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

Abstract

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.

Arginine starvation-associated atypical cellular death involves mitochondrial dysfunction, nuclear DNA leakage, and chromatin autophagy

“…Nutritional starvation often leads to autophagosome induction, and arginine deprivation is no exception (4,18,21,29,31 and this paper). However, the average volume of this chromatin-uptaken vesicle, with its diameter ranging from 2 μm to >10 μm, was several-fold greater than the conventional size [700 nm to 1 μm (28)] in the arginine-starved cell (Fig.…”

“…Investigations by our laboratories and others have established that targeting "arginine addiction" could be exploited therapeutically (16,18,21,29,30), owing to the preferential loss of ASS1 expression in tumor cells. The reduced ASS1 expression renders the cells unable to produce arginine intracellularly and they become addicted to external arginine.…”

“…S1B); all are lacking ASS1 expression and sensitive to ADI-PEG20-induced cell killing. ADI-PEG20-treated Mia and UMUC3 cells exhibited caspase-dependent cell death (14,21). Thus, DNA leakage is not limited to cells undergoing caspase-independent death.…”

A metabolic link between the urea cycle and cancer cell proliferation

“…1) and there are cancers in which both ASL and ASS1 are silenced, we hypothesized that ASS1 silencing in cancer offers an additional metabolic advantage independent of its role in arginine synthesis. 4 Thus, we focused our studies on aspartate, the substrate for ASS1.…”

Arginine deprivation and autophagic cell death in cancer

“…Effectively, ADI-induced autophagy buys tumor cells time before additional mechanisms are activated, such as ASS1 reexpression (7,17). ADI-PEG20-sensitive tumor cell lines exhibit various markers of autophagy, including conversion of cytosolic LC3-1 protein to the lipidated form LC3-II, up-regulation of Beclin 1 (Atg6), Atg genes 5 and 7, appearance of autophagosomes, and degradation of the autophagy substrate protein p62 (18). Moreover, blocking autophagy with the autophagosomal-lysosomal fusion inhibitor, chloroquine, promotes apoptosis and has been suggested as a potential strategy going forward into clinical trial (15,18,19).…”