ANTICANCER RESEARCH 25: 3215-3224 (2005)

Biomarkers of Anticancer Activity of R115777 (Tipifarnib, Zarnestraì) in Human Breast Cancer Models In Vitro*

ELZBIETA IZBICKA1, DAVID CAMPOS1, GILBERT CARRIZALES1 and AMITA PATNAIK2

1Cancer Therapy and Research Center, The Institute for Drug Development, 14960 Omicron Drive, San Antonio, TX, 78245; 2Cancer Therapy and Research Center, 7979 Wurzbach Drive, San Antonio, TX, 78229, U.S.A.

Abstract. Background: Farnesyltransferase inhibitor R115777 Post-translational farnesylation of the Ras oncoprotein, (Tipifarnib, Zarnestraì) is active in breast cancer, but its efficacy catalyzed by farnesyltransferase (FTase), initiates in drug combinations has not been extensively investigated. translocation of Ras to the cell membrane and activation of Materials and Methods: The activity of R115777 and , the Ras signal transduction cascade. Substantial alone and in combination, was studied in the human breast experimental evidence has linked farnesylation of Ras with cancer cell lines, BT-474 (overexpressed HER2/neu) and MDA- a malignant phenotype. Farnesyltransferase inhibitors (FTIs) MB-231 (low HER2/neu), with cell viability and biomarkers for represent a novel class of anticancer drugs, that can inhibit farnesylation (HDJ-2, Rho B), tumor growth (Raf/MEK/ERK), malignant transformation with attenuated toxicity to normal survival (PI3K/Akt) and angiogenesis (VEGF, FGF-2, MMP-1, cells. The mechanism by which FTIs inhibit tumor growth is MMP-2, MMP-9) as the endpoints. Results: The drug more complex than originally thought. Although K-Ras can combination resulted in additive cytotoxicity. R115777 ± escape inhibition of processing by FTIs, tumors with paclitaxel inhibited HDJ-2 farnesylation, up-regulated RhoB, oncogenically mutated K-Ras proteins can still be inhibited transiently lowered (P)ERK/ERK and (P)Akt/Akt, reduced Raf- by FTI treatment. Furthermore, the Ras mutation status 1 and MEK, and inhibited secretion of VEGF and MMP-1. does not necessarily correlate with FTI sensitivity. Ras is Conclusion: The effect of R115777 on prenylation biomarkers is probably not the most important farnesylated protein whose consistent with its mechanism of action. The drug interfered with modification is inhibited as a result of FTI treatment. It is tumor growth, survival and angiogenesis pathways in breast likely that inhibition of the processing of other farnesylated cancer models with low or overexpressed HER2/neu receptor. proteins such as RhoB, a small GTPase, is responsible for The combination of R115777 with paclitaxel might offer clinical the apoptotic and antineoplastic responses of advantage over monotherapies. farnesyltransferase inhibitors (1). The effects of FTIs on the centromere-binding proteins CENP-E and CENP-F have been linked with arrest, apoptosis and tumor regression in preclinical studies (2). Historically, FTIs have *Presented, in part, at the 36th Annual Meeting of the American been shown to suppress farnesylation of proteins DJ-1 and Society of Clinical Oncology, New Orleans, LA, May 2000; 11th DJ-2, homologs of the Escherichia coli heat shock protein Annual Meeting of NCI-AACR/EORTC, Amsterdam, Netherlands, DnaJ, which are ubiquitously expressed and post- November 2000; 92nd Annual Meeting of the American Association translationally modified by farnesylation. Human DJ-2 for Cancer Research, New Orleans, LA, March 2001; and at the AACR/NCI/EORTC International Conference on Molecular Targets (HDJ-2) has been used as one of surrogate biomarkers of and Cancer Therapeutics, Boston, MA, November 17-21, 2003. FTI activity in clinical trials (3). Downstream effectors of Ras, that bind Ras in a GTP- Abbreviations: DTT, dithiothreitol; PBMC, peripheral blood dependent manner through the effector domain loop, are mononuclear cells. likely to be affected by FTIs. They include the Raf family of proteins controlling apoptosis and cell cycle progression, Correspondence to: Elzbieta Izbicka, Cancer Therapy and Research protein kinases MEK and ERK involved in the control of Center, The Institute for Drug Development, 14960 Omicron Drive, San Antonio, TX, 78245, U.S.A. Tel: 210.677.3879, Fax: the transcription pathway, and others. Recently, the 210.677.0058, e-mail: [email protected] phosphoinositide 3-OH kinase/AKT2 pathway has been identified as a critical target for FTI-induced apoptosis. FTI Key Words: Farnesyltransferase inhibitor, R115777, Ras. inhibition of human tumor growth may be explained in

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Table I. Evaluation of cytotoxicity of R115777, R115776 and paclitaxel in human breast cancer cell lines in vitro.

