The field of targeted cancer therapy has progressed in recent years with the approval of new oncology drugs. Coupled with the benefits that these agents provide, has come an appreciation for challenges that occur when attempting to translate successful experiments from the laboratory into effective clinical trials. One such challenge has been predicting the optimal dose and schedule to take into clinical evaluation, given the possibility that certain targeted therapeutics may exhibit maximal anti-tumour efficacy well below maximum tolerated doses. Recent work with a targeted antibody to the mouse vascular endothelial growth factor receptor-2 demonstrated that detection of increased levels of its endogenous ligand in the plasma, namely VEGF, could address this issue, as maximal increases in VEGF paralleled optimal drug activity. The VEGF result has become recognized as a potential class effect for this family of inhibitors. This thesis summarizes experiments designed to build upon this discovery by investigating whether the utility of ligand measurement might also apply to drugs that inhibit the epidermal growth factor receptor (EGFR), which have also received recent regulatory approval, and inhibit angiogenesis as one of their mechanisms of action. In addition, we investigated the potential application of EGFR inhibitors to influence other markers of tumour angiogenesis, specifically their effects on levels of circulating endothelial progenitor cells (CEPs). Finally, we evaluated combination treatment of EGFR inhibition with anti-angiogenic scheduling of chemotherapy. The EGFR ligand TGF-alpha increased in a dose dependent fashion following treatment with cetuximab, and levels in the circulation paralleled anti-tumour activity. This was a host-dependent effect that was not observed with the lower affinity antibody nimotuzumab. Inhibition of host EGFR also reduced plasma CEPs, but at higher doses these drugs increased off target growth factors VEGF and G-CSF, as well as CEPs. In a model of advanced triple negative breast cancer, the combination of nimotuzumab and metronomic cyclophosphamide was efficacious and well tolerated, leading to a potential new treatment strategy for this aggressive disease. Taken together, these studies identify new and useful applications for EGFR-targeted antibodies, and shed further light on their contributions within the field of tumour angiogenesis and antiangiogenic therapy.

The field of targeted cancer therapy has progressed in recent years with the approval of new oncology drugs. Coupled with the benefits that these agents provide, has come an appreciation for challenges that occur when attempting to translate successful experiments from the laboratory into effective clinical trials. One such challenge has been predicting the optimal dose and schedule to take into clinical evaluation, given the possibility that certain targeted therapeutics may exhibit maximal anti-tumour efficacy well below maximum tolerated doses. Recent work with a targeted antibody to the mouse vascular endothelial growth factor receptor-2 demonstrated that detection of increased levels of its endogenous ligand in the plasma, namely VEGF, could address this issue, as maximal increases in VEGF paralleled optimal drug activity. The VEGF result has become recognized as a potential class effect for this family of inhibitors. This thesis summarizes experiments designed to build upon this discovery by investigating whether the utility of ligand measurement might also apply to drugs that inhibit the epidermal growth factor receptor (EGFR), which have also received recent regulatory approval, and inhibit angiogenesis as one of their mechanisms of action. In addition, we investigated the potential application of EGFR inhibitors to influence other markers of tumour angiogenesis, specifically their effects on levels of circulating endothelial progenitor cells (CEPs). Finally, we evaluated combination treatment of EGFR inhibition with anti-angiogenic scheduling of chemotherapy. The EGFR ligand TGF-alpha increased in a dose dependent fashion following treatment with cetuximab, and levels in the circulation paralleled anti-tumour activity. This was a host-dependent effect that was not observed with the lower affinity antibody nimotuzumab. Inhibition of host EGFR also reduced plasma CEPs, but at higher doses these drugs increased off target growth factors VEGF and G-CSF, as well as CEPs. In a model of advanced triple negative breast cancer, the combination of nimotuzumab and metronomic cyclophosphamide was efficacious and well tolerated, leading to a potential new treatment strategy for this aggressive disease. Taken together, these studies identify new and useful applications for EGFR-targeted antibodies, and shed further light on their contributions within the field of tumour angiogenesis and antiangiogenic therapy.

No description available.

