cerivastatin and Breast-Neoplasms

The YAP and TAZ mediators of the Hippo pathway (hereafter called YAP/TAZ) promote tissue proliferation and organ growth. However, how their biological properties intersect with cellular metabolism remains unexplained. Here, we show that YAP/TAZ activity is controlled by the SREBP/mevalonate pathway. Inhibition of the rate-limiting enzyme of this pathway (HMG-CoA reductase) by statins opposes YAP/TAZ nuclear localization and transcriptional responses. Mechanistically, the geranylgeranyl pyrophosphate produced by the mevalonate cascade is required for activation of Rho GTPases that, in turn, activate YAP/TAZ by inhibiting their phosphorylation and promoting their nuclear accumulation. The mevalonate-YAP/TAZ axis is required for proliferation and self-renewal of breast cancer cells. In Drosophila melanogaster, inhibition of mevalonate biosynthesis and geranylgeranylation blunts the eye overgrowth induced by Yorkie, the YAP/TAZ orthologue. In tumour cells, YAP/TAZ activation is promoted by increased levels of mevalonic acid produced by SREBP transcriptional activity, which is induced by its oncogenic cofactor mutant p53. These findings reveal an additional layer of YAP/TAZ regulation by metabolic cues.

Topics: Active Transport, Cell Nucleus; Acyltransferases; Adaptor Proteins, Signal Transducing; Animals; Breast Neoplasms; Cell Proliferation; Drosophila melanogaster; Drosophila Proteins; Female; HCT116 Cells; HEK293 Cells; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hydroxymethylglutaryl-CoA Reductases, NAD-Dependent; Intracellular Signaling Peptides and Proteins; Mevalonic Acid; Mice; Nuclear Proteins; Phosphoproteins; Phosphorylation; Polyisoprenyl Phosphates; Protein Serine-Threonine Kinases; Pyridines; rho GTP-Binding Proteins; RNA Interference; RNA, Small Interfering; Signal Transduction; Sterol Regulatory Element Binding Proteins; Trans-Activators; Transcription Factors; Transcription, Genetic; Tumor Suppressor Proteins; YAP-Signaling Proteins

Bisphosphonates cause apoptosis to various types of cancer cells including breast cancer. Inhibition of the mevalonate pathway was reported to be involved in the apoptosis induced by bisphosphonates, but its precise mechanism has not been unveiled. In the present study, we investigated the molecular mechanism of risedronate, a bisphosphonate, in the apoptosis of the breast cancer cell line MCF-7 in comparison with that of cerivastatin, an HMG CoA reductase inhibitor (statin), since statin has been known to induce apoptosis through an isoprenoid-dependent pathway in these cells. We found that i) risedronate induced MCF-7 cells into apoptosis in a manner similar to cerivastatin with the activation of caspase-9 followed by caspase-6 and -7, that ii) bisphosphonate-induced apoptosis was significantly, but not fully, recovered by the addition of GGOH, an isoprenoid, which completely rescued in case of cerivastatin-induced apoptosis, that iii) risedronate induced G2 arrest with the induction of Bim (BH3-only protein), but that statin induced G1 arrest without it, and that iv) the down-regulation of Bim protein by siRNA significantly attenuated the risedronate-induced apoptosis. These data clearly indicate that both isoprenoid-dependent and -independent pathways might be involved in the apoptosis induced by bisphosphonate, and Bim might be a critical component for the isoprenoid-independent apoptotic pathway.

Topics: Apoptosis; Apoptosis Regulatory Proteins; Bcl-2-Like Protein 11; Bone Density Conservation Agents; Breast Neoplasms; Calcium Channel Blockers; Caspases; Cell Cycle; Cell Line, Tumor; Cell Proliferation; Etidronic Acid; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Membrane Proteins; Proto-Oncogene Proteins; Pyridines; Risedronic Acid; RNA, Small Interfering; Signal Transduction; Terpenes; Tumor Cells, Cultured

Overexpression of RhoA or RhoC in breast cancer indicates a poor prognosis, due to increased tumor cell proliferation and invasion and tumor-dependent angiogenesis. Until now, the strategy of blockage of the Rho-signaling pathway has used either GGTI or HMG-CoA reductase inhibitors, but they are not specific to RhoA or RhoC inhibition. In this study, a new approach with anti-RhoA and anti-RhoC siRNAs was used to inhibit specifically RhoA or RhoC synthesis. Two transfections of either RhoA or RhoC siRNA (8.5 nM) into MDA-MB-231 human breast cancer cells or HMEC-1 endothelial cells induced extensive degradation of the target mRNA and led to a dramatic decrease in synthesis of the corresponding protein. In vitro, these siRNAs inhibited cell proliferation and invasion more effectively than conventional blockers of Rho cell signaling. Finally, in a nude mouse model, intratumoral injections of anti-RhoA siRNA (100 microl at 85 nM) every 3 days for 20 days almost totally inhibited the growth and angiogenesis of xenografted MDA-MB-231 tumors. One may infer from these observations that specific inhibition of the Rho-signaling pathway with siRNAs represents a promising approach for the treatment of aggressive breast cancers.

