ALTERATIONS IN GENE EXPRESSION AS A RESPONSE OF TUMOR CELLS TO STRESSES

219 Pages·2013·4.68 MB·English

The Pennsylvania State University

The Graduate School

Department of Pharmacology

ALTERATIONS IN GENE EXPRESSION AS A RESPONSE
OF TUMOR CELLS TO STRESSES

A Dissertation in

Pharmacology

by

Kathryn Joyce Huber-Keener

 2012 Kathryn Joyce Huber-Keener

Submitted in Partial Fulfillment
of the Requirements
for the Degree of

Doctor of Philosophy

May 2013
The dissertation of Kathryn Joyce Huber-Keener was reviewed and approved* by the following:

Jin-Ming Yang
Professor of Pharmacology
Dissertation Advisor
Chair of Committee

Willard M. Freeman
Associate Professor of Pharmacology

Rongling Wu
Professor of Statistics

Robert G. Levenson
Professor of Pharmacology

Andrea Manni
Professor of Medicine

Jong K. Yun
Director of the Pharmacology Graduate Program

*Signatures are on file in the Graduate School

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ABSTRACT

Solid cancers are the 2nd leading cause of death in adults in the United States.
Understanding the mechanisms by which cancer cells survive under stress is pivotal to
decreasing this statistic. Cancer cells are constantly under stress, a factor which needs to
be taken into consideration during the treatment of solid cancers. Both intrinsic and
extrinsic stresses impact the development and progression of neoplasms, down to the
level of individual proteins and genes. Stresses like nutrient deficiency, hypoxia, acidity,
and the immune response are present during normal tumor growth and throughout
treatment. Tumor cells that survive these stresses are more adept at surviving hostile
conditions and are more resistant to current therapies. Stress, therefore, shapes the tumor
cell population. Gene expression alterations are the driving feature behind the adaptive
ability of cancer cells to these stresses, and therefore, careful examination of these gene
expression changes must be undertaken in order to develop effective therapies for solid
cancers.
In the present investigations, we first explore the global alterations of gene
expression in tamoxifen resistance. Resistance to tamoxifen (Tam), a widely used
antagonist of the estrogen receptor (ER), is a common obstacle to successful breast
cancer treatment. While adjuvant therapy with Tam has been shown to significantly
decrease the rate of disease recurrence and mortality, recurrent disease occurs in one third
of patients treated with Tam within 5 years of therapy. A better understanding of gene
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expression alterations associated with Tam resistance will facilitate circumventing this
problem. Using a next generation sequencing approach and a new bioinformatics model,
we compared the transcriptomes of Tam-sensitive and Tam-resistant breast cancer cells
for identification of genes involved in the development of Tam resistance. We identified
differential expression of 1215 mRNA and 513 small RNA transcripts clustered into ERα
functions, cell cycle regulation, transcription/translation, and mitochondrial dysfunction.
The extent of alterations found at multiple levels of gene regulation highlights the ability
of the Tam-resistant cells to modulate global gene expression. Alterations of small
nucleolar RNA, oxidative phosphorylation, and proliferation processes in Tam-resistant
cells present areas for diagnostic and therapeutic tool development for combating
resistance to this anti-estrogen agent.
After such a global exploration of cancer cell responses to stress, we next
investigated a mechanism of cancer cell survival by exploring the alterations in protein
synthesis inhibitor eEF-2K. Studies show that EF-2K plays a role in cell survival through
this inhibition of protein synthesis and that its protein levels are increased in cancer.
