Farnesyl transferase inhibitors as anticancer agents

Protein farnesylation catalysed by the enzyme farnesyl protein transferase involves the addition of a 15-carbon farnesyl group to conserved amino acid residues at the carboxyl terminus of certain proteins. Protein substrates of farnesyl transferase include several G-proteins, which are critical intermediates of cell signalling and cytoskeletal organisation such as Ras, Rho, PxF and lamins A and B. Activated Ras proteins trigger a cascade of phosphorylation events through sequential activation of the PI3 kinase/AKT pathway, which is critical for cell survival, and the Raf/Mek/Erk kinase pathway that has been implicated in cell proliferation. Ras mutations which encode for constitutively activated proteins are found in 30% of human cancers. Because farnesylation of Ras is required for its transforming and proliferative activity, the farnesyl protein transferase inhibitors were designed as anticancer agents to abrogate Ras function. However, current evidence suggests that the anticancer activity of the farnesyl transferase inhibitors may not be simply due to Ras inhibition. This review will discuss available clinical data on three of these agents that are currently undergoing clinical trials.     

Development of farnesyl transferase inhibitors: a review

Farnesyl transferase inhibitors are a new class of biologically active anticancer drugs. The exact mechanism of action of this class of agents is, however, currently unknown. The drugs inhibit farnesylation of a wide range of target proteins, including Ras. It is thought that these agents block Ras activation through inhibition of the enzyme farnesyl transferase, ultimately resulting in cell growth arrest. In preclinical models, the farnesyl transferase inhibitors showed great potency against tumor cells; yet in clinical studies, their activity was far less than anticipated. Reasons for this disappointing clinical outcome might be found in the drug-development process. In this paper, we outline an algorithm that is potentially useful for the development of biologically active anticancer drugs. The development of farnesyl transferase inhibitors, from discovery to clinical trials, is reviewed on the basis of this algorithm. We found that two important steps of this algorithm were underestimated. First, understanding of the molecular biology of the defective pathway has mainly been focused on H-Ras activation, whereas activation of K-Ras or other farnesylated proteins is probably more important in tumorigenesis. Inhibition of farnesylation is possibly not sufficient, because geranylgeranylation might activate K-Ras and suppress the effect of farnesyl transferase inhibitors. Furthermore, a well-defined proof of concept in preclinical and clinical studies has not been achieved. Integrating the proposed algorithm in future studies of newly developed biologically active anti-cancer drugs might increase the rate of success of these compounds in patients.     

Blocked pathways: FTIs shut down oncogene signals

Ras proteins play fundamental roles in cell signal transduction pathways that regulate cell growth, differentiation, proliferation, and survival. ras mutations are among the most frequently encountered genetic abnormalities in human cancers and play a key role in tumorigenesis. The enzymatic attachment of a 15- or 20-carbon moiety to the Ras protein through farnesylation or geranylgeranylation, respectively, is a required step in the proper localization and activation of Ras. Inhibition of the catalytic enzymes, farnesyl transferase and geranylgeranyl transferase, is a novel, mechanism-based, targeted approach to cancer therapy development. Geranylgeranyl transferase inhibitors suppress tumor growth by accumulating cells in the G(1)/S cell cycle phase. One mechanism by which farnesyl transferase inhibitors suppress tumor growth is by inhibiting bipolar spindle formation, thereby blocking progression from prophase to metaphase. Although the exact molecular target responsible for the antitumor activity of farnesyl transferase inhibitors is unclear, at least in some tumor cells, inhibition of phosphoinositide-3-OH kinase/Akt-mediated cell survival pathways may play a critical role. Identifying the farnesylated proteins that are targeted by farnesyl transferase inhibitors and the tumor molecular signatures that dictate which set of patients will respond to farnesyl transferase inhibitors are critical end points for future mechanistic studies.     

