Post-translational Prenylation Research Articles

On a sugary-relationship between caspases and lamins

On a sugary-relationship between caspases and lamins

Isoprenylcysteine carboxylmethyltransferase regulates mitochondrial respiration and cancer cell metabolism.

Isoprenylcysteine carboxylmethyltransferase (Icmt) catalyzes the last of the three-step posttranslational protein prenylation process for the so-called CaaX proteins, which includes many signaling proteins, such as most small GTPases. Despite extensive studies on Icmt and its regulation of cell functions, the mechanisms of much of the impact of Icmt on cellular functions remain unclear. Our recent studies demonstrated that suppression of Icmt results in induction of autophagy, inhibition of cell growth and inhibition of proliferation in various cancer cell types, prompting this investigation of potential metabolic regulation by Icmt. We report here the findings that Icmt inhibition reduces the function of mitochondrial oxidative phosphorylation in multiple cancer cell lines. In-depth oximetry analysis demonstrated that functions of mitochondrial complex I, II and III are subject to Icmt regulation. Consistently, Icmt inhibition decreased cellular ATP and depleted critical tricarboxylic acid cycle metabolites, leading to suppression of cell anabolism and growth, and marked autophagy. Several different approaches demonstrated that the impact of Icmt inhibition on cell proliferation and viability was largely mediated by its effect on mitochondrial respiration. This previously unappreciated function of Icmt, which can be therapeutically exploited, likely has a significant role in the impact of Icmt on tumorigenic processes.

Human isoprenoid synthase enzymes as therapeutic targets.

In the human body, the complex biochemical network known as the mevalonate pathway is responsible for the biosynthesis of all isoprenoids, which consists of a vast array of metabolites that are vital for proper cellular functions. Two key isoprenoids, farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP) are responsible for the post-translational prenylation of small GTP-binding proteins, and serve as the biosynthetic precursors to numerous other biomolecules. The down-stream metabolite of FPP and GGPP is squalene, the precursor to steroids, bile acids, lipoproteins, and vitamin D. In the past, interest in prenyl synthase inhibitors focused mainly on the role of the FPP in lytic bone diseases. More recently pre-clinical and clinical studies have strongly implicated high levels of protein prenylation in a plethora of human diseases, including non-skeletal cancers, the progression of neurodegenerative diseases and cardiovascular diseases. In this review, we focus mainly on the potential therapeutic value of down-regulating the biosynthesis of FPP, GGPP, and squalene. We summarize the most recent drug discovery efforts and the structural data available that support the current on-going studies.

KRas Localizes to the Plasma Membrane by Spatial Cycles of Solubilization, Trapping and Vesicular Transport

KRas is a major proto-oncogene product whose signaling activity depends on its level of enrichment on the plasma membrane (PM). This PM localization relies on posttranslational prenylation for membrane affinity, while PM specificity has been attributed to electrostatic interactions between negatively charged phospholipids in the PM and basic amino-acids in the C terminus of KRas. By measuring kinetic parameters of KRas dynamics in living cells with a cellular-automata-based data-fitting approach in realistic cell-geometries, we show that charge-based specificity is not sufficient to generate PM enrichment in light of the total surface area of endomembranes. Instead, mislocalized KRas is continuously sequestered from endomembranes by cytosolic PDEδ to be unloaded in an Arl2-dependent manner to perinuclear membranes. Electrostatic interactions then trap KRas at the recycling endosome (RE), from where vesicular transport restores enrichment on the PM. This energy driven reaction-diffusion cycle explains how small molecule targeting of PDEδ affects the spatial organization of KRas.

Thienopyrimidine Bisphosphonate (ThPBP) Inhibitors of the Human Farnesyl Pyrophosphate Synthase: Optimization and Characterization of the Mode of Inhibition

Human farnesyl pyrophosphate synthase (hFPPS) controls the post-translational prenylation of small GTPase proteins that are essential for cell signaling, cell proliferation, and osteoclast-mediated bone resorption. Inhibition of hFPPS is a clinically validated mechanism for the treatment of lytic bone diseases, including osteoporosis and cancer related bone metastases. A new series of thienopyrimidine-based bisphosphonates (ThP-BPs) were identified that inhibit hFPPS with low nanomolar potency. Crystallographic evidence revealed binding of ThP-BP inhibitors in the allylic subpocket of hFPPS. Simultaneous binding of inorganic pyrophosphate in the IPP subpocket leads to conformational closing of the active site cavity. The ThP-BP analogues are significantly less hydrophilic yet exhibit higher affinity for the bone mineral hydroxyapatite than the current N-BP drug risedronic acid. The antiproliferation properties of a potent ThB-BP analogue was assessed in a multiple myeloma cell line and found to be equipotent to the best current N-BP drugs. Consequently, these compounds represent a new structural class of hFPPS inhibitors and a novel scaffold for the development of human therapeutics.