Post-translational modification of translation machinery is important for its regulation
and could be critical for survival of cancer cells encountering stress. Thus, the purpose of
our study is to examine the regulation of EF-2K during stress with a focus on the
phosphorylation status and stability of EF-2K protein in cancer cells. Using two human
glioma cell lines (T98G and LN229), we have found a 2-5 fold increase in EF-2K
expression and activity under stress conditions of nutrient deprivation and hypoxia.
mRNA levels are only transiently increased and shortly return to normal, while EF-2K
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protein levels continue to increase after further exposure to stress. This result could be
explained by decreased turnover of EF-2K protein, which has a normal half-life of ~ 6-8
hours in glioma cells, so cycloheximide experiments were used to examine the effect of
stress on EF-2K protein stability. A seemingly paradoxical decrease in EF-2K stability
(t = 2-4h) was found when glioma cells were subjected to stress despite increased
1/2
protein expression. Phosphorylation may play a role in this altered protein stability as
EF-2K has multiple phosphorylation sites that are phosphorylated by the mTOR/S6
kinase (Ser78 and Ser366) and AMP kinase (Ser398), pathways which would be affected
by stress. Therefore, phosphorylation-defective mutants of EF-2K were made to examine
the effect of phosphorylation at these sites on EF-2K protein stability. We discovered
that the AMP kinase site was pivotal to protein stability as the S398A mutant half-life
increased to greater than 24 hours under both normal and stress conditions. Mutating the
mTOR pathway sites made EF-2K protein more stable under normal conditions (t >
1/2
24h) but decreased to normal levels under stress conditions (t = 8h). Inhibiting the
1/2
mTOR pathway with rapamycin treatment increased protein expression ~ 5 fold and
increased EF-2K stability in these mutants under all culture conditions. These data
indicate that EF-2K is regulated at multiple levels with phosphorylation playing an
important role in protein turnover. The unexpected decrease in EF-2K protein stability
during stress may be a compensatory mechanism for an additional level of regulation at
the post-transcriptional level that increases EF-2K translation. Due to the importance of
translation regulation during stress, it is reasonable to have increased translation of a
regulator of protein synthesis while decreasing the same protein’s stability in order to
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quickly adapt to changing nutrient levels. Further studies will examine the post-
transcriptional regulation of EF-2K during stress as these data demonstrate its complex
and tight regulation. Understanding the regulation of EF-2K could lead to therapeutics
targeting EF-2K that could potentially render cancer cells intolerant to stress and
susceptible to current treatments.
Together, our results represent the global impact that stress can have on cancer
cells. Our NGS study exemplifies the complexity of alterations caused by exogenous
stresses on cancer cells. The wide variety of gene expression changes from genes
involved in proliferation and survival to those that regulate energy metabolism and post-
transcriptional indicate that stress selection can alter the overall functioning of the cancer
cell. The focused eEF-2K study indicates how tightly and complexly the protein
synthesis regulator is controlled, which has broad implications for global and specific
translation of the numerous gene transcript alterations caused by stress. Understanding
how gene expression and proteins are altered in cancer cells by different intrinsic and
extrinsic stresses will help current and future researchers to develop novel and more
effective methods of overcoming cancer cell survival and resistance to current treatments.