Agent BT-474 MDA-MB-231

R115777 5.4±0.2 ÌM 28.9±1.5 ÌM

R115776 4.9±0.1 ÌM 23.9±0.5 ÌM

Paclitaxel 2.7±0.2 nM 1.7±0.1 nM

Figure 1. Structure of R115777. cancer cell lines, BT-474 (HER2/neu overexpression, relatively low EGFR, low levels of activated ras) (9, 10) and MDA-MB-231 (low level of HER2/neu, high EGFR expression, highly activated ras) (10-12), were used as model terms of induction of apoptosis through inhibition of PI systems. We examined the effects of R115777 alone and in 3-kinase/AKT2-mediated cell survival and adhesion pathway combination with paclitaxel on cell viability, prenylation of (4). FTIs have been shown to inhibit angiogenesis in vitro downstream targets, and select biomarkers for tumor growth, and in vivo. It has been proposed that the inhibition of survival and angiogenesis. The study demonstrated the ability oncogenic Ras by FTI can result in two events that alter of R115777 to interfere with tumor signaling pathways host-tumor interactions; up-regulation of Fas, rendering relevant for growth, survival and angiogenesis of cancer cells. tumors more sensitive to immune cytotoxic effector cells, The data suggest that R115777 in combination with paclitaxel and down-regulation of VEGF, which may inhibit tumor might offer an advantage over monotherapies in the angiogenesis (5). treatment of patients with HER2/neu-positive breast cancer. R115777 (Tipifarnib, Zarnestraì; Janssen Research Foundation, Titusville, NJ, USA), an orally bioavailable Materials and Methods quinolone analog of imidazole heterocyclics originally developed as antifungal agents (Figure 1), is a highly Cell culture. BT-474 and MDA-MB-231 human breast carcinoma specific nonpeptidomimetic competitive inhibitor of FTase cells (American Type Culture Collection, Rockville, MD, USA) (6). R115777 does not inhibit prenylation catalyzed by were maintained in a 1:1 mixture of DMEM: F12 (Mediatech Inc., geranylgeranyltransferase type I, which can alternatively Herndon, VA, USA), supplemented with 100 units/ml penicillin, prenylate K-Ras and RhoB. R115777 was shown to inhibit 100 Ìg/ml streptomycin, 2.5 Ìg/ml fungizone (Invitrogen Corp., the growth of a broad spectrum of human tumor models in Carlsbad, CA, USA) and 10% fetal bovine serum (Invitrogen vitro and in vivo, characterized by the wild-type or mutated Corp.) and incubated in a humidified incubator at 37ÆC in 5% CO2 /95% HEPA filtered air. Ras. The drug can be safely administered in clinical trials in patients with advanced solid malignancies and leukemia (7). Drug treatment. R115777, R115776 (Janssen Research Foundation, The most common toxicities include peripheral neuropathy, Titusville, NJ, USA) and paclitaxel (Sigma Chemical Co., St. Louis, fatigue and myelosuppression (8). In recent phase II clinical MO, USA) were prepared as stock solutions in dimethyl sulfoxide trials in patients with metastatic breast carcinoma, the drug (DMSO; Sigma Chemical Co.), diluted into media to the final showed reproducible single-agent activity, mainly in patients concentration of DMSO at 0.1%, and added once to the cells in with HER2-(c-erb-2, neu)-positive disease (7). The the exponential growth phase. HER2/neu receptor, overexpressed in 30% of human breast Growth inhibition assay. A MTS assay (13) was used to evaluate cancers, is associated with a poor prognosis for overall and drug growth inhibitory effects. Briefly, exponentially growing cells disease-free survival. in 100 mL of cell culture medium were plated on Day 0 in 96-well Since the best combinations incorporating trastuzumab microtiter plates at a density of 104 cells/well. On Day 1, the drugs (Herceptinì) yield response of 50%, it is rational to explore or the vehicle controls were added in triplicate to individual wells novel drug combinations as alternative therapeutic of the plates. The vehicle or the drugs at designated concentration approaches. The purpose of the present study was to examine were delivered in 100-ÌL aliquots of cell culture medium plus vehicle. The cells were incubated at 37ÆC and cultured for 72 h. a hypothesis that anticancer combination therapy with Then, 100 ÌL of medium was removed and the cells were incubated R115777 and paclitaxel will be more efficacious than single- with the addition of 20 ÌL MTS (1.9 mg/ml in PBS, pH 6.0), for drug therapy in patients with HER2/neu-expressing breast 1 h at 37ÆC. Absorbance was measured on a Tecan Plus microplate cancers. To mimic the clinical situation, two human breast reader at 490 nm. The IC50 values were determined by using the

