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Department of Oncology, Mayo Clinic (L.M.B., J.N.P., S.H.K., A.A.A.) and Department of Molecular Pharmacology, Mayo Graduate School (S.H.K.), Rochester, Minnesota
Received for publication December 6, 2004.
Accepted for publication May 18, 2005.
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Abstract |
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). This covalent addition of either a farnesyl (15-carbon) or a geranylgeranyl (20-carbon) group to the cysteine residue located in a tetrapeptide CAAX (A = aliphatic, X = methionine or serine) sequence at the carboxyl terminus facilitates membrane attachment of several G proteins that are synthesized in the cytoplasm but perform their functions when anchored in the plasma membrane (Clarke, 1992). One such protein is Ras, which ordinarily requires farnesylation before it can mediate its proliferative functions (Kato et al., 1992). As a consequence of mutations in Ras genes, Ras proteins are constitutively activated in 30% of human tumors. These mutations result in constitutive activation of several Ras-activated intracellular signaling pathways, including the PI-3 kinase/Akt pathway, which is critical for cell survival, and the Raf/MEK/ERK pathway, which has been implicated in cell proliferation (Lange-Carter et al., 1993). Thus, tumors bearing Ras mutations are relatively more aggressive and have a poor clinical outcome (Samowitz et al., 2000).
FTIs were originally developed to inhibit the aberrant Ras-mediated signals that stimulate cell proliferation, apoptosis, invasion, and angiogenesis (Downward, 1998). At least three FTIs discovered through high-throughput screening are currently undergoing clinical testing. These include SCH66336(Lonafarnib, Sarasar), R115777 (Tipi-farnib, Zarnestra), and BMS-214662 (Adjei, 2003). As a class, these agents have shown clinical activity in breast cancer, glioma, head and neck cancer, and a wide range of hematological malignancies, including acute myelogenous leukemia, chronic myelogenous leukemia, and myelodysplastic syndrome (Adjei, 2003).
Despite this potentially promising clinical activity, several questions about the action of these agents remain unanswered. First, the cellular target responsible for the biological activity of FTIs has remained uncertain. Although early studies in Ras-transfected fibroblasts supported the idea that FTIs target Ras, more recent studies suggest that Ras proteins might not be the exclusive target of FTIs. Instead, FTI-induced cytotoxicity has been postulated to involve inhibition of the small GTPase RhoB (Du and Prendergast, 1999), the centromere-binding proteins CENP-E and CENP-F (Ashar et al., 2000), an unidentified polypeptide that acts upstream of the serine/threonine kinases Akt1 and Akt2 (Jiang et al., 2000; Chun et al., 2003), and/or other polypeptides that are yet to be identified (Haluska et al., 2002). Second, the mechanisms that influence the differential sensitivity of various tumors to FTIs are poorly understood. Previous studies have demonstrated that deliberate mutations of FT can produce an enzyme with diminished drug sensitivity (Del Villar et al., 1999); but the occurrence of these mutations in intact cells has not been demonstrated. More recently, Smith et al. (2002) described an R115777-selected cell line but were unable to establish a mechanism for its resistance.
In the present study, we sought to address these issues by selecting a mixed population of HCT 116 colorectal carcinoma cells for resistance to FTIs. This HCT 116R line exhibited 100-fold resistance to various FTIs. Inhibition of protein farnesylation was similar in the parental and resistant cell lines, suggesting that altered drug uptake or FT sensitivity was not the underlying mechanism of resistance. Instead, resistant HCT 116 cells exhibited elevated levels of Akt1 and Akt2 and increased phosphorylation of downstream targets, including the kinase mTOR and its substrate p70 S6 kinase. Further experiments demonstrated that the mTOR inhibitor rapamycin selectively sensitized the resistant cells to SCH66336 These findings not only suggest that resistance of HCT 116R cells is caused by changes in Akt isoforms, but they also provide a potential strategy for overcoming this resistance.
