Figure-S6 PDF file 78K, Figure-S6 ASS1 knockdown promotes cell proliferation, viability, migration, and invasion in LPS510 cells that endogenously express ASS1. (A) Among the 4 liposarcoma cell lines tested, endogenous ASS1 mRNA and protein are only readily detected in LPS510 cells by quantitative RT-PCR (upper) and Western blotting assays (lower), respectively. CCD966SK fibroblasts are run in parallel as a positive control. (B) Stable silencing of ASS1 gene with shASS1 in LPS510 cells show significantly downregulated expression at the mRNA and protein levels as detected by real-time RT-PCR (upper) and Western blotting (lower) assays, respectively. The shLacZ plasmid, POLR2A transcript, and GADPH protein are utilized as controls in RNA interference, quantitative RT-PCR, and Western blotting assays, respectively. (C) Results of Brdu (left upper), XTT (right upper), transwell migration (left lower), and matrigel invasion (right lower) assays reveal that the functions of cell proliferation, cell viability, cell motility, and cell invasiveness are significantly impaired in shLacZ control (all p��0.05) as compared to the LPS510 cells stably silenced against ASS1 expression. All functional experiments are performed in triplicate and results expressed as mean �} SD
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.