Protein Kinase A-dependent Phosphorylation of Rap1 Regulates Its Membrane Localization and Cell Migration

The small G protein Rap1 can mediate "inside-out signaling" by recruiting effectors to the plasma membrane that signal to pathways involved in cell adhesion and cell migration. This action relies on the membrane association of Rap1, which is dictated by post-translational prenylation as well as by a stretch of basic residues within its carboxyl terminus. One feature of this stretch of acidic residues is that it lies adjacent to a functional phosphorylation site for the cAMP-dependent protein kinase PKA. This phosphorylation has two effects on Rap1 action. One, it decreases the level of Rap1 activity as measured by GTP loading and the coupling of Rap1 to RapL, a Rap1 effector that couples Rap1 GTP loading to integrin activation. Two, it destabilizes the membrane localization of Rap1, promoting its translocation into the cytoplasm. These two actions, decreased GTP loading and decreased membrane localization, are related, as the translocation of Rap1-GTP into the cytoplasm is associated with its increased GTP hydrolysis and inactivation. The consequences of this phosphorylation in Rap1-dependent cell adhesion and cell migration were also examined. Active Rap1 mutants that lack this phosphorylation site had a minimal effect on cell adhesion but strongly reduced cell migration, when compared with an active Rap1 mutant that retained the phosphorylation site. This suggests that optimal cell migration is associated with cycles of Rap1 activation, membrane egress, and inactivation, and requires the regulated phosphorylation of Rap1 by PKA.

N6-isopentenyladenosine, an endogenous isoprenoid end product, directly affects cytotoxic and regulatory functions of human NK cells through FDPS modulation

iPA is a naturally occurring nucleoside with an isopentenyl moiety derived from the mevalonate pathway and a well-established anti-tumor activity. In analogy to the unique specificity for phosphoantigens, such as IPP, shown by human Vγ9Vδ2 T cells, here, we report for the first time the ability of iPA to selectively expand and directly target human NK cells. Interestingly, submicromolar doses of iPA stimulate resting human NK cells and synergize with IL-2 to induce a robust activation ex vivo with significant secretion of CCL5 and CCL3 and a large increase in TNF-α and IFN-γ production when compared with IL-2 single cytokine treatment. Moreover, iPA promotes NK cell proliferation and up-regulates the expression of specific NK cell-activating receptors, as well as CD69 and CD107a expression. Accordingly, this phenotype correlates with significantly greater cytotoxicity against tumor targets. At the molecular level, iPA leads to a selective, potent activation of MAPK signaling intermediaries downstream of the IL-2R. The effect results, at least in part, from the fine modulation of the FDPS activity, the same enzyme implicated in the stimulation of the human γδ T cells. The iPA-driven modulation of FDPS can cause an enhancement of post-translational prenylation essential for the biological activity of key proteins in NK signaling and effector functions, such as Ras. These unanticipated properties of iPA provide an additional piece of evidence of the immunoregulatory role of the intermediates of the mevalonate pathway and open novel therapeutic perspectives for this molecule as an immune-modulatory drug.

Tipifarnib and farnesyltransferase inhibitors in the treatment of inflammatory breast cancer: is the story over? A review