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Materials and Methods |
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; Adjei et al., 2000b). Histone H1 antibody was from James Sorace at the Veteran's Affairs Medical Center (Baltimore, MD). Murine monoclonal antibodies that recognize poly(ADP-ribose) polymerase and topo II
were gifts from Drs. Guy Poirier (Laval University, Ste-Foy, QC, Canada) and Udo Kellner (Magdeburg, Germany), respectively. E10 monoclonal phospho-(Thr202/Try204)-ERK; affinity-purified epitope-specific rabbit antisera that recognize phospho-(Ser473)-Akt, phospho-GSK-3/
(Ser21/Ser9), phospho-(Ser2448)-mTOR, phospho-(Thr389)-p70 S6 kinase, and phospho-(Thr183/Tyr185) c-Jun N-terminal kinase; and antisera to Akt, GSK-3/, mTOR, c-Jun N-terminal kinase, and ERK1/2 were from Cell Signaling Technology (Beverly, MA). Antibodies to actin, Akt2, and p70 S6 kinase were from Santa Cruz Biotechnology, Inc. (Santa Cruz, CA). Antibodies to H-Ras, HDJ-2, and PTEN were from ViroMed Biosafety Laboratories (Camden, NJ), Neomarkers (Fremont, CA), and Oncogene Research Products (Cambridge, MA), respectively. Horseradish peroxidase-conjugated secondary antibodies were from Kirkegaard and Perry Laboratories, Inc. (Gaithersburg, MD). Enhanced chemiluminescence reagents were from Amersham Biosciences Inc. (Piscataway, NJ). Cell Culture. HCT 116 cells obtained from the American Type Culture Collection (Manassas, VA) were cultured in RPMI 1640 medium supplemented with 10% fetal bovine serum, 100 U/ml penicillin G, 100 µg/ml streptomycin, and 2 mM glutamine. HCT 116R were developed by exposing HCT 116 cells (IC50 = 50 nM) to increasing concentrations of SCH66336at each passage until concentrations of 1 µM were reached. To begin with, the cells were exposed to 25 nM, and the concentration was doubled approximately once per week until 400 nM was reached. The concentration was then increased by 50 or 100 nM each week until concentrations of 1 µM were achieved. Cells were passed two times per week or when subconfluent monolayers formed.
Colony-Forming Assays. After subconfluent monolayers were trypsinized, 500 cells in 2 ml of medium were added to 35-mm dishes and incubated for 24 h at 37°C to allow cells to attach. Increasing drug concentrations that spanned the range of at least 0.5 to 2.0 times the individual IC50 values or equivalent volumes of diluent were added to triplicate plates, which were then incubated for 7 days to simulate the prolonged drug exposure achieved clinically (Adjei et al., 2000b; Eskens et al., 2001). The resulting colonies were stained with Coomassie blue and counted manually.
Sample Preparation and Immunoblotting. HCT 116 and HCT 116R cells were plated at 1.5 x 106 cells per 100-mm plate and incubated for 72 h. Whole-cell lysates were prepared by washing cells three times with lysis buffer (50 mM Tris-HCl, pH 7.5, 150 mM KCl, 1 mM EDTA, 1 mM EGTA, 1 mM dithiothreitol, 50 mM -glycero-phosphate, 50 mM NaF, 10 mM Na4P3O7 · 10H2O, 0.1 mM Na3VO4, 0.1 mg/ml leupeptin, 0.01 mg/ml aprotinin, 0.001 mg/ml pepstatin, and 0.02 µM microcystin), collecting cells into 200 µl of lysis buffer, and sonicating. After protein concentration was estimated using the Bradford method (Bradford, 1976), serial dilutions of lysates were combined with 4x SDS-PAGE sample buffer (250 mM Tris-HCl, pH 6.8, 4 M urea, 1 mM EDTA, 8% SDS, and 20% 2-mercaptoethanol), heated to 65°C for 20 min, and separated by SDS-PAGE. Otherwise, 100-mm plates containing cells at 50 to 60% confluence were washed with serum-free RPMI 1640 medium containing 10 mM HEPES (pH 7.4 at 21°C) and solubilized in 3 ml of alkylation buffer (6 M guanidine HCl, 250 mM Tris-HCl, pH 8.5 at 21°C, 10 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 1% 2-mercaptoethanol). After the cells were alkylated and dialyzed as described previously (Kaufmann, 1989; Kaufmann et al., 1997), aliquots containing of 50 µg of protein, as determined by the bicinchoninic acid method (Smith et al., 1985), were subjected to SDS-PAGE. After polypeptides were transferred to nitrocellulose, blots were blocked with 5 or 10% (w/v) powdered milk in buffer consisting of 150 mM NaCl, 10 mM Tris-HCl (pH 7.4 at 21°C), and 0.5% (w/v) Tween 20 as described previously (Kaufmann, 2001).
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). After incubation with gentle rotation at 4°C for 1.5 h, the bound antibody-antigen complexes were sedimented, washed three times with lysis buffer, and disrupted by boiling for 5 min in 1x SDS-PAGE sample buffer (4 M urea, 2% SDS, 62.5 mM Tris-HCl, pH 6.8, 1 mM EDTA, and 10% 2-mercaptoethanol). Serial dilutions of immunoprecipitated lysates were separated on 10% gels, transferred to nitrocellulose, and probed for the appropriate proteins.
