Farnesyltransferase and Geranylgeranyltransferase I

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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. Data are representative of three Der, 2003). In this manuscript, we investigated the independent experiments.

Oncogene RhoB upregulation by FTIs and GGTIs FL Delarue et al 635 FTI-277 and GGTI-298induce RhoB protein expression by which GGTIs can induce these opposing effects is not in a wide spectrum of human cancer cell lines known. We next determined whether this induction of RhoB protein expression by FTIs and GGTIs is also observed in other human cancer cell lines of different origins. RhoB induction is blocked by actinomycin D and RhoB Human breast (MDA-MB-231 and MCF-7), colon (HT- transcription is stimulated by FTI-277 and GGTI-298 29), lung (A-549), pancreatic (Panc-1) and brain (U87 The results described above clearly show that incubation and DaOY) cancer cells were treated with either vehicle, of cells with FTI-277 or GGTI-298 resulted in an FTI-277 (15 mM) or GGTI-298 (15 mM) for 48 h and the elevation of RhoB protein levels. The increase of RhoB lysates were processed for immunoblotting as described protein levels could be due to either a stimulation of under Materials and methods. FTI-277 and GGTI-298 RhoB transcription/translation and/or stabilization of induced expression of RhoB in each cell line evaluated RhoB mRNA or protein. To determine whether RhoB is (Figure 2). Furthermore, GGTI-298 was more potent at transcriptionally induced, cells were incubated with increasing the levels of RhoB protein in most of the cell actinomycin D before treatment with FTI-277 and lines. Because protein prenylation in different cancer cell GGTI-298 and the lysates processed for immunoblot- lines could be affected to various degrees, we also ting as described under Materials and methods. RhoB determined the effects of FTI-277 and GGTI-298 on the protein was induced in FTI-277- and GGTI-298- treated prenylation of HDJ-2 and Rap1. Figure 2a shows that samples, compared to vehicle control; however, the FTI-277 blocked the processing of HDJ-2 with little presence of actinomycin D blocked induction of RhoB effect on Rap1 processing. Figure 2b also shows that by either FTI-277 or GGTI-298, indicating that FTI-277 GGTI-298 inhibited Rap1 geranylgeranylation with and GGTI-298 induce RhoB expression at the tran- little effect on HDJ-2 farnesylation. It is interesting to scriptional level (Figure 3). To conﬁrm this directly, we note that inhibition of protein farnesylation affected the analysed the effect of FTI-277 and GGTI-298 on RhoB expression of RhoB without affecting the expression of gene expression by RT–PCR. To this end, Panc-1 cells Rap1A and HDJ2. In contrast, while inhibition of were treated with FTI-277 or GGTI-298 (0–15 mM) for protein geranylgeranylation increased RhoB expression, 48 h, lysed and processed for total RNA extraction, RT– whereas, it decreased the expression of Rap1A and PCR and real-time PCR for quantitation as described HDJ2. Thus, inhibition of protein geranylgeranylation under Materials and methods. The RT–PCR gels of increased the expression of one GTPase (RhoB), Figure 4 show that FTI-277 and GGTI-298 increased whereas it decreased the expression of another (Rap1). the transcription of RhoB. Furthermore, real-time PCR Although this observation is intriguing, the mechanism shows that FTI-277 and GGTI-298 increased RhoB transcription in a dose-dependent manner. Consistent with the induction of RhoB protein levels, GGTI-298 was more potent than FTI-277 at inducing the a Panc-1 MCF-7 U87 HT29 DaOY A549 MDA-MB 231 transcription of the RhoB gene. FTI-277 − + − + − + − + − + − + − +

RhoB FTI-277 and GGTI-298induce histone acetylation in RhoB promoter region HDJ-2 It is well established that the acetylation status of the tail domains of histones H3 and H4 change during transcriptional activation of speciﬁc promoters. Histone Rap1 acetyl transferases like p300/CBP or pCAF are recruited to the promoters by activating transcription factors, b Panc-1MCF-7 U87 HT29 DaOY A549 MDA-MB 231 leading to the acetylation of speciﬁc lysine residues on GGTI-298 − + − + − + − + − + − + − + these histones. Conversely, transcriptional repressors

