Etoposide-induced protein 2.4 homolog promotes argininosuccinate synthase 1 and cancer cell survival upon arginine deprivation

Abstract

Background: Arginine auxotrophy has been reported in a subset of cancers with inherently defective de novo arginine synthesis. However, the use of arginine deprivation therapy seems to be unequally effective, partially owing to the resistance acquired by cancer cells. Study of underlying factors involved in this response thus becomes of utmost importance. Meanwhile, the function of etoposide-induced 2.4 homolog (EI24) in cancer metabolism, and specifically in arginine metabolism, remains unknown.

Methods: EI24 was overexpressed in cancer cells using a doxycycline-inducible system or adenovirus transduction, while siRNA was used to knockdown EI24. Amino acid(s) deprivation medium was exploited with a cell viability assay to check the reliance of cancer cell survival on arginine. Protein expression and activation were examined through western blot and co-immunoprecipitation blot. Furthermore, global and specific protein translation were assessed through the SUnSET assay and polysome fractionation analysis. Gene expression and arginine level were downloaded from public cancer datasets for in silico validation including gene set enrichment and survival analysis to objectively evaluate the association between EI24 and arginine metabolism.

Results: EI24 promoted cancer survival under arginine starvation. Mechanistically, EI24 replenished translation of argininosuccinate synthase 1 (ASS1) by inducing the inactive S-nitrosylated form of phosphatase and tensin homolog (PTEN), leading to release of the phosphoinositide 3-kinase (PI3K)/protein kinase B (AKT) axis. This tumor-promoting action of EI24 could be found in multiple ASS1-deficient cancer cells regardless of p53 status. Furthermore, expression of EI24 was linked to enrichment of arginine metabolism pathway as well as poor survival of patients with cancer across various cancer types, suggesting its role in cancer resistance to arginine deprivation.

Conclusions: This study is the first to report the role of EI24 in promoting cancer survival via translational regulation of the metabolic enzyme ASS1, thus paving a route for further investigation into the link between EI24 and cancer metabolism.

Keywords: ASS1; Arginine; Cancer metabolism; EI24.

Fig. 1

EI24 increased ASS1 upon arginine deprivation in ASS1-deficient breast cancer cells. A Urea cycle-related enzymes (CPS1, carbamoyl phosphate synthetase 1). B Expression of arginine metabolism enzymes in a subset of breast cancer cell lines. C Viability of breast cancer cells upon arginine deprivation with EI24 overexpression (OE) by doxycycline (MDA-MB-231) or adenovirus (Hs 578T). D Expression of ASS1 upon arginine deprivation (left) or lysine supplementation for 24 h (right) in MDA-MB-231 with EI24 OE by doxycycline. E Enzymatic activity of ASS1 upon time-dependent deprivation of arginine with EI24 OE by doxycycline in MDA-MB-231. F Expression of ASS1 upon arginine deprivation in 24 h in Hs 578T cells with EI24 OE by adenovirus (left) and knockdown by siRNA (right). G Arginine level in human breast cancer samples classified by EI24 gene expression. H Correlation of EI24 expression and Kegg_arginine_and_proline_metabolism pathway determined by GSEA in two different breast cancer datasets

Fig. 2

EI24 regulates protein synthesis of ASS1. A Expression of ASS1 upon arginine deprivation and/or cycloheximide (CHX, 10 µg/ml, 24 h), MG132 (10 µM, 6 h), and bafilomycin A1 (Baf, 10 nM, 2 h) treatment in MDA-MB-231 cells; murine double minute 2 (MDM2), hypoxia-inducible factor 1 subunit alpha (Hif1α), and microtubule-associated protein 1 A/1B-light chain 3 (LC3) were included as markers for treatment effects. B Global protein synthesis level with EI24 OE by adenovirus in MDA-MB-231 cells. C Integration of pS6 to ribosome upon EI24 OE by adenovirus in MDA-MB-231 cells examined by Co-IP. D ASS1 mRNA distribution analyzed by qPCR following polysome fractionation in MDA-MB-231 with EI24 OE by doxycycline (upper) and Hs 578T with EI24 OE by adenovirus (lower). E Phosphorylation of AKT upon time-dependent deprivation of arginine in MDA-MB-231 with EI24 OE by doxycycline. F Phosphorylation of AKT upon time-dependent deprivation of arginine in Hs 578T with knockdown of EI24 by siRNA. G ASS1 expression and phosphorylation of AKT upon arginine deprivation and/or PI3K inhibitors LY294002 (LY, 10 µM) and wortmannin (Wort, 50 µM) in 24 h in MDA-MB-231 with EI24 OE by doxycycline

Fig. 3

Activation of PI3K/AKT-regulating ASS1 translation is through nitric oxide (NO)-regulated nitrosylation of PTEN. A Relative nitrite amount in MDA-MB-231 cell lysis upon arginine deprivation with EI24 OE by adenovirus. B Expression of ASS1 and pAKT upon arginine deprivation and/or H₂O₂ and NO donor (deta-NO) treatment in MDA-MB-231 with EI24 OE by adenovirus (NAC, N-acetylcysteine). C ASS1 mRNA distribution in polysome fractions upon NO donor (deta-NO) treatment in MDA-MB-231. D. Effect of L-NAME on ASS1 and pAKT in MDA-MB-231 with EI24 OE by adenovirus. E Cell viability upon arginine deprivation and L-NAME treatment in MDA-MB-231 with EI24 OE by adenovirus. F ASS1 mRNA distribution in polysome fractions upon L-NAME treatment in MDA-MB-231 with EI24 OE by adenovirus. G S-nitrosylation of PTEN upon arginine deprivation and L-NAME treatment in MDA-MB-231 with EI24 OE by adenovirus. H Model of EI24-regulated translation of ASS1 via nitrosylation of PTEN

Fig. 4

EI24 enhanced ASS1 expression in various ASS1-deficient cancers, independent of p53 as a transcription factor for ASS1. A Expression of ASS1 in p53-null H1299 upon transfection of wildtype or point-mutated p53 plasmids; p21 and MDM2 were included as positive control for p53 transfection. B Expression of ASS1 upon arginine deprivation in p53-mutated MDA-MB-231 with wildtype p53 plasmid transfection and EI24 OE by adenovirus. C mRNA expression of urea cycle-related enzymes upon arginine deprivation and/or 10074-G5 (G5) treatment in 24 h in MDA-MB-231. D Expression of ASS1 upon arginine deprivation and/or 10074-G5 (G5) treatment in 24 h in MDA-MB-231. E ASS1 expression in a subset of renal cell carcinoma (RCC) and lung adenocarcinoma (LUAD) cells. F Cell viability upon time-dependent arginine deprivation in Caki-1 and A549 with EI24 OE by adenovirus. G Expression of ASS1 and pAKT upon arginine deprivation for 24 h in Caki-1 and A549 with EI24 OE by adenovirus. H Prognostic value of EI24, and ASS1 in breast cancer determined by survival analysis on GENT2