Chem. Res. Toxicol. 2000, 13, 949-952
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Biological Effects and Mechanism of Action of Farnesyl Transferase Inhibitors Hena R. Ashar, Lydia Armstrong, Linda J. James, Donna M. Carr, Kimberley Gray, Arthur Taveras, Ronald J. Doll, W. Robert Bishop, and Paul T. Kirschmeier* Department of Tumor Biology and Chemistry, Schering-Plough Research Institute, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033 Received June 30, 2000
Introduction. Farnesyl transferase inhibitors (FTIs)1 were developed initially to inhibit the growth of tumors that contained constitutively active ras proteins (1). The three well-characterized forms of these oncoproteins, H-, N-, and K-ras, all contain C-terminal CaaX box motifs that are recognition sequences for posttranslational farnesylation. Results with a number of structurally distinct FTIs show that H-ras-transformed cells revert toward a normal phenotype when exposed to these compounds (1). Additional efficacy studies using transgenic mouse models expressing activated H-ras showed FTIs induced a complete regression of large wellestablished tumor masses (2, 3). Because of these experiments, the FTIs showed promise as chemotherapeutic agents that suppressed ras transformation. In human tumor cells, however, H-ras mutations are not as prevalent as K- or N-ras mutations (4). Experiments characterizing the sensitivity of human tumor cell lines (hTCLs) to FTIs demonstrated that the sensitivity to FTIs did not correlate with the presence of activated ras proteins (5-7). In fact, some cell lines that lack ras mutations were found to be highly sensitive to FTI treatment (5-7). The results of an example of such an experiment are shown in Figure 1A. The effect of SCH66336 on the anchorage-independent growth of a number of hTCLs is shown, and the sensitivity is compared to that of the ras genotype found in the cell. As with other FTIs, there was no correlation between sensitivity to the FTI and the presence of an activating ras mutation. One potential explanation for the lack of sensitivity of some mutant K-ras or N-ras expressing cells is the observation that N-ras and K-ras are alternatively prenylated by geranylgeranyl protein transferase (GGPTI) in cells treated with FTIs. Thus, the membrane localization of N- or K-ras persists in the presence of FTIs (7-9). The experiments exploring the sensitivity of hTCLs to FTI treatment have provided a common platform for comparing the biological effects of structurally distinct compounds. Interestingly, the rank order of sensitivity of the hTCLs to structurally distinct FTIs is essentially the same. For example, the breast carcinoma cell line MCF-7 is sensitive to SCH66336 and to L744,832, whereas another breast carcinoma line, T47D, is resistant to both compounds (5-7). Similarly, HCT-116 is * To whom all correspondence should be addressed: Mail Stop 4950, Schering Plough Research Institute, 2015 Galloping Hill Rd., Kenilworth, NJ 07033. E-mail:
[email protected]. Phone: (908) 740-7327. Fax: (908) 740-7115. 1 Abbreviations: FTIs, farnesyl transferase inhibitors; hTCLs, human tumor cell lines; GGPT-I, geranylgeranyl protein transferase.
Figure 1. Effect of SCH66336 on transformed cells. The indicated cell lines were grown in soft agar. The IC50 of each cell line for SCH66336 was determined. Cells were grouped according to their genomic status (abscissa) and plotted according to their IC50 in nanomolar (ordinate). Cells were grouped depending on their ras status in panel A [(b) cells with H-ras mutations, (9) cells with K-ras mutations, (2) cells with N-ras mutations, and (-) wild-type (wt) ras] and p53 status in panel B [cells with wild-type (wt) p53 (b) and with mutant (mut) p53 (9)].
sensitive to SCH66336 and B956. The observation that a given set of cell lines are all sensitive to structurally distinct FTIs suggests that the sensitivity is mediated via inhibition of farnesyl transferase. This raises two important questions. (1) What are the genetic characteristics of the sensitive cell lines? (2) Which farnesyl transferase substrates determine the sensitivity to FTIs? Role of p53. To approach these questions, the sensitivity of the hTCLs to FTIs was compared to that of another genotype, p53 mutational status. The results of
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Figure 2. FTIs induce the expression of p53 and p21Cip1 (A). Western blot analysis was performed with 150 µg of total protein from the indicated hTCLs grown in the absence (-) or presence (+) of 1 µM SCH66336 and probed with anti-p53 (PAB1801). (B) Western blot analysis was performed with 150 µg of total protein from the indicated hTCLs grown in the absence (-) or presence (+) of 1 µM SCH66336 and probed with anti-p21Cip1 (Cip1/Waf1, clone 70).