The isoprenoid biosynthetic pathway (IBP) has been highly implicated in a number of cellular malignancies, including proliferation, invasion, and migration. Epidemiological studies have found clinically relevant inhibitors of the IBP, such as the statin family and nitrogenous bisphosphonates, reduce the risk of prostate cancer advancement. In vitro work has implicated statin's and nitrogenous bisphosphonate's inhibition of GGPP and protein geranylgeranylation as the components responsible for their reduction of prostate cancer progression. However, their depletion of nearly all isoprenoid intermediates as well as their organ specificities make understanding the specific role of protein geranylgeranylation in prostate cancer metastasis impossible. Consequently, we have developed a novel library of seven alkyl bisphosphonate ethers found to potently reduce GGDPS with little to no activity against the related FDPS enzyme. Inhibition of GGDPS in three human prostate cancer cell lines reduced GGPP and protein geranylgeranylation without affecting protein farnesylation, translating into a reduction in cell migration and invasion. Interestingly, the GGDPS inhibitors reduced protein geranylgeranylation at lower concentrations in the highly metastatic PC3 cell line as compared to the less metastatic LNCaP and 22Rv1 cell lines. Additionally, the PC3 cell line was found to have higher levels of endogenous IBP intermediates as compared to the less metastatic cells. Translation in vivo using two murine models of human prostate cancer metastasis found a reduction in soft tissue tumor burden that corresponded to a biochemical reduction in protein geranylgeranylation. In conclusion, selective reduction of GGPP and protein geranylgeranylation was sufficient to reduce the metastatic potential of prostate cancer in vitro and in vivo.

Despite advances in therapeutic options for head and neck cancer (HNC), treatment-associated toxicities and overall clinical outcomes have remained disappointing. Even with radiation therapy (RT), which remains the primary curative modality for HNC, the most effective regimens achieve local control rates of 45-55%, with disease-free survival rates of only 30-40%. Thus, the development of novel strategies to enhance tumor cell killing, while minimizing damage to the surrounding normal tissues, is critical for improving cure rates with RT. Accordingly, we sought to identify novel radiosensitizing therapies for HNC, exploiting a high-throughput screening (HTS) approach. Initially, a cell-based phenotype-driven HTS of ~2,000 commercially available natural products was conducted, utilizing the short-term MTS cell viability assay. Cetrimonium bromide (CTAB) was identified as a novel anti-cancer agent, exhibiting in vitro and in vivo efficacy against several HNC models, with minimal effects on normal fibroblasts. Two major limitations of our findings, however, were that CTAB did not synergize with radiation, nor was its precise cellular target(s) elucidated. Consequently, an alternative strategy was proposed involving a target-driven RNAi-based HTS. Since the colony formation assay (CFA) is the gold standard for measuring cellular effects of radiation in vitro, an automated high-throughput colony-formation read-out was developed as a more appropriate end-point for radiosensitivity. Although successful as a tool for the discovery of potent anti-cancer cytotoxics, a technical drawback was its limited dynamic range. Thus, the BrdU incorporation assay, which measures replicative DNA synthesis and is a viable CFA alternative, was employed. From an RNAi-based screen of ~7000 human genes, uroporphyrinogen decarboxylase (UROD), a key regulator of heme biosynthesis, was identified as a novel tumor-selective radiosensitizing target against HNC in vitro and in vivo. Radiosensitization appeared to be mediated via tumor-selective enhancement of oxidative stress from perturbation of iron homeostasis and increased ROS production. UROD was significantly over-expressed in HNC patient biopsies, wherein lower pre-RT UROD levels correlated with improved disease-free survival, suggesting that UROD expression could also be a potential predictor for radiation response. Thus, employing a HTS approach, this thesis identified two novel therapeutic strategies with clinical potential in the management of HNC.

Despite advances in therapeutic options for head and neck cancer (HNC), treatment-associated toxicities and overall clinical outcomes have remained disappointing. Even with radiation therapy (RT), which remains the primary curative modality for HNC, the most effective regimens achieve local control rates of 45-55%, with disease-free survival rates of only 30-40%. Thus, the development of novel strategies to enhance tumor cell killing, while minimizing damage to the surrounding normal tissues, is critical for improving cure rates with RT. Accordingly, we sought to identify novel radiosensitizing therapies for HNC, exploiting a high-throughput screening (HTS) approach. Initially, a cell-based phenotype-driven HTS of ~2,000 commercially available natural products was conducted, utilizing the short-term MTS cell viability assay. Cetrimonium bromide (CTAB) was identified as a novel anti-cancer agent, exhibiting in vitro and in vivo efficacy against several HNC models, with minimal effects on normal fibroblasts. Two major limitations of our findings, however, were that CTAB did not synergize with radiation, nor was its precise cellular target(s) elucidated. Consequently, an alternative strategy was proposed involving a target-driven RNAi-based HTS. Since the colony formation assay (CFA) is the gold standard for measuring cellular effects of radiation in vitro, an automated high-throughput colony-formation read-out was developed as a more appropriate end-point for radiosensitivity. Although successful as a tool for the discovery of potent anti-cancer cytotoxics, a technical drawback was its limited dynamic range. Thus, the BrdU incorporation assay, which measures replicative DNA synthesis and is a viable CFA alternative, was employed. From an RNAi-based screen of ~7000 human genes, uroporphyrinogen decarboxylase (UROD), a key regulator of heme biosynthesis, was identified as a novel tumor-selective radiosensitizing target against HNC in vitro and in vivo. Radiosensitization appeared to be mediated via tumor-selective enhancement of oxidative stress from perturbation of iron homeostasis and increased ROS production. UROD was significantly over-expressed in HNC patient biopsies, wherein lower pre-RT UROD levels correlated with improved disease-free survival, suggesting that UROD expression could also be a potential predictor for radiation response. Thus, employing a HTS approach, this thesis identified two novel therapeutic strategies with clinical potential in the management of HNC.

