The farnesyl transferase inhibitor (FTI) SCH66336 (lonafarnib) inhibits Rheb farnesylation and mTOR signaling. Role in FTI enhancement of taxane and tamoxifen anti-tumor activity

Lonafarnib (SCH66336) is a farnesyl transferase inhibitor (FTI) that inhibits the post-translational lipid modification of H-Ras and other farnesylated proteins. K- and N-Ras are also substrates of farnesyl transferase; however, upon treatment with FTIs, they are alternatively prenylated by geranylgeranyl transferase-1. Despite the failure to abrogate prenylation of K- and N-Ras, growth of many tumors in preclinical models is inhibited by FTIs. This suggests that the anti-proliferative action of FTIs is dependent on blocking the farnesylation of other proteins. Rheb (Ras homologue enriched in brain) is a farnesylated small GTPase that positively regulates mTOR (mammalian target of rapamycin) signaling. We found that Rheb and Rheb2 mRNA were elevated in various tumor cell lines relative to normal cells. Peptides derived from the carboxyl termini of human Rheb and Rheb2 are in vitro substrates for farnesyl transferase but not geranylgeranyl transferase-1. Rheb prenylation in cell culture was completely inhibited by SCH66336, indicating a lack of alternative prenylation. SCH66336 treatment also inhibited the phosphorylation of S6 ribosomal protein, a downstream target of Rheb and mTOR signaling. SCH66336 did not inhibit S6 phosphorylation in cells expressing Rheb-CSVL, a mutant construct of Rheb designed to be geranylgeranylated. Importantly, expression of Rheb-CSVL also abrogated SCH66336 enhancement of tamoxifen- and docetaxel-induced apoptosis in MCF-7 breast cancer cells and ES-2 ovarian cancer cells, respectively. Further, inhibition of Rheb signaling by rapamycin treatment, small interfering RNA, or dominant negative Rheb enhanced tamoxifen- and docetaxel-induced apoptosis, similar to FTI treatment. These studies demonstrated that Rheb is modified by farnesylation, is not a substrate for alternative prenylation, and plays a role in SCH66336 enhancement of the anti-tumor response to other chemotherapeutics.     

The farnesyl transferase inhibitor SCH 66336 induces a G(2) --> M or G(1) pause in sensitive human tumor cell lines

SCH 66336 is a potent farnesyl transferase inhibitor (FTI) in clinical development. It efficiently prevents the membrane association of H-ras, but not K- or N-ras. Yet, in soft agar, it reverts the anchorage-independent growth of human tumor cell lines (hTCLs) harboring H-ras, K-ras, and N-ras mutations, implying that blocking farnesylation of proteins besides ras may be responsible for this effect. Experiments show that SCH 66336 altered the cell cycle distribution of sensitive human tumor cells in two distinct ways. Most sensitive hTCLs accumulated in the G(2)-->M phase after the FTI treatment, but those with an activated H-ras accumulated in G(1) phase, suggesting that the biological effects induced by FTIs in cells with an activated H-ras are distinct from other sensitive cells. A careful genotypic comparison of the hTCLs revealed that those cells with wild-type p53 are especially sensitive to the FTIs. In these cells p53 and its downstream target gene p21(Cip1) are induced after treatment with SCH 66336 for 24 h. These data suggest that cell cycle effects, either G(1) or G(2)-->M accumulation, and p53 status are important for mediating the effects of FTIs on tumor cells.     

