Huntington’s disease (HD) is a heritable, neurodegenerative disorder characterized by motor, cognitive, and psychiatric disturbances. An unstable CAG expansion within the gene normally encoding for the Huntingtin protein is responsible. The expanded mutant form of Huntingtin and the putative protein co-factor Rhes interact and cause cell death within the striatum. We hypothesized chronic treatment with simvastatin, a cholesterol lowering drug, would disrupt the biosynthetical pathway which gives both Rhes and its target cells binding sites and render Rhes inactive. Healthy and HD mice were treated with simvastatin or a vehicle. Animals’ motor behavior was assessed with three separate tests over the first four months of life. No significant differences were found between the HD groups; however, the HD treated animals’ performance on the rotarod test, at month 4, was intermediate between healthy mice and HD vehicle treated mice. The results hint at simvastatin’s therapeutic potential, but are interpreted cautiously.

The ErbB family of receptors are key regulators of growth, differentiation, migration and survival of epithelial cells. CI-1033 is an irreversible pan-ErbB tyrosine kinase inhibitor that has the ability to inhibit EGFR function but has shown limited therapeutic efficacy. Lovastatin targets the activity of HMG-CoA reductase, the rate-limiting step in the mevalonate pathway. In this study, the ability of lovastatin to potentiate the cytotoxic effects of CI-1033 was evaluated. The combination of lovastatin and CI-1033 exhibited some cooperative cytotoxic activity in a squamous cell carcinoma–derived cell line. This combination resulted in enhanced cell death by induction of a potent apoptotic response. Furthermore, this drug combination inhibited EGF-induced EGFR autophosphorylation and activation of the downstream signaling effectors, ERK and AKT. These findings suggest that combining lovastatin and tyrosine kinase inhibitors may represent a novel combinational therapeutic approach in squamous cell carcinoma of the head and neck.

Statins, common cholesterol control drugs, are appreciated to have promising anticancer activity through inhibition of the mevalonate pathway. Several lines of evidence suggest that certain tumours are susceptible to statins, but the underlying molecular features arbitrating this sensitivity remain unknown. We hypothesize that (i) not all statins will behave equivalently in the context of anticancer therapy, and (ii) a molecularly-defined subset of tumours are intrinsically sensitive to statins. My objectives have therefore been to further our understanding of functional differences between statins influencing their anticancer effects, and to investigate molecular features associated with statin sensitivity in breast cancer. Specifically, this thesis addresses two aims: (i) to characterize differential interactions between four statins and the xenobiotic transporter P-glycoprotein (P-gp; also known as ABCB1), and (ii) to identify molecular features associated with fluvastatin and lovastatin sensitivity in breast tumour cell lines. We first characterized the interactions of statins with P-gp in vitro and in multidrug-resistant (MDR) tumour cells. While lovastatin could directly bind to P-gp and modulate MDR, no significant interactions were observed with fluvastatin. Fluvastatin may therefore be appropriate for use in unselected patients, to avoid adverse drug interactions with coadministered P-gp substrate chemotherapeutics. Fluvastatin has also shown promise in breast cancer treatment, where molecular features predictive of statin sensitivity would be particularly valuable. A panel of 19 immortalized breast cell lines was therefore characterized for sensitivity to fluvastatin and lovastatin. Relatively statin-sensitive cells underwent apoptosis upon statin treatment, and were more likely to have an estrogen receptor alpha (ERα)-negative, basal-like phenotype. By mining available baseline gene expression data, a candidate 10-gene signature predictive of fluvastatin sensitivity was also generated. Taken together, this research provides insight into molecular markers of statin sensitivity that may facilitate fast-tracking of these drugs to clinical trials in subsets of cancer patients most likely to respond.

