Understanding the biology of transforming growth factor-β in colorectal cancers

400 Pages·2016·12.23 MB·English

by  Harish Reddy

Understanding the biology of
transforming growth factor-β in colorectal
cancers

Harish Reddy Cheruku
Master of Biotechnology, Macquarie University

Faculty of Medicine and Health Sciences
Macquarie University, Sydney, Australia

A thesis submitted to Macquarie University in fulfilment of the
requirements for the degree of Doctor of Philosophy.
July 2015
[ii]
Dedication
With love and gratitude

To my parents,
whose support and encouragement
made this thesis possible

&

To Trúc Thanh Lȇ,
whose love and support
make everything seem possible

[iii]
[iv]
TABLE OF CONTENTS
LIST OF TABLES .................................................................................................................................. VII
LIST OF FIGURES ................................................................................................................................ VII
STATEMENT OF DECLARATION ..................................................................................................... VIII
ACKNOWLEDGEMENTS ..................................................................................................................... IX
ABSTRACT .............................................................................................................................................. X
PUBLICATIONS AND CONTRIBUTION STATEMENT .................................................................... XI
PRESENTATIONS /AWARDS ............................................................................................................. XIII
ABBREVIATIONS ................................................................................................................................. XV
CHAPTER 1 INTRODUCTION .............................................................................................................. 1
1.1 COLORECTAL CANCER ................................................................................................................. 2
1.1.1 CANCER – A GLOBAL BURDEN ............................................................................................................... 2
1.1.2 SIGNIFICANCE OF CRC: STATISTICS ...................................................................................................... 3
1.1.3 AETIOLOGY OF CRC.............................................................................................................................. 4
1.1.4 ANATOMY OF THE COLON AND RECTUM ................................................................................................ 5
1.1.5 SYMPTOMS OF CRC............................................................................................................................... 6
1.1.6 STAGING OF CRC .................................................................................................................................. 7
1.1.7 BIOMARKERS ASSOCIATED WITH CRC .................................................................................................. 9
1.1.8 EARLY DIAGNOSIS AND SCREENING FOR CRC ..................................................................................... 10
1.1.9 TREATMENT OF CRC ........................................................................................................................... 20
1.1.10 CARCINOGENESIS AND GENETIC ALTERATIONS DURING CRC ............................................................ 21
1.1.11 METASTASIS ...................................................................................................................................... 24
The Epithelial to mesenchymal transition ............................................................................................... 24
Invasion ................................................................................................................................................... 25
1.1.12 KEY MOLECULES IN CANCER PROGRESSION ....................................................................................... 26
Integrin αvβ6 as an adhesion molecule ................................................................................................... 27
ECM proteolysis and uPAR .................................................................................................................... 29
Growth and signalling factors ................................................................................................................ 31
1.2 TRANSFORMING GROWTH FACTOR-Β .....................................................................................35
1.2.1 TGFΒ SUPERFAMILY OF LIGANDS ........................................................................................................ 35
1.2.2 TGFΒ RECEPTORS ............................................................................................................................... 36
The TGFβ Type III Receptor ................................................................................................................... 37
The TGFβ Type II Receptor .................................................................................................................... 38
The TGFβ Type I Receptor ...................................................................................................................... 38
1.2.3 CANONICAL TGFΒ RECEPTORS ........................................................................................................... 38
1.2.4 TRANSFORMING GROWTH FACTOR-Β AND CANCER ............................................................................. 41
1.2.5 GENETIC ALTERATIONS IN TGFΒ PATHWAY COMPONENTS IN CRC ..................................................... 41
1.2.6 IMPORTANCE OF UNDERSTANDING THE ROLE OF TGFΒ IN CRC .......................................................... 46
1.3 LITERATURE REVIEWS ................................................................................................................47
REVIEW 1: TRANSFORMING GROWTH FACTOR-Β, MAPK AND WNT SIGNALING INTERACTIONS IN
COLORECTAL CANCER. EUPA OPEN PROTEOMICS (2015). IN PRESS, AVAILABLE ONLINE 2 JULY 2015.
(DOI:10.1016/J.EUPROT.2015.06.004). ......................................................................................................... 47
REVIEW 1 – SUPPLEMENTAL FILES ............................................................................................................... 60
REVIEW 2: THE ΑVΒ6 INTEGRIN SETS THE STAGE FOR COLORECTAL CANCER METASTASIS. ......................... 65
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1.4 MASS SPECTROMETRY BASED CANCER PROTEOMICS ...................................................... 86
1.4.1 WHAT IS PROTEOMICS? ....................................................................................................................... 86
1.4.2 COLORECTAL CANCER PROTEOMICS .................................................................................................... 87