Neoadjuvant Therapy with Drug Arglabin for Breast Cancer with Expression of H-Ras Oncoproteins

Backgrounds: In breast cancer, blocking of Ras signaling and inhibition of H-Ras is quite promising. H-Ras may become a target for farnesyl transferase inhibitors, and in combination with other immunohistochemical factors it will contribute to the progression of a breast tumor. Purpose: The aim of this study was to evaluate the effectiveness of neoadjuvant therapy for breast cancer with the inclusion of farnesyl transferase inhibitor, arglabin interfering with the expression and concentration of H-Ras oncoproteins. Methods: Depending on the presence of H-Ras oncoproteins after Western-blot hybridization, the patients were divided a negative and positive expression of H-Ras groups. Results: Correlation analysis of methods used for determining the expression ability and concentration of H-Ras oncoproteins (immunohistochemistry and Western-blot analysis) demonstrated substantial statistical relationship Rs=0.71, p=0.03. The H-Ras oncoproteins were absent in patients receiving either “Arglabin” or standard AC regimen. However, in the AC + Arglabin group, there was a varying degrees of positive concentration of H-Ras oncoproteins (Kruskal-Wallis=6.92; p=0.03). Conclusion: These results indicate that Arglabin attenuates H-Ras oncoproteins expression which is a promising therapeutic target for breast cancer.     

法尼基转移酶抑制剂--乳腺癌新的治疗药物

原癌基因ras家族编码是与细胞增殖和生长有关的一些重要细胞信号途径的上游产物。ras基因突变导致Ras激活并具有致癌性。翻译后的Ras蛋白的关键环节是经法尼基转移酶的法尼基化。此酶的抑制剂首先被用于抑制Ras突变肿瘤的治疗。但现在发现法尼基转移酶抑制剂(farnesyltransferaseinhibitors,FTIs)还具有不依赖Ras的活性、对缺乏Ras突变的某些肿瘤的治疗也有效。基础资料显示FTIs可以抑制体内、外乳腺癌细胞的生长;Ⅱ期临床研究显示FTI-R115777对进展期乳腺癌的治疗有效。因此,FTIs单药或联合用药有可能成为乳腺癌治疗的新方法。     

Farnesyltransferase inhibitor SCH-66336 downregulates secretion of matrix proteinases and inhibits carcinoma cell migration

The ras oncogenes are among those most frequently found in human cancers. Blocking Ras farnesylation is a promising strategy for arresting cancer growth. Ras activates several signaling pathways with key roles in cellular proliferation, invasion, metastasis and angiogenesis. Furthermore, proteolytic activities of matrix proteinases such as urokinase-type plasminogen activator (uPA) and matrix metalloproteinases (MMPs) are regulated by Ras isoforms. Thus, we investigated the effects of SCH-66336, a farnesyltransferase inhibitor, on secretion of components of the plasminogen activation system as well as on the gelatinases MMP-2 and MMP-9, which play pivotal roles in matrix remodeling. SCH-66336 up to 5 microM did not significantly alter the viability of prostate (PC-3) and renal (Caki-1) cancer cells incubated in serum-depleted medium. SCH-66336 partly inhibited the processing of H-Ras, while levels of mature N-Ras and K-Ras remained unaffected. Under these noncytotoxic conditions, uPA and tPA levels were lowered in culture medium but raised in cell lysates, suggesting inhibition of trafficking pathways. In contrast, SCH-66336 had no effect on uPAR expression or on secreted PAI-1 levels. As expected, the reduction of uPA and tPA activities by SCH-66336 inhibited the conversion of plasminogen to plasmin by about 25% in PC-3 cells. SCH-66336 also inhibited the levels of secreted pro-MMP-2 and pro-MMP-9 as well as the release of their inhibitors TIMP-1 and TIMP-2. SCH-66336 decreased both the adhesion and even more so the migration of PC-3 cells on gelatin. Thus, SCH-66336 inhibited farnesylation in both cancer cell types, and H-Ras functions should be reduced by the drug. In addition, the lower levels of secreted proteinases in the presence of SCH-66336 suggest that reduced matrix remodeling and cell migration should occur in treated tumors.     