Inflammatory breast cancer (IBC) is a rare but aggressive form of breast cancer with unique clinicopathological features and poor prognosis. Its epidemiology is distinct from noninflammatory locally advanced breast cancer (LABC), which points to different etiology. With the advent of multimodality treatment for LABC, the outcomes for women with IBC have improved, but remain significantly inferior to outcomes for noninflammatory LABC. This review focuses on new research in epidemiology and molecular pathways characteristic for IBC. One of the critical carcinogenic events apparently driving the development and progression of IBC is activation of the RhoC protein, which is a part of the Ras oncogene superfamily. These proteins, like other Ras proteins, require posttranslational prenylation for their activation and transfer to cell membranes. Farnesylation is a common type of prenlyation that can be blocked with a new class of drugs - farnesyltransferase inhibitors. The most advanced farnesyltransferase inhibitor in development, tipifarnib, has been evaluated in Phase II clinical trials as monotherapy and in combination with antihormonal agents and trastuzumab in metastatic and locally advanced breast cancer. A few tumor responses have been observed in these trials, but not enough to warrant a Phase III trial. Potential combinations of tipifarnib with other novel agents targeting enzymes downstream to RhoC are reviewed. Some of these drugs, such as imatinib and crizotinib, are already commercially available. Others like perifosine and anti-vascular endothelial growth factor 3 antibody are currently under development. Innovative trial designs to address this rare type of cancer are discussed.

Statins - the Holy Grail for cancer?

Statins or 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitors have been used for primary and secondary prevention of cardiovascular diseases and currently are the most commonly prescribed drug class in the world. Besides cholesterol reduction, pre-clinical studies have shown that statins may exert antineoplastic effects, through both HMG-CoA reductase-dependent and HMG-CoA reductase-independent pathways. By competitive inhibition of HMG-CoA reductase, statins prevent post-translational prenylation of the Ras/Rho superfamily, which are important mediators of cell growth, differentiation and survival (1).

Combination Effects of Nilotinib and Zoledronic Acid On Chronic Myeloid Leukemia Are Canceled in the Co-Culture System Which Mimic the Bone Marrow Microenvironment.

AbstractAbstract 2459Prognosis of chronic myeloid leukemia (CML) has been dramatically improved by the induction of tyrosine kinase inhibitors (TKIs) as a first-line treatment. However, cure is still difficult because of the existence of leukemic stem cell (LSC).Zoledronic acid (ZOL) is a 3rd-generation bisphosphonate that inhibits bone matrix resorption of osteoclasts (OCs), and inhibit the proliferation of several tumor cells by preventing posttranslational prenylation of Ras-related proteins. We have previously reported that ZOL possess anti-leukemic effect that augments synergistically the activity of imatinib mesylate (Kuroda, et al. Blood 2003), and OCs maintain leukemic cells in a quiescent state under newly developed co-culture system (Yokota, et al. Leuk Res 2010). Thus, we hypothesized that the treatment of ZOL combined with nilotinib (Nilo) may be effective to eliminate dormant leukemic cell population, called LSCs, by attacking the OCs. To test this hypothesis, we examined the combined effect of Nilo and ZOL on the human CML cell line BV173 and BaF3 cells expressing p190 wild type BCR-ABL (BaF3/BCR-ABL) in vitro.Firstly, modified MTT assay was performed with ordinary condition (without any other co-cultured cells), resulting that ZOL augmented synergistically in vitro effects of Nilo both on cell lines (combination index < 1 at Fa=0.5) (Figure 1). Next we investigated the anti-leukemic effects of Nilo and ZOL using our developed co-culture system including osteoblasts (OBs), OCs and bone slices (Yokota, et al. Leuk Res 2010). 104 BaF3/BCR-ABL cells were applied to transwell chambers in each well of the co-culture plate (thus, BaF3/BCR-ABL cells did not attach directly to OBs, OCs and bones). Next day, each well was exposed to saline, Nilo 10nM, ZOL 10uM or Nilo 10nM plus ZOL 10uM. 48 hours after treatment, the viable cell number of BaF3/BCR-ABL was counted by trypan blue staining. Cell numbers were as follows; control: 55.79×104/well, Nilo only: 36.17×104/well, ZOL only: 64.46×104/well, Nilo plus ZOL: 39.42×104/well, respectively (Figure 2). These results suggested that combination effect of Nilo plus ZOL in vitro was canceled in the co-culture system. In the same condition, 7-AAD/Annexin V double staining was also performed. The apoptotic rates were as follows; control: 38.05 ± 2.36 %, Nilo only: 29.49 ± 2.59 %, ZOL only: 37.67 ± 1.39 %, Nilo plus ZOL: 29.66 ± 5.67 %. The results indicated that co-culture system of mimic the bone marrow microenvironment changed the drug effect on BaF3/BCR-ABL to resistance.To reveal the mechanisms of this phenomenon, TGF-ƒÀ1 concentration in the co-culture supernatants was measured by ELISA. In the previous report, when BaF3/BCR-ABL were co-cultured with OBs, OCs and bone slices, their proliferation was significantly suppressed and TGF-ƒÀ1 was significantly higher in supernatants from the co-culture system consisted of OB+OC+bone than control. In the present study, TGF-ƒÀ1 ratios (TGF-ƒÀ1 concentration in each well administrated with each agent/that in the medium without any cells) were almost same among wells treated with each agent. These results suggested that TGF-ƒÀ1 concentrations maintained for a while even after OCs were destroyed by ZOL and the remained TGF-ƒÀ1 affected the effects of anti-cancer agents on the proliferation of CML cells.In conclusion, the present study suggested that the evaluation of combined effects in ordinary culture system without any other cells which consist of microenvironment might not be correctly reflected in more physiological system. Therefore, it may be important to utilize the co-culture system used here to evaluate the effects of anti-cancer agents. [Display omitted] [Display omitted] Disclosures:No relevant conflicts of interest to declare.