3216 Izbicka et al: Biomarkers for Farnesyltransferase Inhibitor in Breast Cancer

Prism GraphPad Software, Inc., San Diego, CA, USA. Drug ELISA. Cell culture media were collected at 24-h intervals during combinations were evaluated with cells plated as above in BT-474 4 days of drug treatment with the drugs added once at time=0. The and MDA-MB-231 with 10 nM and 5 nM paclitaxel, respectively. media was centrifuged to remove cellular debris and used for The corresponding concentrations of R115777 were 5 ÌM and 10 analysis of secreted proteins by ELISA using the following human- ÌM. Isoeffect plots were generated as described (14). The data are specific kits: MMP-2 (Amersham Biosciences), MMP-1, MMP-9, representative of at least three separate experiments. VEGF and basic FGF/FGF-2 (R&D Systems, Minneapolis, MN, USA), according to the suppliers’ instructions. All assays were Antibodies. Polyclonal antibodies were obtained from the following performed in duplicate. sources: anti-mitogen activated protein kinase (MAP kinase; ERK1/2), (Sigma Chemical Co.); phospho-p44/42 MAP kinase Results (Thr202/Tyr204) antibody (Cell Signaling Technology, Beverly, MA, USA); MEK1/2 and phospho-MEK1/2 (Ser217/221), Raf-1 (C-12) Cytotoxicity. To assess the drug effects on cell viability, (Santa Cruz Biotechnology, Santa Cruz, CA, USA); phospho-Raf cytotoxicity assays based on formazan metabolism were (Ser259) (Cell Signaling Technology); Akt and phospho-Akt (Ser473) (Cell Signaling Technology), and anti-PI 3-kinase p85 conducted after 72 h of continuous exposure of the cells to (Upstate Biotechnology, Lake Placid, NY, USA). The following R115777, the control enantiomer R115776, or paclitaxel. As monoclonal antibodies were used: RhoB (C-5) (Santa Cruz presented in Table I, R115777 and R115776 showed Biotechnology), HDJ-2/DNAJ (Neomarkers, Fremont, CA, USA) comparable toxicities in BT-474 (5.4 ÌM and 4.9 ÌM, and ‚-actin (Ab-1) (Oncogene Research Products, Boston, MA, respectively) and MDA-MB-231 (28.9 ÌM and 23.9 ÌM, USA). The antibodies were used for immunoblotting following respectively) cells, thus, the BT-474 cells were about 4-fold protocols recommended by the suppliers. more sensitive to both enantiomers than the MDA-MB-231 cells. On the other hand, the paclitaxel toxicity was Immunodetection of biomarkers. Cells were seeded in 6-well Falcon plates (Fisher Scientific, Pittsburgh, PA, USA) and treated with 1 comparable in both cell lines. In the drug combinations (data nM paclitaxel, 10 ÌM R115777, drug combination, or the vehicle not shown), the cytotoxicities of R115777 and paclitaxel were alone for 24 h to 96 h (1 to 4 days). The cells were plated in apparently additive with no evidence of synergy. quadruplicate plates to allow for daily harvests of cells and cell culture media after the initial addition of the drugs. The cell media Prenylation biomarkers. To determine the effects of the drug were collected for quantitative analysis of secreted factors (VEGF, treatment on prenylation of HDJ-2 and RhoB as surrogate FGF-2, MMP-1, MMP-2 and MMP-9) by ELISA. The cells were biomarkers of drug efficacy, cells treated with R115777 or washed with phosphate-buffered saline (PBS) pH 7.4, harvested by paclitaxel, alone or in combination, were harvested at four trypsinization and washed again in PBS by centrifugation in 15 ml conical tubes. Floating cells were removed from the media by consecutive time-points at days 1-4. Representative Western centrifugation and