1.4.3 PROTEIN SAMPLE PREPARATION AND HANDLING ................................................................................. 91
1.4.4 LC MS/MS BASED PROTEOMICS ......................................................................................................... 92
Sample ionization .................................................................................................................................... 92
Mass Analysers ....................................................................................................................................... 93
1.4.5 SHOTGUN QUANTITATIVE PROTEOMICS USING ITRAQ ....................................................................... 94
1.4.6 BIOINFORMATICS TOOLS FOR DATA ANALYSIS .................................................................................... 96
1.5 AIMS OF THE THESIS .................................................................................................................... 98
CHAPTER 2: METHODS .................................................................................................................... 101
CHAPTER 3 .......................................................................................................................................... 103
3.1 - STUDY I: ............................................................................................................................................ 103
3.1.1 - EXPRESSION OF ΑVΒ6 INTEGRIN ENHANCES BOTH PLASMINOGEN AND LATENT-TRANSFORMING
GROWTH FACTOR-Β1 DEPENDANT PROLIFERATION, INVASION AND ERK1/2 SIGNALLING IN COLORECTAL
CANCER CELLS. .......................................................................................................................................... 103
3.1.2 – SUPPLEMENTAL FILES ..................................................................................................................... 132
3.2 - STUDY II: .......................................................................................................................................... 135
3.2.1 - TRANSFORMING GROWTH FACTOR-Β SIGNALLING INDUCES DIFFERENTIAL PROTEIN EXPRESSION IN
COLON CANCER CELLS THAT VARIES WITH THE LEVEL OF INTEGRIN Β6 EXPRESSION. ................................. 135
3.2.2 – SUPPLEMENTAL FILES ..................................................................................................................... 186
CHAPTER 4 .......................................................................................................................................... 209
4.1 - DOES DIFFERENTIAL EXPRESSION OF CELL-SURFACE UPAR ALTER THE EFFECTS ACTIVE TGFΒ HAS ON
THE COLORECTAL CANCER CELL MEMBRANE PROTEOME? ......................................................................... 209
4.2 – SUPPLEMENTAL FILES ........................................................................................................................ 241
CHAPTER 5 .......................................................................................................................................... 255
5.1 - A NOVEL MULTIPLEXED IMMUNOASSAY IDENTIFIES CEA, IL-8 AND PROLACTIN AS PROSPECTIVE
MARKERS FOR DUKES' STAGES A-D COLORECTAL CANCERS. CLIN PROTEOMICS. APR 8; 12(1):10. DOI:
10.1186/S12014-015-9081-X. ECOLLECTION 2015. ................................................................................... 255
5.2 – SUPPLEMENTAL FILES ........................................................................................................................ 268
CHAPTER 6 .......................................................................................................................................... 321
GENERAL DISCUSSION, FUTURE DIRECTIONS AND CONCLUSION ...................................... 321
6.1 GENERAL DISCUSSION .......................................................................................................................... 322
6.2 PROTEINS AS BIOMARKERS FOR CRC ................................................................................................... 328
6.3 FUTURE DIRECTIONS ............................................................................................................................ 330
6.4 CONCLUSION ........................................................................................................................................ 332
REFERENCES ...................................................................................................................................... 333
APPENDICES ....................................................................................................................................... 353
APPENDIX I – EXPRESSION OF TGFΒ RECEPTORS 1 AND 2 IN THE CELL LINES USED IN THIS THESIS ........... 355
APPENDIX II – CHARACTERIZATION OF THE INTERACTION BETWEEN HETERODIMERIC ALPHAVBETA6
INTEGRIN AND UROKINASE PLASMINOGEN ACTIVATOR RECEPTOR (UPAR) USING FUNCTIONAL PROTEOMICS.
................................................................................................................................................................... 357
APPENDIX III – AN IMPROVED METHOD FOR THE DETECTION AND ENRICHMENT OF LOW-ABUNDANT
MEMBRANE AND LIPID RAFT-RESIDING PROTEINS ....................................................................................... 367
APPENDIX IV – ETHICS APPROVAL ............................................................................................................. 377
APPENDIX V – BIOSAFETY CERTIFICATE .................................................................................................... 382
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LIST OF TABLES
§
Table 1 Comparison of Dukes’ and TNM classification of CRC . .................................................... 8
Table 2 Summary of potential CRC biomarkers identified in the last 10 years. .............................. 11
Table 3 Oncogenes and tumour suppressor genes commonly involved in CRC. ............................ 21
§
Table 4 The TGFβ-superfamily ligands, tissue specificity and their major functions . ................... 32
Table 5 Mechanisms of TGFβ activation under different physiological conditions ........................ 36
Table 6 Receptors of the TGFβ superfamily: ................................................................................... 39
§
Table 7 Smad family proteins . ........................................................................................................ 40
Table 8 Genetic alterations in TGFβ signalling components in CRC. ............................................. 42
§
Table 9 Colorectal cancer cell lines used in this thesis ................................................................... 88
Table 10 Summary of recent MS-based proteomic studies performed using the CRC cell lines
employed in this thesis; and CRC patient plasma/serum samples in the recent years. .................... 89

