Peter W. Szlosarek

Arginine deprivation and argininosuccinate synthetase expression in the treatment of cancer

Arginine, a semi‐essential amino acid in humans, is critical for the growth of human cancers, particularly those marked by de novo chemoresistance and a poor clinical outcome. In addition to protein synthesis, arginine is involved in diverse aspects of tumour metabolism, including the synthesis of nitric oxide, polyamines, nucleotides, proline and glutamate. Tumoural downregulation of the enzyme argininosuccinate synthetase (ASS1), a recognised rate‐limiting step in arginine synthesis, results in an intrinsic dependence on extracellular arginine due to an inability to synthesise arginine for growth. This dependence on extracellular arginine is known as arginine auxotrophy. Several tumours are arginine auxotrophic, due to variable loss of ASS1, including hepatocellular carcinoma, malignant melanoma, malignant pleural mesothelioma, prostate and renal cancer. Importantly, targeting extracellular arginine for degradation in the absence of ASS1 triggers apoptosis in arginine auxotrophs. Several phase I/II clinical trials of the arginine‐lowering drug, pegylated arginine deiminase, have shown encouraging evidence of clinical benefit and low toxicity in patients with ASS1‐negative tumours. In part, ASS1 loss is due to epigenetic silencing of the ASS1 promoter in various human cancer cell lines and tumours, and it is this silencing that confers arginine auxotrophy. In relapsed ovarian cancer, this is associated with platinum refractoriness. In contrast, several platinum sensitive tumours, including primary ovarian, stomach and colorectal cancer, are characterised by ASS1 overexpression, which is regulated by proinflammatory cytokines. This review examines the prospects for novel approaches in the prevention, diagnosis and treatment of malignant disease based on ASS1 pathophysiology and its rate‐limiting product, arginine.

Expression and regulation of tumor necrosis factor alpha in normal and malignant ovarian epithelium.

Epidemiologic studies implicate inflammatory stimuli in the development of ovarian cancer. The proinflammatory cytokine tumor necrosis factor α (TNF-α) and both its receptors (TNFRI and TNFRII) are expressed in biopsies of this malignancy. Here, we tested the hypothesis that TNF-α is a regulator of the proinflammatory microenvironment of ovarian cancer. A cancer profiling array showed higher expression of TNF-α in ovarian tumors compared with normal ovarian tissue, and cultured ovarian cancer cells expressed up to 1,000 times more TNF-α mRNA than cultured normal ovarian surface epithelial cells; TNF-α protein was only detected in the supernatant of tumor cell cultures. Treatment with TNF-α induced TNF-α mRNA via TNFRI in both malignant and normal cells with evidence for enhanced TNF-α mRNA stability in tumor cells. TNF-α induced TNF-α protein in an autocrine fashion in tumor but not in normal ovarian surface epithelial cells. The TNF-α neutralizing antibody infliximab reduced the constitutive levels of TNF-α mRNA in tumor cell lines capable of autocrine TNF-α production. Apart from TNF-α mRNA expression, several other proinflammatory cytokines were constitutively expressed in malignant and normal ovarian surface epithelial cells, including interleukin (IL)-1α, IL-6, CCL2, CXCL8, and M-CSF. TNF-α treatment further induced these cytokines with de novo transcription of IL-6 mRNA contrasting with the increased stability of CCL2 mRNA. RNA interference directed against TNF-α was highly effective in abolishing constitutive IL-6 production by ovarian tumor cells. In summary, we show that TNF-α is differentially regulated in ovarian cancer cells compared with untransformed cells and modulates production of several cytokines that may promote ovarian tumorigenesis. Infliximab treatment may have a role in suppressing the TNF-α-driven inflammatory response associated with ovarian cancer. [Mol Cancer Ther 2006;5(2):382-90]

In vivo Loss of Expression of Argininosuccinate Synthetase in Malignant Pleural Mesothelioma Is a Biomarker for Susceptibility to Arginine Depletion

