Andrew D. Hamilton

The Geranylgeranyltransferase-I Inhibitor GGTI-298 Arrests Human Tumor Cells in G0/G1 and Induces p21WAF1/CIP1/SDI1 in a p53-independent Manner

Recently we have shown that in fibroblasts (NIH 3T3 and Rat-1 cells) inhibition of protein geranylgeranylation leads to a G0/G1 arrest, whereas inhibition of protein farnesylation does not affect cell cycle distribution. Here we demonstrate that in human tumor cells the geranylgeranyltransferase-I (GGTase-I) inhibitor GGTI-298 blocked cells in G0/G1, whereas the farnesyltransferase (FTase) inhibitor FTI-277 showed a differential effect depending on the cell line. FTI-277 accumulated Calu-1 and A-549 lung carcinoma and Colo 357 pancreatic carcinoma cells in G2/M, T-24 bladder carcinoma, and HT-1080 fibrosarcoma cells in G0/G1, but had no effect on cell cycle distribution of pancreatic (Panc-1), breast (SKBr 3 and MDAMB-231), and head and neck (A-253) carcinoma cells. Furthermore, treatment of Calu-1, Panc-1, Colo 357, T-24, A-253, SKBr 3, and MDAMB-231 cells with GGTI-298, but not FTI-277, induced the protein expression levels of the cyclin-dependent kinase inhibitor p21WAF. HT-1080 and A-549 cells had a high basal level of p21WAF, and GGTI-298 did not further increase these levels. Furthermore, GGTI-298 also induces the accumulation of large amounts of p21WAF mRNA in Calu-1 cells, a cell line that lacks the tumor suppressor gene p53. There was little effect of GGTI-298 on the cellular levels of another cyclin- dependent kinase inhibitor p27KIP as well as cyclin E and cyclin D1. These results demonstrate that GGTase-I inhibitors arrest cells in G0/G1 and induce accumulation of p21WAF in a p53-independent manner and that FTase inhibitors can interfere with cell cycle events by a mechanism that involves neither p21WAF nor p27KIP. The results also point to the potential of GGTase-I inhibitors as agents capable of restoring growth arrest in cells lacking functional p53.

Design of GFB-111, a platelet-derived growth factor binding molecule with antiangiogenic and anticancer activity against human tumors in mice

We have designed a molecule, GFB-111, that binds to platelet-derived growth factor (PDGF), prevents it from binding to its receptor tyrosine kinase, and blocks PDGF-induced receptor autophosphorylation, activation of Erk1 and Erk2 kinases, and DNA synthesis. GFB-111 is highly potent (IC50 = 250 nM) and selective for PDGF over EGF, IGF-1, aFGF, bFGF, and HRGβ (IC50 values > 100 μM), but inhibits VEGF-induced Flk-1 tyrosine phosphorylation and Erk1/Erk2 activation with an IC50 of 10 μM. GFB-111 treatment of nude mice bearing human tumors resulted in significant inhibition of tumor growth and angiogenesis. The results demonstrate the feasibility of designing novel growth factor–binding molecules with potent anticancer and antiangiogenic activity.

RhoA Prenylation Is Required for Promotion of Cell Growth and Transformation and Cytoskeleton Organization but Not for Induction of Serum Response Element Transcription

The importance of post-translational geranylgeranylation of the GTPase RhoA for its ability to induce cellular proliferation and malignant transformation is not well understood. In this manuscript we demonstrate that geranylgeranylation is required for the proper cellular localization of V14RhoA and for its ability to induce actin stress fiber and focal adhesion formation. Furthermore, V14RhoA geranylgeranylation was also required for suppressing p21WAF transcription, promoting cell cycle progression and cellular proliferation. The ability of V14RhoA to induce focus formation and enhance plating efficiency and oncogenic Ras anchorage-dependent growth was also dependent on its geranylgeranylation. The only biological activity of V14RhoA that was not dependent on its prenylation was its ability to induce serum response element transcriptional activity. Furthermore, we demonstrate that a farnesylated form of V14RhoA was also able to bind RhoGDI-1, was able to induce cytoskeleton organization, proliferation, and transformation, and was just as potent as geranylgeranylated V14RhoA at suppressing p21WAF transcriptional activity. These results demonstrate that RhoA geranylgeranylation is required for its biological activity and that the nature of the lipid modification is not critical.