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HDJ-2 β-actin Figure 2 FTI-277 and GGTI-298 induce RhoB protein levels in a 0 15 15 0 15 15 µM wide spectrum of human cancer cell lines. Human cancer cell lines, 277 298 277 298 Panc-1 (pancreatic), U87 and DaOY (brain), MCF-7 and MDA- MB-231 (breast), A549 (lung) and HT-29 (colon) carcinomas were Figure 3 RhoB induction is blocked by actinomycin D. Panc-1 incubated with 10 mM FTI-277 (a), 10 mM GGTI-298 (b), or vehicle cells were incubated with FTI-277 (15 mM), GGTI-298 (15 mM)or (a, b) for 48 h. Cell lysates were analysed by Western blotting with vehicle in the presence or absence of actinomycin D for 48 h. Cell RhoB, HDJ-2 and Rap1 antibodies as described under Materials lysates were analysed by western blotting with antibodies to RhoB and methods. Data are representative of three independent or b-actin as described under Materials and methods. Data are experiments. representative of two independent experiments.

Oncogene RhoB upregulation by FTIs and GGTIs FL Delarue et al 636 a FTI277 (uM) no RT 0 1 3 10 15 RhoB

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b GGTI298 (uM) no RT 0131015 RhoB

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Relative RhoB mRNA 0 GGTI298(uM) 01 3 10 15 Figure 4 FTI-277 and GGTI-298 stimulate RhoB transcription. Panc-1 cells were treated with either FTI-277 (a) or GGTI-298 (b) with the indicated concentrations and processed for RT–PCR (gels) and real-time PCR (bar graph) as described under Materials and methods. Data are representative of two independent experiments.

recruit histone deacetylases to remove the acetylation there was no HDAC1 associated with the promoter, of the lysine residues on the histone tails to inhibit whereas the histones were acetylated. In addition, transcription. Attempts were made to examine whether acetylation of the histones was accompanied by the the induction of RhoB gene expression by FTIs and recruitment of the histone acetyl transferase p300 to the GGTIs correlates with changes in the acetylation status promoter region; there was p300 associated with the of histones in the promoter region of the human RhoB RhoB promoter from cells treated with GGTI-298 or gene. Chromatin immunoprecipitation (ChIP) assays FTI-277, but not in DMSO-treated cells. This is the were utilized for this purpose. Three different primer pattern expected from promoters that are transcription- sets were synthesized to examine the region correspond- ally active; interestingly, such changes were not observed ing to À368 to þ 85 of the RhoB promoter; primer set A on a control promoter, metallothionein 1G (Figure 5b, spanned residues À368 to À198; primer set B spanned bottom panel). Based on these observations, it appears the region À198 to À51 and set C covered the region likely that FTI-277 and GGTI-298 induce RhoB À51 to þ 85, as shown in Figure 5a. Fragments B and C transcription via altering the histone acetylation pattern contained binding sites for NF-Y, AP2, SP1 and c-myb, in a promoter-speciﬁc manner. as determined by mat-inspector analysis (http://www. genomatic.de). Panc 1 cells were treated with FTI-277 or GGTI-298 for 24 h and ChIP assays were performed using antibodies to acetylated histone H3, HDAC1 as Discussion well as p300. As shown in Figure 5b, HDAC1 was found to be associated with all the three fragments of the RhoB Recent evidence demonstrated that RhoB has tumor promoter when the cells were treated with dimethyl suppressive activity (Du and Prendergast, 1999; Chen sulfoxide (DMSO); in agreement with this, there was no et al., 2000; Fritzand Kaina, 2000; Liu et al., 2000). acetylated histones associated with the promoter, as an First, RhoB is induced as an early response of antibody to acetylated lysine of histone H3 did not eucaryotic cells to genotoxic stress resulting in either a immunoprecipitate the promoter fragment. This pattern prolonged transient block to DNA replication or was reversed in cells treated with FTI-277 or GGTI-298; apoptosis (Fritz et al., 1995; Fritz et al., 1999). Second,