Sequencing of FT. Total RNA was isolated from HCT 116 and HCT 116R cells using the RNeasy Mini Kit from QIAGEN (Valencia, CA). FT - and -subunit cDNA were synthesized using the Super-Script III First-Strand Synthesis Kit from Invitrogen (Carlsbad, CA) according to the manufacturer's instructions and using the following primers: forward, 5'-CTGTCCTGCAGCGTGATGAA-3'; and reverse, 5'-ACCACTCTCGTGTGAAACTC-3' for FT; and forward, 5'-CTGCTGCTCTTCCTGATCATGGCTTCTCC-3'; and reverse, 5'-TCTAGTCGGTTGCAGGCTCTGCCGATGT-3' for FT. Amplification of the sequences from the total cDNA was achieved using the Expand High-Fidelity polymerase chain reaction system from Roche. The amplification cycles were 94°C for 2 min, 40 cycles at 94°C for 15 s, 60°C for 30 s, and 72°C for 2 min, followed by 72°C for 7 min. Sequencing was carried out using the ABI PRISM rhodamine dye terminator reaction kit and analyzed using the ABI PRISM 3700 DNA Analyzer from Applied Biosystems (Foster City, CA). Primers for sequencing are listed in Table 1.
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Transcriptional Profiling Analysis. Labeled complementary DNA was synthesized from total RNA and hybridized to human U133A chips (Affymetrix, Santa Clara, CA). Gene Chip 5.0 (Affymetrix) was used to scan and quantitatively analyze the scanned image. Gene Chip software was used to calculate intensity values for each probe cell. The data were imported into Microsoft Excel (Microsoft, Redmond, CA) and further analyzed with the help of Steve Iturria in the Mayo Clinic Biostatistics Department (Rochester, MN).
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Results |
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Lack of Cross-Resistance of HCT 116R to Standard Cytotoxic Agents. To rule out the possibility that the HCT 116R cells had acquired resistance to a wide array of anticancer agents, colony-forming assays were used to examine the sensitivity of the parental and resistant cells to 7 days of continuous exposure to the cross-linking agent cisplatin, the antimetabolite gemcitabine, and the microtubule poison paclitaxel. As can be seen in Fig. 2, there was much less difference in sensitivity to these agents, suggesting that HCT 116R cells had acquired relatively specific resistance to FTIs.
Accumulation of Prelamin A and Unfarnesylated HDJ-2 in Parental HCT 116 and HCT 116R Cells. In subsequent experiments, parental and resistant cells were treated with varying SCH66336concentrations and then subjected to SDS-PAGE followed by immunoblotting with reagents raised against the FT substrates prelamin A (Adjei et al., 2000a,b) and HDJ-2 (Sinensky et al., 1994; Britten et al., 1999; Adjei et al., 2000a). Increased amounts of a slower migrating species of HDJ-2 were observed after SCH66336treatment in both parental and resistant cell lines (Fig. 3A, third row). Moreover, a small amount of this slower migrating unfarnesylated HDJ-2 was present in HCT 116R cells before FTI exposure. When these same blots were probed with antiserum that specifically recognizes the precursor peptide at the carboxyl terminus of prelamin A (Adjei et al., 2000b), inhibition of farnesylation-dependent prelamin A processing could be detected in a dose-dependent manner in HCT 116 and HCT 116R cells over roughly the same concentration range (Fig. 3A, top row). Similar results were observed when cells were treated with FTI-277 and probed for HDJ-2 (Fig. 3B). Because inhibition of protein farnesylation occurred over the same SCH66336or FTI-277 concentration range in both cell lines, these data suggest that the uptake of FTIs into HCT 116R cells is unimpaired and that FT within these cells remains sensitive to drug-mediated inhibition.
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). For FT, the full-length cDNA could not be amplified because of its high GC content. Therefore, a primer starting at base pair 302 was designed. Full-length FT was sequenced. No difference in the sequence of either the - or -subunit was observed when results obtained in HCT 116R and parental cells were compared with each other and to the consensus sequence published in GenBank (accession no. L10413
[GenBank]
for -subunit and L10414
[GenBank]
for -subunit; data not shown).
In additional experiments, expression of the FT and FT mRNAs was compared in HCT 116R and parental cells. Once again, no differences were seen (data not shown). Likewise, there were no detectable differences in expression of mRNAs encoding farnesyl dipshophate synthase and a variety of FT substrates, including H-Ras, N-Ras, RhoB, and CENP-F. These observations prompted us to search for alternative explanations for the 100-fold resistance in HCT 116R cells.
