Nuclear export and mitochondrial and endoplasmic reticulum localization of IGF-binding protein 3 regulate its apoptotic properties

Abstract

Tumor suppression by IGF-binding protein 3 (IGFBP3) may occur in an IGF-independent manner, in addition to its role as a regulator of IGF bioavailability. After secretion, IGFBP3 is internalized, rapidly localized to the nucleus, and is later detected in the cytoplasm. We identified a putative nuclear export sequence (NES) in IGFBP3 between amino acids 217 and 228, analogous to the leucine-rich NES sequence of p53 and HIV Rev. Mutation of the NES prevents nucleocytoplasmic shuttling of IGFBP3 and blocks its ability to induce apoptosis. Targeting of IGFBP3 to the mitochondria and endoplasmic reticulum (ER) was confirmed by co-localization with organelle markers using fluorescence confocal microscopy and subcellular fractionation. Mitochondrial targeting was also demonstrated in vivo in IGFBP3-treated prostate cancer xenografts. These results show that IGFBP3 shuttles from the nucleus to the mitochondria and ER, and that nuclear export is essential for its effects on prostate cancer apoptosis.

Nuclear actions of insulin-like growth factor binding protein-3

“…The importance of these interactions was suggested by the loss of IGFBP-3-induced apoptosis in Nur77-deficient cells. As noted earlier, mutation of a putative CRM1-binding NES motif on IGFBP-3 also prevented IGFBP-3-induced apoptosis, and also resulted in increased nuclear RXRα-Nur77 retention (Paharkova-Vatchkova and Lee, 2010). Together these date indicate a role for nuclear IGFBP-3 in the CRM1-mediated export of RXRα-Nur77 complexes during IGFBP-3-mediated apoptosis in prostate cancer cells.…”

“…The IGFBP-3 sequence includes several putative NES motifs in its central and C-terminal domains. One such motif, starting at Met190 (excluding the signal peptide) has been reported to serve as a functional NES (Paharkova-Vatchkova and Lee, 2010). In fact this sequence overlaps another putative NES starting at Leu194 (Figure 1).…”

“…In fact this sequence overlaps another putative NES starting at Leu194 (Figure 1). Mutation of Leu197 and Leu200, which are included in both of the overlapping sequences, increased the nuclear retention of IGFBP-3, and abolished the apoptotic effect of IGFBP-3 in 22RV1 prostate cancer cells (Paharkova-Vatchkova and Lee, 2010). Since nuclear entry of IGFBP-3 is believed to precede, and have a role in, its induction of apoptosis (Lee and Cohen, 2002; Santer et al, 2006; Leibowitz et al, 2013), this result suggests that IGFBP-3 may first enter, and then leave, the nucleus for its pro-apoptotic activity.…”

Inhibition of Type I Insulin-Like Growth Factor Receptor Signaling Attenuates the Development of Breast Cancer Brain Metastasis

“…When we examined the IGFBP3 mRNA expression level in 231Br cells, we found that it is expressed 25-30 fold more than in 231P (Figure 2A). While IGFBP3 is traditionally studied as a secreted protein, it is known to carry out some of its functions intracellularly [38,39]. We first analyzed the levels of secreted IGFBP3 by collecting the conditioned medium of 231P and 231Br cells.…”

IGFBP-3 Is a Metastasis Suppression Gene for Prostate Cancer.

“…Molecular mechanisms underlying these effects in CaP cells include rapid cellular internalization via Caveolin-1 binding and nuclear localization (40,41); and activation of the intrinsic apoptosis pathway via association with the nuclear receptors RXRα and Nur77 and nuclear export with subsequent mitochondrial translocation (39,42,43). Importantly, we have established an important physiologic role for IGFBP-3 in apoptosis induced by androgen-deprivation.…”