Receptor tyrosine kinases (RTK) are key regulators of growth, differentiation and survival of epithelial cells and play a significant role in the development and progression of cancers derived from these tissues. In malignant cells, these receptors and their downstream signalling pathways are often deregulated, leading to cell hyper-proliferation, enhanced cell survival and increased metastatic potential. Furthermore, endothelial expressed RTKs regulate tumor angiogenesis allowing for tumor growth and maintenance by promoting their vascularization. Epithelial malignancies such as squamous cell carcinomas (SCC), non-small cell lung (NSCLC) and malignant mesotheliomas have very limited treatment options when presenting as metastatic disease. RTKs, particularly the epidermal growth factor (EGFR) and the vascular endothelial growth factor (VEGFR) receptors, have been shown to play significant roles in the pathogenesis of these tumor types. Statins are potent inhibitors of HMG-CoA reductase, the rate limiting enzyme of the mevalonate pathway, that are widely used as hypercholesterolemia treatments. The mevalonate pathway produces a variety of end products that are critical for many different cellular pathways, thus, targeting this pathway can affect multiple signalling pathways. Our laboratory has previously shown that lovastatin can induce tumor specific apoptosis especially in SCC and that 23% of recurrent SCC patients treated with lovastatin as a single agent showed disease stabilization in our Phase I clinical trial. Subsequently, our lab was able to demonstrate that lovastatin in combination with gefitinib, a potent inhibitor of the EGFR showed co-operative cytotoxicity when combined (Chapter 2). Furthermore, the pro-apoptotic and cytotoxic effects of these agents were found to be synergistic and to be manifested in several types of tumor cell lines including SCC, NSCLC and glioblastoma. I was able to expand upon these important findings and demonstrated that lovastatin, through its ability to disrupt the actin cytoskeleton, inhibited EGFR dimerization and activation (Chapter 3). This novel mechanism targeting this receptor has clinical implications as lovastatin treatment combined with gefitinib showed co-operative inhibitory effects on EGFR activation and downstream signalling. The RTK family of proteins share similar features with respect to activation, internalization and downstream signalling effectors. I further demonstrated that lovastatin can inhibit the VEGFR-2 in endothelial cells and mesotheliomas, where VEGF and its receptor are co-expressed driving their proliferation, and induces synergistic cytotoxicity in mesothelioma cells in combination with VEGFR-2 tyrosine kinase inhibitors (Chapter 4). These findings suggest that statins may augment the effects of a variety of RTK inhibitors in a similar fashion representing a novel combinational therapeutic approach in a wide repertoire of human cancers. More importantly, based on this work, we initiated a Phase I/II study evaluating high dose rosuvastatin and the EGFR inhibitor tarceva in SCC and NSCLC patients at our institute. This clinical evaluation will provide invaluable data that will play a role in developing this novel therapeutic strategy. Together, the work embodied in this thesis provides a model for the regulation of EGFR/VEGFR-2 activation and signalling by targeting the rho family of proteins that demonstrates a novel mechanism that can be exploited to refine current therapeutic paradigms.

Receptor tyrosine kinases (RTK) are key regulators of growth, differentiation and survival of epithelial cells and play a significant role in the development and progression of cancers derived from these tissues. In malignant cells, these receptors and their downstream signalling pathways are often deregulated, leading to cell hyper-proliferation, enhanced cell survival and increased metastatic potential. Furthermore, endothelial expressed RTKs regulate tumor angiogenesis allowing for tumor growth and maintenance by promoting their vascularization. Epithelial malignancies such as squamous cell carcinomas (SCC), non-small cell lung (NSCLC) and malignant mesotheliomas have very limited treatment options when presenting as metastatic disease. RTKs, particularly the epidermal growth factor (EGFR) and the vascular endothelial growth factor (VEGFR) receptors, have been shown to play significant roles in the pathogenesis of these tumor types. Statins are potent inhibitors of HMG-CoA reductase, the rate limiting enzyme of the mevalonate pathway, that are widely used as hypercholesterolemia treatments. The mevalonate pathway produces a variety of end products that are critical for many different cellular pathways, thus, targeting this pathway can affect multiple signalling pathways. Our laboratory has previously shown that lovastatin can induce tumor specific apoptosis especially in SCC and that 23% of recurrent SCC patients treated with lovastatin as a single agent showed disease stabilization in our Phase I clinical trial. Subsequently, our lab was able to demonstrate that lovastatin in combination with gefitinib, a potent inhibitor of the EGFR showed co-operative cytotoxicity when combined (Chapter 2). Furthermore, the pro-apoptotic and cytotoxic effects of these agents were found to be synergistic and to be manifested in several types of tumor cell lines including SCC, NSCLC and glioblastoma. I was able to expand upon these important findings and demonstrated that lovastatin, through its ability to disrupt the actin cytoskeleton, inhibited EGFR dimerization and activation (Chapter 3). This novel mechanism targeting this receptor has clinical implications as lovastatin treatment combined with gefitinib showed co-operative inhibitory effects on EGFR activation and downstream signalling. The RTK family of proteins share similar features with respect to activation, internalization and downstream signalling effectors. I further demonstrated that lovastatin can inhibit the VEGFR-2 in endothelial cells and mesotheliomas, where VEGF and its receptor are co-expressed driving their proliferation, and induces synergistic cytotoxicity in mesothelioma cells in combination with VEGFR-2 tyrosine kinase inhibitors (Chapter 4). These findings suggest that statins may augment the effects of a variety of RTK inhibitors in a similar fashion representing a novel combinational therapeutic approach in a wide repertoire of human cancers. More importantly, based on this work, we initiated a Phase I/II study evaluating high dose rosuvastatin and the EGFR inhibitor tarceva in SCC and NSCLC patients at our institute. This clinical evaluation will provide invaluable data that will play a role in developing this novel therapeutic strategy. Together, the work embodied in this thesis provides a model for the regulation of EGFR/VEGFR-2 activation and signalling by targeting the rho family of proteins that demonstrates a novel mechanism that can be exploited to refine current therapeutic paradigms.