Expression of Ras/Raf/ERK Signaling Pathway Components in HCT 116 and HCT 116R Cells. As indicated in the Introduction, FTIs were initially designed to inhibit the post-translational modification of Ras proteins. The mechanism of cytotoxicity of FTIs is currently unknown but could potentially be related to the interruption of H-ras processing (Gibbs et al., 1993; Kohl et al., 1993; Lerner et al., 1995). Inhibition of MAPK signaling downstream of Ras has been described after treatment with FTIs (Lerner et al., 1995; Karp et al., 2001). To investigate possible alterations involving H-Ras or the MAPK pathway, we initially evaluated the sensitivity of the parental and resistant cell lines to the MEK1/2 inhibitor PD98059 (Dudley et al., 1995). Both cell lines were equally sensitive to this agent (Fig. 4A), suggesting that there were potentially no alterations in MEK or proteins upstream of MEK in the resistant cells. Furthermore, when whole-cell lysates prepared from parental and resistant HCT 116 cells were subjected to SDS-PAGE followed by immunoblotting with reagents that recognize H-Ras, phospho-ERK, and ERK, there were no differences between HCT 116 and HCT 116R cells (Fig. 4B). These results seem to rule out the possibility that altered the expression of H-Ras or enhanced signaling through the Raf/MEK/ERK pathway contributes to resistance in the HCT 116R cells.
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; Chun et al., 2003). To assess the activation of the Akt pathway in parental and resistant HCT 116 cells, whole-cell lysates were analyzed by immunoprecipitation followed by immunoblotting (Fig. 5A) or by direct immunoblotting (Fig. 5, B and C). These analyses demonstrated an increased signal for phospho-Thr389-p70 S6 kinase in HCT 116R cells compared with parental cells (Fig. 5A, top) and an increase in the amount of activating phosphorylation (Nave et al., 1999; Reynolds et al., 2002) on Ser2443 of the intermediary kinase mTOR (Fig. 5B). Although the increased amount of phopsho-Thr389-p70 S6 kinase reflected, at least in part, increased p70 S6 kinase content (Fig. 5A, bottom), the increase in phosphorylated mTOR occurred without any change in mTOR content (Fig. 5B).
Increased Akt Expression in HCT 116R Cells. In an attempt to further delineate components of the Akt pathway contributing to FTI resistance, we performed immunoblotting for polypeptides upstream of mTOR. Akt1 and Akt2 were both increased 2- to 4-fold in the HCT 116R cells (Fig. 5C). Reactivity with anti-phospho-Ser473-Akt, which detects an activating phosphorylation of Akt1 and Akt2, was increased a similar amount in the resistant cells compared with the parental cells (Fig. 5C). In addition, enhanced phosphorylation of GSK-3/, another downstream target of Akt, was detected in the HCT 116R cells. In contrast, the lipid phosphatase PTEN upstream of Akt was unchanged in these cells. Together, these results indicate that Akt isoforms are increased and activated in the HCT 116R cells.
Partial Sensitization of HCT 116R Cells by Rapamycin. Because there were multiple changes in the resistant cells, the role of these changes could not be readily assessed by down-regulating a single polypeptide using antisense or RNA silencing technology. As an alternative, to further explore the relationship between overexpression of Akt and FTI resistance, we initially set out to transfect parental HCT 116 cells with an expression vector encoding myristilated, constitutively active Akt. Pilot studies demonstrated that this construct was expressed for only 2 to 3 days after transient transfection, making it difficult to assess effects on colony-forming assays under these conditions. In four separate transfections, we subsequently attempted to generate stable HCT 116 transfectants. None of the more than 100 clones that grew in selective medium expressed the transfected Akt alleles. Therefore, a pharmacological approach was used to assess the potential importance of the observed changes.
The hypothesis that FTI resistance in the HCT 116R cells results from increased expression of Akt isoforms and a concomitant increase in Akt/mTOR signaling leads to two predictions: 1) depending on the nature of the signaling network, the two cell lines might exhibit similar responses to agents that act upstream of Akt; but 2) mTOR inhibition should diminish the resistance. Further experiments were performed to test these hypotheses.
To assess their sensitivity to agents that act upstream of Akt, cells were treated with increasing concentrations of LY294002, which inhibits PI-3 kinase. The lipid product of PI-3 kinase, phosphatidylinositol-3,4,5-trisphosphate, activates several lipid-dependent kinases. Only part of this signaling impinges on Akt. If the signaling from PI-3 kinase is not the critical input for the resistance, then exposure to LY294002, a PI-3 kinase inhibitor, will not affect HCT 116R cells differentially. Consistent with this hypothesis, there was no difference in sensitivity to this agent (Fig. 6A).
