EFFECTS OF INHIBITING THE MAMMALIAN TARGET OF RAPAMYCIN (mTOR) PATHWAY AND TELOMERASE IN BREAST CANCER CELLS KALPANA GOPALAKRISHNAN (B Sc (Hons.), NUS) A THESIS SUBMITTED FOR THE DEGREE OF MASTER OF SCIENCE DEPARTMENT OF PHYSIOLOGY NATIONAL UNIVERSITY OF SINGAPORE 2011 ACKNOWLEDGEMENTS I take this opportunity to extend my heartfelt gratitude to all those without whom this Masters thesis would not have been possible First and foremost, I would like to thank my supervisor A/P Manoor Prakash Hande for all the guidance, support and inspiration offered to me during my course of research under him I am also grateful for all the valuable advice and encouragement extended by him to me in the context of research and beyond I especially would also like to thank him for having given me the opportunity to attend a number of quality conferences, both international and local The friendships nurtured at Genome Stability Lab, helped to make the work in this thesis both educative and enjoyable I would like to extend my gratitude to all lab members who provided timely assistance and encouragement throughout Last but not the least I would like to express my appreciation to members of other labs in the Department of Physiology who have helped me with advice on techniques and protocols i TABLE OF CONTENTS ACKNOWLEDGEMENTS i TABLE OF CONTENTS .ii SUMMARY viii LIST OF FIGURES x LIST OF ABBREVIATIONS .xiii CHAPTER INTRODUCTION 1.1 THE MAMMALIAN TARGET OF RAPAMYCIN (mTOR): STRUCTURE, FUNCTION AND ROLES IN CANCER 1.1.1 The role of mTOR in cell physiology 1.1.2 mTOR pathway: upstream regulators of mTOR signalling 1.1.3 mTOR in human cancer 1.2 TELOMERES AND TELOMERASE: STRUCTURE, FUNCTION AND ROLES IN CANCER .11 1.2.1 Telomeres: Structure and functions 11 1.2.2 Telomere related functions of telomerase in human cancer 13 ii 1.2.3 Expanding telomerase functions in human cancer 15 1.2.4 Regulation of telomerase activity in human cancer 17 1.3 LINKING mTOR AND TELOMERASE THROUGH RAPAMYCIN 19 1.3.1 Rapamycin as an inhibitor of mTOR 19 1.3.2 The mTOR-telomerase connection via rapamycin 22 1.3.3 The convergence of the mTOR pathway with telomeres and telomerase 24 1.4 BREAST CANCER AS A MODEL OF STUDY .25 1.4.1 mTOR in breast cancer 25 1.4.2 Telomerase in breast cancer 26 1.4.3 Rapamycin in breast cancer .27 1.5 OBJECTIVES, HYPOTHESIS AND SIGNIFICANCE OF STUDY 29 CHAPTER MATERIALS AND METHODS 31 2.1 CELLS AND CELL CULTURE .32 2.2 MOLECULAR CHARACTERISATION OF BREAST CANCER CELLS 33 2.3 DRUG AND DRUG TREATMENT CONDITIONS 33 2.4 SHORT TERM STUDIES 34 2.4.1 Cell treatment 34 2.4.2 Protein expression studies by western blot 35 iii 2.4.3 Telomerase activity measurement by Telomerase Repeat Amplification Protocol (TRAP) .37 2.4.4 Cell Cycle profiling by Propidium Iodide (PI)-assisted Fluorescence Associated Cell Sorting (FACS) 38 2.4.5 DNA damage analysis by alkaline single cell gel electrophoresis (Comet) assay 39 2.4.6 Cell Viability .40 2.4.6.1 MTT 40 2.4.6.2 CellTiter-Glo 40 2.5 LONG TERM STUDIES 41 2.5.1 Cell Treatment 41 2.5.2 Population doubling via Trypan blue dye exclusion assay .42 2.5.3 Telomere length measurement by Telomere Restriction Fragment (TRF) analysis .42 2.6 STATISTICAL ANALYSIS 44 CHAPTER RESULTS 45 3.1 MOLECULAR CHARACTERIZATION OF BREAST CANCER CELLS 46 3.1.1 Breast cancer cells MCF-7 and MDA-MB-231 exhibited concurrent upregulation of phosphorylated mTOR (p-mTOR) and telomerase, albeit to different extents 46 iv 3.2 RAPAMYCIN CHARACTERIZATION: SHORT TERM STUDIES 50 3.2.1 Rapamycin inhibited activation of mTOR pathway in MCF-7 and MDA-MB231 cells……………… 50 3.2.2 Rapamycin did not modulate hTERT