Lonafarnib (SCH66336) improves the activity of temozolomide and radiation for orthotopic malignant gliomas

Malignant gliomas are highly lethal tumors resistant to current therapies. The standard treatment modality for these tumors, surgical resection followed by radiation therapy and concurrent temozolomide, has demonstrated activity, but development of resistance and disease progression is common. Although oncogenic Ras mutations are uncommon in gliomas, Ras has been found to be constitutively activated through the action of upstream signaling pathways, suggesting that farnesyltransferase inhibitors may show activity against these tumors. We now report the in vitro and orthotopic in vivo results of combination therapy using radiation, temozolomide and lonafarnib (SCH66336), an oral farnesyl transferase inhibitor, in a murine model of glioblastoma. We examined the viability, proliferation, farnesylation of H-Ras, and activation of downstream signaling of combination-treated U87 cells in vitro. Lonafarnib alone or in combination with radiation and temozolomide had limited tumor cell cytotoxicity in vitro although it did demonstrate significant inhibition in tumor cell proliferation. In vivo, lonafarnib alone had a modest ability to inhibit orthotopic U87 tumors, radiation and temozolomide demonstrated better inhibition, while significant anti-tumor activity was found with concurrent lonafarnib, radiation, and temozolomide, with the majority of animals demonstrating a decrease in tumor volume. The use of tumor neurospheres derived from freshly resected adult human glioblastoma tissue was relatively resistant to both temozolomide and radiation therapy. Lonafarnib had a significant inhibitory activity against these neurospheres and could potentate the activity of temozolomide and radiation. These data support the continued research of high grade glioma treatment combinations of farnesyl transferase inhibitors, temozolomide, and radiation therapy.     

Manumycin and gliotoxin derivative KT7595 block Ras farnesylation and cell growth but do not disturb lamin farnesylation and localization in human tumour cells.

Recently, many inhibitors of farnesyl protein transferase (FPTase) have been identified. Some of them interrupt cell growth in addition to Ras and nuclear lamin processing of Ras-transformed cells. We have tested the effect of the FPTase inhibitors manumycin, an analogue of farnesyl diphosphate, and KT7595, a gliotoxin derivative, on Ras farnesylation, DNA synthesis and the anchorage-dependent and -independent growth of human colon carcinoma (LoVo), hepatoma (Mahlavu and PLC/PRF/5) and gastric carcinoma (KATO III). Both drugs severely inhibited DNA synthesis, cellular proliferation and Ras farnesylation in LoVo and moderately reduced them in Mahlavu and PLC/PRF/5 but not in KATO III. Complete sequencing of ras genes, however, revealed that LoVo and KATO III have activated Ki-ras and activated N-ras, respectively, whereas Mahlavu and PLC/PRF/5 have no activated ras. We next checked whether the inhibition of the cellular proliferation is due to the blocking of nuclear lamin function. Neither drug disturbed lamin farnesylation and localization, as demonstrated using metabolic labelling, immunoblotting and indirect immunofluorescence. These results indicate that manumycin and KT7595 can inhibit Ras farnesylation and cell growth without disturbing the farnesylation and localization of the lamins on human tumour cell lines.     

RAS and leukemia: from basic mechanisms to gene-directed therapy.

PURPOSE AND DESIGN: The purpose of this review is to provide an overview of the literature linking Ras signaling pathways and leukemia and to discuss the biologic and potential therapeutic implications of these observations. A search of MEDLINE from 1966 to October 1998 was performed. RESULTS: A wealth of data has been published on the role of Ras pathways in cancer. To be biologically active, Ras must move from the cytoplasm to the plasma membrane. Importantly, a posttranslational modification--addition of a farnesyl group to the Ras C-terminal cysteine--is a requisite for membrane localization of Ras. Farnesylation of Ras is catalyzed by an enzyme that is designated farnesyltransferase. Recently, several compounds have been developed that can inhibit farnesylation. Preclinical studies indicate that these molecules can suppress transformation and tumor growth in vitro and in animal models, with little toxicity to normal cells. CONCLUSION: An increasing body of data suggests that disruption of Ras signaling pathways, either directly through mutations or indirectly through other genetic aberrations, is important in the pathogenesis of a wide variety of cancers. Molecules such as farnesyl transferase inhibitors that interfere with the function of Ras may be exploitable in leukemia (as well as in solid tumors) as novel antitumor agents.     

