Nitric oxide (NO) is a crucial mediator of several survival mechanisms in tumor cells. Previous studies demonstrated that ionizing radiation (IR) stimulates NO generation activating pro-survival mechanisms including receptor tyrosine kinase and NF-κB-dependent pathways. In vitro, the IR-induced activation of these pathways survival pathways is inhibited by the NO synthase inhibitor, NG-nitro-L-arginine (L-NNA). Furthermore, the addition of L-NNA to the drinking water of flank xenograft bearing animals slows tumor progression, kills tumor cells and with radiation enhances animal survival. To elucidate the mechanism(s) by which L-NNA acts in such tumors we conducted experiments to study intracelluar signaling pathway activation by L-NNA with and without radiation.

Animals bearing flank xenografts received 10 Gy whole body irradiation either with or without prior L-NNA treatment. Tumors were excised at times post IR up to 30 minutes and protein lysates prepared for analysis by western blot. Cell monolayers were also similarly treated prior to cell lysis and protein analysis.

The activation (phosphorylation) of ERK1 and ERK2 was assayed in cultured monolayers and in flank xenografts using the A431 and FaDu cell lines. In vitro, the addition of L-NNA to the culture medium had little effect upon the activation of either ERK. In vivo however, L-NNA treatment up-regulated the phosphorylation of both ERK isoforms without irradiation and following irradiation elicited a significant enhancement of ERK1 phosphorylation and an inhibition of ERK2.

The current consensus is that ERK1 and ERK2, due to the similarities of their protein sequence, are similarly regulated and in general activate the same substrates. However, recent evidence has emerged to indicate distinct roles for ERK1/2 in addition to their overlapping actions. Indeed, the targeting of ERK2 has been seen to abolish Ras-dependent cell proliferation, an effect not observed with ERK1 targeting. Distinct signaling roles, and the opposite effects we observe upon ERK1 and ERK2 phosphorylation in vivo following L-NNA treatment may explain our previously reported findings of enhanced tumor control. In addition, these findings my shed light upon why ERK inhibitors show only moderate efficacy in clinical trials.

Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1321.