protein, but inhibited telomerase activity in MCF-7 and MDA-MB-31 cells, albeit to different extents 53 3.2.3 Rapamycin induced G1 arrest in MCF-7 and MDA-MB-231 cells at 24 and 48 hours 55 3.2.4 Rapamycin-induced G1 arrest was independent of cyclin D1 and p21 protein expression 57 3.2.5 Rapamycin had limited effect on cell proliferation following 48 hours in MCF-7 and MDA-MB-231 cells .58 3.2.6 Rapamycin had limited effect on DNA damage following 48 hours in MCF-7 and MDA-MB-231 cells .59 3.2.7 Rapamycin led to dose-dependent loss of viability in MCF-10A and IMR-90 cells .60 3.3 RAPAMYCIN THERAPEUTICS: LONG TERM STUDIES .62 3.3.1 Rationale 62 3.3.2 Chronic rapamycin treatment compromised population doubling capacity of MCF-7, MDA-MB-231 and MCF-10A cells, albeit to different extents 63 3.3.3 Chronic rapamycin treatment inhibited the mTOR pathway in MCF-7 and MDA-MB-231 cells, and led to upregulation of p-Akt in MDA-MB-231 cells 65 v 3.3.4 Chronic rapamycin treatment led to slight downregulation of hTERT protein in MCF-7 and MDA-MB-231 cells, with a slight decrease and increase in telomerase activity in MCF-7 and MDA-MB-231 cells, respectively 66 3.3.5 Chronic rapamycin treatment led to reduction of telomere length in MCF-7 and MDA-MB-231 cells, albeit to different extents 68 CHAPTER DISCUSSION 71 4.1 BREAST CANCER CELLS MCF-7 AND MDA-MB-231 ARE A GOOD MODEL TO STUDY THE INHIBITION OF mTOR AND TELOMERASE 72 4.2 UPREGULATION OF P-MTOR AND TELOMERASE DOES NOT NECESSARILY PREDICT RESPONSIVENESS TO RAPAMYCIN .75 4.3 CHRONIC LOW DOSE RAPAMYCIN TREATMENT IN BREAST CANCER CELLS REVEALS A NOVEL MECHANISM OF RAPAMYCIN RESISTANCE INVOLVING AKT AND TELOMERASE 80 CHAPTER CONCLUSIONS, SIGNIFICANCE AND FUTURE DIRECTIONS .84 5.1 CONCLUSIONS AND SIGNIFICANCE 85 5.2 FUTURE DIRECTIONS 86 REFERENCES .88 vi LIST OF CONFERENCES .101 LIST OF PUBLICATIONS .102 LIST OF AWARDS 103 vii SUMMARY This investigation explores the effects of rapamycin on the mTOR pathway and telomerase in breast cancer cells The mTOR pathway, a prototypic survival pathway upregulated in many cancers, integrates various cellular signals serving as a master regulator of protein synthesis, ribosome biogenesis, autophagy, survival and proliferation It also plays a major role in drug resistance, making mTOR an attractive anticancer target The telomerase enzyme maintains telomere length, allowing cells to bypass the antiproliferative barriers of senescence and crisis Telomerase upregulation occurs in more than 90% of human cancers in addition to being critical and specific to cancer cells, making it another attractive anticancer target The macrolide antibiotic rapamycin inhibits the mTOR pathway specifically and potently and exerts anticancer effects in a wide variety of cancers Recent studies also showed that rapamycin inhibited telomerase and induced telomere shortening in some malignancies, although the mechanism is poorly understood Breast cancers exhibit aberrant regulation of both the mTOR pathway and telomerase and hence may be a useful model to study the effects of rapamycin Using this model, the investigation seeks to unravel novel mechanisms by which breast cancer cells may regulate the complex mTOR circuitry and telomerase by as yet uncharacterised mechanisms Our results showed that breast cancer cells MCF-7 and MDA-MB-231 exhibited concurrent upregulation of phosphorylated mTOR (p-mTOR) and hTERT, albeit to different extents In short term studies, we found that rapamycin inhibited viii activation of the mTOR pathway, did not modulate hTERT protein, but significantly inhibited telomerase activity in MCF-7 and MDA-MB-31 cells Rapamycin