ggti-298 and Breast-Neoplasms

Protein geranylgeranylation (GGylation) is an important biochemical process for many cellular signaling molecules. Previous studies have shown that GGylation is essential for cell survival in many types of cancer. However, the molecular mechanism mediating the cell survival effect remains elusive. In this report, we show that the Hippo pathway mediates GGylation-dependent cell proliferation and migration in breast cancer cells. Blockade of GGylation enhanced phosphorylation of Mst1/2 and Lats1, and inhibited YAP and TAZ activity and the Hippo-YAP/TAZ pathway-dependent transcription. The effect of GGylation blockade on inhibition of breast cancer cell proliferation and migration is dependent on the Hippo-YAP/TAZ signaling, in which YAP appears to regulate cell proliferation and TAZ to regulate cell migration. Furthermore, GGylation-dependent cell proliferation is correlated with the activity of YAP/TAZ in breast cancer cells. Finally, Gγ and RhoA are the GGylated proteins that may transduce GGylation signals to the Hippo-YAP/TAZ pathway. Taken together, our studies have demonstrated that the Hippo-YAP/TAZ pathway is essential for GGylation-dependent cancer cell proliferation and migration.

Topics: Atorvastatin; Benzamides; Breast Neoplasms; Cell Movement; Cell Proliferation; Female; HEK293 Cells; Heptanoic Acids; Hippo Signaling Pathway; Humans; MCF-7 Cells; Prenylation; Protein Processing, Post-Translational; Protein Serine-Threonine Kinases; Pyrroles; Signal Transduction; Tumor Cells, Cultured

There is increasing evidence that breast tumors are organized in a hierarchy, with a subpopulation of tumorigenic cancer cells, the cancer stem cells (CSCs), which sustain tumor growth. The characterization of protein networks that govern CSC behavior is paramount to design new therapeutic strategies targeting this subpopulation of cells. We have sought to identify specific molecular pathways of CSCs isolated from 13 different breast cancer cell lines of luminal or basal/mesenchymal subtypes. We compared the gene expression profiling of cancer cells grown in adherent conditions to those of matched tumorsphere cultures. No specific pathway was identified to be commonly regulated in luminal tumorspheres, resulting from a minor CSC enrichment in tumorsphere passages from luminal cell lines. However, in basal/mesenchymal tumorspheres, the enzymes of the mevalonate metabolic pathway were overexpressed compared to those in cognate adherent cells. Inhibition of this pathway with hydroxy-3-methylglutaryl CoA reductase blockers resulted in a reduction of breast CSC independent of inhibition of cholesterol biosynthesis and of protein farnesylation. Further modulation of this metabolic pathway demonstrated that protein geranylgeranylation (GG) is critical to breast CSC maintenance. A small molecule inhibitor of the geranylgeranyl transferase I (GGTI) enzyme reduced the breast CSC subpopulation both in vitro and in primary breast cancer xenografts. We found that the GGTI effect on the CSC subpopulation is mediated by inactivation of Ras homolog family member A (RHOA) and increased accumulation of P27(kip1) in the nucleus. The identification of protein GG as a major contributor to CSC maintenance opens promising perspectives for CSC targeted therapy in basal breast cancer.

Topics: Animals; Antineoplastic Agents; Benzamides; Blotting, Western; Breast Neoplasms; Cell Cycle; Cell Line, Tumor; Cell Survival; Docetaxel; Female; Gene Expression Profiling; Humans; Mevalonic Acid; Mice; Mice, SCID; Neoplasms, Basal Cell; Neoplastic Stem Cells; Taxoids

Inhibitors of protein prenylation, including prenyltransferase inhibitors and aminobisphosphonates such as zoledronic acid, are being investigated intensively as therapeutics in cancer and other diseases. Determining whether prenylation inhibitors directly or indirectly target tumor and/or host cells is key to understanding therapeutic mechanisms.. To determine which cell types can be targeted directly by distinct classes of prenylation inhibitors in vivo, we describe herein the development and implementation of a sensitive and pharmacologically specific bioluminescence-based imaging reporter that is inducible by prenylation inhibitors.. In mouse xenograft models of breast cancer, using reporter-bearing mammary fat pad- or bone-localized tumor cells, we show that a prenyltransferase inhibitor robustly induces reporter activity in vivo. In contrast, zoledronic acid, a bone-associated aminobisphosphonate that exerts adjuvant chemotherapeutic activity in patients with breast cancer, fails to induce reporter activity in tumor cells of either model.. Although a prenyltransferase inhibitor can directly target breast cancer cells in vivo, zoledronic acid and related aminobisphosphonates are likely to exert antitumor activity indirectly by targeting host cells. Accordingly, these findings shift attention toward the goal of determining which host cell types are targeted directly by aminobisphosphonates to exert adjuvant chemotherapeutic activity.