pooled with the trypsinized cells. Combined cell blots at day 4 are shown in Figure 2, while the quantitative harvests were suspended in 1 ml of PBS, transferred to Eppendorf analysis of the data is summarized in Figure 3. As shown by tubes and centrifuged again. The cells were lysed in 400 Ìl of gel-shift farnesylation assays in MDA-MB-231 cell extracts, hypotonic lysis buffer (10 mM HEPES pH 7.9, 10 mM KCl, 0.1 mM paclitaxel had no effect on HDJ-2 post-translational EDTA, 0.1 mM EGTA) with freshly added 2.5 mM DTT and 1% modification by FTase. In contrast, after cell treatment with protease and phosphatase inhibitor cocktails (Sigma Chemical Co.) R115777 alone or with paclitaxel, the ratios of the and incubated on ice for 15 min. Small aliquots of the extracts were used for analysis of protein concentration by micro-BCA assay unfarnesylated (lower mobility protein band at 44 kD) to the (Pierce Biotechnology, Rockford, IL, USA) with bovine serum farnesylated HDJ-2 (higher mobility protein band at 42 kD) albumin as a protein standard. An equal amount (~20-50 Ìg) of were over 8-fold higher than the untreated controls at days 1 protein was boiled in SDS loading buffer containing 0.1 M and 2, and over 5-fold higher at days 3 and 4. In BT-474 cells, dithiothreitol (DTT) and loaded on precast SDS polyacrylamide paclitaxel also had no effect on HDJ-2 farnesylation. gels (Bio-Rad Laboratories, Hercules, CA, USA and BioWhittaker However, R115777, alone or in combination with paclitaxel, Molecular Applications, Rockland, ME, USA). Proteins were increased the ratio of the unfarnesylated to the farnesylated transferred to ImmunBlot PVDF membrane (Bio-Rad Laboratories) and immunoblotted with appropriate primary and HDJ-2 over 3-fold above the control at days 1-3, which later secondary antibodies. The blots were probed with ‚-actin used as decreased to about 2-fold the control level at day 4. The an internal control for assessing protein load. The signal, developed results indicate the greater effect of R115777 on inhibition of by chemiluminescence reaction with SuperSignal West Pico reagent HDJ-2 farnesylation in MDA-MB-231 than in BT-474 cells. (Pierce Biotechnology), was captured by autoradiography on Kodak RhoB, whose isoprenylation is necessary for its rapid BioMax Light-1 film (Sigma Chemical Co.) and quantified by turnover, is a surrogate marker for inhibition of densitometry using PDSI scanner (Molecular Dynamics, Amersham farnesylation. In the presence of FTIs, RhoB does not Biosciences, Piscataway, NJ, USA) and ImageQuaNT software. The densitometric intensity of bands was expressed as a ratio of actin in undergo rapid proteolysis, which leads to accumulation of each sample lane. The ratios of phosphorylated or prenylated the unprenylated protein. As expected, paclitaxel treatment species to the total protein were calculated for select target proteins. of BT-474 or MDA-MB-231 cells had no effect on RhoB The data are representative of at least three separate experiments. stability (Figure 3), while substantial accumulation of RhoB

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Figure 2. Representative images of Western blots of protein biomarkers in MDA-MB-231 and BT-474 cell lysates. Cells were exposed to the vehicle, 10 ÌM R115777, 1 nM paclitaxel, or the drug combination for 4 days. Protein lysate preparation, gel electrophoresis and immunoblotting with human- specific antibodies were performed as described in Materials and Methods.

