The Farnesyltransferase Inhibitor Lonafarnib Induces CCAAT/Enhancer-binding Protein Homologous Protein-dependent Expression of Death Receptor 5, Leading to Induction of Apoptosis in Human Cancer Cells

Abstract

Pre-clinical studies have demonstrated that farnesyltransferase inhibitors (FTIs) induce growth arrest or apoptosis in various human cancer cells independently of Ras mutations. However, the underlying mechanism remains unknown. Death receptor 5 (DR5) is a pro-apoptotic protein involved in mediating the extrinsic apoptotic pathway. Its role in FTI-induced apoptosis has not been reported. In this study, we investigated the modulation of DR5 by the FTI lonafarnib and the involvement of DR5 up-regulation in FTI-induced apoptosis. Lonafarnib activated caspase-8 and its downstream caspases, whereas the caspase-8-specific inhibitor benzyloxycarbonyl-Ile-Glu(methoxy)-Thr-Asp(methoxy)-fluoromethyl ketone or small interfering RNA abrogated lonafarnib-induced apoptosis, indicating that lonafarnib induces caspase-8-dependent apoptosis. Lonafarnib up-regulated DR5 expression, increased cell-surface DR5 distribution, and enhanced tumor necrosis factor-related apoptosis-inducing ligand-induced apoptosis. Overexpression of a dominant-negative Fas-associated death domain mutant or silencing of DR5 expression using small interfering RNA attenuated lonafarnib-induced apoptosis. These results indicate a critical role of the DR5-mediated extrinsic apoptotic pathway in lonafarnib-induced apoptosis. By analyzing the DR5 promoter, we found that lonafarnib induced a CCAAT/enhancer-binding protein homologous protein (CHOP)-dependent transactivation of the DR5 promoter. Lonafarnib increased CHOP expression, whereas silencing of CHOP expression abrogated lonafarnib-induced DR5 expression. These results thus indicate that lonafarnib induces CHOP-dependent DR5 up-regulation. We conclude that CHOP-dependent DR5 up-regulation contributes to lonafarnib-induced apoptosis.Farnesyltransferase inhibitors (FTIs) 3 are a class of agents that suppress the farnesyltransferase enzyme to prevent farnesylation of certain proteins such as the Ras oncoprotein (1, 2). These agents inhibit proliferation and induce apoptosis in various cancer cell lines in culture or suppress the growth of xenografts in nude mice with limited toxicity (1, 2). In the clinic, FTIs are well tolerated and have some positive outcomes in certain settings such as hematological malignancies and breast cancer, although the response rates to FTIs alone are generally poor. When combined with other therapeutic agents or radiotherapy, FTIs exhibit some encouraging clinical responses (1, 2).Lonafarnib (LNF; also called SCH66336 and Sarasar), a nonpeptide tricyclic FTI, was one of the first FTIs to undergo clinical testing and to exhibit significant activity (3). In vitro, this agent, either alone or in combination with other therapeutic agents, inhibits the growth or induces apoptosis of several types of human cancer cells (4 -12). In animal models, LNF demonstrates potent oral activity in a wide array of human tumor xenograft models including tumors of colon, lung, pancreas, prostate, and urinary bladder origin (12). When LNF is combined with other chemotherapeutic agents, enhanced anti...

Oncogenic Ras and B-Raf Proteins Positively Regulate Death Receptor 5 Expression through Co-activation of ERK and JNK Signaling

“…Control (i.e. non-silencing), CHOP, Elk1, and ERK1/2 siRNA and RSK2 shRNA were described previously (15,27). shRSK1 (RHS3979 -9569862) was purchased from Open Biosystems (Huntsville, AL).…”

“…Reporter Plasmids and Luciferase Activity Assay-All DR5 reporter constructs used in this study were described previously (15,27). Plasmid transfection and luciferase assays were the same as described previously (27).…”

DDIT3 and KAT2A Proteins Regulate TNFRSF10A and TNFRSF10B Expression in Endoplasmic Reticulum Stress-mediated Apoptosis in Human Lung Cancer Cells

“…The TNFRSF10A promoter region (Ϫ304/Ϫ1 bp) was amplified from A549 cell genomic DNA and cloned into the pGL3-Basic luciferase reporter vector by using MluI and BglII restriction sites, and the resulting plasmid was designated as pB-TNFRSF10A/AP-1-BS(Ϫ304/Ϫ298)-luc and pB-TNFRSF10A/AP-1-BS(Ϫ304/Ϫ298)(mt)-luc. The TNFRSF10B reporter constructs pB-TNFRSF10B/DDIT3-(Ϫ276/Ϫ264)-BS-luc and pB-TNFRSF10B/DDIT3(Ϫ276/ Ϫ264)(mt)-BS-luc used in this study were described previously (37). Briefly, pB-TNFRSF10B/DDIT3(Ϫ276/Ϫ264)-BS-luc is a pGL3-Basic plasmid containing (Ϫ522/Ϫ1 bp) TNFRSF10B promoter region with a wild type DDIT3 binding site, and pB-TNFRSF10B/DDIT3(Ϫ276/Ϫ264)(mt)-BSluc is the same except that it contains the mutant DDIT3 binding site.…”

CAAT/Enhancer Binding Protein Homologous Protein–Dependent Death Receptor 5 Induction Is a Major Component of SHetA2-Induced Apoptosis in Lung Cancer Cells

“…The procedure for direct antibody staining and subsequent flow cytometric analysis of cell surface proteins was described previously (33). The mean fluorescent intensity (MFI) that represents antigenic density on a per cell basis was used to represent TRAIL receptor expression level.…”

“…It has been recently shown that CHOP, also known as growth arrest and DNA damage gene 153 (GADD153), directly regulates DR5 expression through a CHOP binding site in the 5-flanking region of the DR5 gene (30,31). Thus, certain drugs induce DR5 expression through CHOP-dependent transactivation of the DR5 gene (30)(31)(32)(33)(34).…”