Dissecting the roles of DR 4 , DR 5 and c-FLIP in the regulation of Geranylgeranyltransferase I inhibition-mediated augmentation of TRAIL-induced apoptosis

Background: Geranylgeranyltransferase I (GGTase I) has emerged as a cancer therapeutic target. Accordingly, small molecules that inhibit GGTase I have been developed and exhibit encouraging anticancer activity in preclinical studies. However, their underlying anticancer mechanisms remain unclear. Here we have demonstrated a novel mechanism by which GGTase I inhibition modulates apoptosis. Results: The GGTase I inhibitor GGTI-298 induced apoptosis and augmented tumor necrosis factor-related apoptosis-inducing ligand (TRAIL)-induced apoptosis in human lung cancer cells. GGTI-298 induced DR4 and DR5 expression and reduced c-FLIP levels. Enforced c-FLIP expression or DR5 knockdown attenuated apoptosis induced by GGTI-298 and TRAIL combination. Surprisingly, DR4 knockdown sensitized cancer cells to GGTI298/TRAIL-induced apoptosis. The combination of GGTI-298 and TRAIL was more effective than each single agent in decreasing the levels of I Ba and p-Akt, implying that GGTI298/TRAIL activates NFB and inhibits Akt. Interestingly, knockdown of DR5, but not DR4, prevented GGTI298/TRAIL-induced I Ba and p-Akt reduction, suggesting that DR5 mediates reduction of I Ba and p-Akt induced by GGTI298/TRAIL. In contrast, DR4 knockdown further facilitated GGTI298/ TRAIL-induced p-Akt reduction. Conclusions: Both DR5 induction and c-FLIP downregulation contribute to GGTI-298-mediated augmentation of TRAIL-induced apoptosis. Moreover, DR4 appears to play an opposite role to DR5 in regulation of GGTI/TRAILinduced apoptotic signaling. Background There are two major apoptotic signaling pathways: the intrinsic mitochondria-mediated pathway and the extrinsic death receptor-induced pathway, and these pathways are linked by the truncated proapoptotic protein Bid [1]. The extrinsic apoptotic pathway is negatively regulated primarily by the cellular FLICE-inhibitory protein (cFLIP), including both long (FLIPL) and short (FLIPS) forms, through inhibition of caspase-8 activation, whereas the intrinsic apoptotic pathway is negatively regulated by multiple proteins including survivin [2]. The tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) binds to its receptors: death receptor 4 (DR4, also named TRAIL-R1) and death receptor 5 (DR5, also named TRAIL-R2) to activate the extrinsic apoptotic pathway [3]. Recently TRAIL has received much attention because it preferentially induces apoptosis in transformed or malignant cells, demonstrating potential as a tumor-selective apoptosis-inducing cytokine for cancer therapy [3]. Currently TRAIL is being tested in phase I clinical trials. However, some cancer cells are resistant to TRAIL [4,5]. Thus agents that can sensitize cancer cells to TRAIL are useful to enhance the efficacy of TRAIL-based cancer therapy or to overcome TRAIL-resistance [6]. Protein geranylgeranyltransferase type I (GGTase I) is responsible for the posttranslational modification of CAAX motif-containing proteins such as K-Ras, N-Ras, * Correspondence: ssun@emory.edu Department of Hematology and Medical Oncology, Emory University School of Medicine and Winship Cancer Institute, Atlanta, Georgia, USA Chen et al. Molecular Cancer 2010, 9:23 http://www.molecular-cancer.com/content/9/1/23 © 2010 Chen et al; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. RhoA, RhoC, Rac1, RalA, and Cdc42, which are often involved in cell transformation, tumor development and metastasis [7,8]. Thus, GGTase I has been considered a good cancer therapeutic target and thus great efforts have been made to develop GGTase I inhibitors as anticancer drugs [7,9]. Earlier studies using the GGTase I inhibitor, GGTI-287, showed K-Ras, a substrate for both GGTase I and farnesyltransferase (FTase), was particularly sensitive to GGTase I inhibition [10]. This finding was further supported by a genetic approach which has shown that GGTase I deficiency reduces tumor formation and improves survival in mice with K-Ras-induced lung cancer [11], thus further supporting the importance of GGTase I inhibition as a useful strategy to treat cancer, particularly KRas-induced cancer. It has been documented that GGTase I inhibitors induce apoptosis in human cancer cells [12-16]. However, the underlying mechanisms are largely unknown although it appears to be associated with inhibition of Akt and downregulation of survivin and Mcl-1 [13,14]. The present study focused on examining the effects of the GGTase I inhibitor GGTI-298, as well as GGTIDU40, on induction of apoptosis, particularly induced by TRAIL, in human non-small cell lung cancer (NSCLC) cells and on understanding underlying mechanisms.