Purpose: Malignant pleural mesothelioma (MPM) is an increasing health burden on many societies worldwide and, being generally resistant to conventional treatment, has a poor prognosis with a median survival of <1 year. Novel therapies based on the biology of this tumor seek to activate a proapoptotic cellular pathway. In this study, we investigated the expression and biological significance of argininosuccinate synthetase (AS), a rate-limiting enzyme in arginine production. Experimental Design: Initially, we documented down-regulation of AS mRNA in three of seven MPM cell lines and absence of AS protein in four of seven MPM cell lines. We confirmed that the 9q34 locus, the site of the AS gene, was intact using a 1-Mb comparative genomic hybridization array; however, there was aberrant promoter CpG methylation in cell lines lacking AS expression, consistent with epigenetic regulation of transcription. To investigate the use of AS negativity as a therapeutic target, arginine was removed from the culture medium of the MPM cell lines. Results: In keeping with the cell line data, 63% (52 of 82) of patients had tumors displaying reduced or absent AS protein, as assessed using a tissue microarray. Cell viability declined markedly in the AS-negative cell lines 2591 and MSTO but not in the AS-positive cell line, 28. This response was apparent by day 4 and maintained by day 9 in vitro. Arginine depletion induced BAX conformation change and mitochondrial inner membrane depolarization selectively in AS-negative MPM cells. Conclusions: In summary, we have identified AS negativity as a frequent event in MPM in vivo, leading to susceptibility to cytotoxicity following restriction of arginine. A phase II clinical trial is planned to evaluate the role of arginine depletion in patients with AS-negative MPM.

Targeting Arginine-Dependent Cancers with Arginine-Degrading Enzymes: Opportunities and Challenges

Arginine deprivation is a novel antimetabolite strategy for the treatment of arginine-dependent cancers that exploits differential expression and regulation of key urea cycle enzymes. Several studies have focused on inactivation of argininosuccinate synthetase 1 (ASS1) in a range of malignancies, including melanoma, hepatocellular carcinoma (HCC), mesothelial and urological cancers, sarcomas, and lymphomas. Epigenetic silencing has been identified as a key mechanism for loss of the tumor suppressor role of ASS1 leading to tumoral dependence on exogenous arginine. More recently, dysregulation of argininosuccinate lyase has been documented in a subset of arginine auxotrophic glioblastoma multiforme, HCC and in fumarate hydratase-mutant renal cancers. Clinical trials of several arginine depletors are ongoing, including pegylated arginine deiminase (ADI-PEG20, Polaris Group) and bioengineered forms of human arginase. ADI-PEG20 is furthest along the path of clinical development from combinatorial phase 1 to phase 3 trials and is described in more detail. The challenge will be to identify tumors sensitive to drugs such as ADI-PEG20 and integrate these agents into multimodality drug regimens using imaging and tissue/fluid-based biomarkers as predictors of response. Lastly, resistance pathways to arginine deprivation require further study to optimize arginine-targeted therapies in the oncology clinic.

Epigenetic status of argininosuccinate synthetase and argininosuccinate lyase modulates autophagy and cell death in glioblastoma.

Arginine deprivation, either by nutritional starvation or exposure to ADI-PEG20, induces adaptive transcriptional upregulation of ASS1 and ASL in glioblastoma multiforme ex vivo cultures and cell lines. This adaptive transcriptional upregulation is blocked by neoplasia-specific CpG island methylation in either gene, causing arginine auxotrophy and cell death. In cells with methylated ASS1 or ASL CpG islands, ADI-PEG20 initially induces a protective autophagic response, but abrogation of this by chloroquine accelerates and potentiates cytotoxicity. Concomitant methylation in the CpG islands of both ASS1 and ASL, observed in a subset of cases, confers hypersensitivity to ADI-PEG20. Cancer stem cells positive for CD133 and methylation in the ASL CpG island retain sensitivity to ADI-PEG20. Our results show for the first time that epigenetic changes occur in both of the two key genes of arginine biosynthesis in human cancer and confer sensitivity to therapeutic arginine deprivation. We demonstrate that methylation status of the CpG islands, rather than expression levels per se of the genes, predicts sensitivity to arginine deprivation. Our results suggest a novel therapeutic strategy for this invariably fatal central nervous system neoplasm for which we have identified robust biomarkers and which overcomes the limitations to conventional chemotherapy imposed by the blood/brain barrier.