was noted in both cell lines after treatment with R115777 observed at day 3, followed by a later decline. In comparison alone or the combination with paclitaxel. In MDA-MB-231 with BT-474, the inhibition of prenylation of RhoB was cells, RhoB increased over 20-fold in the presence of greater in MDA-MB-231 cells. R115777 or the combination with paclitaxel at days 1 and 2. Even though a decline in RhoB was seen at later time-points, Biomarkers of tumor growth pathways. The Ras/Raf/MEK at days 3 and 4 the levels of the protein were 10-fold higher (mitogen-activated protein kinase/ERK kinase)/ ERK than the untreated control. In BT-474 cells, the maximum (extracellular-signal-regulated kinase) pathway is in the levels of RhoB (over 6-fold increase above the control) were center of signaling networks that control proliferation,

3218 Izbicka et al: Biomarkers for Farnesyltransferase Inhibitor in Breast Cancer

Figure 3. Quantitative analysis of protein biomarker expression in MDA-MB-231 and BT-474 cell lysates. Cells were exposed to the vehicle, 10 ÌM R115777, 1 nM paclitaxel, or the drug combination for 1-4 days. Protein lysate preparation, gel electrophoresis and immunoblotting with human-specific antibodies was performed as described in Materials and Methods. The protein expression was normalized for ‚-actin. differentiation and tumor cell survival. It was hypothesized transient decrease in ERK signaling was observed at days 2 that inhibition of FTase activity would translate to and 4. In BT-474 cells, Raf phosphorylation was inhibited decreased phosphorylation cascade involving the key by nearly 20% by paclitaxel alone and in combination with proteins; Raf, MEK and ERK. As shown in Figure 3, in R115777 at day 2. At day 4, paclitaxel treatment was the MDA-MB-231 cells, the ratio of the phosphorylated to total least effective, while some inhibition of Raf phosphorylation Raf was unusually high only in the cells treated with could be seen in the presence of R115777 +/– paclitaxel. paclitaxel at day 1. At later time-points, there was no MEK signaling was most efficiently inhibited by paclitaxel. evidence of Raf activation under any experimental The activity of R115777 was similar as a monotherapy or in conditions. Activation of MEK, expressed as the ratio of the combination with paclitaxel. Either case was associated with phosphorylated intermediate to the total MEK protein, was a transient activation, followed by the inhibition and, finally, inhibited by R115777 and brought to a nearly full inhibition the escape from the inhibition at day 4. A similar trend in by the drug combination with paclitaxel. The inhibition the expression of (P)ERK was observed with single agents increased in a time-dependent manner up to 3 days. Beyond and the drug combination. Overall, trends in growth and that time, R115777 alone suppressed MEK phosphorylation survival pathways in MDA-MB-231 were largely paclitaxel- at day 4. However, at that time the cells escaped from the dependent. In BT-474, trends in MEK and ERK activation inhibitory effect of paclitaxel +/– R115777. A small were more R115777-dependent and linked.

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Figure 4. Effects of R115777 and paclitaxel on levels of angiogenic biomarkers in cell culture media. MDA-MB-231 and BT-474 cells were exposed to the vehicle, 10 ÌM R115777, 1 nM paclitaxel, or the drug combination. The cultured media were harvested on days 1-4, and the levels of biomarkers quantified by human-specific ELISA.

Biomarkers of tumor survival pathways. We further shown). As shown in Figure 4, VEGF secretion in MDA- hypothesized that R115777 and paclitaxel may affect the MB-231 cells was not significantly affected by any of the drug expression of biomarkers for tumor survival pathways, the treatment groups. About 20% inhibition was seen in cells PI3K and Akt proteins. Drug treatment of MDA-MB-231 treated with R115777 alone and with the drug combination cells did not significantly change PI3K levels, and had a for up to 3 days. In contrast, single drugs were not effective small transient effect on activation of Akt, as evidenced by in suppressing the production of VEGF in BT-474, but an an increase in the (P)Akt/Akt ratios by day 2. The activation over 2-fold decrease in VEGF levels was observed in the was later inhibited at day 4 by R115777 and its combination cells treated with R115777 in combination with paclitaxel. with paclitaxel. In BT-474 cells, PI3K was largely up- Untreated MDA-MB-231 cells secreted substantial regulated by R115777 plus paclitaxel at day 1, and a similar quantities of MMP-1 after 2 days in culture and all drug "see-saw" pattern was observed at later times with all drugs. treatments dramatically inhibited MMP-1 production. In Akt signaling was dramatically increased by paclitaxel at day BT-474 cells, all drug treatments resulted in nearly 70% 1, but the pattern of near complete inhibition of Akt inhibition of MMP-1 release to the cell culture media. activation was seen at later time-points for all drugs. Discussion Biomarkers of angiogenesis. Since FTIs have been demonstrated to affect tumor signaling pathways responsible FTIs block the main post-translational modification of the for angiogenesis, we examined the effects of monotherapies Ras protein and interfere with subsequent activation of and drug combination on the secretion of pro-angiogenic downstream effectors. Although initially developed to target growth factors to the cell culture media. The levels of Ras in cancer, FTI’s additional and more complex secreted FGF-2, MMP-2 and MMP-9 were under the mechanism of action apparently extends beyond Ras, detection limits of the assay in all treatment groups (data not involving RhoB, centromere-binding proteins and other

