTAP(-) cells exhibiting a 4-fold shift in IC(50) compared to MTAP(+) cells. Several structurally related derivatives of this compound also showed enhanced growth inhibition in MTAP(-) yeast. These compounds were also examined for growth inhibition of isogenic MTAP(+) and MTAP(-) HT1080 fibrosarcoma cells, and 4 of the 5 compounds exhibited evidence of modest but significant increased potency in MTAP(-) cells. In summary, these studies show the feasibility of differential growth screening technology and have identified a novel class of compounds that can preferentially inhibit growth of MTAP(-) cells.     

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.     

Ornithine Decarboxylase Activity in Cerebral Post-Ischemic Reperfusion Damage: Effect of Methionine Sulfoximine

Excessive activation of glutamate receptors via the N-methyl-D-aspartate (NMDA) subtype appears to play a role in the sequence of cellular events which lead to irreversible ischemic damage to neurons. Furthermore, NMDA receptor activation induces a stimulation of ornithine decarboxylase (ODC), the rate-limiting enzyme for polyamine (PA) biosynthesis. In order to better understand the role of PA we have measured ODC activity and the effect of methionine sulfoximine (MSO), a molecule able to stimulate ODC, on a model of transient cerebral ischemia. There was a significant increase in ODC activity in the rat cerebral cortex during post-ischemic reperfusion. The treatment with MSO induced a significant decrease in cerebral glutamine synthetase activity accompained by a marked increase in ODC activity. In MSO-pretreated rats there was a significant decrease in the survival rate when compared to untreated ischemic rats.     

Emerging concepts in targeting the polyamine metabolic pathway in epithelial cancer chemoprevention and chemotherapy

The polyamines are important molecules governing cell proliferation, survival and apoptosis. Consistent with their elevated levels in cancer, they have been shown to mediate tumor promotion and progression. Cellular and tissue polyamine pools and metabolic flux are regulated by a number of processes. Neoplastic transformation is accompanied with an increase in biosynthesis, decreased catabolism and elevated uptake of exogenous polyamines. Effective strategies for cancer chemoprevention and chemotherapy, targeting the polyamine metabolic pathway will likely require a combination of agents acting at multiple sites of this pathway. Genetic variability affecting expression of the ornithine decarboxylase gene suggests an association between ODC expression and cancer risk, and prediction of response to treatment in certain epithelial cancers.     

Elevated ornithine decarboxylase activity promotes skin tumorigenesis by stimulating the recruitment of bulge stem cells but not via toxic polyamine catabolic metabolites

Elevated expression of ornithine decarboxylase (ODC), the regulatory enzyme in polyamine biosynthesis, targeted to the epidermis is sufficient to promote skin tumor development following a single subthreshold dose of dimethylbenz(a)anthracene (DMBA). Since skin tumor promotion involves recruitment of hair follicle bulge stem cells harboring genetic lesions, we assessed the effect of increased epidermal ODC on recruitment of bulge stem cells in ODC-ER transgenic mice in which ODC activity is induced de novo in adult skin with 4-hydroxytamoxifen (4OHT). Bromodeoxyuridine-pulse labeling and use of K15.CrePR1;R26R;ODC-ER triple transgenic mice demonstrated that induction of ODC activity is sufficient to recruit bulge stem cells in quiescent skin. Because increased ODC activity not only stimulates proliferation but also increases reactive oxygen species (ROS) generation via subsequent induction of polyamine catabolic oxidases, we used an inhibitor of polyamine catabolic oxidase activity, MDL72527, to investigate whether ROS generation by polyamine catabolic oxidases contributes to skin tumorigenesis in DMBA-initiated ODC-ER transgenic skin. Newborn ODC-ER transgenic mice and their normal littermates were initiated with a single topical dose of DMBA. To assess tumor development originating from dormant bulge stem cells that possess DMBA-initiated mutations, epidermal ODC activity was induced in ODC-ER mice with 4OHT 5 weeks after DMBA initiation followed by MDL72527 treatment. MDL72527 treatment resulted in a shorter tumor latency time, increased tumor burden, increased conversion to carcinomas, and lower tumor levels of p53. Thus, elevated epidermal ODC activity promotes tumorigenesis by stimulating the recruitment of bulge stem cells but not via ROS generation by polyamine catabolic oxidases.     

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.     

Growth and metastases of human lung cancer are inhibited in mouse xenografts by a transition state analogue of 5'-methylthioadenosine phosphorylase

The S-adenosylmethionine (AdoMet) salvage enzyme 5'-methylthioadenosine phosphorylase (MTAP) has been implicated as both a cancer target and a tumor suppressor. We tested these hypotheses in mouse xenografts of human lung cancers. AdoMet recycling from 5'-methylthioadenosine (MTA) was blocked by inhibition of MTAP with methylthio-DADMe-Immucillin-A (MTDIA), an orally available, nontoxic, picomolar transition state analogue. Blood, urine, and tumor levels of MTA increased in response to MTDIA treatment. MTDIA treatment inhibited A549 (human non-small cell lung carcinoma) and H358 (human bronchioloalveolar non-small cell lung carcinoma cells) xenograft tumor growth in immunodeficient Rag2(-/-)γC(-/-) and NCr-nu mice. Systemic MTA accumulation is implicated as the tumor-suppressive metabolite because MTDIA is effective for in vivo treatment of A549 MTAP(-/-) and H358 MTAP(+/+) tumors. Tumors from treated mice showed increased MTA and decreased polyamines but little alteration in AdoMet, methionine, or adenine levels. Gene expression profiles of A549 tumors from treated and untreated mice revealed only modest alterations with 62 up-regulated and 63 down-regulated mRNAs (≥ 3-fold). MTDIA antitumor activity in xenografts supports MTAP as a target for lung cancer therapy.     

Rat colon ornithine and arginine metabolism: coordinated effects after proliferative stimuli

Ornithine decarboxylase (ODC) catalyzes the first step in the polyamine biosynthetic pathway, a highly regulated pathway in which activity increases during rapid growth. Other enzymes also metabolize ornithine, and in hepatomas, rate of growth correlates with decreased activity of these other enzymes, which thus channels more ornithine to polyamine biosynthesis. Ornithine is produced from arginase cleavage of arginine, which also serves as the precursor for nitric oxide production. To study whether short-term coordination of ornithine and arginine metabolism exists in rat colon, ODC, ornithine aminotransferase (OAT), arginase, ornithine, arginine, and polyamine levels were measured after two stimuli (refeeding and/or deoxycholate exposure) known to synergistically induce ODC activity. Increased ODC activity was accompanied by increased putrescine levels, whereas OAT and arginase activity were reduced by either treatment, accompanied by an increase in both arginine and ornithine levels. These results indicate a rapid reciprocal change in ODC, OAT, and arginase activity in response to refeeding or deoxycholate. The accompanying increases in ornithine and arginine concentration are likely to contribute to increased flux through the polyamine and nitric oxide biosynthetic pathways in vivo.