Epigenetic silencing of argininosuccinate synthetase confers resistance to platinum-induced cell death but collateral sensitivity to arginine auxotrophy in ovarian cancer

Evidence indicates that acquired resistance of cancers to chemotherapeutic agents can occur via epigenetic mechanisms. Down‐regulation of expression of argininosuccinate synthetase (ASS1), the rate‐limiting enzyme in the biosynthesis of arginine, has been associated with the development of platinum resistance in ovarian cancer treated with platinum‐based chemotherapy. The aim of the present study was to analyse epigenetic regulation of ASS1 in ovarian cancer tissue taken at diagnosis and relapse and determine its significance as a predictor of clinical outcome in patients treated with platinum‐based chemotherapy. In addition, expression and epigenetic regulation of ASS1 were analysed in human ovarian cancer cell lines, and ASS1 expression correlated with the ability of the lines to grow in media containing cisplatin, carboplatin or taxol or in arginine‐depleted media. Our results show that aberrant methylation in the ASS1 promoter correlated with transcriptional silencing in ovarian cancer cell lines. ASS1 silencing conferred selective resistance to platinum‐based drugs and conferred arginine auxotrophy and sensitivity to arginine deprivation. In ovarian cancer, ASS1 methylation at diagnosis was associated with significantly reduced overall survival (p = 0.01) and relapse‐free survival (p = 0.01). In patients who relapse, ASS1 methylation was significantly more frequent at relapse (p = 0.008). These data establish epigenetic inactivation of ASS1 as a determinant of response to platinum chemotherapy and imply that transcriptional silencing of ASS1 contributes to treatment failure and clinical relapse in ovarian cancer. The collateral sensitivity of cells lacking endogenous ASS1 to arginine depletion suggests novel therapeutic strategies for the management of relapsed ovarian cancer.

Prognostic and Therapeutic Impact of Argininosuccinate Synthetase 1 Control in Bladder Cancer as Monitored Longitudinally by PET Imaging

Targeted therapies have yet to have significant impact on the survival of patients with bladder cancer. In this study, we focused on the urea cycle enzyme argininosuccinate synthetase 1 (ASS1) as a therapeutic target in bladder cancer, based on our discovery of the prognostic and functional import of ASS1 in this setting. ASS1 expression status in bladder tumors from 183 Caucasian and 295 Asian patients was analyzed, along with its hypothesized prognostic impact and association with clinicopathologic features, including tumor size and invasion. Furthermore, the genetics, biology, and therapeutic implications of ASS1 loss were investigated in urothelial cancer cells. We detected ASS1 negativity in 40% of bladder cancers, in which multivariate analysis indicated worse disease-specific and metastasis-free survival. ASS1 loss secondary to epigenetic silencing was accompanied by increased tumor cell proliferation and invasion, consistent with a tumor-suppressor role for ASS1. In developing a treatment approach, we identified a novel targeted antimetabolite strategy to exploit arginine deprivation with pegylated arginine deiminase (ADI-PEG20) as a therapeutic. ADI-PEG20 was synthetically lethal in ASS1-methylated bladder cells and its exposure was associated with a marked reduction in intracellular levels of thymidine, due to suppression of both uptake and de novo synthesis. We found that thymidine uptake correlated with thymidine kinase-1 protein levels and that thymidine levels were imageable with [(18)F]-fluoro-L-thymidine (FLT)-positron emission tomography (PET). In contrast, inhibition of de novo synthesis was linked to decreased expression of thymidylate synthase and dihydrofolate reductase. Notably, inhibition of de novo synthesis was associated with potentiation of ADI-PEG20 activity by the antifolate drug pemetrexed. Taken together, our findings argue that arginine deprivation combined with antifolates warrants clinical investigation in ASS1-negative urothelial and related cancers, using FLT-PET as an early surrogate marker of response.