this analysis are shown in Figure 1B. Interestingly, the cell lines that are wild-type for p53 are, in general, more sensitive to the FTIs than the cell lines that are mutants for p53. To explore this correlation further, the ability of the FTI SCH66336 to induce p53 was determined. Figure 2 shows that in the tumor cell lines, HCT-116 and NCI H460, that are wild-type for p53, the tricyclic inhibitor clearly induced both p53 and the p53-regulated gene, p21Cip1. It is important to note that p53 and p21Cip1 were also induced with the peptidomimetic FTI, L744,832 (10). Further experiments suggest that p53 is induced 36-48 h after treatment with these inhibitors (7). This was in stark contrast to the rapid induction of p53 and p21Cip1 observed with doxorubicin (7, 8). Since FTIs are not mutagenic, these differences in the kinetics of p53 induction following FTI and doxorubicin treatment suggest a novel mechanism for p53 induction by these compounds. While the exact mechanism of p53 induction by inhibition of farnesylation needs to be explored further, the p53 status appears to influence the sensitivity of tumor cells to the FTIs. Cell Cycle Effects of FTIs. The effect of FTIs on the cell cycle was examined. For these experiments, cells with different genotypes were exposed to the FTI and the distribution of cells in each phase of the cell cycle was determined by propidium iodide staining followed by flow cytometry. FTI treatment of cells that harbor an activated H-ras allele caused an accumulation of cells in G1. This was observed in cells that were engineered to overexpress a mutant H-ras, as shown in Figure 2, as well as the H-ras mutant human bladder carcinoma cell line, T24 (7). The G1 arrest is consistent with the conclusion that FTIs prevent the association of the H-ras protein and therefore interrupt signaling through the ras pathway. Interestingly, T24 cells are mutant at the p53 locus and are quite sensitive to FTIs. This result suggests that the presence of an activated H-ras in the tumor cells is a dominant or governing factor for sensitivity to these compounds. Cell cycle analysis following FTI treatment was also reported for hTCLs that had either a wild-type ras or mutant K-ras genotype. Typical results from these experiments are shown in Figure 3B. In contrast to cells
Figure 3. Cell cycle analysis of hTCLs treated with FTI 66336. Human tumor cells were treated with 1 µM SCH66336 for the indicated period of time and compared with untreated cells that were grown for the same period of time. Cells were collected and examined by flow cytometry, and the data were analyzed and plotted using MODFIT (Verity Software Inc.). For every cell line, the DNA content is plotted on the abcissa and the number of cells on the ordinate. The data from the control and untreated cells are plotted on the left and the data from the FTI-treated cells on the right: H-ras NIH 3T3 at 72 h (A) and NCI-H460 at 72 h (B).
with H-ras mutations, these cells do not accumulate in G1 upon FTI treatment. Rather, FTI-treated NCI-H460 (Figure 3B) and MCF7 (data not shown) accumulate in the G2 f M checkpoint (7). The effect on the cell cycle distribution of cells with these genotypes was observed within 36 h of initiation of treatment with the FTI. Similar results were previously reported for FTI-treated A549 cells (11). These results provide further evidence that the primary effects of the FTIs on sensitive tumor cells with a wild-type ras or mutant K-ras genotype are mediated by effects on cellular processes distinct from the ras signal transduction pathway. Potential Role of CENP-E and CENP-F in Mediating the Cell Cycle Effects of the FTIs. A SWISSPROT database search for candidate CaaX box proteins associated with the regulation of the G2 f M checkpoint identified two centromere-associated proteins, CENP-E and CENP-F (11). CENP-E, a kinesin motor protein, and CENP-F are expressed during the G2 f M phase of the cell cycle (12, 13). CENP-E is localized to the kinetochore at the centromere of condensed chromosomes. This protein has a role in attaching the centromeres to the microtubules during prometaphase at the early stages of the G2 f M phase and was shown to participate in the regulation of this phase of the cell cycle (14, 15). Experiments performed by Rieder have clearly shown
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Figure 4. CENP-E and CENP-F are farnesylated in p-MevDLD-1 cells. (A) Mevalonate labeling of CENP-F and mitosin, CENP-E, and K-ras in the absence (untreated) and presence of 1 µM SCH66336 (FTI). CENP-F and mitosin, and CENP-E, that were mevalonate labeled in p-mev-DLD-1 cells were immunoprecipitated with their specific antibodies and resolved on an 8% Tris glycine gel. The labeled K-ras was resolved on a 14% gel. The gels were dried on a gel dryer with a Whatman paper backing and observed by autoradiography. (B) Western analysis using 60 µg of total protein from DLD-1 cells grown in the absence (untreated) and presence of 1 µM SCH66336 (FTI). To ensure that equal amounts of total protein were used in both lanes, CENP-E blots were probed for actin. Mitosin was probed on a separate blot.