A novel role of farnesylation in targeting a mitotic checkpoint protein,human Spindly,to kinetochores

Kinetochore (KT) localization of mitotic checkpoint proteins is essential for their function during mitosis. hSpindly KT localization is dependent on the RZZ complex and hSpindly recruits the dynein–dynactin complex to KTs during mitosis, but the mechanism of hSpindly KT recruitment is unknown. Through domain-mapping studies we characterized the KT localization domain of hSpindly and discovered it undergoes farnesylation at the C-terminal cysteine residue. The N-terminal 293 residues of hSpindly are dispensable for its KT localization. Inhibition of farnesylation using a farnesyl transferase inhibitor (FTI) abrogated hSpindly KT localization without affecting RZZ complex, CENP-E, and CENP-F KT localization. We showed that hSpindly is farnesylated in vivo and farnesylation is essential for its interaction with the RZZ complex and hence KT localization. FTI treatment and hSpindly knockdown displayed the same mitotic phenotypes, indicating that hSpindly is a key FTI target in mitosis. Our data show a novel role of lipidation in targeting a checkpoint protein to KTs through protein–protein interaction.     

Preventing farnesylation of the dynein adaptor Spindly contributes to the mitotic defects caused by farnesyltransferase inhibitors

The clinical interest in farnesyltransferase inhibitors (FTIs) makes it important to understand how these compounds affect cellular processes involving farnesylated proteins. Mitotic abnormalities observed after treatment with FTIs have so far been attributed to defects in the farnesylation of the outer kinetochore proteins CENP-E and CENP-F, which are involved in chromosome congression and spindle assembly checkpoint signaling. Here we identify the cytoplasmic dynein adaptor Spindly as an additional component of the outer kinetochore that is modified by farnesyltransferase (FTase). We show that farnesylation of Spindly is essential for its localization, and thus for the proper localization of dynein and its cofactor dynactin, to prometaphase kinetochores and that Spindly kinetochore recruitment is more severely affected by FTase inhibition than kinetochore recruitment of CENP-E and CENP-F. Molecular replacement experiments show that both Spindly and CENP-E farnesylation are required for efficient chromosome congression. The identification of Spindly as a new mitotic substrate of FTase provides insight into the causes of the mitotic phenotypes observed with FTase inhibitors.     

Cyclin-like accumulation and loss of the putative kinetochore motor CENP-E results from coupling continuous synthesis with specific degradation at the end of mitosis

CENP-E is a kinesin-like protein that binds to kinetochores through the early stages of mitosis, but after initiation of anaphase, it relocalizes to the overlapping microtubules in the midzone, ultimately concentration in the developing midbody. By immunoblotting of cells separated at various positions in the cell cycle using centrifugal elutriation, we show that CENP-E levels increase progressively across the cycle peaking at approximately 22,000 molecules/cell early in mitosis, followed by an abrupt (> 10 fold) loss at the end of mitosis. Pulse-labeling with [35S]methionine reveals that beyond a twofold increase in synthesis between G1 and G2, interphase accumulation results primarily from stabilization of CENP-E during S and G2. Despite localizing in the midbody during normal cell division, CENP-E loss at the end of mitosis is independent of cytokinesis, since complete blockage of division with cytochalasin has no affect on CENP-E loss at the M/G1 transition. Thus, like mitotic cyclins, CENP-E accumulation peaks before cell division, and it is specifically degraded at the end of mitosis. However, CENP-E degradation kinetically follows proteolysis of cyclin B in anaphase. Combined with cyclin A destruction before the end of metaphase, degradation of as yet unidentified components at the metaphase/anaphase transition, and cyclin B degradation at or after the anaphase transition, CENP-E destruction defines a fourth point in a mitotic cascade of timed proteolysis.     

Pharmacological-based translational induction of transgene expression in mammalian cells

In the quest for the development of pharmacological switches that control gene expression, no system has been reported that regulates at the translational level. To permit small-molecule control of transgene translation, we have constructed a farnesyl transferase inhibitor-responsive translation initiation factor. This artificial protein is a three-component chimaera consisting of the ribosome recruitment core of the eIF4G1 eukaryotic translation initiation factor, the RNA-binding domain of the R17 bacteriophage coat protein and the plasma membrane localization CAAX motif of farnesylated H-Ras. This membrane-delocalized translation factor is inactive unless liberated in the cytosol. Farnesyl transferase inhibitor FTI-277 prevents the membrane association of the CAAX motif and thus increases the cytoplasmic levels of the eIF4G fusion protein, which is then capable of inducing translation of the second cistron of a bicistronic messenger RNA containing an R17-binding site in its intercistronic space. Such direct translational control by farnesyl transferase inhibitors provides a system for fast, graded and reversible regulation of transgene expression.     