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TABLE OF CONTENTS
ABSTRACT ............................................................................................................................. iii
LIST OF FIGURES ................................................................................................................. xi
LIST OF TABLES ................................................................................................................... xii
LIST OF ABBREVIATIONS .................................................................................................. xiii
ACKNOWLEDGEMENTS ..................................................................................................... xvi
EPIGRAPH .............................................................................................................................. xx
Chapter 1 .................................................................................................................................. 1
Cellular stress, gene expression, and cancer development and progression ............................ 1
1.1. Overview of Solid Cancers ...................................................................................... 1
1.1.1. Breast Cancer ................................................................................................. 3
1.1.1.1. Breast cancer statistics 3
1.1.1.2. Breast cancer progression 4
1.1.1.3. Breast cancer etiology 6
1.1.2. Glioma ............................................................................................................ 6
1.1.2.1. Glioma statistics 9
1.1.2.2. Glioma progression 9
1.1.2.3. Glioma etiology 10
1.2. Cellular stress ........................................................................................................... 11
1.2.1. Intrinsic stresses during tumor development and progression ....................... 13
1.2.2. Extrinsic stress caused by cancer treatments .................................................. 18
1.2.2.1. Surgery 19
1.2.2.2. Radiation 19
1.2.2.1. Chemotherapy 20
1.2.2.3. Targeted therapies 22
1.2.3. Molecular markers of stress ........................................................................... 23
1.3. Common mechanisms of cancer therapy resistance ................................................. 28
1.3.1. Multi-drug resistance ...................................................................................... 29
1.3.2. Cancer stem cells ............................................................................................ 29
1.3.3. Autophagy ...................................................................................................... 30
1.3.4. Modifications in signaling pathways and gene expression ............................ 31
1.3.4. Alterations in gene expression ....................................................................... 33
1.4. Regulation of gene expression .................................................................................. 34
1.4.1. Transcriptional control ................................................................................... 34
1.4.2. Regulation of translation ................................................................................ 38
1.4.3. Post-transcriptional and post-translational modifications .............................. 41
1.4.3.1. Covalent additions 41
1.4.3.2. Cleavage reactions 42
1.5. Use of next generation sequencing in gene expression studies ................................. 43
1.5.1. Sequencing Process ........................................................................................ 44
1.5.2. Platforms ........................................................................................................ 45
1.5.2.1. Illumina HiSeq and Genome Analyzer 46
1.5.2.2. Roche 454 Pyrosequencing 47
1.5.2.3. Helicos Heliscope: 47
1.5.2.4. Applied Biosystems SOLiD Sequencing: 47
1.5.2.5. Life Technologies Ion Torrent Sequencing 48
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1.5.3. Advantages and disadvantages of NGS .......................................................... 48
1.5.4. Uses in gene expression studies ..................................................................... 51
1.5.4.1. Chromosomal rearrangements 51
1.5.4.2. Transcriptomes, exomes, and gene signatures 52
1.5.4.3. Epigenome 53
1.5.4.3. Binding sites for transcription factors 54
1.6. Significance of my research project .......................................................................... 54
Chapter 2 Differential gene expression in tamoxifen-resistant breast cancer cells revealed by a
new analytical model of RNA-Seq data ........................................................................... 56
2.1. Abstract ..................................................................................................................... 56
2.2. Introduction ............................................................................................................... 57
2.2.1. Estrogen and Estrogen Receptor .................................................................... 57
2.2.1.1. ER structure 58
2.2.1.2. Regulation and Activity of ER 60
2.2.2. Estrogen receptor in breast cancer .................................................................. 61
2.2.3. Endocrine therapy in breast cancer ................................................................ 62
2.2.3.1. Aromatase Inhibitors (AIs) 62
2.2.3.2. Selective estrogen receptor down-regulators (SERDs) 62
2.2.3.3. Selective estrogen receptor modulators (SERMs) 63
2.2.3. Tam resistance in breast cancer ...................................................................... 64
2.2.5. Technology used for our study of Tam resistance.......................................... 67
2.3. Rationale ................................................................................................................... 69
2.4. Experimental design .................................................................................................. 70
2.4.1. Cell lines and reagents .................................................................................... 70
2.4.2. RNA preparation ............................................................................................ 71
2.4.3. Library preparation for SOLiD™ NGS sequencing ....................................... 71
2.4.4. Library preparation for small RNA sequencing ............................................. 72
2.4.5. Sequencing ..................................................................................................... 73
2.4.6. NGS mapping and expression ........................................................................ 73
2.4.7. qRT-PCR validation ....................................................................................... 74
2.4.8. Statistical models ............................................................................................ 74
2.4.9. Expression Analysis ....................................................................................... 76
2.4.10. SIRT3 expression and cell growth assays .................................................... 77