that mitotic progression is delayed by an inhibitory signal from unattached chromosomes (15, 16). Coincidentally, FTI treatment results in a G2 f M pause (7, 11). A potential role for CENP-E and CENP-F in mediating some of the biological effects of the FTIs was explored further by Ashar et al. (11). Biochemical experiments using peptides derived from the C-terminus of these proteins showed that they were indeed substrates for farnesyl transferase but not for geranylgeranyl transferase. This result implies that, unlike K- or N-ras, neither CENP-E nor CENP-F would be alternatively prenylated in the presence of FTIs. In fact, in DLD-1 cells labeled with [3H]mevalonate (a precursor to the 15- and 20-carbon prenyl pyrophosphates), both CENP-E and CENP-F were shown to incorporate mevalonate, which indicates that they are prenylated proteins. Mevalonate labeling of both CENP-E and CENP-F was blocked by the FTI, supporting the result that both proteins are farnesylated but not geranylgeranylated (11). Figure 4 shows the result of this experiment. The functional consequence of inhibiting the prenylation of CENP-E was examined by exploring the effect of FTI on its attachment to microtubules (11). Microtubules and their associated proteins were isolated from untreated and FTI-treated cells A549 lung carcinoma cells. After separation on SDS gels, the amount of CENP-E in each preparation was probed by Western blotting. Figure 5 shows the results of this experiment. The amount of CENP-E associated with microtubules was significantly reduced in the FTI-treated cultures compared to that in the controls (11). This result suggests that the farnesylation of CENP-E is important for attachment to microtubules. A further implication is that the inhibition of farnesylation of CENP-E would delay the attachment of condensed chromosomes to the spindle during mitosis, leading to a delay at the G2 f M checkpoint (16). Clearly, much more work needs to be done to validate this mechanism. It would be particularly important to show that the primary effects of the FTIs
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Figure 5. Analysis of microtubule-associated proteins in the absence and presence of 1 µM SCH66336 using the CENP-E antibody. MAPs were isolated from A549 cells grown in the absence (untreated) and in the presence of 1 µM SCH66336 for 3 days. The microtubule pellet obtained after two rounds of depolymerization and polymerization was dissolved in sample buffer and resolved on an 8% Tris-glycine gel. Western analysis was performed with the polyclonal anti-CENP-E rabbit antibody on MAPs from A549 cells.
are not on the microtubule but on the associated proteins themselves. Summary and Future Directions. As the farnesyl transferase inhibitors progress in clinical trials for the treatment of cancer, there is an interest in describing their biological effects and mechanism of action. It is particularly important to describe the genetic characteristics of tumor cells that would be sensitive to this class of compounds. Our work and that of others has made progress toward describing these characteristics. Clearly, the presence of a mutated activated H-ras allele is an important factor for sensitivity to FTIs. The biological mechanism, i.e., the inhibition of ras processing, membrane attachment, and signal transduction, is wellunderstood and validated. In the presence of a mutated activated H-ras allele, the p53 status of tumor cells is inconsequential. This has been validated both in vitro and in vivo. However, in tumor cells wild-type for ras or that harbor K- or N-ras mutations, the p53 status appears to be relevant for sensitivity to FTIs. Indeed, the FTIs induce p53 in cells that are wild-type at this locus. This induction and its consequences may account for the increased sensitivity observed in tumor cells that are wild-type for p53. The FTIs also induce accumulation of cells in the G2 f M phase of the cell cycle except for those cells that harbor an activating mutation of H-ras. Although the exact role of the pause in the G2 f M checkpoint is not known, this effect of FTI treatment leads to a reduction in proliferation rates and may lead to other biological consequences such as induction of p53 and apoptosis in cells within appropriate genetic backgrounds. Finally, the proteins that ultimately mediate the chemotherapeutic effects of the FTIs are still being explored. Clearly, the inhibitors will have pleiotropic effects because there are many farnesylated proteins in cells that will be affected. The role of other small GTPases, particularly rhoB, in mediating the cellular effects of FTI is being explored, since they may function in mediating the chemotherapeutic effects of the FTIs
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(17). The centromeric proteins CENP-E and CENP-F are members of the group of nuclear farnesylated proteins, some or all of which may contribute to the cell cycle effects of the FTIs. The role of farnesylation of these proteins in mediating their biological activities is in the early stages of investigation. The following important question remains. How does inhibition of any of the candidate proteins result in selective effects on tumor cells? No doubt there will be other farnesylated proteins that will merit investigation for their potential contributions to the chemotherapeutic effects of the FTIs.
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