Nifedipine prevents etoposide-induced caspase-3 activation, prenyl transferase degradation and loss in cell viability in pancreatic β-cells

Emerging evidence implicates novel roles for post-translational prenylation (i.e., farnesylation and geranylgeranylation) of various signaling proteins in a variety of cellular functions including hormone secretion, survival and apoptosis. In the context of cellular apoptosis, it has been shown previously that caspase-3 activation, a hallmark of mitochondrial dysregulation, promotes hydrolysis of several key cellular proteins. We report herein that exposure of insulin-secreting INS 832/13 cells or normal rat islets to etoposide leads to significant activation of caspase-3 and subsequent degradation of the common α-subunit of farnesyl/geranylgeranyl transferases (FTase/GGTase). Furthermore, the above stated signaling steps were prevented by Z-DEVD-FMK, a known inhibitor of caspase-3. In addition, treatment of cell lysates with recombinant caspase-3 also caused FTase/GGTase α-subunit degradation. Moreover, nifedipine, a calcium channel blocker, markedly attenuated etoposide-induced caspase-3 activation, FTase/GGTase α-subunit degradation in INS 832/13 cells and normal rat islets. Further, nifedipine significantly restored etoposide-induced loss in metabolic cell viability in INS 832/13 cells. Based on these findings, we conclude that etoposide induces loss in cell viability by inducing mitochondrial dysfunction, caspase-3 activation and degradation of FTase/GGTase α-subunit. Potential significance of these findings in the context of protein prenylation and β-cell survival are discussed.

Rab-geranylgeranyl transferase regulates glucose-stimulated insulin secretion from pancreatic β cells

A growing body of evidence implicates essential roles for small molecular weight G-proteins (e.g., Cdc42, Rac1, Arf6 and Rab3A and Rab27A) in islet β-cell function including glucose-stimulated insulin secretion (GSIS). One of the known mechanisms for optimal activation of small G-proteins involves post-translational prenylation, which is mediated by farnesyltransferase (FTase) and geranylgeranyl transferases (GGTases I and II). The FTase catalyzes incorporation of a 15-carbon farnesyl group while the GGTase mediates incorporation of a 20-carbon geranylgeranyl group into the C-terminal cysteines of G-proteins. The FTase, GGTase I and GGTase II prenylate Ras, Cdc42/Rac1, and Rab G-proteins, respectively. While considerable evidence exists on FTase/GGTase I-mediated regulation of GSIS, very little is known about GGTase II (also referred to as Rab GGTase; RGGT) and its regulatory proteins in the cascade of events leading to GSIS. Herein, we provide the first immunological evidence to suggest expression of α- and β-subunits of RGGT in clonal INS 832/13 β-cells, normal rat islets and human islets. Furthermore, Rab escort protein1 (REP1), which has been shown to be critical for prenylation of Rab G-proteins, is also expressed in these cells. Furthermore, evidence is presented to suggest that siRNA-mediated knockdown of α- or β-subunits of RGGT and REP1 markedly attenuates GSIS in INS 832/13 cells. These findings provide the first evidence in support of key roles for RGGT and its regulatory proteins in GSIS.