3220 Izbicka et al: Biomarkers for Farnesyltransferase Inhibitor in Breast Cancer farnesylated proteins (15). R115777 is historically the first may be model-dependent. Further studies are warranted to FTI to be evaluated in human clinical trials. To date, single- determine if the schedule of drug combination could agent phase I/II studies have demonstrated that the enhance the biological activities of R115777 and paclitaxel. compound can be safely administered with a favorable To date, several in vitro and in vivo studies have examined therapeutic index, allowing the administration of biologically the efficacy of other FTIs in combination with paclitaxel active doses of the drug. Dose-limiting myelotoxicity and and, in some instances, the combinations resulted in neurotoxicity were observed, and intermittent schedules were synergy. SCH66336, an orally active FTI, demonstrated generally well tolerated. Antitumor activity was observed, synergy in combination with paclitaxel in 10 out of 11 tumor particularly in breast cancer and hematological malignancies. cells lines originating from breast, colon, lung, ovary, Recent phase II clinical trials in patients with metastatic prostate and pancreas. In the NCI-H460 lung cancer breast carcinoma have shown that R115777 has reproducible xenograft model, oral SCH66336 and intraperitoneal single-agent activity, with the activity being predominantly paclitaxel caused greater tumor growth inhibition compared seen in patients with HER2-positive disease. The single to paclitaxel alone (21). The combination of SCH 66336 and agent phase II studies have shown feasibility of chronic paclitaxel in non-small cell lung cancer cell lines resulted in dosing, which may be relevant for future combination studies a schedule-dependent synergy when SCH 66336 followed (3, 7). However, two phase III trials of R115777 in colorectal paclitaxel treatment, but antagonism was found when SCH and pancreatic cancer did not show a clear survival benefit. 66336 preceded paclitaxel treatment (22). In a triple The future clinical development of R115777 is likely to be combination of SCH66336, paclitaxel and a recombinant focused on combination therapies (16-18). adenovirus that expressed the human p53 gene, each two A number of combination studies with R115777 have drug interaction displayed synergy in a panel of human cell been carried out. A recent study demonstrated that the lines in vitro (23). ER-51785, a new peptidomimetic combination of R115777 with was well inhibitor of farnesyl transferase, synergized with paclitaxel tolerated. Biologically relevant plasma concentrations of in human pancreatic cancer cells in vitro (24). Manumycin R115777 were achieved (3). Many and FTI enhanced the cytotoxic effect of paclitaxel on anaplastic combinations are being evaluated because preclinical thyroid carcinoma cells in vitro. However, the combination studies suggest that these classes of anticancer agents may was not synergistic in vivo (25) and it is not clear if the be synergistic. Of particular interest may be randomized preclinical studies on FTI synergy will translate to similar clinical studies investigating the clinical benefits of R115777, clinical responses. One should also keep in mind that with or without a taxane, in patients with treatment-naive endpoints other than cell viability may be more relevant for HER2-positive metastatic breast carcinoma (7). targeted therapies. The present study examined a hypothesis that combination R115777, alone and with paclitaxel, has been shown to therapy with R115777 and paclitaxel will be more efficacious inhibit prenylation of two biomarkers, HDJ-2 and RhoB. than single-drug therapy in patients with HER2/neu- The inhibition of prenylation was more pronounced in the expressing breast cancers. Amplification of the HER2 gene breast cancer model with a low expression of HER2/neu and overexpression of the HER2 protein induces cell than the overexpressing cells. The extent of inhibition most transformation and has been demonstrated in 10% to 40% of probably reflects relative stoichiometry between the enzyme human breast cancer. HER2 overexpression has been (FTase) and the inhibitor (R115777) in the cell suggested to associate with tumor aggressiveness, prognosis environment. In either breast cancer cell line, no inhibition and responsiveness to hormonal and cytotoxic agents in was observed with paclitaxel alone, consistent with the breast cancer patients. The reason for this association is still expected mechanism of action of R115777. unclear, although it has been suggested to result in increased In the past, several studies investigated HDJ-2 as the proliferation, vessel formation and/or invasiveness (19). endpoint of FTI acitivity (3, 26-28). We have demonstrated The cell viability studies demonstrated that R115777 and effects of R115777 on farnesylation of HDJ-2 in peripheral its enantiomer are more toxic in human breast cancer cells blood mononuclear cells at biologically relevant plasma overexpressing HER2/neu than in cells with low HER2/neu concentrations of R115777 (3). The use of HDJ-2 in PBMC, levels. Given the resistance of HER2/neu-overexpressing as a surrogate pharmacodynamic assay, has been shown to cancers to many therapies, this finding might be of be feasible in the context of phase I evaluations of R115777, relevance for R115777 monotherapy in the clinic. On the but the significance the finding was unclear with respect to other hand, combination of the drug with paclitaxel was the drug effect in tumor tissue. The present observation additive but not synergistic. Yet, the activity of R115777 suggests that assessment of the prenylation status of HDJ-2, alone and in combination with purine nucleoside analogs on RhoB and/or other substrates for FTI in tumor biopsies acute myeloid leukemia progenitors in vitro has been shown might be important to confirm drug activity in the target to be more than additive (20), suggesting that the synergy tissue, not only in the surrogate tissue such as PBMCs.

