the role of amp-activated protein kinase (ampk) in mediating radiation responses in cancer cells

279 Pages·2012·5.72 MB·English

by  Toran Sanli

THE ROLE OF AMPK IN MEDIATING RADIATON RESPONSES IN CANCER
THE ROLE OF AMP-ACTIVATED PROTEIN KINASE (AMPK) IN MEDIATING
RADIATION REPONSES IN CANCER CELLS

By TORAN SANLI, B.Sc., M.Sc.

A Thesis Submitted to the school of Graduate Studies in Partial Fulfillment of the
Requirements for the Degree Doctor of Philosophy

McMaster University © Copyright by Toran Sanli, April 2012
McMaster University DOCTOR OF PHILOSPHY (2012) Hamilton, Ontario (Medical
Science)
TITLE: The role of AMP-activated protein kinase (AMPK) in mediating radiation
responses in cancer cells
AUTHOR: Toran Sanli, B.Sc. (Honours with 1st Class Standing) (Brock University),
M.Sc. (Brock University)
SUPERVISOR: Professor Gurmit Singh, Ph.D.
NUMBER OF PAGES: v, 273
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Abstract
One of the hallmark features of cancer is altered metabolism, whereby rates of
glucose and fatty acid turnover are constitutively elevated to support uncontrolled
propagation. The key regulator of energy metabolism is the enzyme AMP-activated
protein kinase (AMPK), which suppresses anabolic pathways that increase proliferation
and enhanced catabolic pathways that liberate energy, all in an attempt to maintain energy
homeostasis in the cell. In addition to regulating metabolism, AMPK has also been
implicated as a tumour suppressor and we have suggested that it may be a modulator of
radiation responses in cancer cells in vitro. Moreover, we investigated the molecular
mechanisms that facilitate ionizing radiation (IR)-induced AMPK activation, as well as
demonstrated that certain AMPK activating drugs can work as radiation sensitizers in a
variety of cancer cell lines. Stemming from this framework, we also provided
experimental evidence that suggests AMPK is centrally involved in pathways that
regulate DNA damage and proliferation at the basal level, and in response to IR. One of
the targets involved in these pathways that can also influence AMPK regulation is the
stress-activated Sestrin 2 protein. We have provided evidence that Sestrin 2 mediates IR-
induced activation and expression of AMPK. Taken together, this work has provided
novel insight into the ability of IR to modulate the activity and expression of AMPK,
which in turn is required to facilitate the appropriate stress-response in cancer cells. Given
its emerging interest in the cancer field, AMPK may become an important biomarker for
evaluating clinical outcomes in patients undergoing radiation therapy.

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Preface
This doctoral work has been prepared as a “sandwich” Ph.D. thesis, and consists
of four manuscripts, all of which have now been published, or accepted for publication.
Many of the experimental protocols were oriented around the general focus on AMPK
signalling in response to ionizing radiation, and as such, the reader may encounter some
repetition in experimental design between articles. Following the introduction and
background information, each paper is presented as a separate chapter that highlights a
particular theme within the overall framework of the thesis. Supporting, unpublished data
may also be included following each paper to strengthen the materials and objectives of
each individual study. The figures are denoted so that the chapter number is indicated
first, followed by the actual figure number that corresponds to the appropriate paper (e.g.
Figure 1 for chapter 2 is labeled as Figure 2.1).
The last chapter will contain a conclusion that will summarize the major findings
of the author’s thesis, as well as provide future directions to expand on this work.
References contained within the body of the sandwich thesis are formatted in accordance
to the journal in which they were published. Literature cited within the introduction and
conclusion of the thesis will adhere to the American Psychological Association (5th
edition) style, with the bibliography of references appearing at the end of the dissertation.
Additional information pertaining to general methodologies and supporting experiments
are provided in Appendix 1 and 2 respectively.
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Acknowledgements
I would like to thank my supervisor, Dr. Gurmit Singh, for his guidance and
support of my research project throughout the years. I would also like to acknowledge
and thank Dr Theodoros Tsakiridis for initially taking me on as a graduate student and
exposing me to the translational work with AMPK and radiotherapy at the Juravinski
Cancer Centre (JCC). Dr. Tsakiridis has been a long-time mentor throughout my graduate
training at both the Master’s and Doctoral level. In addition, Dr. Hal Hirte is also a
member on my committee and has provided helpful feedback throughout my training.
I would also like to thank the kind and supportive staff and graduate students at
the JCC. The input and feedback from the many members of our lab has greatly aided me
with my research projects and has contributed to my knowledge and experience as a
scientist. I am greatly indebted to Dr. Katja Linher-Melville, who has help train me in
many aspects of basic cell biology, as well as contribute to a couple of our published
papers. Helpful advice was also given by Dr. Eric Seidlitz, who is knowledgeable in all
aspects of research conducted at the JCC. Sarah Hopmans and Yaryna Storozhuk also
aided me in conducting experiments, as well as the radiation therapy staff at the JCC.
Importantly, I would like to thank my family for their continued support of my
ongoing education. My parents have always encouraged me to have high academic
standards, and it is from them where I have gained my strong work ethic. Equally
essential is the love and support of my wife, Elizabeth, whom I can confide in and look to
for guidance as a fellow graduate student. My goal is to now put this Ph.D degree to good
use to benefit society and provide for our future.
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Ph.D. Thesis – Toran Sanli – McMaster University – Medical Sciences