Design and Synthesis of Active Site Inhibitors of the Human Farnesyl Pyrophosphate Synthase: Apoptosis and Inhibition of ERK Phosphorylation in Multiple Myeloma Cells

Human farnesyl pyrophosphate synthase (hFPPS) controls intracellular levels of FPP and post-translational prenylation of small GTPase proteins, which are essential for cell signaling and cell proliferation. Clinical investigations provide evidence that N-BP inhibitors of hFPPS are disease modifying agents that improve survival of multiple myeloma (MM) patients via mechanisms unrelated to their skeletal effects. A new series of N-BPs was designed that interact with a larger portion of the GPP subpocket, as compared to the current therapeutic drugs, and rigidify the (364)KRRK(367) tail of hFPPS in the closed conformation in the absence of IPP. An analogue of this series was used to demonstrate inhibition of the intended biological target, resulting in apoptosis and down-regulation of ERK phosphorylation in human MM cell lines.

Prenylation of Rho G-Proteins: a Novel Mechanism Regulating Gene Expression and Protein Stability in Human Trabecular Meshwork Cells

Endogenous prenylation with sesquiterpene or diterpene isoprenoids facilitates membrane localization and functional activation of small monomeric GTP-binding proteins. A direct effect of isoprenoids on regulation of gene expression and protein stability has also been proposed. In this study, we determined the role of sesquiterpene or diterpene isoprenoids on the regulation of Rho G-protein expression, activation, and stability in human trabecular meshwork (TM) cells. In both primary and transformed human TM cells, limiting endogenous isoprenoid synthesis with lovastatin, a potent HMG-CoA reductase inhibitor, elicited marked increases in RhoA and RhoB mRNA and protein content. The effect of lovastatin was dose-dependent with newly synthesized inactive protein accumulating in the cytosol. Supplementation with geranylgeranyl pyrophosphate (GGPP) prevented, while inhibition of geranylgeranyl transferase-I mimicked, the effects of lovastatin on RhoA and RhoB protein content. Similarly, lovastatin-dependent increases in RhoA and RhoB mRNA expression were mimicked by geranylgeranyl transferase-I inhibition. Interestingly, GGPP supplementation selectively promoted the degradation of newly synthesized Rho proteins which was mediated, in part, through the 20S proteasome. Functionally, GGPP supplementation prevented lovastatin-dependent decreases in actin stress fiber organization while selectively facilitating the subcellular redistribution of accumulated Rho proteins from the cytosol to the membrane and increasing RhoA activation. Post-translational prenylation with geranylgeranyl diterpenes selectively facilitates the expression, membrane translocation, functional activation, and turnover of newly synthesized Rho proteins. Geranylgeranyl prenylation represents a novel mechanism by which active Rho proteins are targeted to the 20S proteasome for degradation in human TM cells.

Geranyl modification on the tryptophan residue of ComXRO-E-2 pheromone by a cell-free system

ComX pheromone is an isoprenoidal oligopeptide containing a modified tryptophan residue, which stimulates natural genetic competence in the gram-positive bacterium Bacillus. Since posttranslational prenylation on the tryptophan residue has not been reported except in ComX pheromone, the universality of this modification has not yet been elucidated. In this paper, we established a cell-free system, whereby the tryptophan residue in peptides is modified with a geranyl group by modifying enzyme ComQ. In addition, we investigated enzymatic reaction conditions using an in vitro enzyme reaction system. This is the first report of in vitro geranylation on the tryptophan residue. This system is potentially a useful tool for elucidating the universality of prenylation on the tryptophan residue.

Geranylgeranyl transferase 1 modulates autophagy and apoptosis in human airway smooth muscle

Geranylgeranyl transferase 1 (GGT1) is involved in the posttranslational prenylation of signaling proteins, such as small GTPases. We have shown that blocking the formation of isoprenoids with statins regulates survival of human lung mesenchymal cells; thus, we tested the hypothesis that GGT1 may specifically modulate programmed cell death pathways in these cells. To this end, human airway smooth muscle (HASM) cells were treated with the selective GGT1 inhibitor GGTi-298. Apoptosis was seen using assays for cellular DNA content and caspase activation. Induction of autophagy was observed using transmission electron microscopy, immunoblotting for LC3 lipidation and Atg5-12 complex content, and confocal microscopy to detect formation of lysosome-localized LC3 punctae. Notably, GGT1 inhibition induced expression of p53-dependent proteins, p53 upregulated modulator of apoptosis (Noxa), and damage-regulated autophagy modulator (DRAM), this was inhibited by the p53 transcriptional activation inhibitor cyclic-pifithrin-α. Inhibition of autophagy with bafilomycin-A1 or short-hairpin RNA silencing of Atg7 substantially augmented GGTi-298-induced apoptosis. Overall, we demonstrate for the first time that pharmacological inhibition of GGT1 induces simultaneous p53-dependent apoptosis and autophagy in HASM. Moreover, autophagy regulates apoptosis induction. Thus, our findings identify GGT1 as a key regulator of HASM cell viability.