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The tumor growth and survival pathways represented in Acknowledgements this study included the Raf/MEK/ERK and PI3K/Akt proteins and the respective phosphorylated intermediates. We thank Dr. S. Rani for help with the Western blot and ELISA We demonstrated a transient but reproducible suppression of assays. the Raf/MEK/ERK activation, as evidenced by a lower phospho- rylation state of the representative proteins. References Interestingly, the drug combination was highly effective in inhibiting ERK activation in HER2/neu-overexpressing cells. 1 Rowinsky EK, Windle JJ and Von Hoff DD: Ras protein HER2/neu overexpression can activate PI3K and Ras/Raf/ farnesyltransferase: a strategic target for anticancer therapeutic MEK/ERK pathways, resulting in reduction of wild-type p53 development. J Clin Oncol 17(11): 3631-3652, 1999. protein expression. This may be the molecular mechanism 2 Cox AD: Farnesyltransferase inhibitors: potential role in the responsible for the poor prognosis and therapeutic non- treatment of cancer. Drugs 61(6): 723-732, 2001. 3 Patnaik A, Eckhardt SG, Izbicka E, Tolcher AA, Hammond responsiveness in HER2/neu-overexpressed breast cancer LA, Takimoto CH, Schwartz G, McCreery H, Goetz A, Mori M patients (29). Other studies suggest that the intracellular et al: A phase I, pharmacokinetic, and biological study of the signaling pathway of HER2 involves Ras-MAPK, MAPK- farnesyltransferase inhibitor tipifarnib in combination with independent S6 kinase and phospholipase C-gamma signaling gemcitabine in patients with advanced malignancies. Clin pathways. However, the biological consequences of the Cancer Res 9(13): 4761-4771, 2003. activation of these pathways are completely known (30). Our 4 Jiang K, Coppola D, Crespo NC, Nicosia SV, Hamilton AD, results suggest that R115777, and the combination thereof Sebti SM and Cheng JQ: The phosphoinositide 3-OH kinase/AKT2 pathway as a critical target for farnesyltransferase with paclitaxel, may actually overcome the inherent resistance inhibitor-induced apoptosis. Mol Cell Biol 20(1): 139-148, 2000. of the HER2/neu-overexpressing breast cancer cells and 5 Zhang B, Prendergast GC and Fenton RG: Farnesyltransferase result in a clear clinical benefit. inhibitors reverse Ras-mediated inhibition of Fas gene The combination of R115777 plus paclitaxel inhibited the expression. Cancer Res 62(2): 450-458, 2002. production of pro-angiogenic growth factors in HER2/neu 6 Venet M, End D and Angibaud P: Farnesyl protein transferase low and overexpressing breast cancer cells. This finding may inhibitor ZARNESTRA R115777 – history of a discovery. Curr be consequential for future clinical studies. Overexpression Top Med Chem 3(10): 1095-1102, 2003. of HER2 in human tumor cells is closely associated with 7 de Bono JS, Tolcher AW and Rowinsky EK: Farnesyltransferase inhibitors and their potential in the treatment of breast increased angiogenesis and expression of VEGF. Up- carcinoma. Semin Oncol 30(5 Suppl 16): 79-92, 