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In a final series of experiments, we explored whether the effect of rapamycin resulted from increased inhibition of FT. HCT 116 and HCT 116R cells treated with 100 nM SCH66336in the absence or presence of increasing concentrations of rapamycin were immunoblotted for HDJ-2. Rapamycin had no discernible effect on the ability of SCH66336to inhibit FT as manifested by the appearance of unfarnesylated HDJ-2 (Fig. 6E). Thus, it seems that rapamycin sensitizes the HCT 116R cells downstream of FT inhibition rather than at the level of FT.
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To further delineate the cause of this resistance, the effects of SCH66336on protein farnesylation in parental and HCT 116R were compared. Previous studies have demonstrated that the prenylated protein HDJ-2 demonstrates a shift in mobility upon treatment with FTIs (Britten et al., 1999; Adjei et al., 2000a; Karp et al., 2001). In addition, work from several laboratories demonstrated previously that prelamin A is a farnesylated polypeptide (Adjei et al., 2000a; Sinensky et al., 1994). The present study demonstrated that farnesylation of prelamin A and HDJ-2 was inhibited at similar SCH66336concentrations in both parental and resistant HCT 116 cells. HDJ-2 farnesylation was also inhibited at similar concentrations by FTI-277 in the parental and resistant cells. These observations suggest that neither alterations in drug transport nor alterations in the sensitivity of FT to inhibition by SCH66336would explain the observed resistance. These results are consistent with the observations of Smith et al. (2002), who also failed to detect an alteration in FT in an R115777-selected colorectal carcinoma cell line.
Even though changes in FT sensitivity do not seem to explain the resistance, a slight alteration in protein farnesylation was observed in the HCT 116R cells (Fig. 3). In particular, the presence of unfarnesylated polypeptides in the resistant cells before FTI exposure raised the possibility that the catalytic ability of FT may be slightly reduced in these cells. Further experiments designed to explore this possibility failed to demonstrate any change in the sequence or expression of either FT subunit. Even if this analysis missed a small difference in FT activity, it is important to emphasize that decreased FT activity in HCT 116R cells would be expected to sensitize the cells to FT catalytic inhibition rather than convey resistance.
In further studies, we focused on the Ras/MAPK and PI-3 kinase/Akt pathways that have been implicated in the cytotoxicity of FTIs (Gibbs et al., 1993; Kohl et al., 1993; Lerner et al., 1995; Jiang et al., 2000; Chun et al., 2003). The results in Fig. 4 fail to implicate alterations in the Ras/Raf/MAPK pathway in the resistance of HCT 116R cells. In particular, the parental and resistant HCT 116R cells were equally sensitive to the cytotoxicity of the MEK inhibitor PD98059. Moreover, the expression of H-ras and the phosphorylation of ERK, a readout of pathway activation, were indistinguishable in the two cell lines. Therefore, the cause of the FTI resistance was believed to lie elsewhere.
We next examined the PI-3 kinase/Akt pathway. Signaling through this pathway inhibits apoptosis by phosphorylation of substrates such as Bad and forkhead transcription family members (Datta et al., 1999). Akt activity, which is constitutive and PI-3 kinase-dependent in a number of tumor cells, does not correlate directly with Ras status (Brognard et al., 2001). Instead, Akt also mediates survival signals from polypeptide growth factor receptors and other tyrosine kinases as well as focal adhesion kinase, which is activated by integrin-mediated signaling. It is interesting to note that several previous studies have indicated that engagement of adhesion and growth factor receptors can rescue cells from FTI-induced apoptosis (Lebowitz et al., 1997; Suzuki et al., 1998), providing indirect evidence that Akt pathway activation might cause resistance to FTIs. This conclusion was further strengthened by subsequent results showing that forced overexpression of Akt1 or Akt2 results in resistance to FTI-induced apoptosis (Jiang et al., 2000; Chun et al., 2003).
In the present study, comparison of the HCT 116 and HCT 116R cells revealed multiple changes in the Akt signaling pathway. Phosphorylation of p70 S6 kinase, a downstream target of the Akt pathway, was elevated (Fig. 5A). This change, which reflected enhanced mTOR activity, occurred without any change in mTOR levels. Instead, increased phosphorylation of mTOR was detected (Fig. 5B). In addition, increased phosphorylation of GSK-3 and - was observed in HCT 116R cells (Fig. 5C). Increased mTOR and GSK-3/ phosphorylation in turn reflected increased levels of phosphorylated Akt1 and Akt2, as well as increased levels of total Akt1 and Akt2 protein in the resistant cells (Fig. 5C). These changes are summarized in Fig. 7.