Inhibitors of farnesyl protein transferase and MEK1,2 induce apoptosis in fibroblasts transformed with farnesylated but not geranylgeranylated H-Ras

Farnesyl protein transferase inhibitors (FTIs) reverse the transformed phenotype of fibroblasts expressing activated H-Ras and block anchorage-independent growth and tumorigenesis of tumor cell lines independent of their Ras mutational status. FTIs induce significant tumor regression accompanied by apoptosis in several transgenic mouse tumor models. FTI treatment of tumor cells in vitro is proapoptotic under certain cell culture conditions. Induction of apoptosis by FTIs in vitro generally requires a second death-promoting signal. To better understand FTI-induced apoptosis we analyzed the effect of SCH 66336, a tricyclic FTI, on apoptosis of Ras-transformed Rat2 fibroblasts. Treatment of H-Ras-CVLS-transformed fibroblasts with MEK1,2 inhibitors provides a pharmacological second signal to enhance FTI-induced apoptosis. Simultaneous treatment of these cells with a MEK1,2 inhibitor markedly enhanced caspase-3 activity and the apoptotic response to SCH 66336. The combination treatment resulted in a more complete and sustained inhibition of MAPK pathway activity than observed with either drug alone. Surprisingly, after treatment with either agent alone or in combination, no apoptotic response was observed in Rat2 cells transformed with a geranylgeranylated form of H-Ras (H-Ras-CVLL). Differences were also observed when SCH 66336 treatment was combined with forced suspension growth or serum withdrawal, in that an increase in drug-induced apoptosis was observed in H-Ras-CVLS-transformed Rat2 cells but not H-Ras-CVLL-transformed Rat2 cells. The lack of apoptotic effect of SCH 66336 and MEK inhibitor, alone or in combination, in H-Ras-CVLL-transformed cells suggests a difference in the reliance of cells transformed with farnesylated and geranylgeranylated forms of H-Ras on the MAPK signal transduction cascade for survival. K-Ras-transformed cells underwent apoptosis upon MEK1,2 inhibition but not in response to SCH 66336 treatment. The apoptotic response induced by MEK1,2 inhibitors is much greater in magnitude in H-Ras-transformed cells than in K-Ras-transformed cells, also pointing to differences in pathway utilization and/or dependence for these two Ras isoforms.     

Novel reverse-turn mimics inhibit farnesyl transferase

Reverse-turn inducing bicyclic lactams were incorporated into the substrate sequence recognized by farnesyl transferase to create inhibitors of RAS farnesylation. While the free peptides did not show any effect on the farnesylation, their Fmoc-protected counterparts impede the transformation of RAS with IC50's in the low micromolar range.     

The Microtubule Binding Properties of CENP-E's C-Terminus and CENP-F

CENP-E (centromere protein E) and CENP-F (centromere protein F), also known as mitosin, are large, multi-functional proteins associated with the outer kinetochore. CENP-E features a well-characterized kinesin motor domain at its N-terminus and a second microtubule-binding domain at its C-terminus of unknown function. CENP-F is important for the formation of proper kinetochore–microtubule attachment and, similar to CENP-E, contains two microtubule-binding domains at its termini. While the importance of these proteins is known, the details of their interactions with microtubules have not yet been investigated. We have biochemically and structurally characterized the microtubule-binding properties of the amino- and carboxyl-terminal domains of CENP-F as well as the carboxyl-terminal (non-kinesin) domain of CENP-E. CENP-E's C-terminus and CENP-F's N-terminus bind microtubules with similar affinity to the well-characterized Ndc80 complex, while CENP-F's C-terminus shows much lower affinity. Electron microscopy analysis reveals that all of these domains engage the microtubule surface in a disordered manner, suggesting that these factors have no favored binding geometry and may allow for initial side-on attachments early in mitosis.     

