GCN2 (EIF2AK4) Is a Critical Regulator of Bladder Cancer Cell Viability in Response to Arginine Deprivation Therapy
Sounak Gupta, Maria Mir, John Bomalaski, Kamini Singh, Jyoti Harwalkar, Paula Carver, Alex Almasan, Donna E Hansel. Cleveland Clinic Foundation, Cleveland, OH; Polaris Group, San Diego, CA; Memorial Sloan-Kettering Cancer Center, New York, NY
Background: Arginine is biosynthesized from citrulline, with the enzyme argininosuccinate synthetase (ASS1) catalyzing the rate limiting step. Arginine deprivation therapy, utilizing pegylated arginine deiminase (ADI-PEG20) is emerging as a therapeutic modality in cancer subtypes deficient for ASS1. ADI-PEG20 further exacerbates arginine auxotrophy in a background of ASS1 deficiency by degrading circulating arginine. Previously we screened multiple bladder cancer subtypes for basal ASS1 expression and identified pure small cell and squamous cell carcinomas as being deficient for ASS1 expression. We therefore investigated the effect of ADI-PEG20 therapy in-vitro, utilizing bladder cancer-derived cell-lines that were ASS1 deficient.
Design: Multiple bladder cancer cell-lines were screened for ASS1 expression by western blotting. Quantitative PCRs and immunoblotting was used to assess gene and protein expression changes in response to arginine deprived media or ADI-PEG20 treatment. Corresponding changes in cell viability was assessed using clonogenic and MTT assays. Analysis of cell cycle progression and cell death were done by propidium iodide (PI) staining alone or PI /Annexin V double staining followed by flow cytometry, respectively.
Results: Several ASS1-deficient cell lines were identified; the urothelial carcinoma derived J82 and squamous cell carcinoma derived Scaber cells were chosen for further analysis. Arginine deficient media in these cells led to GCN2 activation, a kinase known to be activated by amino acid deprivation. Downstream changes included phosphorylation of its substrate eIF2α and an induction of the pro-apoptotic gene: CHOP. These changes were reproduced in a dose dependent manner using ADI-PEG20 and this was correlated with reduced cell viability with an IC50 of 0.16ug/ml and 0.24ug/ml in Scaber and J82 cells, respectively. The reduction in cell viability was due to increased apoptosis.
Conclusions: Our proposed model suggests that ADI-PEG20 therapy in ASS1 deficient bladder cancer cells activates GCN2 dependent eIF2α phosphorylation and an induction of the CHOP gene, leading to apoptotic cell death. As limited therapies are currently available for rarer variants of bladder cancer such as small cell carcinoma and squamous cell carcinoma, ADI-PEG20 represents a novel therapeutic avenue for these subtypes.
Category: Genitourinary (including renal tumors)
Monday, March 4, 2013 1:00 PM
Poster Session II # 178, Monday Afternoon