induced G1 arrest in both cells independently of cyclin D1 and p21 expression Rapamycin had limited effect on cell proliferation and DNA damage in MCF-7 and MDA-MB231 cells, and led to dose-dependent loss of viability only in MCF-10A and IMR-90 cells Altogether these results suggest that while breast cancer cells may be a useful model to study the dual inhibition of the mTOR pathway and telomerase, the activation of these two players alone cannot predict the responsiveness of these cells to short term rapamycin treatment Long term studies showed that low dose rapamycin treatment compromised population doubling capacity of MCF-7, MDA-MB-231 and MCF-10A cells and inhibited 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Mol Cell Biol 29(9): 2390-2397 100 LIST OF CONFERENCES Kalpana Gopalakrishnan, Grace Kah Mun Low, Aloysius Poh Leong Ting, Prarthana Srikanth, Predrag Slijepcevic, M Prakash Hande Genomic instability and telomere attrition in Xeroderma Pigmentosum type-B deficient cells under oxidative stress 36th Annual Conference of the Environmental and Mutagen Society of India (EMSI) and International symposium on environmental exposures to mutagens and carcinogens on human health‟ Vellore, India February 2011 Kalpana Gopalakrishnan, Aloysius Poh Leong Ting, Grace Kah Mun Low, M Prakash Hande Telomere attrition and genomic instability in Xeroderma Pigmentosum type-B deficient cells under oxidative stress Keystone Symposium on Telomere Biology and DNA Repair Queensland, Australia October 2009 Aloysius Poh Leong Ting, Kalpana Gopalakrishnan, Grace Kah Mun Low, M Prakash Hande Genomic instability and telomere attrition in Xeroderma Pigmentosum type-B deficient fiborblasts under oxidative stress Keystone Symposium on Stem Cells, Cancer and Aging Singapore September – October 2008 101 LIST OF PUBLICATIONS Gopalakrishnan K, Low GK, Ting AP, Srikanth P, Slijepcevic P, Hande MP (2010) “Hydrogen peroxide induced genomic instability in nucleotide excision repairdeficient lymphoblastoid cells.” Genome Integr 1(1): 16 Mistry H, Tamblyn L, Butt H, Sisgoreo D, Gracias A, Larin M, Gopalakrishnan K, Hande MP, McPherson JP (2010) “UHRF1 is a genome caretaker that facilitates the DNA damage response to gamma-irradiation.” Genome Integr 1(1): Ting AP, Low GK, Gopalakrishnan K, Hande MP (2010) “Telomere attrition and genomic instability in xeroderma pigmentosum type-b deficient fibroblasts under oxidative stress.” J Cell Mol Med 14(1-2): 403-416 102 LIST OF AWARDS Best presentation award for „Genomic instability and telomere attrition in Xeroderma Pigmentosum type-B deficient cells under oxidative stress’ at the 36th Annual Conference of EMSI and International symposium on environmental exposures to mutagens and carcinogens on human health Vellore, India February 2011 Scholarship for „Genomic instability and telomere attrition in Xeroderma Pigmentosum type-B deficient cells under oxidative stress’ at the Keystone Symposium on Telomere Biology and DNA Repair Queensland, Australia October 2009 103 ... objectives of the study are to: Validate breast cancer cells as a model to study the dual inhibition of mTOR and telomerase Investigate rapamycin? ??s inhibition of the mTOR pathway and telomerase in breast. .. test the efficacy of mTOR kinase domain inhibitors in preclinical and clinical studies (Menon and Manning 2008) These will be discussed in detail in the context of rapamycin? ??s status in preclinical... other than mTOR, specifically telomerase, in mediating the anticancer effects of rapamycin Further, while rapamycin may function as a dual inhibitor of mTOR and telomerase, sustained rapamycin