Figure-S2 PDF file 129K, Figure-S2 ADI-PEG20 treatment in myxofibrosarcoma cell lines and xenografts: (A) Pharmacokinetic curve of ADI-PEG20 demonstrates its dose-dependent capability in deprivation of arginine in culture medium of OH931 cells within the range from 0 to 100 ng/ml. (B) Macroscopic appearances of xenograft specimens treated with PBS versus various doses of ADI-PEG20. After 28 days of treatment, mice are scarified and tumor sizes are measured, demonstrating apparent regressing fibrotic appearance in the ADI-PEG20-treated groups even at a dose as low as 2.5 IU/kg. (C) ADI-PEG20 treatment impairs cell proliferation of myxofibrosarcoma and causes S phase arrest of cell cycle. (a) By using an ELISA-based, colorimetric assay to assess the rate of BrdU uptake, cell proliferation is significantly reduced in OH931 (upper), NMFH-1 (middle), and NMFH-2 (right) cell lines incubated with 150 IU of ADI-PEG20 for 72 h, as compared to the corresponding PBS-treated controls. (b) As analyzed by flow cytometry for OH931 cell line, cell cycle distribution in one each representative experiment is demonstrated for treatment with vehicle alone (upper), 75 IU of ADI-PEG20 (middle), and 150 IU of ADI-PEG20 (lower), showing dose-dependent S phase arrest induced by ADI-PEG20
ARTICLE ABSTRACT
Purpose: The principal goals were to identify and validate targetable metabolic drivers relevant to myxofibrosarcoma pathogenesis using a published transcriptome.Experimental Design: As the most significantly downregulated gene regulating amino acid metabolism, argininosuccinate synthetase (ASS1) was selected for further analysis by methylation-specific PCR, pyrosequencing, and immunohistochemistry of myxofibrosarcoma samples. The roles of ASS1 in tumorigenesis and the therapeutic relevance of the arginine-depriving agent pegylated arginine deiminase (ADI-PEG20) were elucidated in ASS1-deficient myxofibrosarcoma cell lines and xenografts with and without stable ASS1 reexpression.Results:ASS1 promoter hypermethylation was detected in myxofibrosarcoma samples and cell lines and was strongly linked to ASS1 protein deficiency. The latter correlated with increased tumor grade and stage and independently predicted a worse survival. ASS1-deficient cell lines were auxotrophic for arginine and susceptible to ADI-PEG20 treatment, with dose-dependent reductions in cell viability and tumor growth attributable to cell-cycle arrest in the S-phase. ASS1 expression was restored in 2 of 3 ASS1-deficient myxofibrosarcoma cell lines by 5-aza-2′-deoxycytidine, abrogating the inhibitory effect of ADI-PEG20. Conditioned media following ASS1 reexpression attenuated HUVEC tube-forming capability, which was associated with suppression of MMP-9 and an antiangiogenic effect in corresponding myxofibrosarcoma xenografts. In addition to delayed wound closure and fewer invading cells in a Matrigel assay, ASS1 reexpression reduced tumor cell proliferation, induced G1-phase arrest, and downregulated cyclin E with corresponding growth inhibition in soft agar and xenograft assays.Conclusions: Our findings highlight ASS1 as a novel tumor suppressor in myxofibrosarcomas, with loss of expression linked to promoter methylation, clinical aggressiveness, and sensitivity to ADI-PEG20. Clin Cancer Res; 19(11); 2861–72. ©2013 AACR.