Inhibition of geranylgeranylation mediates sensitivity to CHOP-induced cell death of DLBCL cell lines

Prenylation is a post-translational hydrophobic modification of proteins, important for their membrane localization and biological function. The use of inhibitors of prenylation has proven to be a useful tool in the activation of apoptotic pathways in tumor cell lines. Rab geranylgeranyl transferase (Rab GGT) is responsible for the prenylation of the Rab family. Overexpression of Rab GGTbeta has been identified in CHOP refractory diffuse large B cell lymphoma (DLBCL). Using a cell line-based model for CHOP resistant DLBCL, we show that treatment with simvastatin, which inhibits protein farnesylation and geranylgeranylation, sensitizes DLBCL cells to cytotoxic treatment. Treatment with the farnesyl transferase inhibitor FTI-277 or the geranylgeranyl transferase I inhibitor GGTI-298 indicates that the reduction in cell viability was restricted to inhibition of geranylgeranylation. In addition, treatment with BMS1, a combined inhibitor of farnesyl transferase and Rab GGT, resulted in a high cytostatic effect in WSU-NHL cells, demonstrated by reduced cell viability and decreased proliferation. Co-treatment of BMS1 or GGTI-298 with CHOP showed synergistic effects with regard to markers of apoptosis. We propose that inhibition of protein geranylgeranylation together with conventional cytostatic therapy is a potential novel strategy for treating patients with CHOP refractory DLBCL.     

The synthesis and biological evaluation of a series of potent dual inhibitors of farnesyl and geranyl-Geranyl protein transferases

We have prepared a series of potent, dual inhibitors of the prenyl transferases farnesyl protein transferase (FPTase) and geranyl-geranyl protein transferase I (GGPTase). The compounds were shown to possess potent activity against both enzymes in cell culture. Mechanistic analysis has shown that the compounds are CAAX competitive for FPTase inhibition but geranyl-geranyl pyrophosphate (GGPP) competitive for GGPTase inhibiton.     

Inhibition of purified p21ras farnesyl:protein transferase by Cys-AAX tetrapeptides

We report the identification, purification, and characterization of a farnesyl:protein transferase that transfers the farnesyl moiety from farnesyl pyrophosphate to a cysteine in p21ras proteins. The enzyme was purified approximately 60,000-fold from rat brain cytosol through use of a chromatography step based on the enzyme's ability to bind to a hexapeptide containing the consensus sequence (Cys-AAX) for farnesylation. The purified enzyme migrated on gel filtration chromatography with an apparent molecular weight of 70,000-100,000. High resolution SDS-polyacrylamide gels showed two closely spaced approximately 50 kd protein bands in the final preparation. The enzyme was inhibited competitively by peptides as short as 4 residues that contained the Cys-AAX motif. These peptides acted as alternative substrates that competed with p21H-ras for farnesylation. Effective peptides included the COOH-terminal sequences of all known p21ras proteins as well as those of lamin A and B.     

A Novel Time-Dependent CENP-E Inhibitor with Potent Antitumor Activity

Centromere-associated protein E (CENP-E) regulates both chromosome congression and the spindle assembly checkpoint (SAC) during mitosis. The loss of CENP-E function causes chromosome misalignment, leading to SAC activation and apoptosis during prolonged mitotic arrest. Here, we describe the biological and antiproliferative activities of a novel small-molecule inhibitor of CENP-E, Compound-A (Cmpd-A). Cmpd-A inhibits the ATPase activity of the CENP-E motor domain, acting as a time-dependent inhibitor with an ATP-competitive-like behavior. Cmpd-A causes chromosome misalignment on the metaphase plate, leading to prolonged mitotic arrest. Treatment with Cmpd-A induces antiproliferation in multiple cancer cell lines. Furthermore, Cmpd-A exhibits antitumor activity in a nude mouse xenograft model, and this antitumor activity is accompanied by the elevation of phosphohistone H3 levels in tumors. These findings demonstrate the potency of the CENP-E inhibitor Cmpd-A and its potential as an anticancer therapeutic agent.     