LIST OF FIGURES
Figure 1 - Global cancer statistics. ..................................................................................................... 3
Figure 2 - Colorectal cancer statistics. ............................................................................................... 4
Figure 3 - (a) Basic anatomy of the colon and rectum (b) and the layers of colon and rectum wall. 6
Figure 4 - The Dukes’ staging system in the context of CRC. ........................................................... 7
Figure 5 - The colorectal adenoma-carcinoma sequence. ................................................................ 23
Figure 6 - Concept of the Epithelial to Mesenchymal Transition (EMT) ........................................ 25
Figure 7 - Key molecules in cancer progression. ............................................................................. 26
Figure 8 - The uPAR-integrin β6-TGFβ signalling pathway. .......................................................... 29
Figure 9 - Basic workflow of LC-MS/MS-based label-free shotgun proteomics. ........................... 87
Figure 10 - Basic workflow of iTRAQ based proteomics experiments. .......................................... 95

Appendix I Figure 1 – Expression TGFβ receptor 1 ................................................................. 355
Appendix I Figure 2 – Expression TGFβ receptor 2 ...................................................................... 356

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STATEMENT OF DECLARATION

I certify that the work presented in this thesis titled “Understanding the biology of
transforming growth factor-beta in colorectal cancers” has not previously been submitted, in
either whole or part, for obtaining any other degree to any other university or institution
other than Macquarie University. The work in this thesis has been carried out by the author,
unless otherwise acknowledged.

Harish R Cheruku
41629191

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ACKNOWLEDGEMENTS

The thesis could not have been completed without the help and support of my family, friends
and colleagues. Firstly, I would like to thank my supervisor Professor Mark S Baker for
providing me the opportunity to work on this project. However, this would not have been
possible without the scholarship (iMQRES) from Macquarie University. I would also like to
thank Edouard Nice my adjunct supervisor and Shoba Ranganathan my co-supervisor for
their support.
I also acknowledge the my colleagues and I would like to extend my earnest gratitude to the
current and past members of the Baker research team including Abidali Mohamedali, Sadia
Mahboob, David Cantor, Charlie (Seong Beom) Ahn, Nurul Jufri, Sock Hwee Tan, Alison
Kan, Samyuktha Anand, Ilze Simpson, Susan Fanayan, Manveen Sethi and Ling Lee for
their professional camaraderie, approachability and for providing a supportive environment
of cooperation and collegiality. I would also like to thank Mehdi Mirzaei for his valuable
discussions on various general experimental methods.
I would like to acknowledge the technical expertise provided by APAF staff including Dylan
Xavier, Natasha Care and Thiri Zaw for assistance with mass spectrometry. I would alsolike
to specially thank Dana Pascovici for all the help and guidance for statistical analysis for all
the manuscripts.
I would also like to thank my friends outside of university for their continual support in
everything. I would also like to acknowledge my friend Manoj Prajwal Bhattaram for proof-
reading my thesis.
Most importantly, I would like to thank my parents, sister, my in-laws and especially my
wife Thanh Truc Le for their support and understanding without whom this Ph.D. thesis
would not have been possible.

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ABSTRACT
Colorectal cancer (CRC) affects millions of people every year globally. Abnormal cells
utilize several mutated proteins and perturbed pathways to progress from a benign tumour
to malignant cancer. Expression of proteins such as uPAR, integrin β6 and TGFβ have been
extensively implicated by us and others in CRC.
The primary aim of this thesis was to contribute additional knowledge to regarding the role
TGFβ in CRC through investigation of the proposed hypothetical uPAR•αvβ6•TGFβ1
interactome. This was achieved by using six CRC cell lines as model systems where
Mock
expression levels of two known activator systems of TGFβ, namely integrin 6 (SW480 ,
β6OE Mock β6AS WT
SW480 , HT29 , HT29 ) and the uPA protease receptor uPAR (HCT116 and
uPARAS
HT29 ) have been artificially expressed or down regulated. The changes in these model
systems following active TGFβ1 treatment were investigated using state-of-the-art
proteomics and a cell signalling assay (i.e., AlphaScreen® SureFire® Assay) technologies
β6OE
in conjunction with sophisticated bioinformatics. The cells expressing β6 (SW480 ,
Mock β6AS
HT29 , HT29 ) exhibited increased proliferation, invasion and wound healing upon
treatment with TGFβ1. The cells with higher uPAR expression did not respond to
WT
(HCT116 ) TGFβ treatments. These results determined that malignancy was attained in a
TGFβ-dependent manner when β6 was expressed or in a TGFβ-independent manner when
uPAR was expressed. Additionally, the proteomic data presented in this thesis identified
several perturbed proteins and biomolecular pathways that could be associated with CRC
and has given important clues to understanding the role of TGFβ and the proposed
hypothetical uPAR•αvβ6•TGFβ1 interactome.
Additionally, an Olink Proseek study using Dukes’ stage A-D CRC patient plasma samples
(1μL of plasma) identified CEA, IL-8 and prolactin were determined to differentiate
unaffected controls from non-malignant (Dukes’ A + B) and malignant (Dukes’ C + D)
stages and were published as potential plasma Dukes’-stage CRC biomarkers.
This thesis has demonstrated the immense power of high-throughput modern proteomic and
multiplexing technologies to gain insights into the TGFβ associated CRC pathogenesis at
detailed molecular level and to identify avenues for disease biomarker exploration.

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