Identification of a Novel Cell Death Receptor Mediating IGFBP-3-induced Anti-tumor Effects in Breast and Prostate Cancer

Insulin-like growth factor-binding protein-3 (IGFBP-3), a major regulator of endocrine actions of IGFs, is a p53-regulated potent apoptotic factor and is significantly suppressed in a variety of cancers. Recent epidemiologic studies suggest that IGFBP-3 contributes to cancer risk protection in a variety of cancers, and a polymorphic variation of IGFBP-3 influences cancer risk, although other studies vary in their conclusions. Some antiproliferative actions of IGFBP-3 have been reported to be independent of IGFs, but the precise biochemical/molecular mechanisms of IGF-independent, antiproliferative actions of IGFBP-3 are largely unknown. Here we report a new cell death receptor, IGFBP-3R, that is a single-span membrane protein and binds specifically to IGFBP-3 but not other IGFBP species. Expression analysis of IGFBP-3 and IGFBP-3R indicates that the IGFBP-3/IGFBP-3R axis is impaired in breast and prostate cancer. We also provide evidence for anti-tumor effect of IGFBP-3R in vivo using prostate and breast cancer xenografts in athymic nude mice. Further in vitro studies demonstrate that IGFBP-3R mediates IGFBP-3-induced caspase-8-dependent apoptosis in various cancer cells. Knockdown of IGFBP-3R attenuated IGFBP-3-induced caspase activities and apoptosis, whereas overexpression of IGFBP-3R enhanced IGFBP-3 biological effects. IGFBP-3R physically interacts and activates caspase-8, and knockdown of caspase-8 expression or activity inhibited IGFBP-3/IGFBP-3R-induced apoptosis. Here, we propose that IGFBP-3R represents a novel cell death receptor and is essential for the IGFBP-3-induced apoptosis and tumor suppression. Thus, the IGFBP-3/IGFBP-3R axis may provide therapeutic and prognostic value for the treatment of cancer.     

p53 Is Required for Cisplatin-induced Processing of the Mitochondrial Fusion Protein L-Opa1 That Is Mediated by the Mitochondrial Metallopeptidase Oma1 in Gynecologic Cancers

Mitochondria are highly dynamic organelles, and mitochondrial fission is a crucial step of apoptosis. Although Oma1 is believed to be responsible for long form Opa1 (L-Opa1) processing during mitochondrial fragmentation, whether and how Oma1 is involved in L-Opa1 processing and participates in the regulation of chemoresistance is unknown. Chemosensitive and chemoresistant ovarian (OVCA) and cervical (CECA) cancer cells were treated with cisplatin (CDDP). Mitochondrial dynamics and protein contents were assessed by immunofluorescence and Western blot, respectively. The requirements of Oma1 and p53 for CDDP-induced L-Opa1 processing, mitochondrial fragmentation, and apoptosis were examined by siRNA or cDNA. CDDP induces L-Opa1 processing and mitochondrial fragmentation in chemosensitive but not in chemoresistant cells. CDDP induced Oma1 40-kDa form increases in OV2008 cells, not in C13* cells. Oma1 knockdown inhibited L-Opa1 processing, mitochondrial fragmentation, and apoptosis. Silencing p53 expression attenuated the effects of CDDP in Oma1 (40 kDa) increase, L-Opa1 processing, mitochondrial fragmentation, and apoptosis in chemosensitive OVCA cells, whereas reconstitution of p53 in p53 mutant or null chemoresistant OVCA cells induced Oma1 (40 kDa) increase, L-Opa1 processing, mitochondrial fragmentation, and apoptosis irrespective of the presence of CDDP. Prohibitin 1 (Phb1) dissociates from Opa1-Phb1 complex and binds phosphorylated p53 (serine 15) in response to CDDP in chemosensitive but not chemoresistant CECA cells. These findings demonstrate that (a) p53 and Oma1 mediate L-Opa1 processing, (b) mitochondrial fragmentation is involved in CDDP-induced apoptosis in OVCA and CECA cells, and (c) dysregulated mitochondrial dynamics may in part be involved in the pathophysiology of CDDP resistance.     

Involvement of insulin-like growth factor-binding protein-3 in the effects of histone deacetylase inhibitor MS-275 in hepatoma cells

Insulin-like growth factor-binding protein-3 (IGFBP-3) expression is frequently suppressed in liver cancers and can be reactivated by histone deacetylase (HDAC) inhibition. This study examined the role of IGFBP-3 in mediating the effects of the HDAC inhibitor MS-275 in liver cancer cells and identified IGFBP-3-dependent proteins that regulate proliferation and migration. In HepG2 cells, MS-275 inhibited DNA synthesis, cell cycle activity, and cell viability concomitantly with increased binding of acetylated histone H3 to IGFBP-3 promoter sequences and induction of IGFBP-3 expression. IGFBP-3 down-regulation by siRNA significantly reversed the inhibition of cell viability and DNA synthesis by MS-275, indicating an intermediary role for IGFBP-3. Induction of the cyclin-dependent kinase inhibitor p21 by MS-275 was attenuated by IGFBP-3 down-regulation, providing an explanation for IGFBP-3-dependent effects of MS-275 on cell cycle activity. In contrast, MS-275 stimulated HepG2 cell migration, an effect also inhibited by IGFBP-3 down-regulation. Among genes whose induction by MS-275 was attenuated by IGFBP-3 down-regulation, LYVE1 and THBS2 (thrombospondin-2) were identified as mediators of IGFBP-3-dependent effects of MS-275. Silencing of either protein had no effect on the inhibition of HepG2 viability by MS-275 but reversed its stimulatory effect on cell migration. We conclude that among genes up-regulated by MS-275, IGFBP-3 is a key mediator of effects on hepatoma cell growth and migration, involving IGFBP-3-dependent proteins p21 (proliferation) and LYVE1 and THBS2 (migration). The enhanced cell motility that accompanies reactivation of IGFBP-3 expression in liver cancer by HDAC inhibition suggests the possibility of increased metastatic spread despite inhibited cell proliferation.