Isoprenoids are the largest family of biologically active compounds, synthesized by five carbon subunits namely isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP). For long time the mevalonate-dependent (MVA) pathway has been considered as the sole source of IPP and DMAPP, until recently a new non-mevalonte dependent (NMVA) pathway was discovered. This new pathway utilizes entirely different set of enzymes for isoprenoids synthesis and don't have any homologues in humans. NMVA pathway is the only source of isoprenoids for certain eubacteria, parasite and plants. Absence of the NMVA pathway in higher organisms has opened a new platform for the development of novel antibiotics and antimalarials. 1-deoxy-D-xylulose-5-phosphate synthase (DXS), the first enzyme in NMVA pathway has been reported as the rate limiting enzyme in the synthesis of IPP and DMAPP and has been the center of interest for inhibitor development. Reaction mechanism of thiamine pyrophosphate (TPP) and Mg2+ dependent DXS enzyme has been studied in this report. Using steady state kinetics analysis, product inhibition and dead end inhibitor, the mechanism of substrate (pyruvate and D-glyceraldehyde-3-phosphate) addition was studied. Due to different domain organization in DXS as compared to theother TPP dependent enzyme, the mechanism of addition was found to be random sequential rather than ping-pong mechanism. Based on bioinformatics tool and in vitro studies it has been established that NMVA exists in all the plasmodium species, thus making the enzymes involved in NMVA as an alluring target for new antimalarial drugs. All the plasmodium species and other member of the phylum apicomplexa harbor apicoplast an organelle which is homologous to the chloroplast of plants and algae. All the enzymes from NMVA pathway translocate to apicoplast from nucleus through a secretory pathway using signaling and transit peptide. In this study DXS from P. vivax has been cloned and expressed in E. coli using genomic DNA and codon optimized synthetic DNA as a source. Expression of full length DXS with signal and transit peptide as well as mature protein without these peptide using serial deletion has been studied. Kinetic parameters of P.vivax DXS have been calculated and found to be comparable to the DXS from other species.

Statins, common cholesterol control drugs, are appreciated to have promising anticancer activity through inhibition of the mevalonate pathway. Several lines of evidence suggest that certain tumours are susceptible to statins, but the underlying molecular features arbitrating this sensitivity remain unknown. We hypothesize that (i) not all statins will behave equivalently in the context of anticancer therapy, and (ii) a molecularly-defined subset of tumours are intrinsically sensitive to statins. My objectives have therefore been to further our understanding of functional differences between statins influencing their anticancer effects, and to investigate molecular features associated with statin sensitivity in breast cancer. Specifically, this thesis addresses two aims: (i) to characterize differential interactions between four statins and the xenobiotic transporter P-glycoprotein (P-gp; also known as ABCB1), and (ii) to identify molecular features associated with fluvastatin and lovastatin sensitivity in breast tumour cell lines. We first characterized the interactions of statins with P-gp in vitro and in multidrug-resistant (MDR) tumour cells. While lovastatin could directly bind to P-gp and modulate MDR, no significant interactions were observed with fluvastatin. Fluvastatin may therefore be appropriate for use in unselected patients, to avoid adverse drug interactions with coadministered P-gp substrate chemotherapeutics. Fluvastatin has also shown promise in breast cancer treatment, where molecular features predictive of statin sensitivity would be particularly valuable. A panel of 19 immortalized breast cell lines was therefore characterized for sensitivity to fluvastatin and lovastatin. Relatively statin-sensitive cells underwent apoptosis upon statin treatment, and were more likely to have an estrogen receptor alpha (ERα)-negative, basal-like phenotype. By mining available baseline gene expression data, a candidate 10-gene signature predictive of fluvastatin sensitivity was also generated. Taken together, this research provides insight into molecular markers of statin sensitivity that may facilitate fast-tracking of these drugs to clinical trials in subsets of cancer patients most likely to respond.

