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Farnesyltransferase and Geranylgeranyltransferase I Oncogene (2007) 26, 633–640 & 2007 Nature Publishing Group All rights reserved 0950-9232/07 $30.00 www.nature.com/onc ORIGINAL ARTICLE Farnesyltransferase and geranylgeranyltransferase I inhibitors upregulate RhoB expression by HDAC1 dissociation, HAT association and histone acetylation of the RhoB promoter FL Delarue1,2,4, J Adnane1,2,4, B Joshi1,2,5, MA Blaskovich1, D-A Wang1,2, J Hawker1,2, F Bizouarn1,2, J Ohkanda3, K Zhu1,2, AD Hamilton3, S Chellappan1,2 and SM Sebti1,2 1Drug Discovery Program at H Lee Moffitt Cancer Center and Research Institute, Tampa, FL, USA; 2Departments of Oncology and Biochemistry and Molecular Biology, University of South Florida, Tampa, FL, USA and 3Department of Chemistry, Yale University, New Haven, CT, USA Recently, we have shown that RhoB suppresses EGFR-, nylation is required for the oncogenic properties of some ErbB2-, Ras- and Akt-mediated malignant transforma- GDP/GTP binding GTPases such as Ras and Rho (Cox tion and metastasis. In this paper, we demonstrate that and Der, 1997; Gibbs and Oliff, 1997; Sebti and Der, the novel antitumor agents farnesyltransferase inhi- 2003). Protein prenylation involves C-terminal addition bitors (FTIs) and geranylgeranyltransferase I inhibitors of C15 (farnesyl) by FTase or C20 (geranylgeranyl) by (GGTIs) upregulate RhoB expression in a wide spectrum GGTase I (Zhang and Casey, 1996). FTase and GGTase of human cancer cells including those from pancreatic, I mediate prenylation of members of the Ras and Rho breast, lung, colon, bladder and brain cancers. RhoB families of small GTPases that end with a C-terminal induction by FTI-277 and GGTI-298 occurs at the CAAX prenylation motifs, where C is cysteine, A is any transcriptional level and is blocked by actinomycin D. aliphatic amino acid and X is any amino acid (Zhang Reverse transcription–PCR experiments documented that and Casey, 1996). Because prenylation is required for the increase in RhoB protein levels is due to an increase in Ras and Rho protein-mediated oncogenesis, metastasis RhoB transcription. Furthermore, treatment with FTIs and invasiveness, we and others have designed CAAX and GGTIs of cancer cells results in HDAC1 dissociation, peptidomimetics that are potent and selective inhibitors HAT association and histone acetylation of the RhoB of FTase (FTIs) and GGTase I (GGTIs), respectively, as promoter. Thus, promoter acetylation is a novel mechan- potential anticancer drugs (Sebti and Hamilton, 2000). ism by which RhoB expression levels are regulated FTIs and GGTIs are potent inhibitors of Ras and Rho following treatment with the anticancer agents FTIs and processing, respectively, and suppress the growth of GGTIs. murine and human tumors in various animal models Oncogene (2007) 26, 633–640. doi:10.1038/sj.onc.1209819; (Kohl et al., 1994; Nagasu et al., 1995; Barrington et al., published online 14 August 2006 1998; Liu et al., 1998; Sun et al., 1998; Hunt et al., 2000). Recent investigations into the biological mechanisms Keywords: RhoB; farnesyltransferase inhibitors; geranyl- that underlie FTI anti-transforming effects have raised geranyltransferase inhibitors; HDAC1 dissociation; questions about their exact mode of action (Cox and HAT association; histone acetylation Der, 1997; Gibbs and Oliff, 1997; Sebti and Hamilton, 2000; Sebti and Der, 2003). For example, there was no correlation between the Ras mutation status and Introduction reversal of transformation as some cancer cells that do not express oncogenic Ras were sensitive to FTI, and Farnesyltransferase (FTase) and geranylgeranyltransfer- inversely, some cancer cells containing activated Ras ase I (GGTase) inhibitors (FTIs and GGTIs) are novel were resistant to FTI (Cox and Der, 1997; Gibbs and classes of antitumor agents whose development was Oliff, 1997; Sebti and Hamilton, 2000; Sebti and Der, based upon the discovery that post-translational pre- 2003). In addition, in vitro and in vivo experiments have shown that K-Ras was geranylgeranylated in cells treated with FTIs (Rowell et al., 1997; Whyte et al., Correspondence: Dr SM Sebti, Drug Discovery Program, H Lee 1997; Sun et al., 1998). Thus, both FTase and GGTase I Moffitt Cancer Center and Research Institute, 12902 Magnolia Drive, SRB3-DRDIS, Tampa, FL 33612, USA. need to be inhibited to fully block K-Ras isoprenylation E-mail: sebti@moffitt.usf.edu (Lerner et al., 1997). Whether inhibition of RhoB 4These authors contributed equally to this work. farnesylation is involved in the mechanism of action of 5Current address: Department of Cellular and Physiological Sciences, FTIs is unclear at this time (Lebowitzand Prendergast, University of British Columbia, 2177 Wesbrook Mall, Vancouver, British Columbia, Canada V6T 1Z3. 