Oncogene RhoB upregulation by FTIs and GGTIs FL Delarue et al 637

Figure 5 GGTI- and FTI- induced RhoB promoter activation correlates with histone acetylation. (a) Schematic diagram showing the minimal functional region À504 to þ 85 bp of RhoB promoter. Arrows and A, B, C represent primer sets and three contiguous regions of promoter used in the ChIP assay. Location and orientation of potential binding sites for Ap2, NFY, SP1 and c-myb are also shown. (b) ChIPassays showing RhoB promoter acetylation upon GGTI and FTI treatment. Input indicates an aliquot of total DNA. Antibodies used for immunoprecipitation are indicated above the lanes. An anti-acetylated histone H3 and anti-p300 antibodies immunoprecipitated the RhoB promoter fragment in the GGTI and FTI-treated lysates, indicating recruitment of p300 and histone acetylation. HDAC1 was associated with the RhoB promoter in DMSO-treated control cells but not in GGTI- and FTI- treated cells. Metallothionein 1G promoter was used as a control. An irrelevant antibody (IR-Ab) did not immunoprecipitate the promoter region in these experiments; ÀVe indicates a lane with no input DNA. Data are representative of three independent experiments.

RhoB prevents oncogenes such as EGFR, ErbB2, Ras human cancer-derived cell lines, suggesting that RhoB and Akt from inducing tumor survival, malignant induction is not tumor type specific. Our results are transformation, invasion and metastasis (Jiang et al., consistent with those of Pan et al. (2005), who showed 2004a, b). The expression of RhoB is critical for FTI- that FTIs induce reactive oxygen species that damage induced apoptosis in transformed murine embryo DNA and upregulate RhoB levels. The results are also fibroblasts, as targeted deletion in the RhoB gene makes consistent with a report showing that lovastatin (HMG- these cells resistant to FTI-induced apoptosis (Liu et al., CoA reductase inhibitor), which inhibits protein farne- 2000). Third, when overexpressed in Ras-transformed sylation and geranylgeranylation by depleting FPP and cells, RhoB was shown to induce cell growth inhibition GGPP, also upregulated RhoB expression (Holstein and activation of the cell cycle kinase inhibitor et al., 2002a). However, another study by the same p21WAF1 (Du and Prendergast, 1999). Fourth, we group showed that FTIs and GGTIs do not upregulate have shown that RhoB overexpression inhibits onco- RhoB (Holstein et al., 2002b). The reasons for this genic signaling and cell proliferation in cultured human inconsistency are not clear at this moment. One possible cancer cell lines and tumor growth in nude mice as well explanation is that different types of cells were used in as induces apoptosis (Chen et al., 2000). Furthermore, the two different studies. This is highly unlikely, as we we and others have recently shown that RhoB expres- have shown upregulation of RhoB in seven different sion is suppressed during tumor progression in human cancer cell lines from five different cancer types. A more head and neck, lung and brain cancer patients (Adnane likely reason for this inconsistency is that the concen- et al., 2002; Mazieres et al., 2004). Finally, RhoB trations used were very low (100 nM for FTI-277 and expression was found to be very low in 130 human 2 mM for GGTI-286) compared to our concentrations cancer cell lines (Wang et al., 2003), giving further that ranged from 1 to 15 mM. support to RhoB tumor suppressive activity. RhoB required 24–48 h of exposure to FTI-277 or FTIs and GGTIs were shown to be potent inhibitors GGTI-298 for full induction, suggesting that inhibition of cell proliferation and tumor growth in various animal of the prenylation of farnesylated and geranylgerany- models (Kohl et al., 1994; Nagasu et al., 1995; lated proteins that have long half-lives is responsible for Barrington et al., 1998; Liu et al., 1998; Sun et al., RhoB induction. Likely candidates include Ras and Rho 1998; Hunt et al., 2000). However, the mechanism by small GTPases, which are prenylated and have half-lives which this occurs is not known (Prendergast, 2001; Sebti ranging from 20 to 24 h. We found the FTI-277 and and Der, 2003). Here, we show that human cancer cells GGTI-298 increases in RhoB protein levels to be due to that are treated with FTI-277 or GGTI-298 express high a transcriptional activation. RhoB expression was levels of RhoB protein. The induction of RhoB by FTI- stimulated by FTI-277 and GGTI-298 as documented 277 and GGTI-298 was observed in different types of by RT–PCR, and actinomycin D blocked RhoB protein