Integrin-dependent Leukocyte Adhesion Involves Geranylgeranylated Protein(s)

Integrin-dependent leukocyte adhesion is modulated by alterations in receptor affinity or by post-receptor events. Pretreatment of Jurkat T-cells with the 3-hydroxymethylglutaryl-coenzyme A reductase inhibitor, lovastatin, markedly reduced (IC50 ≈1–2 μm) α4β1-dependent adhesion to fibronectin (FN) stimulated by phorbol 12-myristate 13-acetate (PMA) which modulates post-receptor events. In contrast, lovastatin did not inhibit Jurkat cell adhesion to FN induced by the β1integrin-activating monoclonal antibody (mAb) 8A2, which directly modulates β1 integrin affinity. Similarly, pretreatment of U937 cells with lovastatin inhibited PMA-stimulated, but not mAb 8A2-stimulated, α6β1-dependent leukocyte adhesion to laminin. The inhibition of lovastatin on PMA-stimulated leukocyte adhesion was not mediated by mitogen-activated protein kinase or phosphatidylinositol 3-kinase pathway. The inhibitory effect of lovastatin on PMA-stimulated leukocyte adhesion was reversed by co-incubation with geranylgeraniol, but not with farnesol, with concurrent reversal of the inhibition of protein prenylation as shown by protein RhoA geranylgeranylation. The selective inhibition of protein geranylgeranylation by the specific protein geranylgeranyltransferase-I inhibitor, GGTI-298, blocked PMA-stimulated leukocyte adhesion but not mAb 8A2-induced leukocyte adhesion. The protein farnesyltransferase inhibitor, FTI-277, had no effect on leukocyte adhesion induced by either stimulus. These results demonstrate that protein geranylgeranylation, but not farnesylation, is required for integrin-dependent post-receptor events in leukocyte adhesion.

The Geranylgeranyltransferase I Inhibitor GGTI-298 Induces Hypophosphorylation of Retinoblastoma and Partner Switching of Cyclin-dependent Kinase Inhibitors A POTENTIAL MECHANISM FOR GGTI-298 ANTITUMOR ACTIVITY

The geranylgeranyltransferase I inhibitor GGTI-298 has recently been shown to arrest human tumor cells in the G1 phase of the cell cycle, induce apoptosis, and inhibit tumor growth in nude mice. In the present manuscript, we provide a possible mechanism by which GGTI-298 mediates its tumor growth arrest. Treatment of the human lung carcinoma cell line Calu-1 with GGTI-298 results in inhibition of the phosphorylation of retinoblastoma protein, a critical step for G1/S transition. The kinase activities of two G1/S cyclin-dependent kinases, CDK2 and CDK4, are inhibited in Calu-1 cells treated with GGTI-298. Furthermore, GGTI-298 has little effect on the expression levels of CDK2, CDK4, CDK6, cyclins D1 and E, but decreases the levels of cyclin A. GGTI-298 increases the levels of the cyclin-dependent kinase inhibitors p21 and p15 and had little effect on those of p27 and p16. Most interesting is the ability of GGTI-298 to induce partner switching for several CDK inhibitors. GGTI-298 promotes binding of p21 and p27 to CDK2 while decreasing their binding to CDK6. Reversal of partner switching and G1 block was observed after removal of GGTI-298. Furthermore, GGTI-298 treatment results in an increased binding of p15 to CDK4, which is paralleled with decreased binding to p27. The results demonstrate that the GGTI-298-mediated G1 block in Calu-1 cells involves increased expression and partner switching of CDK inhibitors resulting in inhibition of CDK2 and CDK4, and retinoblastoma protein phosphorylation.

Anticancer activity of farnesyltransferase and geranylgeranyltransferase i inhibitors: prospects for drug development

Inhibition of farnesyltransferase (FTase) has been thoroughly investigated as a strategy to discover novel anticancer drugs because the oncoprotein Ras, requires farnesylation for its cancer-causing activity. Several highly potent and selective FTase inhibitors have been made and show excellent antitumour activity against human tumours in animal models without toxicity to normal cells. However, resistance of the most frequently mutated form of Ras, K-Ras, to FTase inhibitors and its alternative prenylation by geranylgeranyltransferase I (GGTase I), has cast doubts on whether K-Ras is the target for FTase inhibitors. This monthly update focuses on issues of critical importance to the further development of FTase inhibitors as anticancer agents. Alternative prenylation of K-Ras by GGTase I as a mechanism of resistance to FTase inhibitors, targets for FTase inhibitors other than K-Ras and the relevance of GGTase I inhibitors as antitumour agents will be discussed.