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To our knowledge, this is the first report showing Akt/mTOR pathway alterations in FTI-selected cells. The cause of the increased Akt1 and Akt2 activation and expression in the resistant cells is not currently known and is under investigation. Further studies in additional FTI-selected cell lines and in clinical material are also required to determine how commonly alterations in this pathway contribute to FTI resistance.
If enhanced signaling downstream of Akt were contributing to the FTI resistance, then inhibition of this signaling should sensitize the resistant cells. Because mTOR is activated in the HCT 116R cells (Fig. 5B), and previous studies have demonstrated that cells with enhanced Akt/mTOR signaling as a consequence of PTEN deletion are particularly sensitive to mTOR inhibitors (Neshat et al., 2001; Bjornsti and Houghton, 2004), we hypothesized that inhibition of mTOR might reverse the resistance of HCT 116R cells. Consistent with this prediction, rapamycin treatment enhanced SCH66336sensitivity in HCT 116R cells (Fig. 6C). It is important that the effect of rapamycin seemed to occur downstream of FT inhibition (Fig. 6E). Moreover, rapamycin failed to sensitize HCT 116R cells to cisplatin (Fig. 6D), gemicitabine, or paclitaxel (data not shown), demonstrating the specificity of this effect. Nonetheless, rapamycin did not completely restore SCH66336sensitivity to that of parental cells (compare Figs. 6C and 1A), perhaps because Akt signals through other pathways or additional alterations also contribute to FTI resistance. Even with this limitation, the IC50 values of the resistant cells in the presence of rapamycin falls well within therapeutically achievable SCH66336plasma levels, which exceed 1 µM for weeks at a time on the twice-daily oral-dosing schedule (Eskens et al., 2001). Thus, the results in Fig. 6C not only provide pharmacological confirmation that the observed changes in the Akt pathway contribute to resistance in the HCT 116R cells but also suggest that the combination of FTIs with rapamycin analogs or Akt inhibitors currently under development might warrant further preclinical and possible clinical testing.
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Acknowledgements |
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antibody. We also thank Raquel Ostby and Deb Strauss for expert secretarial assistance and Steve Iturria for assistance with data analysis. |
Footnotes |
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Article, publication date, and citation information can be found at http://molpharm.aspetjournals.org.
ABBREVIATIONS: PI-3, phosphatidylinositol-3; ERK, extracellular signal-regulated kinase; FT, farnesyl protein transferase; FTI, farnesyltransferase inhibitor; GSK, glycogen synthase kinase; MAPK, mitogen activated protein kinase; mTOR, molecular target of rapamycin; MEK, mitogen-activated protein kinase kinase; topo, topoisomerase; PAGE, polyacrylamide gel electrophoresis; SCH6636, 4-(2-(4-(8-chloro-3,10-dibromo-6,11-dihydro-5H-benzo-(5,6)-cyclohepta(1,2-b)-pyridin-11(R)-yl)-1-piperidinyl)-2-oxo-ethyl)-1-piperidinecarboxamide; PD98059, 2'-amino-3'-methoxyflavone; LY294002, 2-(4-morpholinyl)-8-phenyl-1(4H)-benzopyran-4-one hydrochloride; R115777, (R)-6-(amino(4-chlorophenyl)(1-methyl-1H-imidazol-5-yl)methyl)-4-(3-chlorophenyl)-1-methyl-2(1H)-quinolinone; BMS-214662, (R)-7-cyano-2,3,4,5-tetrahydro-1-(1H-imidazole-4-ylmethyl)-3-(phenylmethyl)-4-(2-thienylsulfonyl)-1H,4-benzodiazepine; FTI-277, methyl {N-[2-phenyl-4-N[2(R)-amino-3-mecaptopropylamino] benzoyl]}-methionate, TFA; GGTI-286, N-4-[2(R)-amino-3-mercaptopropyl]amino-2-phenylbenzoyl-(L)-leucine methyl ester, TPA.
Address correspondence to: Dr. Alex A. Adjei, Division of Medical Oncology, Mayo Clinic, 200 First Street SW, Rochester, MN 55905. E-mail: adjei.alex{at}mayo.edu.
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References |
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TopAbstractMaterials and MethodsResultsDiscussionReferences |
|---|
Adjei AA, Davis JN, Erlichman C, Svingen PA, and Kaufmann SH (2000a) Comparison of potential markers of farnesyltransferase inhibition. Clin Cancer Res 6: 2318-2325.