Table of Contents
List of Diagrams, Figures, and Tables ................................................................................ 5
List of Abbreviations ........................................................................................................... 9
Chapter 1: Introduction ..................................................................................................... 12
Cellular Energy Metabolism .......................................................................................... 12
Energy Metabolism in Cancer Cells .......................................................................... 13
Regulating Energy Balance and the Identification of AMPK ....................................... 16
AMPK Structure and Function ...................................................................................... 18
The AMPK Subunits .................................................................................................. 18
Modes of AMPK Activation ...................................................................................... 21
Downstream Targets of AMPK ................................................................................. 26
Pharmaceutical Modulators of AMPK Activity as Anti-Cancer Agents ....................... 31
AICAR ....................................................................................................................... 32
Metformin .................................................................................................................. 32
Resveratrol ................................................................................................................. 34
Statins ......................................................................................................................... 34
The Stress-Responsive Sestrin Proteins ........................................................................ 35
The Identification of Sestrins ..................................................................................... 36
Antioxidant Function of Sestrins ............................................................................... 37
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Ph.D. Thesis – Toran Sanli – McMaster University – Medical Sciences

Sestrins and Suppression of mTOR Signalling .......................................................... 38
Overview of Radiobiology and its Application in Cancer ............................................ 39
Types and Classifications of IR ................................................................................. 39
Cellular and Molecular Events in Response to Ionizing Radiation ........................... 40
Radiotherapy and Fractionation of Tumours ............................................................. 47
Hypothesis and Objectives ............................................................................................ 52
Chapter 2: Ionizing Radiation Activates AMPK ............................................................... 55
Context and Background Information ........................................................................... 55
Paper: Ionizing Radiation Activates AMP-Activated Kinase (AMPK): A Target for
Radiosensitization of Human Cancer Cells. .................................................................. 60
Supporting Experiments ................................................................................................ 88
Chapter 3: Lovastatin Sensitizes Lung Cancer Cell to Ionizing Radiation ....................... 96
Context and Background Information ........................................................................... 96
Paper: Lovastatin sensitizes lung cancer cells to ionizing radiation. Modulation of
molecular pathways of radioresistance and tumour suppression. ................................ 100
Supporting Experiments .............................................................................................. 136
Chapter 4: Ionizing Radiation Regulates AMPK Expression in Cancer Cells ............... 139
Context and Background Information ......................................................................... 139
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Ph.D. Thesis – Toran Sanli – McMaster University – Medical Sciences

Paper: Ionizing radiation regulates the expression of AMP-activated protein kinase
(AMPK) in epithelial cancer cells. Modulation of cellular signals regulating cell cycle
and survival. ................................................................................................................. 143
Supporting Experiments .............................................................................................. 169
Chapter 5: Sestrin2 Modulates AMPK Activity and Expression .................................... 174
Context and Background Information ......................................................................... 174
Paper: Sestrin2 modulates AMPK subunit expression and its response to ionizing
radiation in breast cancer cells. .................................................................................... 178
Supporting Experiments .............................................................................................. 207
Chapter 6: Conclusions ................................................................................................... 212
Future Directions ......................................................................................................... 218
Summary ...................................................................................................................... 224
Reference List ................................................................................................................. 226
Appendix 1: General Methodology ................................................................................. 244
Cell Culture Technique ................................................................................................ 244
IR and Cell Treatments ................................................................................................ 245
Measuring Cell Proliferation and Survival .................................................................. 245
Clonogenic Survival Assays .................................................................................... 245
Crystal Violet Assay ................................................................................................ 246
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Ph.D. Thesis – Toran Sanli – McMaster University – Medical Sciences

Hoechst 33258 Assay ............................................................................................... 247
Western blotting .......................................................................................................... 248
Protein Assay ............................................................................................................... 250
Immunoprecipitation ................................................................................................... 252
Real Time Polymerase Chain Reaction (RT-PCR) ..................................................... 253
SESN2 Overexpression using a Flag-tagged Vector ................................................... 257
siRNA-Mediated Gene Silencing ................................................................................ 258
Immunofluorescence Protocol ..................................................................................... 259
Cell Cycle Analysis ..................................................................................................... 261
Statistical Analyses ...................................................................................................... 261
Appendix 2: Supporting Experiments ............................................................................. 263
Appendix 3: Copyright Licence Agreements for Papers ................................................ 268

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