Receptor tyrosine kinases (RTK) are key regulators of growth, differentiation and survival of epithelial cells and play a significant role in the development and progression of cancers derived from these tissues. In malignant cells, these receptors and their downstream signalling pathways are often deregulated, leading to cell hyper-proliferation, enhanced cell survival and increased metastatic potential. Furthermore, endothelial expressed RTKs regulate tumor angiogenesis allowing for tumor growth and maintenance by promoting their vascularization. Epithelial malignancies such as squamous cell carcinomas (SCC), non-small cell lung (NSCLC) and malignant mesotheliomas have very limited treatment options when presenting as metastatic disease. RTKs, particularly the epidermal growth factor (EGFR) and the vascular endothelial growth factor (VEGFR) receptors, have been shown to play significant roles in the pathogenesis of these tumor types. Statins are potent inhibitors of HMG-CoA reductase, the rate limiting enzyme of the mevalonate pathway, that are widely used as hypercholesterolemia treatments. The mevalonate pathway produces a variety of end products that are critical for many different cellular pathways, thus, targeting this pathway can affect multiple signalling pathways. Our laboratory has previously shown that lovastatin can induce tumor specific apoptosis especially in SCC and that 23% of recurrent SCC patients treated with lovastatin as a single agent showed disease stabilization in our Phase I clinical trial. Subsequently, our lab was able to demonstrate that lovastatin in combination with gefitinib, a potent inhibitor of the EGFR showed co-operative cytotoxicity when combined (Chapter 2). Furthermore, the pro-apoptotic and cytotoxic effects of these agents were found to be synergistic and to be manifested in several types of tumor cell lines including SCC, NSCLC and glioblastoma. I was able to expand upon these important findings and demonstrated that lovastatin, through its ability to disrupt the actin cytoskeleton, inhibited EGFR dimerization and activation (Chapter 3). This novel mechanism targeting this receptor has clinical implications as lovastatin treatment combined with gefitinib showed co-operative inhibitory effects on EGFR activation and downstream signalling. The RTK family of proteins share similar features with respect to activation, internalization and downstream signalling effectors. I further demonstrated that lovastatin can inhibit the VEGFR-2 in endothelial cells and mesotheliomas, where VEGF and its receptor are co-expressed driving their proliferation, and induces synergistic cytotoxicity in mesothelioma cells in combination with VEGFR-2 tyrosine kinase inhibitors (Chapter 4). These findings suggest that statins may augment the effects of a variety of RTK inhibitors in a similar fashion representing a novel combinational therapeutic approach in a wide repertoire of human cancers. More importantly, based on this work, we initiated a Phase I/II study evaluating high dose rosuvastatin and the EGFR inhibitor tarceva in SCC and NSCLC patients at our institute. This clinical evaluation will provide invaluable data that will play a role in developing this novel therapeutic strategy. Together, the work embodied in this thesis provides a model for the regulation of EGFR/VEGFR-2 activation and signalling by targeting the rho family of proteins that demonstrates a novel mechanism that can be exploited to refine current therapeutic paradigms.