Prenyltransferase Inhibitors as Radiosensitizers

Radiation therapy is frequently used in the treatment of a number of different tumors. However, the effectiveness of radiotherapy is limited by the ability of normal tissues adjacent to tumors to tolerate radiation in the doses required to kill or sterilize tumor cells. This limitation is compounded by the presence in tumors of radiation-resistant cells that may arise as a result of environmental factors, such as hypoxic regions in tumors, the expression of growth factors that can reduce radiation sensitivity, or tumor cell intrinsic radiation resistance that may be imparted through the activation of certain oncogenes. Ras oncogenes in particular may contribute to radiation resistance, because they have been shown to increase radiation resistance in many experimental systems, and are mutated in an estimated 30% of all human tumors. Basic fibroblast growth factor (bFGF) has also been implicated in increased radiation resistance and is over-expressed in certain tumors, particularly glioblastomas.

Farnesyltransferase and Geranylgeranyltransferase I Inhibitors as Novel Agents for Cancer and Cardiovascular Diseases

Cancer is believed to result from an accumulation of genetic alterations that cause loss of function of tumor suppressor genes and gain of function of oncogenes. Among the most thoroughly studied oncogenes are those that encode Ras proteins. Ras proteins are guanosine triphosphate/guanosine diphosphate (GTP/GDP) binding guanosine triphosphate phosphatases (GTPases) that play a critical role in a variety of signal transduction pathways in the cell (1–7). The three mammalian ras genes encode four highly homologous plasma membrane-bound G-proteins (H-, N-, KA- and KB-Ras) that cycle between their GTP (active)- and GDP (inactive)- forms to switch on and off signals from the cell surface to the nucleus (4–8). For example, binding of platelet-derived growth factor (PDGF) to its receptor tyrosine kinase results in autophosphorylation, which creates phosphotyrosines that serve as binding sites for several key signaling molecules such as phospholipase C-γl and phosphatidyl inositol-3 kinase (PI-3 Kinase) (9–15). Most important for Ras activation is the recruitment of the growth factor receptor binding protein 2 (GRB-2), which is complexed to a ras GTP/GDP exchanger, mammalian son of seven less-1 (m-SOS-1), (6,7). The exchanger m-SOS-1 binds GDP Ras and catalyzes the exchange of GDP for GTP, which results in Ras activation (6,7). GTP-bound Ras can then activate several effectors that result in triggering a variety of signaling pathways, such as the PI-3 kinase/AKT-2 survival pathway and the mitogen-activated protein (MAP) kinase cascade. In the latter, GTP-Ras recruits a ser/thr kinase, c-raf- 1, to the plasma membrane where it gets activated and in turn activates a series of MAP kinases (5,8). These Ras-dependent signals are turned off by hydrolysis of the bound GTP, which returns Ras to its GDP-bound state. The GTPase activity is intrinsic to the Ras protein but requires the GTPase activating protein, Ras-GAP (4). Thus, Ras is activated by guanine nucleotide exchange factors, such as m-SOS-1, and turned off by GTPase activating proteins such as GAP. Mutations in the Ras sequence (i.e., amino acids 12, 13, and 61) that lock Ras in its GTP-bound form result in a growth factor-independent, constitutively activated signal that leads to uncontrolled growth (16–19). Mutated Ras no longer needs exchange factors for activation and GAP does not turn it off. This Ras-dependent uncontrolled growth is believed to be directly implicated in a large number of human cancers, because 30% of human tumors express ras oncogenes with these transforming mutations (1,2).

Peptidomimetic-based inhibitors of farnesyltransferase

Human H-, K-, and N-ras genes encode four structurally related proteins with 188 or 189 amino acids and a molecular weight of 21 kDa (1,2). Analysis of oncogenes in human tumors showed that mutated Ras existed in approx 30% of all human tumors, particularly in over 90% of human pancreatic carcinomas and 50% of human colon cancers (1). The frequency of mutation is dependent on tumor type, with breast, ovary, and stomach carcinomas showing the lowest frequency Ras mutations (3). Ras proteins play a crucial role as a molecular switch transducing signals from receptor tyrosine kinases to the cell nucleus (4). Normal Ras exists in an equilibrium between inactive GDP- and active GTP-bound forms, with a strong preference for Ras-GDP (5). When mutated at positions 12, 13, and 61, Ras proteins lose their ability to hydrolyze GTP to GDP and so deactivate the switch (6). Consequently, the mutated Ras is locked in the GTP-bound form, causing uncontrolled proliferation.