Sustained Activation of Extracellular Signal‐Regulated Kinase (ERK) Signaling in Human Prostate Cancer LNCaP Cells Depleted of Androgen

Objectives: The mitogen-activated protein kinase (MAPK) cascade is involved in the control of cell growth and differentiation. In this study, we have investigated the effect of androgen withdrawal on this pathway and its potential role in the induction of neuroendocrine (NE) differentiation. For this purpose, we used the androgen-sensitive human prostate cancer LNCaP cells as an in vitro model. Methods and Results: The incubation of LNCaP cells for 6 days in medium, either free of serum or supplemented with serum depleted of steroids (i.e., charcoal-stripped serum), resulted in NE differentiation as determined by growth arrest, the formation of neurites, and an increase in neuron-specific enolase protein expression. Sustained extracellular-regulated kinase (ERK) phosphorylation/activity and enhanced ERK/MAPK kinase (MEK) activity also were observed on serum or steroid withdrawal. A synthetic androgen, mibolerone, blocked both NE differentiation and ERK phosphorylation induced by the incubation of the cells in steroid-depleted medium, thus confirming androgen specificity. Furthermore, a culture of LNCaP cells in complete medium supplemented with a 5-α-reductase inhibitor, finasteride, also induced NE differentiation and ERK phosphorylation. This implicates depletion of the principal prostatic androgen, dihydrotestosterone, as the specific mediator of these effects. In contrast to ERK, the phosphorylation status of the stress-activated MAPK members c-Jun N-terminal kinase and p38 was not altered by steroid withdrawal. The MEK inhibitor U0126 was used to study the potential role of ERK in regulating NE differentiation. However, U0126 did not reverse NE differentiation associated with steroid depletion, even though ERK phosphorylation was suppressed. The role of erb B tyrosine kinase receptors in mediating ERK phosphorylation during steroid depletion also was investigated. erb B1 protein levels decreased, erb B3 protein levels and phosphorylation remained unaltered, and erb B2 phosphoprotein levels increased after steroid depletion. Stable expression of an intracellular antibody to erb B2, however, did not prevent the up-regulation of ERK phosphorylation that is associated with steroid depletion. Conclusions: Androgen depletion induces sustained erb B-independent ERK signaling in LNCaP cells, however, this pathway is not essential for the associated NE differentiation.