Recent advances in understanding the antineoplastic mechanisms of farnesyltransferase inhibitors

Farnesyltransferase (FTase) inhibitors (FTI) have broad antineoplastic actions targeting both cancer cells and mesenchymal cells involved in tumor angiogenesis. The small GTPases H-Ras, Rheb, and RhoB and the centromere proteins CENP-E and CENP-F are relevant targets of farnesylation inhibition; however, their relative importance in the antineoplastic effect of FTIs may vary in different cell types at different stages of the cell cycle and at different stages in oncogenesis. Three recent studies argue that Ras-independent and perhaps even FTase-independent properties are important to the antineoplastic action of this class of drugs. In mice, genetic ablation of FTase does not abolish the oncogenic activity of Ras, limiting the original conception of FTIs as an effective means to target Ras in cancer cells. FTase may not be the sole molecular target of these agents, and one study has suggested that FTIs act by targeting geranylgeranyl transferase II. Lastly, we have obtained evidence that induction of reactive oxygen species and reactive oxygen species-mediated DNA damage by FTIs may be critical for their antineoplastic action as a class. Together, these findings may alter thinking about how to apply FTIs in the clinic.     

Normoxic and hypoxic regulation of vascular endothelial growth factor (VEGF) by astrocytoma cells is mediated by Ras

Vascular endothelial growth Factor (VEGF) has been identified as a key angiogenic factor involved in the growth and malignant progression of tumours. Glioblastoma multiforme (GBM) are the most common primary human brain tumours, histo-pathologically characterized by intense tumour angiogenesis. GBMs do not harbour oncogenic Ras mutations, but there is a functional up-regulation of Ras signaling through activation of receptor tyrosine kinases overexpressed by these tumours. We demonstrate that Ras pathway activation regulates VEGF secretion in astrocytoma cell lines. Ras pathway inhibition was carried out using genetic and pharmacologic techniques. Astrocytoma cells that were transfected to express the dominant inhibitory mutant H-Ras(N17) demonstrated a reduction in VEGF secretion under both normoxic and hypoxic conditions. Cells treated with the farnesyl transferase inhibitor L-744,832 demonstrated similar reductions in VEGF secretion. Furthermore, astrocytoma cells expressing a constitutively phosphorylated and truncated EGF-R common in GBMs (EGFRvIII or p140(EGF-R)) demonstrate further elevations in Ras activation, resulting in a further increase in VEGF secretion. We have previously demonstrated that activation of Ras plays a vital role in transducing mitogenic signals in human malignant astrocytoma cells. Our present results further extend the role of Ras activation in modulating tumour angiogenesis in these tumours. We propose that Ras may contribute to the angiogenic switch in astrocytomas.     

Farnesyl transferase inhibitors: current developments and future perspectives

Ras oncogenes play an important role in carcinogenesis and are frequently found in various human tumour types. Cellular activity of Ras oncoprotein, regulated through the enzyme farnesyl transferase, is crucial in the process of ras -dependent carcinogenesis, and therefore, specific inhibition of this enzyme is an attractive goal in anticancer treatment. Specific inhibitors of farnesyl transferase have been developed in recent years, many of them showing in vitro and in vivo growth inhibitory or cytostatic activity. Recently, results of the first clinical studies with various farnesyl transferase inhibitors have been presented. In the design of phase I and II studies, either single-agent or combination studies, new endpoints have to be defined in order to properly assess feasibility, antitumour activity and clinical valuability.     