Oncogene RhoB upregulation by FTIs and GGTIs FL Delarue et al 638 induction. Furthermore, the induction of RhoB by repressed RhoB expression. The studies presented here GGTI-298 was more potent than that by FTI-277. show that small molecule inhibitors of cell proliferation Among the proteins that are post-translationally mod- can affect the acetylation status as well as transcription ified by geranylgeranylation are the small GTPases of the of the RhoB promoter. It remains to be seen whether the Rho family (Cox and Der, 1997). Rho proteins, such as opposite changes take place on promoters that are Rac1, RhoA and cdc42, were shown to mediate cell repressed by GGTIs and FTIs; such experiments will transformation, either by themselves or in cooperation ascertain whether altering the acetylation status of with oncogenic Ras (Zohn et al., 1998). Furthermore, histones is a general mechanism for transcriptional these Rho proteins were shown to promote G1-S control mediated by these inhibitors. traverse and their inhibition resulted in induction of Taken together, our results demonstrate that FTI-277 p21waf and a G1 block (Zohn et al., 1998). RhoB was and GGTI-298 induce the transcription of RhoB by also shown to stimulate p21WAF1/CIP1 expression, and the inducing HDAC1 dissociation and promoting histone increase in p21waf and G1 block by GGTIs (Vogt et al., acetylation of the RhoB promoter. The induction of 1997) could be mediated by RhoB (Mazieres et al., 2004). RhoB transcription coupled with previous reports using The observation that FTI-277 and GGTI-298 affect RhoB (À/À) cells showing that RhoB is critical for FTI- the acetylation status of histones is novel and intriguing. induced apoptosis suggests that FTI antitumor activity Although many transcriptional repressors like Rb are may be mediated at least in part by an induction of the known to recruit histone deacetylases to repress RhoB promoter, which is independent of inhibition of transcription, the induction of acetylation by FTIs and RhoB farnesylation. GGTIs has not been reported before. Further, it appears that the changes on the RhoB promoter are brought about by differential recruitment of histone deacetylases Materials and methods and histone acetyl transferases. The fact that both FTI- 277 and GGTI-298 increase RhoB transcription through Tissue culture acetylation/deacetylation is intriguing. However, we do The human pancreatic carcinoma-derived cell line Panc-1, the not believe that the biochemical events leading to this breast adenocarcinoma-derived cell lines MCF7 and MDA- are common. The mechanisms that mediate the dis- MB-231 and the medulloblastoma-derived cell line DaOY sociation of HDAC1 from the RhoB promoter upon were grown in Dulbecco’s modified Eagle’s medium supple- FTI-277 and GGTI-298 treatment remain to be mented with 10% fetal bovine serum (FBS). The human lung carcinoma-derived cell line A549 (ATCC) was grown in F12 elucidated. Our earlier studies had shown that FTIs medium supplemented with 10% FBS. The glioblastoma- could enhance the tumor growth factor b (TGFb) derived cell line U87 was grown in minimal essential medium receptor