Topics: Active Transport, Cell Nucleus; Animals; beta Catenin; Breast Neoplasms; Cell Line, Tumor; Cell Nucleus; Cell Proliferation; Collagen; Cytoskeletal Proteins; Down-Regulation; Drug Combinations; Enzyme Inhibitors; Gene Expression Regulation, Neoplastic; Humans; Laminin; Mice; Neoplasm Invasiveness; Neovascularization, Pathologic; Proteoglycans; Pyridines; ras Proteins; rho GTP-Binding Proteins; rhoA GTP-Binding Protein; rhoC GTP-Binding Protein; RNA Interference; RNA, Messenger; RNA, Small Interfering; Trans-Activators; Transfection

Competitive inhibitors of 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase are commonly used in the clinic to treat hypercholesterolemia and have been reported to exert antitumor effects. Cerivastatin is a novel, synthetic and the most pharmacologically potent inhibitor of HMG-CoA reductase. We decided to examine the cytostatic/cytotoxic activity of cerivastatin against human breast cancer cell lines and to test whether the effects of cerivastatin could be potentiated by doxorubicin and cisplatin. Cytostatic/cytotoxic effects of cerivastatin used alone or in the combination with chemotherapeutics were measured with MTT assay. The cell cycle distribution and apoptosis induction were evaluated with flow cytometer. The expression of p21 and p27 cyclin-dependent kinase inhibitors was measured with Western blotting. Isobologram analysis was performed to study the drug interactions. We observed that cerivastatin exerts cytostatic/cytotoxic effects against four human tumor cell lines (T-47D, T4-2, MDA-MB-231, MCF-7). We also demonstrated that cerivastatin exerts growth inhibitory effect through induction of p21 cyclin-dependent kinase inhibitor and inhibition of cell cycle progression. In the two tumor cell lines studied, one sensitive (MDA-MB-231) and one moderately resistant (T4-2) to the cytostatic/cytotoxic effects of cerivastatin we examined the effects of combined treatment with cerivastatin and either doxorubicin or cisplatin. Cerivastatin potentiated cytostatic/cytotoxic effects of cisplatin against T4-2 cells and those of doxorubicin against both cell lines. In T4-2 cells the interaction between doxorubicin and cerivastatin and between cisplatin and cerivastatin was found to be synergistic. Altogether, these studies indicate that cerivastatin is another HMG-CoA reductase inhibitor with potent antitumor effects.

Topics: Antineoplastic Combined Chemotherapy Protocols; Apoptosis; Blotting, Western; Breast Neoplasms; Carrier Proteins; Cell Cycle; Cisplatin; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Doxorubicin; Drug Interactions; Drug Synergism; Female; Humans; Intracellular Signaling Peptides and Proteins; Pyridines; Tumor Cells, Cultured

Statins are currently used for the treatment of hypercholesterolemia. Recently, we demonstrated that cerivastatin also reduces the proliferation and invasion of aggressive breast cancer cells, MDA-MB-231. In this report, a molecular mechanism to explain its anti-cancer action is proposed by combining the study of cerivastatin effect on both gene expression (microarray) and signal transduction pathways. Firstly, the expression of 13 genes was modified by cerivastatin and confirmed at protein level. They could contribute to the inhibition of both cell proliferation (down-regulation of cyclin D1, PCNA, c-myc and up-regulation p21(Waf1), p19(INK4d), integrin beta8) and cell invasion, either directly (decrease in u-PA, MMP-9, u-PAR, PAI-1 and increase in anti-oncogenes Wnt-5a and H-cadherin) or indirectly by stimulating an anti-angiogenic gene (thrombospondin-2). The anti-angiogenic activity was confirmed by in vivo experiments. Secondly, we demonstrated that the biochemical mechanism of its anti-cancer action could be mainly explained by the inhibition of RhoA-dependent cell signalling. This hypothesis was supported by the fact that a RhoA inhibitor (C3 exoenzyme) or a dominant negative mutant RhoA (N19RhoA) induced similar effects to those of cerivastatin. In conclusion, cerivastatin, by preventing RhoA prenylation, inhibits (i) the RhoA/ROCK pathway, leading to defective actin stress fibres formation responsible for the loss of traction forces required for cell motility and (ii) the RhoA/FAK/AKT signalling pathway that could explain the majority of cancer-related gene modifications described above. Thus, the inhibition of RhoA cell signalling could be a good strategy in therapy of aggressive forms of breast cancer.