Adjei AA, Erlichman C, Davis JN, Cutler DL, Sloan JA, Marks RS, Hanson LJ, Svingen PA, Atherton P, Bishop WR, et al. (2000b) A Phase I trial of the farnesyltransferase inhibitor SCH66336: evidence for biological and clinical activity. Cancer Res 60: 1871-1877.
Ashar HR, James L, Gray K, Carr D, Black S, Armstrong L, Bishop WR, and Kirschmeier P (2000) Farnesyl transferase inhibitors block the farnesylation of CENP-E and CENP-F and alter the association of CENP-E with the microtubules. J Biol Chem 275: 30451-30457.
Bjornsti MA and Houghton PJ (2004) The TOR pathway: a target for cancer therapy. Nat Rev Cancer 4: 335-348.[CrossRef][Medline]
Bradford MM (1976) A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248-254.[CrossRef][Medline]
Britten CD, Rowinsky E, Yao S, Soignet S, Rosen N, Eckhardt SG, Drengler R, Hammond L, Siu LL, Smith L, et al. (1999) The farnesyl protein transferase (FPTase) inhibitor L-778,123 in patients with solid cancers (Abstract 597). Proc Am Soc Clin Oncol 18: 155A
Brognard J, Clark AS, Ni Y, and Dennis PA (2001) Akt/protein kinase B is constitutively active in non-small cell lung cancer cells and promotes cellular survival and resistance to chemotherapy and radiation. Cancer Res 61: 3986-3997.
Chun KH, Lee HY, Hassan K, Khuri F, Hong WK, and Lotan R (2003) Implication of protein kinase B/Akt and Bcl-2/Bcl-XL suppression by the farnesyl transferase inhibitor SCH66336 in apoptosis induction in squamous carcinoma cells. Cancer Res 63: 4796-4800.
Clarke S (1992) Protein isoprenylation and methylation at carboxyl-terminal cysteine residues. Annu Rev Biochem 61: 355-386.[Medline]
Datta SR, Brunet A, and Greenberg ME (1999) Cellular survival: a play in three Akts. Genes Dev 13: 2905-2927.
Del Villar K, Urano J, Guo L, and Tamanoi F (1999) A mutant form of human protein farnesyltransferase exhibits increased resistance to farnesyltransferase inhibitors. J Biol Chem 274: 27010-27017.
Downward J (1998) Ras signalling and apoptosis. Curr Opin Genet Dev 8: 49-54.[CrossRef][Medline]
Du W and Prendergast GC (1999) Geranylgeranylated RhoB mediates suppression of human tumor cell growth by farnesyltransferase inhibitors. Cancer Res 59: 5492-5496.
Dudley DT, Pang L, Decker SJ, Bridges AJ, and Saltiel AR (1995) A synthetic inhibitor of the mitogen-activated protein kinase cascade. Proc Natl Acad Sci USA 91: 7686-7689.
Eskens FA, Awada A, Cutler DL, de Jonge MJ, Luyten GP, Faber MN, Statkevich P, Sparreboom A, Verweij J, Hanauske AR, et al. (2001) Phase I and pharmacokinetic study of the oral farnesyl transferase inhibitor SCH 66336 given twice daily to patients with advanced solid tumors. J Clin Oncol 19: 1167-1175.
Gibbs JB, Pompliano DL, Mosser SD, Rands E, Lingham RB, Singh SB, Scolnick EM, Kohl NE, and Oliff A (1993) Selective inhibition of farnesyl-protein transferase blocks ras processing in vivo. J Biol Chem 268: 7617-7620.
Haluska P, Dy GK, and Adjei AA (2002) Farnesyl transferase inhibitors as anticancer agents. Eur J Cancer 38: 1685-1700.
Harlow E and Lane D (1988) Antibodies: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.
Jiang K, Coppola D, Crespo NC, Nicosia SV, Hamilton AD, Sebti SM, and Cheng JQ (2000) The phosphoinositide 3-OH kinase/AKT2 pathway as a critical target for farnesyltransferase inhibitor-induced apoptosis. Mol Cell Biol 20: 139-148.
Karp JE, Lancet JE, Kaufmann SH, End DW, Wright JJ, Bol K, Horak I, Tidwell ML, Liesveld J, Kottke TJ, et al. (2001) Clinical and biologic activity of the farnesyltransferase inhibitor R115777 in adults with refractory and relapsed acute leukemias: a phase 1 clinical-laboratory correlative trial. Blood 97: 3361-3369.