2-Arylindole-3-acetamides: FPP-competitive inhibitors of farnesyl protein transferase

A series of 2-arylindole-3-acetamide farnesyl protein transferase inhibitors has been identified. The compounds inhibit the enzyme in a farnesyl pyrophosphate-competitive manner and are selective for farnesyl protein transferase over the related enzyme geranylgeranyltransferase-I. A representative member of this series of inhibitors demonstrates equal effectiveness against HDJ-2 and K-Ras farnesylation in a cell-based assay when geranylgeranylation is suppressed.     

Elevating the level of Cdc34/Ubc3 ubiquitin-conjugating enzyme in mitosis inhibits association of CENP-E with kinetochores and blocks the metaphase alignment of chromosomes

Cdc34/Ubc3 is a ubiquitin-conjugating enzyme that functions in targeting proteins for proteasome-mediated degradation at the G1 to S cell cycle transition. Elevation of Cdc34 protein levels by microinjection of bacterially expressed Cdc34 into mammalian cells at prophase inhibited chromosome congression to the metaphase plate with many chromosomes remaining near the spindle poles. Chromosome condensation and nuclear envelope breakdown occurred normally, and chromosomes showed oscillatory movements along mitotic spindle microtubules. Most injected cells arrested in a prometaphase-like state. Kinetochores, even those of chromosomes that failed to congress, possessed the normal trilaminar plate ultrastructure. The elevation of Cdc34 protein levels in early mitosis selectively blocked centromere protein E (CENP-E), a mitotic kinesin, from associating with kinetochores. Other proteins, including two CENP-E-associated proteins, BubR1 and phospho-p42/p44 mitogen-activated protein kinase, and mitotic centromere-associated kinesin, cytoplasmic dynein, Cdc20, and Mad2, all exhibited normal localization to kinetochores. Proteasome inhibitors did not affect the prometaphase arrest induced by Cdc34 injection. These studies suggest that CENP-E targeting to kinetochores is regulated by ubiquitylation not involving proteasome-mediated degradation.     

Kinesin-7 CENP-E is essential for chromosome alignment and spindle assembly of mouse spermatocytes

Genome stability depends on chromosome congression and alignment during cell division. Kinesin-7 CENP-E is critical for kinetochore-microtubule attachment and chromosome alignment, which contribute to genome stability in mitosis. However, the functions and mechanisms of CENP-E in the meiotic division of male spermatocytes remain largely unknown. In this study, by combining the use of chemical inhibitors, siRNA-mediated gene knockdown, immunohistochemistry, and high-resolution microscopy, we have found that CENP-E inhibition results in chromosome misalignment and metaphase arrest in dividing spermatocyte during meiosis. Strikingly, we have revealed that CENP-E regulates spindle organization in metaphase I spermatocytes and cultured GC-2 spd cells. CENP-E depletion leads to spindle elongation, chromosome misalignment, and chromosome instability in spermatocytes. Together, these findings indicate that CENP-E mediates the kinetochore recruitment of BubR1, spindle assembly checkpoint and chromosome alignment in dividing spermatocytes, which finally contribute to faithful chromosome segregation and chromosome stability in the male meiotic division.     

Isoprenylation of proteins: what is its role?]

