Biphasic activation of extracellular signal-regulated kinase (ERK) 1/2 in epidermal growth factor (EGF)-stimulated SW480 colorectal cancer cells

Abstract

Cancer cells have different characteristics due to the genetic differences where these unique features may strongly influence the effectiveness of therapeutic interventions. Here, we show that the spontaneous reactivation of extracellular signalregulated kinase (ERK), distinct from conventional ERK activation, represents a potent mechanism for cancer cell survival. We studied ERK1/2 activation in vitro in SW480 colorectal cancer cells. Although ERK signaling tends to be transiently activated, we observed the delayed reactivation of ERK1/2 in epidermal growth factor (EGF)-stimulated SW480 cells. This effect was observed even after EGF withdrawal. While phosphorylated ERK1/2 translocated into the nucleus following its primary activation, it remained in the cytoplasm during late-phase activation. The inhibition of primary ERK1/2 activation or protein trafficking, blocked reactivation and concurrently increased caspase 3 activity. Our results suggest that the biphasic activation of ERK1/2 plays a role in cancer cell survival; thus, regulation of ERK1/2 activation may improve the efficacy of cancer therapies that target ERK signaling. [BMB Reports 2016; 49(4): 220-225].

Fig. 1.. Patterns of ERK activation in EGF-stimulated SW480 cells. (A) SW480 cells were treated with EGF (10 ng/ml) for the indicated time periods and the levels of ERK1/2 and pERK1/2 were measured by Western blot analysis. (B) SW480 cells treated with 10 ng/ml EGF for five min were labeled with anti-pERK1/2 antibodies (red) and DAPI (blue). Then, the localization of pERK1/2 was monitored by confocal microscopy. Scale bar, 10 μm. (C, D) Following stimulation with EGF for five min, either EGF stimulation continued (C) or the EGF-containing medium was replaced with serum-free medium (D). Protein extracts were immunoblotted with anti-pERK1/2 and anti-ERK1/2 antibodies.

Fig. 2.. Localization of pERK1/2 during the second activation phase and its effects on IL-8 expression. (A) SW480 cells were stimulated with EGF (10 ng/ml) for the indicated periods of time, then split into nuclear and cytosolic fractions using Nuclear and Cytoplasmic Extraction Reagents as described in the Materials and Methods section. The extracts were then immunoblotted with anti-pERK1/2 and anti-ERK1/2 antibodies. (B) SW480 cells were stimulated with EGF (10 ng/ml) for the indicated periods. pERK1/2 was detected using anti-phospho ERK1/2 antibodies and Alexa Fluor^Ⓡ 647-conjugated anti-rabbit IgG. Cell nuclei were stained with DAPI. (C) SW480 cells were stimulated with EGF (10 ng/ml) and the induction of IL-8 RNA was analyzed by RT-PCR assay as described in the Materials and Methods section.

Fig. 3.. Activation of ERK signaling following the inhibition of primary ERK activation or protein transport. (A) SW480 cells were incubated with or without U0126 (20 μM) for 60 min and then stimulated with EGF (10 ng/ml) for 10 or 300 min. (B) SW480 cells, pretreated with or without U0126, were stimulated with EGF for the indicated time periods. *Media was replaced with 20% FBS at 300 min and cells were lysed 10 min later. (C) SW480 cells were pretreated with or without monensin for 60 min and then stimulated with EGF (10 ng/ml). Protein extracts were immunoblotted using anti-ERK1/2 and anti-pERK1/2 antibodies.

Fig. 4.. Caspase 3 activity during the second activation phase and activation of AKT signaling in EGF-stimulated SW480 cells pretreated with U0126. (A) SW480 cells were incubated with or without U0126 (10 μM) for 60 min and then stimulated with EGF (10 ng/ml) for 30 or 300 min. Lanes 7 and 9: samples from cells treated with U0126 (10 μM) for 60 min prior to protein extraction. Protein extracts (50 μg) were incubated with Ac-DEVD-AMC at 37℃ for 30 min. (B) SW480 cells were incubated with or without U0126 (10 μM) for 60 min and then stimulated with EGF (10 ng/ml) for 10 or 300 min. Protein extracts were immunoblotted using anti-ERK1/2, anti-pERK1/2, anti-AKT, and anti-pAKT antibodies. *indicates P value ≤ 0.05.