Kato K, Cox AD, Hisaka MM, Graham SM, Buss JE, and Der CJ (1992) Isoprenoid addition to Ras protein is the critical modification for its membrane association and transforming activity. Proc Natl Acad Sci USA 89: 6403-6407.
Kaufmann SH (1989) Additional members of the rat liver lamin polypeptide family: structural and immunological characterization. J Biol Chem 264: 13946-13955.
Kaufmann SH (2001) Reutilization of immunoblots after chemiluminescent detection. Anal Biochem 296: 283-286.[CrossRef][Medline]
Kaufmann SH, Svingen PA, Gore SD, Armstrong DK, Cheng YC, and Rowinsky EK (1997) Altered formation of topotecan-stabilized topoisomerase I-DNA adducts in human leukemia cells. Blood 89: 2098-2104.
Kohl NE, Mosser SD, deSolms SJ, Giuliani EA, Pompliano DL, Graham SL, Smith RL, Scolnick EM, Oliff A, and Gibbs JB (1993) Selective inhibition of rasdependent transformation by a farnesyltransferase inhibitor. Science (Wash DC) 260: 1934-1937.
Lange-Carter CA, Pleiman CM, Gardner AM, Blumer KJ, and Johnson GL (1993) A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf. Science (Wash DC) 260: 315-319.
Lebowitz PF, Sakamuro D, and Prendergast GC (1997) Farnesyl transferase inhibitors induce apoptosis of Ras-transformed cells denied substratum attachment. Cancer Res 57: 708-713.
Lerner EC, Qian Y, Blaskovich MA, Fossum RD, Vogt A, Sun J, Cox AD, Der CJ, Hamilton AD, and Sebti SM (1995) Ras CAAX peptidomimetic FTI-277 selectively blocks oncogenic Ras signaling by inducing cytoplasmic accumulation of inactive Ras-Raf complexes. J Biol Chem 270: 26802-26806.
Nave BT, Ouwens M, Withers DJ, Alessi DR, and Shepherd PR (1999) Mammalian target of rapamycin is a direct target for protein kinase B: identification of a convergence point for opposing effects of insulin and amino-acid deficiency on protein translation. Biochem J 344: 427-431.
Neshat MS, Mellinghoff IK, Tran C, Stiles B, Thomas G, Petersen R, Frost P, Gibbons JJ, Wu H, and Sawyers CL (2001) Enhanced sensitivity of PTEN deficient tumors to inhibition of FRAP/mTOR. Proc Natl Acad Sci USA 98: 10314-10319.
Reynolds THT, Bodine SC, and Lawrence CJ Jr (2002) Control of Ser2448 phosphorylation in the mammalian target of rapamycin by insulin and skeletal muscle load. J Biol Chem 277: 17657-17662.
Samowitz WS, Curtin K, Schaffer D, Robertson M, Leppert M, and Slattery ML (2000) Relationship of Ki-ras mutations in colon cancers to tumor location, stage and survival: a population-based study. Cancer Epidemiol Biomarkers Prev 9: 1193-1197.
Sinensky M, Fantle K, and Dalton M (1994) An antibody which specifically recognizes prelamin A but not mature lamin A: application to detection of blocks in farnesylation-dependent protein processing. Cancer Res 54: 3229-3232.
Smith PK, Krohn RI, Hermanson GT, Mallia AK, Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, and Klenk DC (1985) Measurement of protein using bicinchoninic acid. Anal Biochem 150: 76-85.[CrossRef][Medline]
Smith V, Rowlands MG, Barrie E, Workman P, and Kelland LR (2002) Establishment and characterization of acquired resistance to the farnesyl protein transferase inhibitor R115777 in a human colon cancer cell line. Clin Cancer Res 8: 2002-2009.
Suzuki N, Urano J, and Tamanoi F (1998) Farnesyltransferase inhibitors induce cytochrome c release and caspase 3 activation preferentially in transformed cells. Proc Natl Acad Sci USA 95: 15356-15361.
Zhang FL and Casey PJ (1996) Protein prenylation: molecular mechanisms and functional consequences. Annu Rev Biochem 65: 241-269.[CrossRef][Medline]
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) and HCT 116R cells (
) grown for 7 days in the presence of continuous exposure to SCH66336(A) or FTI-277 (C). B, in a pilot experiment, HCT 116R cells were cultured for 3 months in drug-free medium and then subjected to 7 days of continuous exposure to SCH66336in a colony-forming assay. Some error bars in C are smaller than the symbols.
). D, HCT 116R cells were treated with cisplatin (