Differential Sensitivity of Chemoresistant Neuroblastoma Subtypes to MAPK-targeted Treatment Correlates with ERK, P53 Expression, and Signaling Response to U0126

Purpose: Neuroblastoma tumors are comprised of neuroblastic (N), substrate-adherent (S), and intermediate (I) cells. Because cell growth and differentiation often involve p44/p42 mitogen-activated protein kinase (MAPK) pathway signaling, we explored MAPK signaling and growth response in three NB cell types after MAPK kinase (MEK) inhibition to evaluate the feasibility of MAPK-targeted treatment strategies.

Methods: Three human NB cell cultures, SH-SY5Y (N-type), BE(2)-C (I-type), and SK-N-AS (S-type), were treated in monolayer cultures with increasing concentrations of the MEK inhibitor U0126. MAPK pathway intermediates MEK and ERK, their activated (phosphorylated) forms p-MEK and p-ERK, and p53 expression were assessed by Western blot at 1 and 24 hours. At 72 hours, cell counts determined growth inhibition and DNA fragmentation ELISA assessed apoptosis.

Results: Among all three lines, total ERK and MEK expression were unaffected by U0126. However, constitutive total ERK and p53 expression were significantly greater in BE(2)-C (I-type) cells than in SH-SY5Y (N-type) and SK-N-AS (S-type). Active ERK (p-ERK) levels decreased in dose response to U0126 at 1 and 24 hours in all lines. Conversely, p-MEK levels increased with increasing U0126 concentrations at 1 hour in SH-SY5Y (N-type) and at 24 hours in all lines. BE(2)-C (I-type) cell counts decreased in concentration-dependent fashion with U0126, whereas SH-SY5Y (N-type) and SK-N-AS (S-type) showed a biphasic response with increased cell counts at 1 micromol/L U0126 and slightly decreased cell counts at 10 mumol/L U0126.

Conclusion: This study demonstrates that BE(2)-C (I-type) cells exhibit greater constitutive total ERK and p53 expression than SH-SY5Y (N-type) and SK-N-AS (S-type). Although all three lines exhibit p-ERK decreases with MEK inhibition, only BE(2)-C (I-type) cells significantly decrease their proliferation with U0126 treatment. Although MEK inhibition holds promise in targeting I-type NB cells, successfully treating this heterogeneous tumor may require combining agents against N- and S-type cells.