Topics: Alkyl and Aryl Transferases; Animals; Benzamides; Blotting, Western; Bone Density Conservation Agents; Breast Neoplasms; Cell Line, Tumor; Clodronic Acid; Diphosphonates; Female; Genes, Reporter; Green Fluorescent Proteins; HEK293 Cells; Humans; Imidazoles; Luciferases; Luminescent Measurements; Mammary Neoplasms, Experimental; Mice; Mice, Nude; Microscopy, Confocal; Mutation; Protein Prenylation; Recombinant Fusion Proteins; Transplantation, Heterologous; Zoledronic Acid

Isoprenoid inhibitors are being evaluated as agents for the treatment of cancer. Their antitumor activity is attributed to inhibition of post-translational modification of Ras, which is crucial for its translocation and attachment to the plasma membrane, and ultimate involvement in signal transduction. However, whether blocking of Ras is solely responsible for the observed antitumor activity is unresolved. In this report, we propose an alternate mechanism. Using breast tumor models, we show that agents possessing a lactone moiety, including statins (such as lovastatin) and the isoprenoid inhibitors (such as FTI-277 and GGTI-298), mediate their cell cycle inhibitory activities by blocking the chymotrypsin activity of the proteasome in vitro. This results in the accumulation of cyclin-dependent kinase inhibitors p21 and p27 with subsequent G(1) arrest. Cells devoid of p21 were refractory to the growth-inhibitory activity of lovastatin, FTI-277, and GGTI-298. However, in these p21 null cells, isoprenylation of key substrates of farnesyl transferase (such as Ras) and of geranylgeranyl transferase (such as RAP-1) were inhibited by FTI-277 and GGTI-298, respectively, suggesting that although both these isoprenoid inhibitors reached and inhibited their intended targets, inhibition of the isoprenylation of Ras and RAP-1A are not sufficient to mediate G(1) arrest. We also show that the cell cycle effects can be attributed to the functional lactone moiety of the aforementioned agents. Collectively, our data suggest that FTI and GGTI and other agents containing an active lactone moiety mediate G(1) arrest via inhibition of the proteasome and up-regulation of p21, independent of the inhibition of isoprenylation of Ras or RAP-1.

Topics: Antineoplastic Agents; Benzamides; Breast Neoplasms; Cyclin-Dependent Kinase Inhibitor p21; Enzyme Inhibitors; G1 Phase; Humans; Lactic Acid; Lovastatin; Methionine; Proteasome Endopeptidase Complex; Protein Prenylation; rap1 GTP-Binding Proteins; ras Proteins; Tumor Cells, Cultured; Up-Regulation

Rho family GTPases are frequently overexpressed in breast cancers, which regulate cancer cell migration and invasion. They require prenylation, a lipid post-translational modification, for full biological functions. We examined the effects of 3-Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitor (fluvastatin), a selective farnesyltransferase inhibitor (FTI-277) and a selective geranylgeranyltransferase type I inhibitor (GGTI-298) on in vitro invasive capacity of MDA-MB-231 human breast cancer cells into the endothelial cell monolayer in a transendothelial migration assay. Although, at a maximal dose of 5 microM, fluvastatin did not affect the integrity of endothelial cell monolayer, the transendothelial migration of MDA-MB-231 cells was inhibited potently by fluvastatin in a dose-dependent manner. The transendothelial migration of MDA-MB-231 cells was also inhibited potently by GGTI-298 in a dose-dependent manner but weakly by FTI-277. The inhibitory effects of fluvastatin, GGTI-298 and FTI-277 on MDA-MB-231 cell invasion were shown to correlate well with inhibition of the membrane localization of RhoA and RhoC, but not with Ras. These results suggest that geranylgeranylation step of RhoA and RhoC could be a good therapeutic target for the prevention of invasion and metastasis of breast cancer cells.

Topics: Alkyl and Aryl Transferases; Animals; Benzamides; Breast Neoplasms; Cattle; Cell Adhesion; Cell Line, Tumor; Cell Movement; Cell Shape; Coculture Techniques; Collagen; Drug Combinations; Endothelial Cells; Farnesyltranstransferase; Fatty Acids, Monounsaturated; Female; Fluvastatin; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Indoles; Laminin; Methionine; Neoplasm Invasiveness; Protein Prenylation; Protein Processing, Post-Translational; Protein Transport; Proteoglycans; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; rhoC GTP-Binding Protein

