Altered Cholesterol Metabolism In Human Cancers Unraveled By Label-Free Spectroscopic Imaging

179 Pages·2017·5.25 MB·English

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Open Access Dissertations Theses and Dissertations
Fall 2013
Altered Cholesterol Metabolism In Human
Cancers Unraveled By Label-Free Spectroscopic
Imaging
Shuhua Yue
Purdue University
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Yue, Shuhua, "Altered Cholesterol Metabolism In Human Cancers Unraveled By Label-Free Spectroscopic Imaging" (2013).Open
Access Dissertations. 11.
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ALTERED CHOLESTEROL METABOLISM IN HUMAN CANCERS UNRAVELED
BY LABEL-FREE SPECTROSCOPIC IMAGING
A Dissertation
Submitted to the Faculty
of
Purdue University
by
Shuhua Yue
In Partial Fulfillment of the
Requirements for the Degree
of
Doctor of Philosophy
December 2013
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Purdue University
West Lafayette, Indiana
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To my parents, my husband, and my son.

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ACKNOWLEDGEMENTS
First and foremost, I would like to take this opportunity to express my heartfelt
gratitude to my academic advisor Prof. Ji-Xin Cheng, for his constant guidance and
continuous support during the past six years. His passion to science and rigorous
scholarship set an excellent example for me to pursue my academic career.
I sincerely thank my graduate committee members: Prof. Ji-Xin Cheng, Prof.
Sophie. Lelièvre, Prof. Ignacio Camarillo, Prof. Zheng Ouyang, and Prof. Young Kim,
for their supports and kindly help in my Ph.D. study and future career.
Special thanks to Prof. Timothy Ratliff and Prof. Xiaoqi Liu for their advice on
prostate cancer biology; and Prof. Sophie A. Lelièvre for her advice on breast cancer
biology.
In addition, I’d like to thank all the current and former members in Cheng’s group,
who I have had the great pleasure of working with. In particular, I am grateful to Dr.
Haifeng Wang, Dr. Thuc Le, Dr. Mihail Slipchenko, Dr. Li Li, Dr. Hongtao Chen, Dr.
Brandon Huff, Dr. Yan Fu, Dr. Yunzhou shi, Dr. Ling Tong, Dr. Hanwei Wang, Dr.
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Jiabin Zhu, and Dr. Yookyung Jung for all of their insightful comments on my research. I
also thank team colleagues Junjie Li, Hyeon Jeong Lee, Seung-Young Lee, Bing Song,
Leelyn Chong, Pu Wang, Delong Zhang, Wei Wu, Nan Lin, Hunter Cooke, Anna
Sullivan, and many of my friends for all the great knowledge we have shared with.
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Furthermore, I greatly appreciate Prof. Timothy Ratliff, Prof. Sophie Lelièvre,
Prof. Xiaoqi Liu, Prof. Michael Koch, Prof. Timothy Masterson, Dr. Liang Cheng, Dr.
Juan Manuel Cárdenas-Mora, and Dr. Tiao Shao for great collaborations that lead to
fruitful research achievements. I thank Prof. Kimberly Buhman, Prof. Chang-Deng Hu,
Prof. Scott Crist, Prof. James Fleet, Prof. Beth Pflug, and Prof. Graham Cooks for their
constructive comments on my PhD work.
I thank Mrs. Sandra Torregrosa-Allen from Purdue Cancer Center; Mrs. Debra
Sherman from Life Science Microscopy Facility; Tracy and Carol from Histopathology
Laboratory, and Dr. Amber Jannasch from Bindley Bioscience Center for their technical
support.
Finally, I give my greatest appreciation to my husband, Yanjie Zhao, for his
endless support and love. I also thank my parents and parents-in-law for their unlimited
love. I would also thank my son for bringing all the happiness to my life.

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TABLE OF CONTENTS
Page
LIST OF TABLES ............................................................................................................. ix
LIST OF FIGURES ............................................................................................................ x
NOMENCLATURE ........................................................................................................ xix
ABSTRACT ................................................................................................................... xxiv
CHAPTER 1. INTRODUCTION ............................................................................. 1
1.1 Fat and Cancer ........................................................................................... 3
1.1.1 Whole-body Fat Metabolism and Cancer ........................................... 3
1.1.2 Intracellular Fatty Acid Metabolism and Cancer ............................... 4
1.1.2.1 De Novo Fatty Acid Synthesis in Cancer Cells ................................. 5
1.1.2.2 Lipid Hydrolysis in Cancer Cells ....................................................... 8
1.1.2.3 FFAs Promote Cancer Development ................................................. 8
1.1.2.4 Neutral Fat Storage in Cancer Cells................................................... 9
1.2 Cholesterol and Cancer ........................................................................... 10
1.2.1 Whole-body Cholesterol Metabolism and Cancer ........................... 10
1.2.1.1 Dietary Cholesterol Intake and Cancer Risk .................................... 11
1.2.1.2 Blood Cholesterol Levels and Cancer Risk ..................................... 12
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1.2.1.3 Statin Use and Cancer Risk.............................................................. 15
1.2.2 Intracellular Cholesterol Metabolism and Cancer ............................ 17
1.2.2.1 Cholesterol Synthesis, Uptake, and Efflux in Cancer Cells............. 18
1.2.2.2 Cholesterol Homeostasis in Cancer Cells ........................................ 19
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1.2.2.3 Cholesterol as a Precursor of Steroidogenesis in Cancer Cells ....... 20
1.2.2.4 Cholesterol-rich Membrane Domains (Lipid Raft) in Cancer Cells 20
1.2.2.5 Cholesterol Esterification and Storage in Cancer Cells ................... 21
1.3 Current Analytical Tools for Lipid Research .......................................... 22
1.4 Label-free Spectroscopic Imaging of Lipids in Single Live Cells .......... 23
CHAPTER 2. ALTERED CHOLESTEROL METABOLISM: NEW AVENUE TO
DIAGNOSIS AND TREATMENT OF HUMAN PROSTATE CANCER ..................... 26
2.1 Introduction ............................................................................................. 27
2.2 Experimental Section .............................................................................. 29
2.2.1 Human Prostate Tissue Specimens .................................................. 29
2.2.2 Cell Culture ...................................................................................... 29
2.2.3 Chemicals ......................................................................................... 30
2.2.4 Label-free Spectroscopic Imaging ................................................... 30
2.2.5 Fluorescence Imaging of LDL Uptake ............................................. 31
2.2.6 Cell Viability Assay ......................................................................... 32
2.2.7 Cell Cycle Analysis .......................................................................... 32
2.2.8 Migration and Invasion Assays ........................................................ 32
2.2.9 ESI-MS Measurement of Lipid Extraction ...................................... 33
2.2.10 Biochemical Measurement of Cellular Lipids ................................. 33 vi