expression and modulate TGFb-mediated cell supplemented with 10% FBS. The bladder carcinoma-derived proliferation and transcriptional regulation (Adnane cell line T24 and the colorectal adenocarcinoma HT-29 were et al., 2000), and similar mechanisms could be opera- grown in McCoy’s 5a medium supplemented with 10% FBS. tional here also. Although the actual transcription factor binding sites mediating the response to FTIs and GGTIs Western blot analysis are not known, it has been shown that Ras/Rho The ability of FTI-277 and GGTI-298 to inhibit protein signaling can affect the activity of many factors, prenylation and to affect RhoB and RhoA protein levels was including AP2 and myb. This raises the possibility that determined as described previously (Lerner et al., 1995). FTIs and GGTIs may block pathways that prevent the Briefly, for effects on prenylation aliquots of cell lysate were binding of transcription factors like AP2 that could be analysed by SDS–polyacrylamide gel electrophoresis (SDS– recruiting HDAC1 to the promoter. Such an inhibition PAGE) followed by immunoblotting with anti-HDJ-2 antibody (MS-325 Lab Vision Corp., Fremont, CA, USA) or anti- of transcription factor binding will prevent the recruit- Rap1 antibody (sc-65, Santa CruzBiotechnology, Santa Cruz, ment of HDAC1, facilitating histone acetylation and CA, USA). HDJ-2 is strictly farnesylated, whereas Rap1 is enhancing transcription of the RhoB promoter selec- only geranylgeranylated. For analysis of the effects of FTI-277 tively. Indeed, it is well established that transcription and GGTI-298 on the levels of RhoB and RhoA proteins, cells factors and associated cofactors can be recruited and were lysed in HEPES lysis buffer, then 50 mg proteins were dissociated from promoters in a signal-dependent separated by SDS–PAGE (12.5%), followed by nitrocellulose manner (Dasgupta et al., 2004). membrane blotting. RhoB protein was detected by mouse Our results show that whereas histone deacetylases monoclonal anti-RhoB antibody (sc-8048, Santa Cruz) and are dissociated from the RhoB promoter upon treatment RhoA (sc-418, Santa Cruz). Blots were stripped and re-probed with FTI-277 and GGTI-298, there is enhanced with antibodies to b-actin (clone AC-15, Sigma-Aldrich, St Louis, MO, USA). acetylation of the histones, facilitating transcription. This appears to be mediated through p300, a histone acetyl transferase that is known to promote transcrip- ChIP assays tion from a variety of cellular promoters. Interestingly, ChIP assays were performed as described previously (Wein- mann et al., 2001; Fusaro et al., 2003). To test at promoter these changes are specific to the RhoB promoter; they level whether GGTI-298 and FTI-277 treatment of Panc-1 were not observed in the MT1G promoter. This is leads to the RhoB activation, we performed ChIP assays using consistent with work by Wang et al. (2003) where HDAC1, Ac-H3 and p300 antibodies. Briefly, after appro- inhibition of HDAC1 by TPX or antisense resulted in priate incubations, Panc-1 cells were treated with 1% RhoB induction, whereas HDAC1 ectopic expression formaldehyde for 10 min at room temperature in order to

