T-LAK Cell-originated Protein Kinase (TOPK) Phosphorylation of Prx1 at Ser-32 Prevents UVB-induced Apoptosis in RPMI7951 Melanoma Cells through the Regulation of Prx1 Peroxidase Activity

Protein kinases are potential targets for the prevention and control of UV-induced skin cancer. T-cell-originated protein kinase (TOPK) is highly expressed in skin cancer cells, but its specific function is still unknown. We investigated the role of TOPK in UVB-induced apoptosis in RPMI7951 human melanoma cells. Liquid chromatography-tandem mass spectrometry analysis was used to identify proteins that bind with TOPK. Immunofluorescence, Western blot, and flow cytometry were used to assess the effect of UVB on TOPK, peroxiredoxin 1 (Prx1), and apoptosis in RPMI7951 cells. TOPK binds with Prx1 and its phosphorylation of Prx1 at Ser-32 is important for regulation of H₂O₂-mediated signal transduction. Analysis of the CD spectra of Prx1 and mutant Prx1 (S32A) proteins showed that the secondary structure of Prx1 was significantly altered by phosphorylation of Prx1 at Ser-32. UVB irradiation induced phosphorylation of TOPK in RPMI7951 human melanoma cells and phosphorylated TOPK co-localized with Prx1 in the nucleus. UVB induced the peroxidase activity of Prx1 in vitro and ex vivo. Following treatment with UVB, H₂O₂ levels and apoptosis were increased in RPMI7951 cells stably expressing TOPK siRNA or stably mutant Prx1 (S32A). Phosphorylation of Prx1 (Ser-32) by TOPK prevents UVB-induced apoptosis in RPMI7951 melanoma cells through regulation of Prx1 peroxidase activity and blockade of intracellular H₂O₂ accumulation.     

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.     

Neuropilin-1 Mediates Divergent R-Smad Signaling and the Myofibroblast Phenotype

The transforming growth factor-beta (TGF-β) superfamily is one of the most diversified cell signaling pathways and regulates many physiological and pathological processes. Recently, neuropilin-1 (NRP-1) was reported to bind and activate the latent form of TGF-β1 (LAP-TGF-β1). We investigated the role of NRP-1 on Smad signaling in stromal fibroblasts upon TGF-β stimulation. Elimination of NRP-1 in stromal fibroblast cell lines increases Smad1/5 phosphorylation and downstream responses as evidenced by up-regulation of inhibitor of differentiation (Id-1). Conversely, NRP-1 loss decreases Smad2/3 phosphorylation and its responses as shown by down-regulation of α-smooth muscle actin (α-SMA) and also cells exhibit more quiescent phenotypes and growth arrest. Moreover, we also observed that NRP-1 expression is increased during the culture activation of hepatic stellate cells (HSCs), a liver resident fibroblast. Taken together, our data suggest that NRP-1 functions as a key determinant of the diverse responses downstream of TGF-β1 that are mediated by distinct Smad proteins and promotes myofibroblast phenotype.     

Glycosylphosphatidylinositol Anchor Analogues Sequester Cholesterol and Reduce Prion Formation

A hallmark of prion diseases is the conversion of the host-encoded prion protein (PrP^C where C is cellular) into an alternatively folded, disease-related isoform (PrP^Sc, where Sc is scrapie), the accumulation of which is associated with synapse degeneration and ultimately neuronal death. The formation of PrP^Sc is dependent upon the presence of PrP^C in specific, cholesterol-sensitive membrane microdomains, commonly called lipid rafts. PrP^C is targeted to these lipid rafts because it is attached to membranes via a glycosylphosphatidylinositol anchor. Here, we show that treatment of prion-infected neuronal cell lines (ScN2a, ScGT1, or SMB cells) with synthetic glycosylphosphatidylinositol analogues, glucosamine-phosphatidylinositol (glucosamine-PI) or glucosamine 2-O-methyl inositol octadecyl phosphate, reduced the PrP^Sc content of these cells in a dose-dependent manner. In addition, ScGT1 cells treated with glucosamine-PI did not transmit infection following intracerebral injection to mice. Treatment with glucosamine-PI increased the cholesterol content of ScGT1 cell membranes and reduced activation of cytoplasmic phospholipase A₂ (PLA₂), consistent with the hypothesis that the composition of cell membranes affects key PLA₂-dependent signaling pathways involved in PrP^Sc formation. The effect of glucosamine-PI on PrP^Sc formation was also reversed by the addition of platelet-activating factor. Glucosamine-PI caused the displacement of PrP^C from lipid rafts and reduced expression of PrP^C at the cell surface, putative sites for PrP^Sc formation. We propose that treatment with glucosamine-PI modifies local micro-environments that control PrP^C expression and activation of PLA₂ and subsequently inhibits PrP^Sc formation.