Collaboration Between RSK-EphA2 and Gas6-Axl RTK Signaling in Arginine Starvation Response That Confers Resistance to EGFR Inhibitors

Abstract

Many human malignancies require extracellular arginine (Arg) for survival because the key enzyme for de novo Arg biosynthesis, argininosuccinate synthetase 1 (ASS1), is silenced. Recombinant arginine deiminase (ADI-PEG20), which digests extracellular Arg, has been in clinical trials for treating ASS1-negative tumors. Reactivation of ASS1 is responsible for the treatment failure. We previously demonstrated that ASS1 reactivation is transcriptionally regulated by c-Myc via the upstream Gas6-Axl tyrosine kinase (RTK) signal. Here, we report that another RTK EphA2 is coactivated via PI3K-ERK/RSK1 pathway in a ligand-independent mechanism. EphA2 is also regulated by c-Myc. Moreover, we found that knockdown Axl upregulates EphA2 expression, demonstrating cross-talk between these RTKs. ADI^R cell lines exhibits enhanced sensitivities to nutrient deprivation such as charcoal-stripped FBS and multiple RTK inhibitor foretinib but resistance to EGFR inhibitors. Knockdown EphA2, and to lesser extent, Axl, overcomes EGFRi resistance. c-Myc inhibitor JQ1 can also sensitize ADI^R cells to ADI-PEG20. This study elucidates molecular interactions of multiple RTKs in Arg-stress response and offers approaches for developing strategies of overcoming ADI-PEG20 resistance.

Figure 1

ADI^Rcells are sensitive to nutrient stress and RTK inhibitor foretinib. (A) Phase-contrast morphology of five ADI^R cells (58R1 to 58R5) and ADI-sensitive cells (A2058) cultured in medium containing charcoal-stripped FBS for 96 hours; (B) and (C) proliferation measurements of cells cultured under charcoal-stripped FBS for 96 hours by DNA fragmentation and by SRB assays, respectively; (D) and (E) cell proliferation measurements of A2058 and ADI^R cells treated with foretinib (2 nM, 48 hours) and determined by SRB assay and by DNA fragment assay, respectively. RTK, receptor tyrosine kinase; SRB, sulforhodamine B.

Figure 2

Analyses of RTK expression inADI^Rcells. (A) Western blots of ADI^R and A2058 cell lysates using anti-phosphorylated Tyr antibody; (B) determination of activation of various RTKs by phosphor-RTK array; (C) Western blots showing elected expression of ASS1 and various RTK in A2058 and in five ADI^R cells; (D) time-dependent activation of Axl and EphA2 in A2058 cells treated with ADI (0.5 μg/ml) for the time as indicated. RTK, receptor tyrosine kinase.

Figure 3

Activation of EphA2 by ADI is mediated by RSK1. (A) Time-dependent activation of p-RSK1 by ADI; (B) knockdown of RSK1 using two siRNAs shows suppression of EphA2 induction by ADI; (C) inhibition of EphA2 activation by RSK1 inhibitor BI-D1870; (D) and (E) inhibitions of ADI-induced activation of RSK1 and EphA2 by PI3K inhibitors PI-103 and Ly294002, respectively; (F) effects of AKT inhibitor (perifosine) on RSKs and EphA2 treated with ADI. (B–F) A2058 cells were treated with the indicated drugs for 24 hours. ADI, arginine deiminase.

Figure 4

Interactions between Axl and EphA2 in response to ADI. (A) Inhibition of Axl activates RSK1 and EphA2 using dominant-negative (DN) Axl and soluble-Axl (s-Axl) recombinants in A2058 cells treated with ADI; (B) time-course suppression of TK-Mer and Tyro3 expression by ADI; (C) knockdown of Axl in two ADI^R (58R3 and 58R4) cells induces p-RSK1 and p-EphA2(S897) but downregulates TK-Mer and Tyro3, si-scr refers to scramble siRNA; (D) marginal regulation of Axl and RSK1 by EphA2 siRNA in A2058 cells; (E) Western blot assays showing that knockdown of EphA2 in two ADI^R cells moderately increase Axl expression.

Figure 5

Effects of Axl and EphA2 knockdown by siRNAs on sensitivities of ADI^R cells to EGFRi. (A) and (B) Cell killing assays of five ADI^R cell lines treated with lapatinib or gefitinib as indicated; (C) and (D) effects of cell sensitivities to lapatinib or gefitinib, respectively, by Axl-knockdown, respectively; (E) and (F) effects of cell sensitivities to lapatinib or gefitinib, respectively, by EphA2-knockdown cells in ADI^R 58R3 and 58R4 cells. * denotes significant (P < 0.05).

Figure 6

Effects of c-Myc knockdown on the growth of ADI^R (58R3 and 58R4) cells to EGFRi. (A) Western blots showing reduced expression of Axl, EphA2, and ASS1 in two ADI^R cells; (B) and (C) effects of c-Myc knockdown on cell proliferation of A2058 and ADI^R treated with lapatinib and gefitinib, respectively; (D) and (E) increases of cell killing effects by JQ1 in combination therapy with ADI on 58R3 and 58R4, respectively.

Figure 7

Schematic diagram depicting Axl and EphA2 signaling to Arg starvation response in Arg auxotrophic cells. Arg depletion using ADI induces ligand (Gas)-dependent activation of Axl and its downstream signal leading to induced c-Myc expression described previously [20] and ligand-independent activation of EphA2 via the PI3K-MEK-RSK axis. c-Myc transcriptionally induces ASS1 expression resulting in arginine synthesis and hence ADI resistance. c-Myc also transcriptionally regulates Axl to amply the Axl-PI3K-AKT loop. c-Myc upregulates EphA2 expression (perhaps by transcriptional mechanism as denoted by “?”), leading to acquired resistance to EGFRi. Horizontal bar denotes interaction between Axl and EphA2. ADI, arginine deiminase.