The role of mevalonate in the control of DNA synthesis during the cell cycle has been studied and has lead to the detection of isoprenylated proteins. These proteins are modified by a polyisoprenoid (farnesyl or geranylgeranyl) moiety via a thioether linkage. This modification is required for the following steps of the post-translational maturation of these proteins: proteolysis of the last three C-terminal amino-acids and carboxymethylation of the Cysteine-COOH. The isoprenylation could play a role in the membrane localisation of these proteins. Farnesylated proteins present a C-terminal CAAX domain. Moreover, the farnesylation is required for their biological activity independently of the membrane localization (Prelamine A, p21ras(Val 12)). Among geranylgeranyl proteins, two types of C-terminal sequences have been found: one with the motif CAAX, the other with the motif CC or CXC. In the last type, both Cysteines are geranylgeranylated. The hydrophobicity of the geranylgeranyl moiety leads to the membrane attachment, without any specificity. Moreover, geranylgeranylation as well as farnesylation seem important for protein-protein interactions. Among the identified isoprenylated proteins, the lamins, gamma-subunits of G proteins and the numerous (if not all) members of the Ras superfamily were characterized. The exact role of isoprenylation is still uncertain but it seems to affect the membrane localization and the protein-protein interactions.     

The farnesyltransferase inhibitor, FTI-2153, blocks bipolar spindle formation and chromosome alignment and causes prometaphase accumulation during mitosis of human lung cancer cells

Even though farnesyltransferase inhibitors (FTIs), a novel class of therapeutic agents presently in clinical trials, have preclinically outstanding anticancer activity and impressive lack of toxicity, their mechanism of action is not well understood. To enhance our understanding of how FTIs inhibit the growth of tumors, we have investigated their effects on cell cycle progression of two human lung cancer cell lines, A-549 and Calu-1. In this report, we show in synchronized A-549 and Calu-1 cells that FTI-2153 treatment resulted in a large accumulation of cells in the mitosis phase of the cell division cycle, with some cells in the G(0)/G(1) phase. Furthermore, microtubule immunostaining and 4,6-diamidino-2-phenylindole DNA staining demonstrated that the FTI-2153-induced accumulation in mitosis is due to the inability of these cells to progress from prophase to metaphase. FTI-2153 inhibited the ability of A-549 and Calu-1 cells to form bipolar spindles and caused formation of monoasteral spindles. Furthermore, FTI-2153 induced a ring-shaped chromosome morphology and inhibited chromosome alignment. Time-lapse videomicroscopy confirmed this result by showing that FTI-2153-treated cells are unable to align their chromosomes at the metaphase plate. FTI-2153 did not affect the localization to the kinetochores of two farnesylated centromeric proteins, CENP-E and CENP-F. Thus, a mechanism by which FTIs inhibit progression through mitosis and tumor growth is by blocking bipolar spindle formation and chromosome alignment.     

Interaction of Skp1 with CENP-E at the midbody is essential for cytokinesis

Centromere-associated protein E (CENP-E) is a kinesin-related microtubule motor protein that is essential for chromosome congression during mitosis. Our previous studies show that microtubule motor CENP-E represents a link between attachment of spindle microtubules and the mitotic checkpoint signaling cascade. However, the molecular function of CENP-E at the midbody had remained elusive. Here we show that CENP-E interacts with Skp1 at the midbody and participates in cytokinesis. CENP-E interacts with Skp1 in vitro and in vivo via its coiled-coil domain. Our yeast two-hybrid assays mapped the binding interfaces to the central stalk region of CENP-E (955-1571 aa) and the C-terminal 33 amino acids of Skp1, respectively. Our immunocytochemical studies revealed that CENP-E targets to the midbody prior to Skp1 and the midbody localization of CENP-E becomes diminished as Skp1 arrives at the midbody. Suppression of Skp1 in mitotic HeLa cells by siRNA resulted in accumulation of telophase cells with elongated inter-cell bridges and with midbodies stretched 2-3 times longer than that of normal cells. These Skp1-eliminated or -suppressed cells accumulate higher level of CENP-E, suggesting that spatiotemporal regulation of CENP-E degradation at the midbody is essential for cytokinesis. Over-expression of Skp1 lacking the CENP-E-binding domain confirmed that Skp1-CENP-E interaction is essential for faithful cytokinesis. We hypothesize that CENP-E degradation is essential for faithful mitotic exit and the proteolysis of CENP-E is mediated by SCF via a direct Skp1 link.