p75 neurotrophin receptor and fenretinide-induced signaling in neuroblastoma

Abstract

Purpose: Neuroblastoma is the most common extracranial solid tumor of childhood. The retinoic acid analogue, fenretinide (4-hydroxyphenyl retinamide; 4-HPR), induces apoptosis in neuroblastoma cells in vitro and is currently in clinical trials for children with refractory neuroblastoma. We have previously shown that expression of the p75 neurotrophin receptor (p75NTR) enhances apoptosis induction and mitochondrial accumulation of reactive oxygen species by 4-HPR in neuroblastoma cells. We now examine the signaling events that underlie this effect.

Methods: Systematic examination of pro- and anti-apoptotic signaling effectors was performed by Western blot. Specific inhibitors of JNK phosphorylation and scavengers of mitochondrial reactive oxygen species were used to demonstrate the roles of these phenomena in the enhancement of fenretinide efficacy.

Results: The present studies demonstrate that enhancement of 4-HPR-induced apoptosis by p75NTR is dependent upon p38MAPK phosphorylation, JNK phosphorylation, caspase 3 activation, Akt cleavage, and decreased Akt phosphorylation. In addition, treatment with 4-HPR results in upregulation of MKK4 and MEKK1, and phosphorylation of MKK3/6. Efforts to enhance the efficacy of 4-HPR and to identify those tumors most likely to respond to it might exploit these effectors of 4-HPR-induced apoptosis.

Conclusions: Pharmacological agents that enhance MKK4 or MEKK1 expression or JNK expression or phosphorylation may enhance efficacy of 4-HPR in neuroblastomas that do not express high levels of p75NTR.

Figure 1

Downregulation of p75NTR expression (50% relative to cells transfected with a scrambled construct (SCR; [7], Fig. 3) in SH-EP1 human S-type neuroblastoma cells (American Type Culture Collection, Rockville, MD) causes a decrease in 4-HPR-induced (Sigma Chemical Corp, St. Louis, MO) JNK and p38 phosphorylation. A. 4-HPR treatment of p75shRNA and scrambled control (SCR) transfected cells causes an increase in JNK phosphorylation which is inhibited by knockdown of p75NTR. A representative Western blot is shown. For this and all subsequent graphs, error bars signify the SEM of three or more independent values. *p<0.05; **p<0.01 relative to control; Student's t-test. B. Time course of 4-HPR treatment (6 μM) in p75shRNA and SCR transfected cells shows a significant decrease in JNK phosphorylation when p75NTR is knocked down. A representative Western blot is shown and the associated graph depicts mean ± SEM for three independent determinations (Scion Image) of optical density of pJNK. *p<0.05; **p<0.01 relative to control. C. Optical densitometric data from Western blots (n=3) showing no change in phosphorylation of p38 in p75shRNA transfected cells, but a significant increase in SCR transfected cells when treated for 72 h with different concentrations of 4-HPR. *p<0.05; **p<0.01 relative to control; Student's t-test.

Figure 2

JNK phosphorylation is a necessary intermediate step in 4-HPR-induced apoptosis in SH-EP1 neuroblastoma cells. A. Alamar blue assay (Invitrogen, Carlsbad, CA) absorbance demonstrating the protective effect of inhibiting JNK phosphorylation in 72 h treatment of 4-HPR cells. Differential results are observed with SP600125 (Selleck Chemicals, Houston, TX; white circles) and without SP6000125 (black circles) pretreatment followed by 4-HPR treatment (0–20 μM). At all concentrations of 4-HPR, SH-EP1 cell metabolic viability with JNK inhibitor pretreatment differs from that without pretreatment with p<0.01. B. Caspase-3 cleavage (normalized to β-actin loading control) quantified from Western blots of SH-EP1 cells treated with 4-HPR (0-16 μM). *p<0.05; **p<0.01 relative to control; Student's t-test. C. Time course of JNK phosphorylation in p75shRNA and SCR transfected cells pretreated with the mitochondrial ROS scavenger, DHA (Sigma Chemical Corp., St. Louis, MO; 400 M; 24 h), then treated with 4-HPR (6 μM) for 72 h. As is the case for Figure 1B, in the absence of DHA, quantified Western blots showing a significant decrease in JNK phosphorylation in p75shRNA transfected relative to SCR transfected SH-EP1 cells with *p<0.05; **p<0.01; ***p<0.001. However, DHA pretreatment does not alter JNK phosphorylation in either transfectant (Compare Figure 1B and Figure 2C. Note that the time course in Figure 1B is normalized actin at each time point and then normalized to t=0 for each transfectant, while that in Figure 2C is simply normalized to actin at each time point.). D. MitoSOX™ (Molecular Probes, Inc., Eugene, OR) staining of SH-EP1 cells treated with 4-HPR alone, SP600125 alone, and SP600125 pretreatment followed by 4-HPR treatment. A representative set of sister culture micrographs is shown of three independent sets performed. Fluorescence imaging of MitoSOX™ stain (seen as bright red dots on the otherwise black background) demonstrates accumulation of superoxide in mitochondria in 4-HPR treated SH-EP1 cells but significantly reduced MitoSOX™ staining with SP600125 treatment. The images shown are representative results of three experiments performed.

Figure 3

MAPK signaling pathways activated in SH-EP1 neuroblastoma cells during 4-HPR-induced apoptosis. A. Time course of 4-HPR treatment of SCR and p75shRNA transfected SH-EP1 cells treated with 6 μM 4-HPR. Western blots showing MEKK1, MEK3/6, MEK4, and β-actin protein expression for early and late time points following 4-HPR treatment. B. Quantification of Western blots shown in (A) demonstrating a significant increase in MEKK1 phosphorylation after 4-HPR treatment of SCR transfected cells. *p<0.05; **p<0.01; ***p<0.001 relative to control. Note that an approximately 2-fold increase in MEKK1 phosphorylation is seen in SCR relative to p75shRNA transfected cells. C. Quantification of Western blots shown in (A) demonstrating a significant increase in MEK4 phosphorylation after 4-HPR treatment of SCR transfected cells. **p<0.01 relative to control. Note that a 2-3 fold increase in MEK4 phosphorylation is seen in SCR relative to p75shRNA transfected cells. D. Quantification of Western blots shown in (A) demonstrating a significant increase in MEK3/6 phosphorylation after 4-HPR treatment of SCR transfected cells. *p<0.05; **p<0.01 relative to control. Note that an approximately 2-fold increase in MEK3/6 phosphorylation is seen in SCR relative to p75shRNA transfected cells.

Figure 4

Akt phosphorylation protects SH-EP1 cells transfected with p75shRNA from and is cleaved during 4-HPR-induced apoptosis. A. Time course of Akt phosphorylation following 8 μM 4-HPR treatment showing a significant and sustained increase in Akt phosphorylation in SH-EP1 cells transfected with p75shRNA with *p<0.05; **p<0.01 relative to SCR transfected cells. A representative Western blot is shown and quantified across 3 repetitions. B. Akt cleavage was detected by Western blot using anti-Akt1/2 antibody from Santa Cruz (sc-1619), which detects both holo- and the N-terminal fragment of Akt. Akt cleavage at the later time points (24-72 h) following 4-HPR treatment of SH-EP1 cells was significantly higher in SCR transfected with **p<0.01; ***p<0.001 relative to p75shRNA transfected cells.