No description available.

La diminution de la biodisponibilité de la sérotonine (5-HT) en inhibant sa biosynthèse peut représenter un traitement complémentaire efficace de l'hypertension artérielle pulmonaire (HTAP). Nous avons évalué cette hypothèse en utilisant LP533401, qui inhibe la tryptophane hydroxylase 1 (TPH1), enzyme limitante de la biosynthèse de la sérotonine, exprimée dans l'intestin et les poumons, sans inhiber Tph2 exprimée dans les neurones. Méthodes et résultats - Les souris traitées à plusieurs reprises avec LP533401 (30-250 mg / kg par jour) ont présenté une diminution marquée du contenu en 5-HT dans le tube digestif, dans les poumons et dans le sang, mais pas dans le cerveau. Après une seule dose LP533401 (250 mg / kg), le contenu en 5-HT du poumon et l'intestin a diminué de 50%, tandis que les niveaux 5-HT sanguins sont restés inchangés, suggérant une synthèse de 5-HT dans l'intestin et des poumons. Le traitement par l'inhibiteur du transporteur de la 5-HT (5-HTT), le citalopram, a diminué le contenu en 5-HT dans le sang et les poumons, mais pas dans l'intestin. En transgénique, les souris SM22-5-HTT+, surexprimant 5-HTT dans les cellules musculaires lisses, développent spontanément de l'HTAP. Le traitement par 250 mg / kg par jour de LP533401 ou 10 mg / kg de citalopram par jour pendant 21 jours diminue nettement les taux de 5-HT dans les poumons et dans le sang, ainsi que la pression systolique du ventricule droit (VD), l'hypertrophie du VD, la muscularisation distale de l'artère pulmonaire et les cellules Ki67-positives (P< 0,001). Le traitement combiné avec les deux médicaments était plus efficace dans l'amélioration des paramètres hémodynamiques que chacun des médicaments seuls. Un traitement par LP533401 ou par citalopram empêche partiellement le développement de l'HTAP chez les souris de type sauvage exposées à l'hypoxie chronique. Le niveau de 5-HT dans les poumons et dans le sang était plus faible dans les souris hypoxiques que dans souris normoxiques et est encore diminué après traitement par LP533401 ou par citalopram. / Decreasing the bioavailability of serotonin (5-HT) by inhibiting its biosynthesis may represent a useful adjunctive treatment of pulmonary hypertension (PH). We assessed this hypothesis using LP533401, which inhibits the rate-limiting enzyme tryptophan hydroxylase 1 (Tph1) expressed in the gut and lung, without inhibiting Tph2 expressed in neurons. Mice treated repeatedly with LP533401 (30–250 mg/kg per day) exhibited marked 5-HT content reductions in the gut, lungs, and blood, but not in the brain. After a single LP533401 dose (250 mg/kg), lung and gut 5-HT contents decreased by 50%, whereas blood 5-HT levels remained unchanged, suggesting gut and lung 5-HT synthesis. Treatment with the 5-HT transporter (5-HTT) inhibitor citalopram decreased 5-HT contents in the blood and lungs but not in the gut. In transgenic SM22-5-HTT+ mice, which overexpress 5-HTT in smooth muscle cells and spontaneously develop PH, 250 mg/kg per day LP533401 or 10 mg/kg per day citalopram for 21 days markedly reduced lung and blood 5-HT levels, right ventricular (RV) systolic pressure, RV hypertrophy, distal pulmonary artery muscularization, and vascular Ki67-positive cells. Combined treatment with both drugs was more effective in improving PH-related hemodynamic parameters than either drug alone. LP533401 or citalopram treatment partially prevented PH development in wild-type mice exposed to chronic hypoxia. Lung and blood 5-HT levels were lower in hypoxic than in normoxic mice and decreased further after LP533401 or citalopram treatment. These results provide proof of concept that inhibiting Tph1 may represent a new therapeutic strategy for human PH.