Oncogene RhoB upregulation by FTIs and GGTIs FL Delarue et al 639 crosslink protein–DNA complexes. Cells were harvested, RhoB promoter F2 (forward primer) 50-GCA GCA GCA washed three times in 1 Â phosphate-buffered saline (PBS) GCG CGG ACT-30 and lysed in a SDS, 50 mM Tris-HCI (pH 8.1) and 1 mM RhoB promoter R2 (reverse primer) 50-ATA TAG CCG ethylenediaminetetraacetic acid (EDTA). Cells were sonicated TCC AAT GGG-30 two times at 10 W for 15 s, cell debris pelleted at 41C and the RhoB promoter F3 (forward primer) 50-CCC ATT GGA supernatant evenly distributed for incubation with primary CGG CTA TAT-30 antibodies. One aliquot of the supernatant was reserved to RhoB promoter R3 (reverse primer) 50-ACT CGG CCT serve as the input reaction. Reactions were performed in a final AGC TCT CTC-30 volume of 400 ml in dilution buffer (0.01% SDS, 16.7 mM Tris- hMT- I G promoter (forward primer) 50-TGC GCT CAA HCI (pH 8.1), 1.2 mM EDTA, 150 mM NaCl and 1% Triton X- GGG ACC TTG CA-30 100) plus 3 mg of the appropriate primary antibody and rotated hMT-IG promoter (reverse primer) 50-CTC GAG CCC overnight at 41C. HDAC1 and p300 antibodies were pur- AAC AGC CA-30 chased from Santa CruzBiotechnologies, anti-acetylated- histone H3 monoclonal antibodies were purchased from Upstate Inc. (Chicago, IL, USA) (Benjamin and Jost, 2001; RT–PCR and real time PCR Chaya et al., 2001; Gui and Dean, 2001; Dey et al., 2003) RT–PCR After Panc-1 cells were treated with FTI-277 or whereas secondary anti-mouse antibody was purchased from GGTI-298 (0, 1, 3, 10 and 15 mM) for 48 h, cells were washed Pierce Biotechnology Inc. (Rockford, IL, USA). Four hundred with PBS and lysed with 1 ml Trizol (Invitrogen Corp., microliters of Protein A Sepharose beads (30 mg/ml in dilution Carlsbad, CA, USA). Total RNA was purified by the Trizol buffer) was then added to each reaction and rotated for 2 h at kit (Life Technologies, Gaithersburg, MD, USA), then the first 41C. Beads were washed three times in dilution buffer, cDNA strand was synthesized by SuperScript First Strand followed by one additional wash in dilution buffer containing cDNA Synthesis system (Life Technologies, Gaithersburg, 500 mM NaCl, incubated with 300 ml elution buffer (1% SDS, MD, USA). RhoB primers used were F-231CGACGTCATTC 0.1 M Na2CO3) and rotated for 20 min at room temperature. TCATGTCT and R-CGAGGTAGTCGTAGGCTTGG-472. Eluted supernatants as well as input DNA samples were then Actin primers used were F-844ATCATGAAGTGTGACGTG de-crosslinked by incubating at 651C for 4 h. DNA was then GAC and R-GACTCGTCATACTCCTGCTTG-1074. Thirty ethanol precipitated, and treated with Proteinase-K for 30 min PCR cycles, each consisting of 20 s at 941C, 30 s at 551C and at 371C. The samples were then extracted in phenol:chloro- 1 min at 721C, were performed. form, ethanol precipitated and re-suspended in 20 ml PCR- grade water (Sigma). PCR reactions, using the following three primer sets (A, B, C) that flank the entire À504 to þ 85 bp Real-time PCR The above PCR primers were designed using region of RhoB promoter, were then performed using 5 mlof the Primer Express software (Applied Biosystems, Foster City, DNA from the IP reactions or 1 ml of DNA from the input CA, USA). PCR reactions were performed using an ABI Prism reaction as template. Metallothionein 1G promoter was used 7700 Sequence Detection System (Applied Biosystems, Foster as a negative control in order to test the specificity of all ChIP City, CA, USA). The SYBR Green PCR core reagents kit assays and PCR reactions. The sequences of primers used in (Applied Biosystems, Foster City, CA, USA) was used for the the PCR reactions were as follows: PCR reaction. The reaction contained 10 ml of SYBR reagent, 2 ml of cDNA temple and 30 nM primers in a 20 ml volume. The RhoB promoter F1 (forward primer) 50-TCT GAA TGG thermal cycling conditions involved an initial denaturation GAG TCG CCA AC AC-30 step at 951C for 10 s and an extension step at 651C for 1 min. RhoB promoter R1 (reverse primer) 50-AGT CCG CGC The data are representative of two experiments each performed TGC TGC TGC-30 in triplicate for each data point.

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