ABSTRACT
Autophagy as a Survival Mechanism for Tumor Cells Exposed to Chemotherapy or Hypoxia, and its Inhibition by Pantoprazole to Improve
Outcome of Treatment
QIAN TAN
Doctor of Philosophy
Graduate Department of Medical Biophysics
University of Toronto
2015
Chemotherapy is widely used as treatment for patients with metastatic cancer, and is also used to prevent disease recurrence and metastatic spread after local treatment. Several factors limit the effectiveness of chemotherapy. Resistance to anti-cancer agents may occur because of molecular changes in single cells, or through mechanisms related to the microenvironment of solid tumors such as limited drug distribution, and the presence of slowly-proliferating cells that are resistant to cycle-active drugs. Our group has worked in the field of chemotherapy resistance studying mechanisms oriented to improve drug penetration in solid tumors. The work described in this thesis aims to gain insight as to whether the clinically used proton pump inhibitor pantoprazole (PTP) inhibits autophagy and/or improves penetration and activity of chemotherapy drugs in solid tumors, thereby enhancing the effectiveness of chemotherapy.
Autophagy allows recycling of cellular components and may facilitate cell survival after chemotherapy. Pantoprazole inhibits proton pumps, including that maintaining low pH in endosomes, and is reported to inhibit autophagy. The first objective of my work was to evaluate effects of pantoprazole to modify cytotoxicity of the anticancer drug docetaxel, and to determine underlying mechanisms. I found that pantoprazole (i) increased toxicity of docetaxel for cultured cells; increased docetaxel-induced expression of DNA damage (evaluated by expression of γH2AX using immunohistochemistry, IHC) and apoptosis (evaluated by expression of cleaved caspase-3) and decreased cell proliferation (evaluated by expression of Ki67) in tumors; (iii) increased docetaxel-induced growth-delay of four human xenografts of low, moderate and high sensitivity to docetaxel, with minimal increase in toxicity. Docetaxel led to increased autophagy throughout tumor sections as evaluated by IHC showing increased LC3 and decreased P62 expression. Pantoprazole inhibited autophagy, and the effects of pantoprazole were reduced against genetically-modified cells with decreased ability to undergo autophagy. Overall autophagy is a mechanism of resistance to docetaxel chemotherapy that may be modified by pantoprazole to improve therapeutic index.
The second objective of my work addressed whether induction of autophagy is a general mechanism of resistance to chemotherapy, and whether autophagy and hence drug resistance can be inhibited by proton pump inhibitors. It was found that seven clinically-used anti-cancer drugs induced autophagy in cultured PC3, LnCAP and MCF7 cells, as assessed by western blot analysis of the autophagy markers LC3 and P62, and by fluorescence microscopy assay using a special construct that links LC3 to genes encoding GFP and RFP. Anti-cancer drugs including doxorubicin, docetaxel and paclitaxel also induced autophagy in PC3, LnCAP and MCF7 xenografts, as evaluated by IHC using the autophagy biomarkers LC3 and p62. Induction of autophagy was inhibited by pre-treatment with pantroprazole and lansoprazole.
The third objective of my work was to determine whether autophagy is a survival mechanism for hypoxic cells. I exposed several cancer cell lines, including those selected by shRNA to be deficient in the autophagy genes BECLIN-1 and ATG-7, to the presence or absence of pantoprazole under aerobic and hypoxic (<1% O2, 24h) conditions, , and evaluated autophagy by expression of LC3 and P62 by western blot assay. I also quantified rates of cell death under hypoxic conditions. Xenografts were generated from the autophagy deficient cells; their growth rate and extent of hypoxia in the tumor (following injection of the hypoxia marker EF5 and IHC) were compared to controls. It was found that (i) hypoxia induced autophagy in cultured PC3, LnCaP and MCF7 cells and this was inhibited in the presence of pantoprazole. ii) hypoxia-induced cell death was more rapid for autophagy deficient cells, and was increased in wild-type cells in the presence of pantoprazole. (iii) Xenografts generated from autophagy-deficient cells grew more slowly than wild type tumors. (iv) Autophagy co-localized with hypoxic regions in wild-type tumors, and the proportion of hypoxia cells was markedly decreased in xenografts generated from single knockdowns and absent in double knockdown tumors.
The work presented in this thesis highlights the importance of the tumor microenvironment in determining response to chemotherapy and suggests that induction of autophagy might be a common mechanism of drug resistance. Effects of anti-cancer drugs might be improved by using proton pump inhibitors to inhibit autophagy. Based on these data, a phase I trial of pantoprazole +doxorubicin has been undertaken in patients with advanced solid tumors and a phase II study of pantoprazole + docetaxel is underway in men with castrate-resistant prostate cancer.