1998; Prendergast, 2001; Sebti and Der, 2003). Whereas Received 12 August 2005; revised 19 May 2006; accepted 22 May 2006; most Rho proteins (e.g., RhoA, Rac1 and Cdc42) are published online 14 August 2006 modified by geranylgeranylation only, RhoB exists RhoB upregulation by FTIs and GGTIs FL Delarue et al 634 normally in vivo in either a farnesylated form (RhoB-F) effects of FTI-277 and GGTI-298 on RhoB expression. or geranylgeranylated form (RhoB-GG) (Lebowitz To this end, human pancreatic carcinoma-derived cells, et al., 1997; Baron et al., 2000). We have shown that Panc-1, were treated with vehicle or various concentra- RhoB prenylation mutants with mutations in the CAAX tions of FTI-277 or GGTI-298, and the cell lysates sequence that force RhoB to be either farnesylated were processed for RhoB Western immunoblotting (RhoB-F) or geranylgeranylated (RhoB-GG) are both as described under Materials and methods. Cells treated equally efficient in inhibiting human cancer cell growth, with vehicle contained barely detectable levels of transformation, oncogenic signaling and tumor growth Rho B. However, following treatment with FTI-277 or in nude mice (Chen et al., 2000). These results suggested GGTI-298, RhoB expression was induced in a concen- that elevated expression of RhoB rather than inhibition tration-dependant manner (Figure 1a). At the highest of RhoB farnesylation may mediate some of FTIs’ concentration used (15 mM), GGTI-298 shows a 114-fold effects. Furthermore, oncogenic Ras-transformed RhoB increase in Rho B expression, whereas FTI-277 causes a null (À/À) mouse embryo fibroblasts were resistant to 16-fold increase in Rho B (Figure 1a). By contrast, both FTI-induced apoptosis, but their growth in soft agar drugs showed much less induction of RhoA (Figure 1b), was sensitive to FTIs (Liu et al., 2000). Elevation of a closely related family member that shares 86% amino- RhoB protein level is an immediate early response acid identity with RhoB. The ability of FTIs and GGTIs following different types of stresses including UV and to induce RhoB expression correlated with their ability DNA-damaging agents (Lebowitzand Prendergast, to inhibit prenylation. FTI-277 (1 mM) inhibited HDJ-2 1998). Interestingly, it was found that induction of farnesylation by 60% and induced RhoB by 2.6-fold. At RhoB resulted in either a prolonged transient block 10 mM, FTI-277 inhibited HDJ-2 farnesylation by 77% to DNA replication or apoptosis, upon treatment with little effect on Rap1 geranylgeranylation and with DNA-damaging drugs (Fritzand Kaina, 2000). induced RhoB by 10-fold (Figure 1a). GGTI-298 was Recently, we demonstrated that oncogenes such as a more potent inducer of RhoB than FTI-277. At 10 mM, epidermal growth factor receptor (EGFR), ErbB2 and GGTI-298 inhibited Rap1 geranylgeranylation by 76% Ras suppress RhoB by a mechanism involving the with little effect on HDJ and induced RhoB by 76-fold phosphatidylinositol 30-kinase (PI3K)/Akt pathway (Figure 1a). (Jiang et al., 2004a; Jiang et al., 2004b). Furthermore, Time-course experiments revealed that induction of we found that ectopic expression of RhoB suppresses RhoB with both drugs began 16 h after drug addition oncogene-mediated malignant transformation, tumor and peaked at 24–48 h (Figure 1c). These results suggest survival, invasion and metastasis (Jiang et al., that the effects of FTI-277 and GGTI-298 on RhoB 2004a, b). Thus, RhoB may be a regulatory factor that expression are most likely not mediated by inhibition of quickly triggers subsequent protective responses such as prenylation of a protein with a short half-life such as cell cycle arrest, through induction of the cell cycle RhoB itself. kinase inhibitor p21WAF1/CIP1 or apoptosis (Du and Prendergast, 1999; Fritzand Kaina, 2000; Liu et al., 2000). Here we found that FTI- and GGTI-treated a RhoB human cancer cells contain elevated levels of RhoB protein. The FTI- and GGTI-mediated increase of U Rap1 RhoB occurred at the transcriptional level as RhoB P D induction was blocked by actinomycin and both U HDJ-2 inhibitors stimulated RhoB expression as documented P by Reverse transcription–PCR (RT–PCR). Finally, we β-actin found that FTI-277 and GGTI-298 induce RhoB 0 1 3 10 15 0 1 3 10 15 µM expression by a mechanism involving histone acetylation FTI-277 GGTI-298 of the RhoB promoter. b RhoA 0 1 3 10 15 0 1 3 10 15 µM Results FTI-277 GGTI-298 c 15 µM RhoB RhoB protein expression is induced in a concentration- GGTI-298 and time-dependant manner by FTI-277 and GGTI-298 15 µM FTIs and GGTIs are pharmacological agents that RhoB FTI-277 inhibit tumor cell proliferation in vitro and tumor growth in nude mice (Cox and Der, 1997; Gibbs and 0 8 16 24 48 Oliff, 1997; Sebti and Der, 2003). However, the time (hr) mechanism by which FTIs and GGTIs inhibit tumor Figure 1 FTI-277 or GGTI-298 treatment increases RhoB protein growth is not known. We and others have recently levels. Panc-1 cells were incubated with various concentrations of FTI-277, GGTI-298 or vehicle for 48 h. Cell lysates were analysed shown that RhoB plays an important role in suppressing by Western blotting with antibodies to Rho B, Rap1, HDJ or malignant transformation (Prendergast, 2001; Sebti and b-actin as indicated in the figure.
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