Inflammatory cytokine signaling contributes to Erlotinib resistance in head and neck squamous cell

129 Pages·2017·3.79 MB·English

by  Aditya Stanam

University of Iowa
Iowa Research Online
Theses and Dissertations
Spring 2016
Inflammatory cytokine signaling contributes to
Erlotinib resistance in head and neck squamous cell
carcinoma
Aditya Stanam
University of Iowa
Copyright 2016 Aditya Stanam
This dissertation is available at Iowa Research Online: https://ir.uiowa.edu/etd/3194
Recommended Citation
Stanam, Aditya. "Inflammatory cytokine signaling contributes to Erlotinib resistance in head and neck squamous cell carcinoma." PhD
(Doctor of Philosophy) thesis, University of Iowa, 2016.
https://doi.org/10.17077/etd.n8bdd9wz
Follow this and additional works at:https://ir.uiowa.edu/etd
Part of thePathology Commons
INFLAMMATORY CYTOKINE SIGNALING CONTRIBUTES TO ERLOTINIB
RESISTANCE IN HEAD AND NECK SQUAMOUS CELL CARCINOMA

by
Aditya Stanam

A thesis submitted in partial fulfillment
of the requirements for the Doctor of Philosophy
degree in Human Toxicology in the
Graduate College of
The University of Iowa

May 2016

Thesis Supervisor: Assistant Professor Andrean Simons-Burnett
Graduate College
The University of Iowa
Iowa City, Iowa

CERTIFICATE OF APPROVAL

____________________________

PH.D. THESIS

_________________

This is to certify that the Ph.D. thesis of

Aditya Stanam

has been approved by the Examining Committee for
the thesis requirement for the Doctor of Philosophy degree
in Human Toxicology at the May 2016 graduation.

Thesis Committee: ____________________________________________
Andrean L. Simons-Burnett, Thesis Supervisor

____________________________________________
Weizhou Zhang

____________________________________________
Katherine Gibson-Corley

____________________________________________
Bryan G. Allen

____________________________________________
Peter S. Thorne
To my wife Shivani, parents Gopal and Hymavathi, sister Anusha, and uncle Raghu for their
everlasting love, support, and encouragement to help me accomplish my dissertation goals.
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ACKNOWLEDGEMENTS

This dissertation would not have been possible without the help of so many people in so
many ways. Foremost, I would like to express my sincere gratitude to my thesis supervisor Dr.
Andrean Simons-Burnett whose continuous support, guidance, valuable comments and
suggestions benefitted me in the successful completion of my thesis work.
I thank my committee members: Dr. Weizhou Zhang: for allowing me to participate in
his lab meetings, journal clubs and for providing useful suggestions; Dr. Katherine Gibson-
Corley: for providing valuable insights into tumor histopathology both through her course (on
translational histopathology) and personal discussions; Drs. Peter Thorne and Bryan Allen for
sharing their precious time and positive insights.
I thank my fellow labmates in Dr. Simons-Burnett lab: Laurie, Madelyn, and Adam, for
their help with several experiments and for promoting positive atmosphere of the lab. Especially
Laurie and Madelyn for our discussions on wide range of topics, exchanges of knowledge, skills
which helped enrich my experience.
Special thanks to my dear friends Jae Hong Park, Xuefang Jing, Ryan Kolb, and Nerwana
Metwali, who were always willing to help and gave their best suggestions.
I would also like to thank Dr. Tom Peters and Pam Geyer for sharing their success
principles, which helped me to meet my academic objectives, enjoy the beauty of science and
creativity.
Last but not the least, I would like to thank my family: my wife Shivani, my parents
Gopal and Hymavathi, my sister Anusha for their everlasting love, support, and encouragement
throughout my life.