2.5. Results and Discussion .............................................................................................. 77
2.5.1. Clustering of gene expression data ................................................................. 77
2.5.1.1. Rationale behind mathematical model 78
2.5.1.2. Validation and comparison of gene expression levels between Tam-
sensitive and Tam-resistant breast cancer cells. 79
2.5.1.3. Phenotypic plasticity clustering analysis. 85
2.5.1.4. Effects of Tam resistance on smRNA expression and clustering. 90
2.5.1.5. Gene ontology and clustering analysis of mRNA expression. 92
2.5.2. Comparison to traditional analysis methods and previous studies ................. 97
2.5.3. Effects of SIRT3 expression on Tam resistance ............................................ 102
2.6. Conclusions ............................................................................................................... 104
Chapter 3 .................................................................................................................................. 107
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Phosphorylation of elongation factor-2 kinase and the stability of the enzyme under various stress
conditions ......................................................................................................................... 107
3.1. Abstract ..................................................................................................................... 107
3.2. Introduction ............................................................................................................... 109
3.2.1. Eukaryotic elongation factor-2 kinase (eEF-2K) ........................................... 109
3.2.1.1. eEF-2K structure 111
3.2.1.2. Regulation of protein synthesis by eEF-2K 112
3.2.1.3 Regulation of eEF-2K 112
3.2.1.3.1. Calcium/Calmodulin and autophosphorylation 112
3.2.1.3.2. cAMP-dependent protein kinase (PKA) regulation of eEF-2K 114
3.2.1.3.3. mammalian target of rapamycin (mTOR) regulation of eEF-2K 115
3.2.1 3.4. Adenosine monophosphate-activated protein kinase (AMPK)
pathway regulation of eEF-2K 117
3.2.1.3.5. Multiple stress response pathways regulation of eEF-2K 118
3.2.2. Stability of eEF-2K protein ............................................................................ 121
3.2.3. eEF-2K expression ......................................................................................... 123
3.2.3.1. eEF-2K expression in cancer 123
3.2.3.2. Correlation with stress and cellular energy 124
3.3. Rationale ................................................................................................................... 126
3.4. Experimental design .................................................................................................. 127
3.4.1. Cell lines and culture. ..................................................................................... 127
3.4.2. Reagents and antibodies ................................................................................. 128
3.4.3. Stress Conditions ............................................................................................ 128
3.4.4. Real time RT-PCR ......................................................................................... 128
3.4.5. EF-2K phosphorylation-defective mutants .................................................... 129
3.4.6. Preparation of cellular extracts and Western blot analysis ............................. 129
3.4.7. Mining of mRNA functional elements ........................................................... 130
3.5. Results ....................................................................................................................... 130
3.5.1. eEF-2K levels are increased by metabolic stress in glioma cells ................... 130
3.5.2. eEF-2K protein turnover is increased by metabolic stress ............................. 132
3.5.4. Phosphorylation sites differentially regulate eEF-2K turnover ...................... 134
3.5.5. Effects of inhibition of upstream signaling cascades on eEF-2K stability ..... 135
3.5.6. Determination of RNA elements important for translation of eEF-2K .......... 138
3.6. Discussion ................................................................................................................. 139
Chapter 4: Discussion of the current Studies; .......................................................................... 146
Altered signaling and gene expression in cancer cells under stress ......................................... 146
4.1. Preamble.................................................................................................................... 146
4.2. Tam resistance in breast cancer ................................................................................. 147
4.2.1. Clinical Studies .............................................................................................. 147
4.2.2. Potential biomarkers of Tam resistance ......................................................... 149
4.2.3. Current findings: major gene expression changes found by next generation
sequencing ........................................................................................................ 150
4.2.3.1. The importance of analytical methods in determining differential gene
expression. 151
4.2.3.2. Important ontological groups altered by Tam resistance 152
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4.2.3.3. Importance of small RNA in Tam resistance 153
4.2.5. Directions for future studies ........................................................................... 155
4.2.5.1. Moving from preclinical to clinical studies: the use of NGS in Tam
resistant breast cancer tumor samples 157
4.2.5.2. SIRT3 findings and future experiments 159
4.2.5.3. Mitochondria and energy metabolism 161
4.3. Regulation of eEF-2K in response to metabolic stress ............................................. 162
4.3.1. Protein synthesis in cancer cells ..................................................................... 163
4.3.2. Metabolic stress effects on protein phosphorylation ...................................... 163
4.3.3. Current findings: Phosphorylation at specific sites of eEF-2K differentially
modulates its turnover ...................................................................................... 164
4.3.3.1. Cellular stress alters eEF-2K protein stability 165
4.3.3.2. Identification of upstream signaling pathways and phosphorylation sites
affecting eEF-2K protein turnover 165
4.3.4. Directions for further studies .......................................................................... 167
4.3.4.1. Identification of additional upstream pathways that phosphorylate eEF-2K
under stressful conditions 168
4.3.4.2. Examination of discordant mRNA and protein levels 169
4.3.4.3. Effect of eEF-2K phosphorylation on specific versus global protein
synthesis 170
4.4. Epilogue: The importance of stress response in cancer research .............................. 171
REFERENCES................................................................................................................. 174

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