2003. regulation of VEGF in cancer epithelial cells is likely to 8 Punt CJ, van Maanen L, Bol CJ, Seifert WF, Wagener DJ: support angiogenesis, sustaining and promoting survival and Phase I and pharmacokinetic study of the orally administered metastasis of tumor cells, while inhibition of the VEGF farnesyl transferase inhibitor R115777 in patients with advanced pathway has been shown to suppress tumor growth (31). solid tumors. Anticancer Drugs 12(3): 193-197, 2001. Measurements of angiogenesis and lymphangiogenesis may 9 Moulder SL, Yakes FM, Muthuswamy SK, Bianco R, Simpson be important for breast cancer pathology, particularly for JF and Arteaga CL: Epidermal growth factor receptor (HER1) tyrosine kinase inhibitor ZD1839 (Iressa) inhibits HER2/neu estimation of metastatic risk (32). HER2 overexpression (erbB2)-overexpressing breast cancer cells in vitro and in vivo. seems to be a significant predictor of response to . Cancer Res 61(24): 8887-8895, 2001. Recent evidence confirms the independent prognostic value 10 Ogata H, Sato H, Takatsuka J and De Luca LM: Human breast of VEGF, UPA and PAI-1 in women with early breast cancer MDA-MB-231 cells fail to express the neurofibromin cancer and suggests that such parameters may have a role protein, lack its type I mRNA isoform and show accumulation of in selecting systemic therapy (33). P-MAPK and activated Ras. Cancer Lett 172(2): 159-164, 2001. In conclusion, the activity of R115777 in vitro on 11 Kunisue H, Kurebayashi J, Otsuki T, Tang CK, Kurosumi M, Yamamoto S, Tanaka K, Doihara H, Shimizu N and Sonoo H: prenylation biomarkers is consistent with the expected drug Anti-HER2 antibody enhances the growth inhibitory effect of mechanism of action. In addition, R115777 in combination anti-oestrogen on breast cancer cells expressing both oestrogen with paclitaxel was shown to interfere with tumor growth, receptors and HER2. Br J Cancer 82(1): 46-51, 2000. survival and angiogenesis pathways in breast cancer models 12 Fitzpatrick SL, LaChance MP and Schultz GS: Characterization with low or overexpressed HER2/neu. Pharmacokinetic of epidermal growth factor receptor and action on human breast analysis demonstrated that peak plasma concentrations of cancer cells in culture. Cancer Res 44(8): 3442-3447, 1984. 881+/–393 ng/mL, corresponding to low micromolar 13 Mossman T: Rapid colorimetric assay for cellular growth and concentrations of R115777, were reached within 1-5 h (8). The survival: application to proliferation and cytotoxicity assays. J Immunol Methods 65: 55-63, 1983. observed biological activity in vitro was seen at R115777 14 Weitman S, Barrera H, Moore R, Gonzalez C, Marty J, concentrations that are achievable in the clinic. Thus, Hilsenbeck S, MacDonald JR, Waters SJ and Von Hoff D: combination therapy might offer further clinical advantage MGI 114: augmentation of antitumor activity when combined over R115777 and paclitaxel monotherapies. with . J Pediatr Hematol Oncol 22(4): 306-314, 2000.

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