Positron Emission Tomography Imaging of Platinum Resistant Ovarian Cancer And Drug Modulation

Epithelial ovarian cancer is the commonest cause of death from gynaecological malignancy. Platinum based chemotherapy remains the cornerstone of first-line therapy for ovarian cancer, however relapse is common and acquired resistance is frequently observed on subsequent lines of platinum based treatment. Because activation of the PI3K/AKT pathway has been shown to play a role in acquired platinum resistance phenotype of ovarian cancer, combination of platinum with AKT pathway inhibitors holds promise for resensitising tumours to platinum. The aims of this thesis were i) to evaluate the effect of pharmacological AKT inhibitors AKT on the resensitisation of clinically-derived platinum resistant ovarian cancer cells to platinum, and ii) establish molecular imaging approaches for monitoring resensitisation to platinum in vivo combined with biochemical profiling of pathway activity. Treatment of platinum sensitive PEO1 ovarian cancer cells with cisplatin induced growth inhibition and apoptosis. The AKT inhibitor, API-2, resensitised paired platinum resistant PEO4 cells to cisplatin. These in vitro studies established 1h pre-treatment time point with API-2 followed by cisplatin as the most appropriate schedule for further studies. Resensitisation to platinum appeared to be a pathway effect rather than a specific effect of API-2. In order to establish the utility of PET imaging for therapy response, a bilateral tumour xenograft model comprising of platinum sensitive PEO1 and the platinum resistant cell line PEO4 was developed. Both [18F]fluorothymidine ([18F]FLT)-PET, which measures proliferation, and [18F]fluorodeoxyglucose ([18F]FDG)-PET, which measures glucose metabolism detected responses to cisplatin alone in PEO1 tumours and the combination of cisplatin and API-2 in both PEO1 and PEO4 tumours. Correlative reduction in phosphorylated PRAS40, a direct downstream marker of AKT activity indicated that in vivo changes in imaging variables of the combination treatment resulted from AKT inhibition. The imaging changes were also linked to histological reductions in Ki67 labelling index. AKT Biochemical profiling of tumours obtained after imaging confirmed that the changes in tumour [18F]FDG and [18F]FLT uptake were due, at least in part, to reductions in the expression of glucose transporter Glut-1 and hexokinase activity, as well as decreases in TK1 expression, respectively. These studies demonstrated that [18F]FDG-and [18F]FLT-PET hold promise for clinical evaluation of platinum resensitisation in patients with platinum resistant ovarian cancer. Given also that combination treatment with cisplatin and API-2 induced apoptosis in PEO4 cells in vitro, further studies were conducted with the aim of establishing [18F]ICMT11 PET, a specific cleaved caspase-3/7 radiotracer, for monitoring re-sensitisation to platinum in PEO4 tumours. [18F]ICMT11 uptake detected by PET increased after combination treatment but not with cisplatin alone. Parallel increases in TUNEL and cleaved caspase-3 staining by immunohistochemistry were observed consistent with the PET outcome. Thus, [18F]ICMT11 PET could also find utility in monitoring early responses to platinum therapy in combination with AKT inhibition. In addition to the 3 imaging agents described above, analysis of baseline levels of integrin αvβ3/5 expression, an index of angiogenesis, as well as myo-inositol uptake were investigated as potential discriminators of platinum resistant phenotype. These preliminary experiments were inconclusive. In summary PET imaging of proliferation, glucose metabolism and apoptosis were shown to be promising techniques for early detection of resensitisation to platinum therapy in platinum resistant ovarian cancer and warrant further investigation

Positron emission tomography imaging of platinum resistant ovarian cancer and drug modulation

Epithelial ovarian cancer is the commonest cause of death from gynaecological malignancy. Platinum based chemotherapy remains the cornerstone of first-line therapy for ovarian cancer, however relapse is common and acquired resistance is frequently observed on subsequent lines of platinum based treatment. Because activation of the PI3K/AKT pathway has been shown to play a role in acquired platinum resistance phenotype of ovarian cancer, combination of platinum with AKT pathway inhibitors holds promise for resensitising tumours to platinum. The aims of this thesis were i) to evaluate the effect of pharmacological AKT inhibitors AKT on the resensitisation of clinically-derived platinum resistant ovarian cancer cells to platinum, and ii) establish molecular imaging approaches for monitoring resensitisation to platinum in vivo combined with biochemical profiling of pathway activity. Treatment of platinum sensitive PEO1 ovarian cancer cells with cisplatin induced growth inhibition and apoptosis. The AKT inhibitor, API-2, resensitised paired platinum resistant PEO4 cells to cisplatin. These in vitro studies established 1h pre-treatment time point with API-2 followed by cisplatin as the most appropriate schedule for further studies. Resensitisation to platinum appeared to be a pathway effect rather than a specific effect of API-2. In order to establish the utility of PET imaging for therapy response, a bilateral tumour xenograft model comprising of platinum sensitive PEO1 and the platinum resistant cell line PEO4 was developed. Both [18F]fluorothymidine ([18F]FLT)-PET, which measures proliferation, and [18F]fluorodeoxyglucose ([18F]FDG)-PET, which measures glucose metabolism detected responses to cisplatin alone in PEO1 tumours and the combination of cisplatin and API-2 in both PEO1 and PEO4 tumours. Correlative reduction in phosphorylated PRAS40, a direct downstream marker of AKT activity indicated that in vivo changes in imaging variables of the combination treatment resulted from AKT inhibition. The imaging changes were also linked to histological reductions in Ki67 labelling index. AKT Biochemical profiling of tumours obtained after imaging confirmed that the changes in tumour [18F]FDG and [18F]FLT uptake were due, at least in part, to reductions in the expression of glucose transporter Glut-1 and hexokinase activity, as well as decreases in TK1 expression, respectively. These studies demonstrated that [18F]FDG-and [18F]FLT-PET hold promise for clinical evaluation of platinum resensitisation in patients with platinum resistant ovarian cancer. Given also that combination treatment with cisplatin and API-2 induced apoptosis in PEO4 cells in vitro, further studies were conducted with the aim of establishing [18F]ICMT11 PET, a specific cleaved caspase-3/7 radiotracer, for monitoring re-sensitisation to platinum in PEO4 tumours. [18F]ICMT11 uptake detected by PET increased after combination treatment but not with cisplatin alone. Parallel increases in TUNEL and cleaved caspase-3 staining by immunohistochemistry were observed consistent with the PET outcome. Thus, [18F]ICMT11 PET could also find utility in monitoring early responses to platinum therapy in combination with AKT inhibition. In addition to the 3 imaging agents described above, analysis of baseline levels of integrin αvβ3/5 expression, an index of angiogenesis, as well as myo-inositol uptake were investigated as potential discriminators of platinum resistant phenotype. These preliminary experiments were inconclusive. In summary PET imaging of proliferation, glucose metabolism and apoptosis were shown to be promising techniques for early detection of resensitisation to platinum therapy in platinum resistant ovarian cancer and warrant further investigation.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Glucose Metabolism Measured by [18F]Fluorodeoxyglucose Positron Emission Tomography Is Independent of PTEN/AKT Status in Human Colon Carcinoma Cells1