We showed in a previous study that prenylated proteins play a role in estradiol stimulation of proliferation. However, these proteins antagonize the ability of estrogen receptor (ER) alpha to stimulate estrogen response element (ERE)-dependent transcriptional activity, potentially through the formation of a co-regulator complex. The present study investigates, in further detail, how prenylated proteins modulate the transcriptional activities mediated by ERalpha and by ERbeta.. The ERE-beta-globin-Luc-SV-Neo plasmid was either stably transfected into MCF-7 cells or HeLa cells (MELN cells and HELN cells, respectively) or transiently transfected into MCF-7 cells using polyethylenimine. Cells deprived of estradiol were analyzed for ERE-dependent luciferase activity 16 hours after estradiol stimulation and treatment with FTI-277 (a farnesyltransferase inhibitor) or with GGTI-298 (a geranylgeranyltransferase I inhibitor). In HELN cells, the effect of prenyltransferase inhibitors on luciferase activity was compared after transient transfection of plasmids coding either the full-length ERalpha, the full-length ERbeta, the AF-1-deleted ERalpha or the AF-2-deleted ERalpha. The presence of ERalpha was then detected by immunocytochemistry in either the nuclei or the cytoplasms of MCF-7 cells. Finally, Clostridium botulinum C3 exoenzyme treatment was used to determine the involvement of Rho proteins in ERE-dependent luciferase activity.. FTI-277 and GGTI-298 only stimulate ERE-dependent luciferase activity in stably transfected MCF-7 cells. They stimulate both ERalpha-mediated and ERbeta-mediated ERE-dependent luciferase activity in HELN cells, in the presence of and in the absence of estradiol. The roles of both AF-1 and AF-2 are significant in this effect. Nuclear ERalpha is decreased in the presence of prenyltransferase inhibitors in MCF-7 cells, again in the presence of and in the absence of estradiol. By contrast, cytoplasmic ERalpha is mainly decreased after treatment with FTI-277, in the presence of and in the absence of estradiol. The involvement of Rho proteins in ERE-dependent luciferase activity in MELN cells is clearly established.. Together, these results demonstrate that prenylated proteins (at least RhoA, RhoB and/or RhoC) antagonize the ability of ERalpha and ERbeta to stimulate ERE-dependent transcriptional activity, potentially acting through both AF-1 and AF-2 transcriptional activities.

Topics: Benzamides; Breast Neoplasms; Dimethylallyltranstransferase; Enzyme Inhibitors; Estrogen Receptor alpha; Estrogen Receptor beta; HeLa Cells; Humans; Immunohistochemistry; Luciferases; Methionine; Mutation; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; rhoB GTP-Binding Protein; rhoC GTP-Binding Protein; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Activation of estrogen receptors (ERs) by estrogens triggers both ER nuclear transcriptional activity and Src/Ras/Erks pathway-dependent mitogenic activity. The present study implicates prenylated proteins in both estrogenic actions. The farnesyltransferase and geranylgeranyltransferase I inhibitors (FTI-277 and GGTI-298, respectively) antagonize estradiol-stimulated cell cycle progression, progesterone receptor, cyclin D1, and c-Myc expression. In contrast, the inhibitors markedly stimulate transcription from two genes containing estrogen response elements, both in the absence and presence of estradiol. The pure antiestrogen ICI 182,780 inhibits by more than 85% these effects on transcription. We demonstrate that both FTI-277 and GGTI-298 increase the association of steroid receptor coactivator-1 with ER alpha and FTI-277 decreases the association of ER alpha with the histone deacetylase 1, a known transcriptional repressor. In addition, FTI-277 has no marked effect on the association of the two corepressors, nuclear receptor corepressor and silencing mediator of retinoid and thyroid receptor with ER alpha, whereas GGTI-298, similar to tamoxifen, clearly increased these associations. Together, these results demonstrate that prenylated proteins play a role in estradiol stimulation of proliferation and progesterone receptor expression. However, they antagonize the ability of ER alpha to stimulate estrogen response element-dependent transcriptional activity, acting presumably through coregulator complex formation.

Topics: Alkyl and Aryl Transferases; Benzamides; Breast Neoplasms; Cell Cycle; Cell Division; Cyclin D1; Dimethylallyltranstransferase; Enzyme Inhibitors; Estradiol; Estrogen Antagonists; Estrogen Receptor alpha; Farnesyltranstransferase; Gene Expression; Humans; Methionine; Proto-Oncogene Proteins c-myc; Receptors, Estrogen; Receptors, Progesterone; Response Elements; RNA, Messenger; Transcription, Genetic; Tumor Cells, Cultured

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.

Topics: Alkyl and Aryl Transferases; Benzamides; Breast Neoplasms; Cell Cycle; Cells, Cultured; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Enzyme Inhibitors; Female; Fibrosarcoma; G1 Phase; Head and Neck Neoplasms; Humans; Lovastatin; Methionine; Oligopeptides; Pancreatic Neoplasms; Protein Prenylation; Resting Phase, Cell Cycle; Tumor Suppressor Protein p53; Urinary Bladder Neoplasms