The juvenile hormones (JHs) are sesquiterpenoid compounds that play a central role in insect reproduction, development and behavior. They are synthesized and secreted by a pair of small endocrine glands, the corpora allata (CA), which are intimately connected to the brain. The enzymes involved in the biosynthesis of JH are attractive targets for the control of mosquito populations. This dissertation is a comprehensive functional study of five Aedes aegypti CA enzymes, HMG-CoA synthase (AaHMGS), mevalonate kinase (AaMK), phosphomevalonate kinase (AaPMK), farnesyl diphosphate synthase (AaFPPS) and farnesyl pyrophosphate phosphatase (AaFPPase). The enzyme AaHMGS catalyzes the condensation of acetoacetyl-CoA and acetyl-CoA to produce HMG-CoA. The enzyme does not require any co-factor, although its activity is enhanced by addition of Mg2+. The enzyme AaMK is a class I mevalonate kinase that catalyzes the ATP-dependent phosphorylation of mevalonic acid to form mevalonate 5-phosphate. Activity of AaMK is inhibited by isoprenoids. The enzyme AaPMK catalyzes the cation-dependent reversible reaction of phosphomevalonate and ATP to form diphosphate mevalonate and ADP. The enzyme AaFPPS catalyzes the condensation of isopentenyl diphosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to form geranyl diphosphate (GPP) and farnesyl pyrophosphate (FPP). The enzyme AaFPPS shows an unusual product regulation mechanism, with chain length final product of 10 or 15 C depending on the metal cofactor present. The enzymes AaFPPase-1 and AaFPPase-2 efficiently hydrolyze FPP into farnesol, although RNAi experiments demonstrate that only AaFPPase-1 is involved in the catalysis of FPP into FOL in the CA of A. aegypti. This dissertation also explored the inhibition of the activity of some of the JH biosynthesis enzymes as tools for insect control. We described the effect of N-acetyl-S-geranylgeranyl-L-cysteine as a potent inhibitor of AaFPPase 1 and AaFPPase-2. In addition, inhibitors of AaMK and AaHMGS were also investigated using purified recombinant proteins. The present study provides an important contribution to the characterization of recombinant proteins, the analysis of enzyme kinetics and inhibition constants, as well as the understanding of the importance of these five enzymes in the control of JH biosynthesis rates.

Receptor tyrosine kinases (RTK) are key regulators of growth, differentiation and survival of epithelial cells and play a significant role in the development and progression of cancers derived from these tissues. In malignant cells, these receptors and their downstream signalling pathways are often deregulated, leading to cell hyper-proliferation, enhanced cell survival and increased metastatic potential. Furthermore, endothelial expressed RTKs regulate tumor angiogenesis allowing for tumor growth and maintenance by promoting their vascularization. Epithelial malignancies such as squamous cell carcinomas (SCC), non-small cell lung (NSCLC) and malignant mesotheliomas have very limited treatment options when presenting as metastatic disease. RTKs, particularly the epidermal growth factor (EGFR) and the vascular endothelial growth factor (VEGFR) receptors, have been shown to play significant roles in the pathogenesis of these tumor types. Statins are potent inhibitors of HMG-CoA reductase, the rate limiting enzyme of the mevalonate pathway, that are widely used as hypercholesterolemia treatments. The mevalonate pathway produces a variety of end products that are critical for many different cellular pathways, thus, targeting this pathway can affect multiple signalling pathways. Our laboratory has previously shown that lovastatin can induce tumor specific apoptosis especially in SCC and that 23% of recurrent SCC patients treated with lovastatin as a single agent showed disease stabilization in our Phase I clinical trial. Subsequently, our lab was able to demonstrate that lovastatin in combination with gefitinib, a potent inhibitor of the EGFR showed co-operative cytotoxicity when combined (Chapter 2). Furthermore, the pro-apoptotic and cytotoxic effects of these agents were found to be synergistic and to be manifested in several types of tumor cell lines including SCC, NSCLC and glioblastoma. I was able to expand upon these important findings and demonstrated that lovastatin, through its ability to disrupt the actin cytoskeleton, inhibited EGFR dimerization and activation (Chapter 3). This novel mechanism targeting this receptor has clinical implications as lovastatin treatment combined with gefitinib showed co-operative inhibitory effects on EGFR activation and downstream signalling. The RTK family of proteins share similar features with respect to activation, internalization and downstream signalling effectors. I further demonstrated that lovastatin can inhibit the VEGFR-2 in endothelial cells and mesotheliomas, where VEGF and its receptor are co-expressed driving their proliferation, and induces synergistic cytotoxicity in mesothelioma cells in combination with VEGFR-2 tyrosine kinase inhibitors (Chapter 4). These findings suggest that statins may augment the effects of a variety of RTK inhibitors in a similar fashion representing a novel combinational therapeutic approach in a wide repertoire of human cancers. More importantly, based on this work, we initiated a Phase I/II study evaluating high dose rosuvastatin and the EGFR inhibitor tarceva in SCC and NSCLC patients at our institute. This clinical evaluation will provide invaluable data that will play a role in developing this novel therapeutic strategy. Together, the work embodied in this thesis provides a model for the regulation of EGFR/VEGFR-2 activation and signalling by targeting the rho family of proteins that demonstrates a novel mechanism that can be exploited to refine current therapeutic paradigms.