Modulation of Anthracycline-Induced Cytotoxicity by Targeting the Prenylated Prenylome in Myeloid Leukemia Cells

Abstract 1418 Background:Deregulation of Ras/ERK signaling in myeloid leukemias makes this pathway an interesting target for drug development. Design and Methods:Myeloid leukemia cell lines (HL60, KG1a, THP-1, K562) were screened for idarubicin-induced apoptosis, cell cycle progression, cell-cycle dependent MAP kinase kinase (MEK-1/2) activation, and Top2 expression. Cell-cycle dependent activation of MEK/ERK signaling was blocked using farnesyltransferase inhibitor (FTI) BMS-214,662 and dual prenyltransferase inhibitor (DPI) L-778,123 to disrupt Ras signaling. Analysis of synergy was done by the method of Chou and Talalay using the CompuSyn program. Analysis of the prenylated proteome was performed using a tagging-via-substrate approach. Knock-down of K-Ras and Cdc42 protein levels was done by RNA interference (RNAi) using lentiviral shRNA constructs. Results:Idarubicin caused a G2/M cell-cycle arrest characterized by elevated diphosphorylated MEK-1/2 and Top2a expression levels. Although cells were most sensitive to idarubicin, DPI L-778,123 and FTI BMS-214,662 also potently inhibited leukemia cell growth with IC50 values in the micromolar and sub-micromolar range. The FTI/DPIs elicited distinct effects on Ras signaling, protein prenylation, cell cycling, Top2 expression and apoptosis. Combining these FTI/DPIs with idarubicin synergistically inhibited proliferation of leukemia cell lines (KG1a, THP-1 and K562), but the L-778,123+idarubicin combination exhibited synergistic growth inhibition over a greater range of drug concentrations. Both BMS-214,662 and L-778,123 effectively inhibited prenylation of the majority of larger molecular weight proteins (e.g. Lamins, NAP1L1, HDJ-2), and some smaller molecular weight proteins (e.g. H-Ras, N-Ras). Additionally, only DPI L-778,123 blocked prenylation of smaller molecular weight proteins such as K-Ras and Cdc42. Interestingly, combined FTI/DPI treatment synergistically inhibited cell proliferation, induced apoptosis and nearly completely blocked protein prenylation in all cell lines tested. However, RhoB and RhoC remained prenylated even upon combined FTI/DPI treatment. Inhibition of K-Ras expression by RNA interference or blockade of its post-translational prenylation led to increased BMS-214,662-induced apoptosis. Conclusion:Our results suggest that nearly complete inhibition of protein prenylation using an FTI+DPI combination is the most effective method to induce apoptosis and to block anthracycline induced activation of ERK signaling. Disclosures:Reuter:Sanofi: Honoraria; Amgen: Honoraria.