Promoter methylation of argininosuccinate synthetase-1 sensitises lymphomas to arginine deiminase treatment, autophagy and caspase-dependent apoptosis

Tumours lacking argininosuccinate synthetase-1 (ASS1) are auxotrophic for arginine and sensitive to amino-acid deprivation. Here, we investigated the role of ASS1 as a biomarker of response to the arginine-lowering agent, pegylated arginine deiminase (ADI-PEG20), in lymphoid malignancies. Although ASS1 protein was largely undetectable in normal and malignant lymphoid tissues, frequent hypermethylation of the ASS1 promoter was observed specifically in the latter. A good correlation was observed between ASS1 methylation, low ASS1 mRNA, absence of ASS1 protein expression and sensitivity to ADI-PEG20 in malignant lymphoid cell lines. We confirmed that the demethylating agent 5-Aza-dC reactivated ASS1 expression and rescued lymphoma cell lines from ADI-PEG20 cytotoxicity. ASS1-methylated cell lines exhibited autophagy and caspase-dependent apoptosis following treatment with ADI-PEG20. In addition, the autophagy inhibitor chloroquine triggered an accumulation of light chain 3-II protein and potentiated the apoptotic effect of ADI-PEG20 in malignant lymphoid cells and patient-derived tumour cells. Finally, a patient with an ASS1-methylated cutaneous T-cell lymphoma responded to compassionate-use ADI-PEG20. In summary, ASS1 promoter methylation contributes to arginine auxotrophy and represents a novel biomarker for evaluating the efficacy of arginine deprivation in patients with lymphoma.

NT5E (CD73) is epigenetically regulated in malignant melanoma and associated with metastatic site specificity

Background:Novel prognostic biomarkers and therapeutic strategies are urgently required for malignant melanoma. Ecto-5-prime-nucleotidase (NT5E; CD73) overexpression has been reported in several human cancers. The mechanism(s) underlying deregulated expression and the clinical consequences of changes in expression are not known.Methods:We used RT–PCR, qPCR, methylation-specific PCR and pyrosequencing to analyse expression and regulation of NT5E in malignant melanoma cell lines and primary and metastatic melanomas.Results:NT5E is subject to epigenetic regulation in melanoma. NT5E mRNA is downregulated by methylation-dependent transcriptional silencing in the melanoma cell lines SKMel2, SKMel23, WM35, Mel501, Mel505 and C81–61 and expression is reactivated by azacytidine. In contrast, the CpG island is unmethylated and the gene expressed in cultured normal melanocytes. In clinical cases of melanoma, methylation in the NT5E CpG island occurs in both primary and metastatic melanomas and correlates with transcriptional downregulation of NT5E mRNA. Relapse with metastatic disease, particularly to the visceral sites and brain, is more common in primary melanomas lacking NT5E methylation. Primary melanomas with methylation in NT5E show limited metastatic potential or more commonly metastasise predominantly to nodal sites rather than viscera and brain (P=0.01).Conclusion:Deregulation of NT5E expression in melanoma occurs via epigenetic changes in the NT5E CpG island. Confirmation of our results in larger clinical series would support the candidacy of NT5E as a clinical biomarker in melanoma, which could be applied in both primary and relapsed disease. Inhibition of NT5E may have therapeutic potential in melanoma, particularly in patients with more aggressive disease metastatic to viscera or the brain.

Reversed argininosuccinate lyase activity in fumarate hydratase-deficient cancer cells

BackgroundLoss of function of fumarate hydratase (FH), the mitochondrial tumor suppressor and tricarboxylic acid (TCA) cycle enzyme, is associated with a highly malignant form of papillary and collecting duct renal cell cancer. The accumulation of fumarate in these cells has been linked to the tumorigenic process. However, little is known about the overall effects of the loss of FH on cellular metabolism.MethodsWe performed comprehensive metabolomic analyses of urine from Fh1- deficient mice and stable isotopologue tracing from human and mouse FH-deficient cell lines to investigate the biochemical signature of the loss of FH.ResultsThe metabolomics analysis revealed that the urea cycle metabolite argininosuccinate is a common metabolic biomarker of FH deficiency. Argininosuccinate was found to be produced from arginine and fumarate by the reverse activity of the urea cycle enzyme argininosuccinate lyase (ASL), making these cells auxotrophic for arginine. Depleting arginine from the growth media by the addition of pegylated arginine deiminase (ADI-PEG 20) decreased the production of argininosuccinate in FH-deficient cells and reduced cell survival and proliferation.ConclusionsThese results unravel a previously unidentified correlation between fumarate accumulation and the urea cycle enzyme ASL in FH-deficient cells. The finding that FH-deficient cells become auxotrophic for arginine opens a new therapeutic perspective for the cure of hereditary leiomyomatosis and renal cell cancer (HLRCC).