The phosphoinositide 3-kinase (PI3K) signaling pathway is one of the most altered in cancer, leading to a range of cellular responses including enhanced proliferation, survival, and metabolism, and is thus an attractive target for anticancer drug development. Stimulation of the PI3K pathway can be initiated by alterations at different levels of the signaling cascade including growth factor receptor activation, as well as mutations in PIK3CA, PTEN, and AKT genes frequently found in a broad range of cancers. Given its role in glucose metabolism, we investigated the utility of [18F]fluorodeoxyglucose positron emission tomography ([18F]FDG PET) as a pharmacodynamic biomarker of PI3K pathway-induced glucose metabolism. PTEN deletion in human colon carcinoma cells led to constitutive AKT activation but did not confer a phenotype of increased cell proliferation or glucose metabolism advantage in vivo relative to isogenic tumors derived from cells with a wild-type allele. This was not due to the activation context, that is, phosphatase activity, per se because PIK3CA activation in xenografts derived from the same lineage failed to increase glucose metabolism. Acute inhibition of PI3K activity by LY294002, and hence decreased activated AKT expression, led to a significant reduction in tumor [18F]FDG uptake that could be explained at least in part by decreased membrane glucose transporter 1 expression. The pharmacodynamic effect was again independent of PTEN status. In conclusion, [18F]FDG PET is a promising pharmacodynamic biomarker of PI3K pathway inhibition; however, its utility to detect glucose metabolism is not directly linked to the magnitude of activated AKT protein expression

Adaptation to HIF-1 deficiency by upregulation of the AMP/ATP ratio and phosphofructokinase activation in hepatomas

Background HIF-1 deficiency has marked effects on tumour glycolysis and growth. We therefore investigated the consequences of HIF-1 deficiency in mice, using the well established Hepa-1 wild-type (WT) and HIF-1β-deficient (c4) model. These mechanisms could be clinically relevant, since HIF-1 is now a therapeutic target. Methods Hepa-1 WT and c4 tumours grown in vivo were analysed by 18FDG-PET and 19FDG Magnetic Resonance Spectroscopy for glucose uptake; by HPLC for adenine nucleotides; by immunohistochemistry for GLUTs; by immunoblotting and by DIGE followed by tandem mass spectrometry for protein expression; and by classical enzymatic methods for enzyme activity. Results HIF-1β deficient Hepa-1 c4 tumours grew significantly more slowly than WT tumours, and (as expected) showed significantly lower expression of many glycolytic enzymes. However, HIF-1β deficiency caused no significant change in the rate of glucose uptake in c4 tumours compared to WT when assessed in vivo by measuring fluoro-deoxyglucose (FDG) uptake. Immunohistochemistry demonstrated less GLUT-1 in c4 tumours, whereas GLUT-2 (liver type) was similar to WT. Factors that might upregulate glucose uptake independently of HIF-1 (phospho-Akt, c-Myc) were shown to have either lower or similar expression in c4 compared to WT tumours. However the AMP/ATP ratio was 4.5 fold higher (p < 0.01) in c4 tumours, and phosphofructokinase-1 (PFK-1) activity, measured at prevailing cellular ATP and AMP concentrations, was up to two-fold higher in homogenates of the deficient c4 cells and tumours compared to WT (p < 0.001), suggesting that allosteric PFK activation could explain their normal level of glycolysis. Phospho AMP-Kinase was also higher in the c4 tumours. Conclusions Despite their defective HIF-1 and consequent down-regulation of glycolytic enzyme expression, Hepa-1 c4 tumours maintain glucose uptake and glycolysis because the resulting low [ATP] high [AMP] allosterically activate PFK-1. This mechanism of resistance would keep glycolysis functioning and also result in activation of AMP-Kinase and growth inhibition; it may have major implications for the therapeutic activity of HIF inhibitors in vivo. Interestingly, this control mechanism does not involve transcriptional control or proteomics, but rather the classical activation and inhibition mechanisms of glycolytic enzymes