2.2.11 LC-MS Analysis of AA .................................................................... 33
2.2.12 Immunoblotting ................................................................................ 34
2.2.13 IHC ................................................................................................... 34
2.2.14 RNA Interference ............................................................................. 34
2.2.15 Treatment of PCa in a Mouse Xenograft Model .............................. 34
2.2.16 Statistical Analysis ........................................................................... 35
2.3 Results ..................................................................................................... 36
2.3.1 Aberrant Accumulation of Esterified Cholesterol in Advanced
Human PCa Revealed by Raman Spectromicroscopy ..................... 36
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2.3.2 CE Accumulation in PCa Is Not Correlated with Androgen
Signaling .......................................................................................... 42
2.3.3 CE Accumulation Is Driven by Loss of PTEN and Consequent
Upregulation of PI3K/AKT/mTOR/SREBP Pathway ..................... 45
2.3.4 CE Accumulation in PCa cells Arises from Enhanced Uptake of
Exogenous LDL ............................................................................... 48
2.3.5 CE Accumulation in PCa Cells Requires Cholesterol
Esterification by ACAT-1 ................................................................ 50
2.3.6 Pharmacological Depletion of CE Storage Impairs PCa
Aggressiveness ................................................................................. 52
2.3.7 CE Depletion Impairs PCa Growth by Limiting Uptake of
Essential Fatty Acids........................................................................ 57
2.4 Discussion ............................................................................................... 59
CHAPTER 3. LABEL-FREE ANALYSIS OF BREAST TISSUE POLARITY BY
RAMAN IMAGING OF LIPID PHASE .......................................................................... 66
3.1 Introduction ............................................................................................. 67
3.2 Experimental Section .............................................................................. 72
3.2.1 3D Culture of Human Mammary Acini ........................................... 72
3.2.2 Immunostaining ................................................................................ 73
3.2.3 Compound Raman Microscopy and Spectrum Analysis .................. 74
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3.2.4 Statistical Analysis ........................................................................... 78 i
3.3 Results and Discussion ............................................................................ 79
CHAPTER 4. LABEL-FREE IDENTIFICATION OF MAMMARY ADENOMA
AND ADENOCARCINOMA BY COHERENT ANTI-STOKES RAMAN
SCATTERING AND SUM FREQUENCY GENERATION IMAGING ........................ 95
4.1 Introduction ............................................................................................. 96
4.2 Experimental Section ............................................................................ 100
4.2.1 Animal Model and Tissue Specimens ............................................ 100
4.2.2 Histological Analysis ..................................................................... 100
4.2.3 CARS/SFG Imaging System .......................................................... 101
4.2.4 Imaging Conditions ........................................................................ 102
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4.2.5 Quantitative Analysis of Collagen Density Surrounding Tumor
Mass ............................................................................................... 103
4.3 Results ................................................................................................... 103
4.3.1 Significant Components of Mammary Tissues Visualized with
Label-free CARS/SFG Imaging ..................................................... 103
4.3.2 CARS/SFG Imaging of Normal Tissue, Adenoma, and
Adenocarcinoma of Mammary Glands in Histology Samples ...... 105
4.3.3 CARS/SFG Imaging of Intact Normal tissue, Adenoma, and
Adenocarcinoma of Mammary Glands .......................................... 106
4.3.4 Quantitative Analysis of Collagen Density Surrounding Tumor
Mass ............................................................................................... 110
4.4 Discussion ............................................................................................. 111
CHAPTER 5. OUTLOOK .................................................................................... 114
5.1 Cholesterol Metabolism in Cancer ........................................................ 114
5.2 Membrane Lipids in Cancer .................................................................. 115
LIST OF REFERENCES ................................................................................................ 118
VITA ............................................................................................................................... 147
PUBLICATIONS ............................................................................................................ 148

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