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ABSTRACT

Resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR TKIs) is
a major obstacle in the success of head and neck cancer therapy. Despite efforts by several
groups to understand the mechanisms of resistance to tyrosine kinase inhibitors such as erlotinib,
there has been little success in improving the patient survival. Given that there are a number of
ongoing clinical trials testing the efficacy of erlotinib in head and neck cancer, it is essential to
investigate the novel mechanisms of erlotinib resistance to improve its efficacy and patient
survival. This dissertation addresses this issue of erlotinib resistance in head and neck cancer,
underscoring the role of inflammatory cytokine signaling. Chapter 1 introduces the problem of
erlotinib resistance and discusses the potential link between inflammatory signaling and cancer
progression and erlotinib resistance in head and neck squamous cell carcinoma. Chapter 2
discusses the role of the cytokine interleukin-6 signaling in acquired resistance to erlotinib in
head and neck squamous cell carcinoma. Chapter 3 describes the role of IL-1 signaling in
acquired resistance to erlotinib in head and neck squamous cell carcinoma. Chapter 4 discusses
the specific role of IL-1M (an agonistic ligand for IL-1 signaling) in acquired resistance to
erlotinib in head and neck squamous cell carcinoma. Chapter 5 discusses ideas to test for future
work in this field. Altogether, this dissertation endeavors to emphasize the contributory role of
inflammatory cytokine signaling in erlotinib resistance in head and neck squamous cell
carcinoma so that it helps in the development of effective anti-cancer therapies and biomarkers
of resistance and/or response in HNSCC.

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PUBLIC ABSTRACT

The majority (up to 90%) of head and neck cancers (HNCs) have increased levels and
activity of a molecule called ‘epidermal growth factor receptor’ (EGFR). Based on this, drugs
that inhibit EGFR have been development. Erlotinib (Tarceva®) is an EGFR-inhibitor to which,
surprisingly, only a small subset (~5%) of head and neck cancer patients showed response.
However, the majority of respondents develop resistance to erlotinib relatively quickly.
Therefore, in order to improve the efficacy of erlotinib, it is essential to understand the
mechanisms of erlotinib resistance in HNC patients. This thesis work addresses the issue of
erlotinib resistance in HNC. Inflammation is known to play a critical role in the progression of
various cancers including HNC. Chapter 1 introduces the problem of erlotinib resistance and
discusses the potential link between inflammation, cancer progression and erlotinib resistance in
HNC. Chapter 2 discusses the role of an inflammatory molecule called interleukin-6 (IL-6) in
acquired resistance to erlotinib in HNC. Chapter 3 describes the signaling role of another
inflammatory molecule called interleukin-1 (IL-1; fever molecule) in acquired resistance to
erlotinib in HNC. Chapter 4 discusses the specific role of an inflammatory molecule called
interleukin-1 alpha (IL-1M), which triggers IL-1 signaling in acquired resistance to erlotinib in
HNC. Chapter 5 discusses ideas to test for future work in this field. Overall, this thesis work
uncovers the contributory role of inflammation (or inflammatory molecules) in resistance to
erlotinib in HNC. We hope these results would propel the development of effective anti-cancer
therapies in HNC patients.