Bisphosphonates: Beyond Prevention of Bone Metastases

Bisphosphonates have traditionally been indicated for treatment and control of osteoporosis because they inhibit osteoclastmediated bone resorption and reduce the release of calcium into the blood stream (1). They are currently being used by millions of people (mostly women) worldwide (2). Accumulating evidence over the past 10 years has demonstrated that the use of third-generation nitrogen-containing bisphosphonates can effectively treat bone metastases in women with breast cancer (3). It has been hypothesized that bisphosphonates can also prevent bone metastases altogether and could therefore be implemented in an adjuvant setup in women with breast cancer (4,5). A recent randomized controlled study failed to demonstrate such an effect using third-generation bisphosphonate (6). If bisphosphonate use is associated with a generalized reduced risk of malignancy, one would expect to see reduced risk of developing breast cancer or other cancers in regular users of secondgeneration bisphosphonates, such as osteoporotic women. One would also expect to see reduced risks of developing contralateral breast cancer in women with breast cancer who were further treated with bisphosphonates. Three descriptive studies have thus far shown a strong and similar negative association between the use of common second-generation oral bisphosphonates and the risk of breast cancer (7–9). These findings raised the possibility of con founding by indication. Bisphosphonates are used by osteoporotic women, and osteoporosis is the result of reduced circulating estrogen levels. Estrogen is strongly associated with risk of breast cancer, and women with low estrogen levels are expected to have lower breast cancer rates. Thus, the negative association with bisphosphonate use could actually reflect a negative association with low estrogen levels in the body. However, evidence that the degree of breast cancer risk reduction among bisphosphonate users was not correlated with the degree of bone density loss (7), that risk reduction was seen only after a year of treatment (8), and that postmenopausal bisphosphonates users also demonstrated a reduced risk of colorectal cancer (10,11), all suggest that the association between bisphosphonate use and reduced risk of cancer is real. Mechanistically, nitrogen-containing bisphosphonates inhibit protein prenylation by blocking the mevalonate pathway. Selective inhibition of farensyl pyrophosphate synthase by bisphosphonates can lead to diminished posttranslational prenylation of small GTPase proteins (including signaling molecules such as proteins of the Ras and Rho families) that promote tumorigenesis and metastases (12). In a mouse model, use of bisphosphonates was associated with the suppression of Rho and Ras pathways (13). Further support for a possible role of bisphosphonates in primary prevention of cancer comes from numerous previous reports on the negative association between the use of statins and the risk of cancer in various organs [eg, (14–20)]. Statins, which use the same mevalonate pathway as do bisphosphonates (acting upstream of the farensylation–geranylation step), have been shown to be associated with cancer risk reduction and with possibly improved survival of patients with cancers in multiple sites (21).

A Role for Rac3 GTPase in the Regulation of Autophagy

The process of autophagy is situated at the intersection of multiple cell signaling pathways, including cell metabolism, growth, and death, and hence is subject to multiple forms of regulation. We previously reported that inhibition of isoprenylcysteine carboxylmethyltransferase (Icmt), which catalyzes the final step in the post-translational prenylation of so-called CAAX proteins, results in the induction of autophagy which enhances cell death in some cancer cells. In this study, using siRNA-mediated knockdown of a group of small GTPases that are predicted Icmt substrates, we identify Rac3 GTPase as a negative regulator of the process of autophagy. Knockdown of Rac3, but not the closely related isoforms Rac1 and Rac2, results in induction of autophagy. Ectopic expression of Rac3, significantly rescues cells from autophagy and cell death induced by Icmt inhibition, strengthening the notion of an isoform-specific autophagy regulatory function of Rac3. This role of Rac3 was observed in multiple cell lines with varying Rac subtype expression profiles, suggesting its broad involvement in the process. The identification of this less-studied Rac member as a novel regulator provides new insight into autophagy and opens opportunities in identifying additional regulatory inputs of the process.

Modulation of anthracycline-induced cytotoxicity by targeting the prenylated proteome in myeloid leukemia cells

Deregulation of Ras/ERK signaling in myeloid leukemias makes this pathway an interesting target for drug development. Myeloid leukemia cell lines were screened for idarubicin-induced apoptosis, cell-cycle progression, cell-cycle-dependent MAP kinase kinase (MEK-1/2) activation, and Top2 expression. Cell-cycle-dependent activation of MEK/ERK signaling was blocked using farnesyltransferase inhibitor (FTI) BMS-214,662 and dual prenyltransferase inhibitor (DPI) L-778,123 to disrupt Ras signaling. Idarubicin caused a G2/M cell-cycle arrest characterized by elevated diphosphorylated MEK-1/2 and Top2α expression levels. The FTI/DPIs elicited distinct effects on Ras signaling, protein prenylation, cell cycling and apoptosis. Combining these FTI/DPIs with idarubicin synergistically inhibited proliferation of leukemia cell lines, but the L-778,123+idarubicin combination exhibited synergistic growth inhibition over a greater range of drug concentrations. Interestingly, combined FTI/DPI treatment synergistically inhibited cell proliferation, induced apoptosis and nearly completely blocked protein prenylation. Inhibition of K-Ras expression by RNA interference or blockade of its post-translational prenylation led to increased BMS-214,662-induced apoptosis. Our results suggest that nearly complete inhibition of protein prenylation using an FTI + DPI combination is the most effective method to induce apoptosis and to block anthracycline-induced activation of ERK signaling.