Suppression of Human Pancreatic Cancer Growth in BALB/c Nude Mice by Manumycin, a Farnesyl:protein Transferase Inhibitor

Activating mutations of Ki- ras have been detected in most human pancreatic adenocarcinomas. Since Ras protein requires farnesylation to function, we investigated the effects of manumycin, a potent farnesyl:protein transferase inhibitor, on the growth in nude mice of a human pancreatic cancer cell line, MIA PaCa-2, with a point mutation in the Ki- ras gene. Tumor-bearing mice received intraperitoneal injection of 1 or 5 mg/kg manumycin daily for 5 days, or 2 mg/kg manumycin daily for 2 weeks. Growth of inoculated tumors was significantly inhibited by the treatment. The treatment significantly (P<0.05) lowered the numbers of bromodeoxyuridine-incorporating tumor cells. Manumycin did not have apparent hepatotoxicity in vivo . Farnesyl:protein transferase inhibitors could offer a new approach for cancer chemotherapy.     

Ras pathway is required for the activation of MMP-2 secretion and for the invasion of src-transformed 3Y1

To search for the signaling pathway critical for tumor invasion, we examined the effects of dominant negative ras (S17N ras) expression on the activation of matrix metalloproteinase-2 (MMP-2) in src-transformed 3Y1, SR3Y1, under the control of conditionally inducible promoter. In SR3Y1 clones transfected with S17N ras, augmented secretion and proteolytic activation of MMP-2 were dramatically suppressed by S17N Ras expression, while tyrosine phosphorylation of cellular proteins was not suppressed. We found that invasiveness of SR3Y1 cells assayed by the modified Boyden Chamber method was strongly suppressed by S17N Ras expression. In contrast, cell morphology reverted partially and glucose uptake remained unchanged by S17N Ras expression. In addition, treatment of SR3Y1 with manumycin A, a potent inhibitor of Ras farnesyltransferase, strongly suppressed both augmented secretion and proteolytic activation of MMP-2. Contrary, treatment of SR3Y1 with wortmannin or TPA showed no clear effect on MMP-2 activation. Thus, these results strongly suggest that Ras-signaling, but neither P13 kinase- nor protein kinase C-signalings, plays a critical role in activation of MMP-2 and, subsequently, in the invasiveness of src-transformed cells.     

Manunycin对人肝癌HepG2细胞的生长抑制作用与Ras通路的关系

背景与目的:目前肝癌药物治疗的临床疗效还不尽人意。在肝癌的发生发展过程中,Ras起着重要的作用。Ras必须在法尼基转移酶的作用下,经过法尼基化修饰才具有生物活性。我们观察了法尼基转移酶抑制剂Manumycin对人肝癌HepG2细胞株生长的抑制作用,并探讨其对Ras通路的影响。方法:应用氚标胸腺嘧啶脱氧核苷掺入实验观察法尼基转移酶抑制剂Manumycin对人肝癌HepG2细胞株生长的抑制作用,Westernblot技术检测Manumycin对HepG2细胞Ras通路相关蛋白,包括pan-Ras、N-Ras、membrane-pan-Ras、ERK1/2、p-ERK1/2、AKT、p-AKT及MKP-1表达的影响。结果:Manumycin(5、10、20、40、80μmol/L)处理24h对肝癌HepG2细胞株生长具有明显的抑制作用,并呈浓度依赖性,其IC50为(17.1±2.6)μmol/L。同时Westernblot分析显示,Manumycin对肝癌HepG2细胞pan-Ras蛋白和N-Ras蛋白及细胞膜pan-Ras蛋白表达均有抑制作用,且对细胞膜pan-Ras蛋白的抑制作用更明显。进一步研究发现Manumycin对Ras蛋白下游ERK1/2和AKT活性也有抑制作用,表现为ERK1/2和AKT磷酸化水平降低。Manumycin对AKT活性的影响与PI3K抑制剂Wortmannin作用相似,两者联用可加强对AKT的抑制。此外,Manumycin诱导MKP-1的表达具有浓度依赖性。结论:Manumycin抑制肝癌HepG2细胞株     

Galectin-1(L11A) predicted from a computed galectin-1 farnesyl-binding pocket selectively inhibits Ras-GTP

