A novel function of differentiation revealed by cDNA microarray profiling of p75 NTR-regulated gene expression

Abstract

The expression of the p75 neurotrophin receptor (p75 NTR) is diminished in epithelial cells during progression of prostate cancer in vivo and in vitro. Previous studies have demonstrated a role for p75 NTR as a tumor suppressor in prostate growth. To better understand the molecular mechanism of p75 NTR on tumor suppression, we utilized a complementary deoxyribonucleic acid microarray composed of ∼6,000 human cancer-related genes to determine the gene expression pattern altered by re-introduction of p75 NTR into PC-3 prostate tumor cells. Comparison of the transcripts in the neo and p75 NTR-transfected cells revealed 52 differentially expressed genes, of which 21 were up-regulated and 31 were down-regulated in the presence of p75 NTR. Based on the known biological functions of the p75 NTR-regulated genes, we observed that p75 NTR modulated the expression of genes that are critically involved in the regulation of differentiation as well as cell adhesion, signal transduction, apoptosis, tumor cell invasion, and metastasis. Several differentially expressed genes identified by microarray were selected for confirmation using quantitative real-time polymerase chain reaction. Immunoblot analysis further confirmed increased cellular retinoic acid-binding protein I (CRABPI) and IGFBP5 protein levels and decreased level of PLAUR protein with increasing p75 NTR protein expression. As CRABPI was elevated far more than any other genes, we observed that the retinoids, all- trans retinoic acid and 9- cis retinoic acid, that bind CRABPI, promoted nitroblue tetrazolium-associated functional cell differentiation in p75 NTR PC-3 cells, but not in neo control PC-3 cells. Subsequent examination of the retinoic acid receptors (RARs) expression levels demonstrated an absence of RAR-β in the neo control cells and re-expression in the p75 NTR expressing cells, consistent with previous findings where RAR-β is believed to play a critical role as a tumor suppressor gene that is lost during de-differentiation of prostate epithelial cells. Whereas the RAR-α and -γ protein levels remained unchanged, retinoid X receptor (RXR)-α and -β also exhibited increasing protein levels with re-expression of the p75 NTR protein. Moreover, the ability of p75 NTR siRNA to knockdown levels of RAR-β, RXR-α, and RXR-β supports the specificity of the functional involvement of p75 NTR in differentiation. Hence, re-expression of the p75 NTR appears to partially reverse de-differentiation of prostate cancer cells by up-regulating the expression of CRABPI for localized sequestration of retinoids that are available to newly up-regulated RAR-β, RXR-α, and RXR-β.