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TABLE OF CONTENTS
LIST OF TABLES ...........................................................................................................................x
LIST OF FIGURES ....................................................................................................................... xi
CHAPTER 1. INTRODUCTION ....................................................................................................1
1.1. Head and neck cancer ........................................................................................................ 1
1.1.1. Definition ......................................................................................................................1
1.1.2. Problem statement ........................................................................................................1
1.1.3. Risk factors ...................................................................................................................1
1.1.4. HNSCC classification ...................................................................................................2
1.1.5. Staging and grading of HNSCC ...................................................................................2
1.1.6. Treatment of HNSCC ...................................................................................................2
1.1.7. Targeted therapy in HNSCC ........................................................................................3
1.2. Problem with EGFR inhibitors .......................................................................................... 4
1.2.1. Current update on the problem of erlotinib resistance .................................................5
1.3. Role of inflammation in cancer .......................................................................................... 5
1.3.1. Definition of inflammation ...........................................................................................5
1.3.2. Inflammation and cancer progression ...........................................................................6
1.3.3. Inflammation and erlotinib resistance ...........................................................................7
1.4. Prior work in our laboratory .............................................................................................. 7
1.4.1. IL-6 signaling and its role in cancer .............................................................................8
1.4.2. IL-1 signaling and its role in cancer ...........................................................................10
1.5. Hypothesis and specific aims ........................................................................................... 13
1.6. References ........................................................................................................................ 13
CHAPTER 2: UPREGULATED INTERLEUKIN-6 EXPRESSION CONTRIBUTES TO
ERLOTINIB RESISTANCE IN HEAD AND NECK SQUAMOUS CELL CARCINOMA ......27
2.1. Abstract ............................................................................................................................ 27
2.2. Introduction ...................................................................................................................... 28
2.3. Materials and Methods ..................................................................................................... 29
2.3.1. Cell lines and cell culture ............................................................................................29
2.3.2. Drugs ...........................................................................................................................29
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2.3.3. Establishment of erlotinib-resistant HNSCC cell lines ...............................................30
2.3.4. Cell viability assay ......................................................................................................30
2.3.5. RNA isolation and gene expression profiling .............................................................30
2.3.6. Quantitative reverse transcription polymerase chain reaction (q RT-PCR) ...............31
2.3.7. IL-6 ELISA .................................................................................................................31
2.3.8. Western blot analysis ..................................................................................................32
2.3.9. Tumor cell implantation ..............................................................................................32
2.3.10. Tumor measurements ................................................................................................32
2.3.11. In vivo drugs administration .....................................................................................32
2.3.12. Statistical analysis .....................................................................................................33
2.4. Results .............................................................................................................................. 33
2.4.1. Validation of erlotinib resistance ................................................................................33
2.4.2. Hierarchical cluster analysis .......................................................................................34
2.4.3. Enrichment analysis ....................................................................................................34
2.4.4. Network analysis .........................................................................................................35
2.4.5. Differential gene expression in erlotinib-resistant vs sensitive HNSCC cells ............36
2.4.6. Combined inhibition of IL-6 and EGFR signaling to overcome erlotinib
resistance ....................................................................................................................36
2.5. Discussion ........................................................................................................................ 37
2.6. Grant support ................................................................................................................... 40
2.7. Acknowledgments............................................................................................................ 40
2.8. References ........................................................................................................................ 40
CHAPTER 3: INTERLEUKIN-1 BLOCKADE OVERCOMES ERLOTINIB RESISTANCE
IN HEAD AND NECK SQUAMOUS CELL CARCINOMA ......................................................59
3.1. Abstract ............................................................................................................................ 59
3.2. Introduction ...................................................................................................................... 60
3.3. Materials and Methods ..................................................................................................... 60
3.3.1. Cell lines and culture ..................................................................................................62
3.3.2. Drugs ...........................................................................................................................62
3.3.3. Cell viability assay ......................................................................................................63
3.3.4. Gene expression profiling ...........................................................................................63
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3.3.5. Enrichment analysis ....................................................................................................63
3.3.6. Reverse transcription PCR ..........................................................................................63
3.3.7. ELISA .........................................................................................................................64
3.3.8. Xenograft experiments ................................................................................................64
3.3.9. In vivo drug administration .........................................................................................64
3.3.10. Tumor histopathology and Immunohistochemistry ..................................................65
3.3.11. Bio-Plex assay ...........................................................................................................65
3.3.12. TCGA analysis ..........................................................................................................66
3.3.13. Statistical analysis .....................................................................................................66
3.4. Results .............................................................................................................................. 67
3.4.1. A proinflammatory gene signature is associated with erlotinib resistance .................67
3.4.2. Increased expression of IL-1 pathway genes is associated with erlotinib
resistance ....................................................................................................................68
3.4.3. Increased expression of IL-1 pathway ligands may be associated with erlotinib
resistance ....................................................................................................................68
3.4.4. IL-1 blockade does not affect cell viability in vitro ....................................................69
3.4.5. IL-1 blockade overcomes erlotinib resistance in vivo. ...............................................69
3.4.6. The anti-tumor effect of anakinra is associated with a reduction in angiogenesis .....70
3.4.7. Anakinra modulates circulating levels of proinflammatory cytokines in mice
bearing ER-SQ20B xenografts ...................................................................................71
3.4.8. IL1A and IL1RAP are association with HNSCC patient survival ..............................71
3.5. Discussion ........................................................................................................................ 72
3.6. Acknowledgements .......................................................................................................... 72
3.7. References ........................................................................................................................ 74
CHAPTER 4. ROLE OF IL-1M ON ERLOTINIB RESISTANCE IN HEAD AND NECK
SQUAMOUS CELL CARCINOMA .............................................................................................94
4.1. Introduction ...................................................................................................................... 94
4.2. Methods............................................................................................................................ 95
4.2.1. Cell viability assay ......................................................................................................95
4.2.2. IL-6 and IL-8 ELISA ..................................................................................................95
4.2.3. Xenograft experiment .................................................................................................96
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