Ras biological activity necessitates membrane anchorage that depends on the Ras farnesyl moiety and is strengthened by Ras/galectin-1 interactions. We identified a hydrophobic pocket in galectin-1, analogous to the Cdc42 geranylgeranyl-binding cavity in RhoGDI, possessing homologous isoprenoid-binding residues, including the critical L11, whose RhoGDI L77 homologue changes dramatically on Cdc42 binding. By substituting L11A, we obtained a dominant interfering galectin-1 that possessed normal carbohydrate-binding capacity but inhibited H-Ras GTP-loading and extracellular signal-regulated kinase activation, dislodged H-Ras(G12V) from the cell membrane, and attenuated H-Ras(G12V) fibroblast transformation and PC12-cell neurite outgrowth. Thus, independently of carbohydrate binding, galectin-1 cooperates with Ras, whereas galectin-1(L11A) inhibits it.     

Characterization of a R115777-resistant human multiple myeloma cell line with cross-resistance to PS-341.

The farnesyl transferase inhibitor R115777 has been found to have clinical activity in diverse hematopoietic tumors. Clinical efficacy, however, does not correlate with Ras mutation status or inhibition of farnesyl transferase. To further elucidate the mechanisms by which R115777 induces apoptosis and to investigate drug resistance, we have identified and characterized a R115777-resistant human myeloma cell line. 8226/R5 cells were found to be at least 50 times more resistant to R115777 compared with the parent cell line 8226/S. K-Ras remained prenylated in both resistant and sensitive cells after R115777 treatment; however, HDJ-2 farnesylation was inhibited in both lines, implying that farnesyl transferase (the drug target) has not been mutated. Whereas many 8226 lines that acquire drug resistance have elevated expression of P-glycoprotein, we found that P-glycoprotein expression is not increased in the 8226/R5 line and intracellular accumulation of R115777 was not reduced. In fact, 8226/R5 cells were insensitive to a diverse group of antitumor agents including PS-341, and multidrug resistance did not correlate with the expression of heat shock proteins. Comparison of gene expression profiles between resistant and sensitive cells revealed expression changes in several genes involved in myeloma survival and drug resistance. Future experiments will attempt to identify genes that are directly linked to the resistant phenotype. Identification of molecules associated with R115777 and PS-341 resistance is clinically relevant because both compounds are being tested in solid tumors and hematopoietic malignancies.     

Farnesyltransferase inhibitors and anti-Ras therapy

Summary The oncoprotein encoded by mutantras genes is initially synthesized as a cytoplasmic precursor which requires posttranslational processing to attain biological activity; farnesylation of the cysteine residue present in the CaaX motif located at the carboxy-terminus of all Ras proteins is the critical modification. Once farnesylated and further modified, the mature Ras protein is inserted into the cell's plasma membrane where it participates in the signal transduction pathways that control cell growth and differentiation. The farnesylation reaction that modifies Ras and other cellular proteins having an appropriate CaaX motif is catalyzed by a housekeeping enzyme termed farnesyl-protein transferase (FPTase). Inhibitors of this enzyme have been prepared by several laboratories in an effort to identify compounds that would block Ras-induced cell transformation and thereby function as Ras-specific anticancer agents. A variety of natural products and synthetic organic compounds were found to block farnesylation of Ras proteinsin vitro. Some of these compounds exhibit antiproliferative activity in cell culture, block the morphological alterations associated with Ras-transformation, and can block the growth of Ras-transformed cell lines in tumor colony-forming assays. By contrast, these compounds do not affect the growth or morphology of cells transformed by the Raf or Mos oncoproteins, which do not require farnesylation to achieve biological activity. The efficacy and lack of toxicity observed with FPTase inhibitors in an animal tumor model suggest that specific FPTase inhibitors may be useful for the treatment of some types of cancer.Presented at the symposium "New Approaches in the Therapy of Breast Cancer", Georgetown University Medical Center, Washington DC, October 1994, generously supported by an education grant from Bristol-Myers Squibb.