NKX3.1 Activates Expression of Insulin-like Growth Factor Binding Protein-3 to Mediate Insulin-like Growth Factor-I Signaling and Cell Proliferation

Abstract

NKX3.1 is a homeobox gene that codes for a haploinsufficient prostate cancer tumor suppressor. NKX3.1 protein levels are down regulated in the majority of primary prostate cancer tissues. NKX3.1 expression in PC-3 cells increased IGFBP-3 mRNA expression 10-fold as determined by expression microarray analysis. In both stably and transiently transfected PC-3 cells and in LNCaP cells NKX3.1 expression increased IGFBP-3 mRNA and protein expression. In prostates of Nkx3.1 gene-targeted mice Igfbp-3 mRNA levels correlated with Nkx3.1 copy number. NKX3.1 expression in PC-3 cells attenuated the ability of IGF-I to induce phosphorylation of IGF-IR, IRS-1, PI3-kinase, and AKT. The effect of NKX3.1 on IGF-I signaling was not seen when cells were exposed to long-R3-IGF-I, an IGF-1 variant peptide that does not bind to IGFBP-3. Additionally, siRNA-induced knock down of IGFBP-3 expression partially reversed the attenuation of IGF-1R signaling by NKX3.1 and abrogated NKX3.1 suppression of PC-3 cell proliferation. Thus there is a close relationship in vitro and in vivo between NKX3.1 and IGFBP-3. The growth suppressive effects of NKX3.1 in prostate cells are mediated, in part, by activation of IGFBP-3 expression.

Variant NKX3.1 and Serum IGF-1: Investigation of Interaction in Prostate Cancer

“…4 In early human prostate cancer we have found that NKX3.1 expression is down-regulated over a broad range 4,24 NKX3.1 is known to have a broad effect on transcriptional targets, both increasing and decreasing expression of many genes. 21,25 However, our experiments with reporter constructs containing the cognate NKX3.1 DNA binding sequence have shown that promoter binding by NKX3.1 fails to activate transcription directly and only was observed to down-regulate gene expression. 26 NKX3.1 is able to cooperate with other transcription factors such as serum response factor to function as a coactivator and enhance transcription.…”

“…The effect of NKX3.1 on IGFR-I activation was not seen when Long R-IGF-I, an IGFR-I ligand that does not bind to IGFBP-3 was used, or when cells were pretreated with IGFBP-3 siRNA. 21 In contrast, neither NKX3.1(R52C) nor NKX3.1(S48A) had an effect on IGFR-I signaling ( Fig. 2A and B).…”

“…PC-3 and A172 cell lines were grown as previously described. 21 Plasmids and transfection full length NKX3.1, NKX3.1 (R52C), and NKX3.1(S48A) were expressed from constructs in the mammalian expression vector, pcDNA3.1 (Invitrogen, Carlsbad, CA) as previously described. 20 Transient and stable transfections were carried out as described earlier.…”

“…20 Transient and stable transfections were carried out as described earlier. 21 Western blot analysis. Immunoblotting was done as previously described.…”

“…Immunoblotting was done as previously described. 21 Antibody reagents were as follows: β-actin (Sigma, St. Louis, MO) 1:10,000; NKX3.1 21:2,000; IGFBP-3 (sc-9028, Santa Cruz Biotechnology, Santa Cruz, CA) 1:8,000 at 4°C overnight, followed by 3 washes in phosphate-buffered saline. Horseradish peroxidase conjugated goat-anti-rabbit and goat-anti-mouse (ImmunoPure antibodies, Pierce Biotechnology, Rockford, IL) secondary antibodies in 1% milk or 1% bovine serum albumin were applied for 1 hour at room temperature.…”