Topics: ADP Ribose Transferases; Animals; Antineoplastic Agents; Botulinum Toxins; Breast Neoplasms; Cell Division; Cell Membrane; Cytosol; Gene Expression Regulation, Neoplastic; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Mice; Mice, Nude; Neoplasm Invasiveness; Neovascularization, Pathologic; Oligonucleotide Array Sequence Analysis; Protein Prenylation; Pyridines; rhoA GTP-Binding Protein; Signal Transduction; Tumor Cells, Cultured; Xenograft Model Antitumor Assays

Topics: Animals; Anticarcinogenic Agents; Anticholesteremic Agents; Breast Neoplasms; Canada; Cell Cycle; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Endopeptidases; Female; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Incidence; Lovastatin; Microfilament Proteins; Multienzyme Complexes; Muscle Proteins; Neoplasms; Proteasome Endopeptidase Complex; Pyridines; Signal Transduction

Statin induced myopathy is the most commonly seen side effect in users of this family of drugs. Their different forms present with either creatine phosphokinase (CK) elevation or not, signs of in vivo oxidation injury or not or a combination of both. The pathogenetic background, however, still remains obscure. As MIBI, beside myocardial and tumour scintigraphy, is useful in detecting muscle metabolic abnormalities, an increased uptake of MIBI in the diseased muscular segments could be expected. We investigated seven patients (five males, two females; aged 36-56 years) with statin induced myopathy with either elevated CK, isoprostanes or muscle pains at varying combinations. MIBI whole-body imaging was done immediately, the patients still being on the respective statin. Sixteen patients (six males, 10 females) suffering from lung or breast cancer and being on statins served as controls. No uptake abnormalities in any muscular segment either in the patients or the control group were seen. Thus, MIBI scintigraphy is not useful, apparently, in diagnosing and eventually localizing statin induced myopathy. These findings indicate that MIBI scintigraphy is of no help for diagnosis and gaining further insight into statin induced myopathy.

Topics: Adult; Anticholesteremic Agents; Atorvastatin; Breast Neoplasms; Fatty Acids, Monounsaturated; Female; Fluvastatin; Heptanoic Acids; Heterozygote; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Hyperlipoproteinemia Type II; Indoles; Lovastatin; Male; Middle Aged; Muscle, Skeletal; Muscular Diseases; Pain; Pravastatin; Pyridines; Pyrroles; Radionuclide Imaging; Radiopharmaceuticals; Simvastatin; Technetium Tc 99m Sestamibi

Cerivastatin is used in the treatment of hypercholesterolemia to inhibit 3-hydroxy 3-methylglutaryl coenzyme A reductase and thus prevent the synthesis of cholesterol precursors, such as farnesyl pyrophosphate (FPP) and geranylgeranyl pyrophosphate (GGPP), responsible, respectively, for translocation of Ras and Rho to the cell membrane, a step required for their cell signaling, leading to cell proliferation and migration. Recently, it has been suggested that non lipid-related effects of statins could play a beneficial role in cancer therapy. In this study, we have investigated the mechanisms by which statins inhibit cancer and the types of cancers which could benefit from this therapy. In MDA-MB-231 cells, an aggressive breast cancer cell line with spontaneous activation of Ras and NFkappaB and overexpression of RhoA, cerivastatin induced inhibition of both cell proliferation and invasion through Matrigel. This anti-proliferative effect was related to G(1)/S arrest due to an increase in p21(Waf1/Cip1). The anti-invasive effect was observed from 18 h and could be explained by RhoA delocalization from the cell membrane, resulting in disorganization of the actin fibers and disappearance of focal adhesion sites. The importance of RhoA inactivation in both these inhibitory effects was proved by their reversion by GGPP but not by FPP. Moreover, cerivastatin was also shown to induce inactivation of NFkappaB, in a RhoA inhibition-dependent manner, resulting in a decrease in urokinase and metalloproteinase-9 expression, two proteases involved in cell migration. The participation of Ras inactivation is considered a subsidiary mechanism for the effects of cerivastatin, as they were not rescued by FPP. Prolonged treatment of MDA-MB-231 cells with high doses of cerivastatin induced a loss of cell attachment. Interestingly, the effect of cerivastatin was considerably lower on poorly invasive MCF-7 cells. In conclusion, our results suggest that cerivastatin inhibits cell signaling pathways involved in the invasiveness and metastatic properties of highly invasive cancers.

Topics: Apoptosis; Base Sequence; Breast Neoplasms; Cell Cycle; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; DNA Primers; Gene Expression Regulation, Neoplastic; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; I-kappa B Proteins; In Vitro Techniques; Matrix Metalloproteinase 9; Neoplasm Invasiveness; Neoplasm Metastasis; NF-kappa B; Pyridines; RNA, Messenger; Signal Transduction; Thromboplastin; Tumor Cells, Cultured; Urokinase-Type Plasminogen Activator