Characterization of TMEFF2

322 Pages·2013·21.62 MB·English

Characterization of TMEFF2;
its role in tumour progression and development of targeting
strategies for anti-cancer therapy.
by
Katarzyna Gaweł-Bęben
Extracellular Matrix (ECM) in Repair & Remodelling
Cardiff University School of Dentistry
Cardiff, UK

A thesis submitted to the Cardiff University for the degree of Doctor of Philosophy

PhD Project Supervisors:
Dr Vera Knäuper
Dr Ann Ager

August 2013
I
DECLARATION
This work has not been submitted in substance for any other degree or award at this or
any other university or place of learning, nor is being submitted concurrently in
candidature for any degree or other award.

Signed ………………………………….......... (candidate) Date …………………………

II
STATEMENT 1
This thesis is being submitted in partial fulfilment of the requirements for the degree of
Doctor of Philosophy.
Signed ………………………………….......... (candidate) Date …………………………

STATEMENT 2
This thesis is the result of my own independent work/investigation, except where
otherwise stated. Other sources are acknowledged by explicit references. The views
expressed are my own.
Signed ………………………………….......... (candidate) Date …………………………

STATEMENT 3
I hereby give consent for my thesis, if accepted, to be available for photocopying and for
inter-library loan, and for the title and summary to be made available to outside
organisations.

Signed ………………………………….......... (candidate) Date …………………………

STATEMENT 4: PREVIOUSLY APPROVED BAR ON ACCESS
I hereby give consent for my thesis, if accepted, to be available for photocopying and for
inter-library loans after expiry of a bar on access previously approved by the
Academic Standards & Quality Committee.

Signed ………………………………….......... (candidate) Date …………………………

III
Acknowledgments

I would like to thank my supervisors, Vera Knäuper and Ann Ager
for motivating and inspiring me from the very first day of my PhD.

This work could not be completed without the help and support of my great colleagues:
Magdalena Adamczyk, Lea Bauer, Andreas Heil and Tim Wanger – thank you very much.

I would also like to thank Łukasz for encouraging me to accept every challenge and
supporting me during difficult times.

IV
Abstract

TMEFF2 (transmembrane protein with EGF-like and two follistatin motifs 2) is a
transmembrane protein expressed in brain and prostate and over-expressed in prostate
cancer (Uchida et al. 1999; Horie et al. 2000). The role of TMEFF2 in prostate cancer is
controversial. Several data indicate that TMEFF2 has cancer-promoting activity (Glynne-
Jones et al. 2001; Ali and Knäuper 2007), while others suggest that TMEFF2 inhibits
progression of cancer (Gery et al. 2002; Gery and Koeffler 2003). TMEFF2 is cleaved by
membrane-anchored proteases, including disintegrin and metalloproteases (ADAMs) and
γ-secretase (Ali and Knäuper 2007), but the biological meaning of TMEFF2 shedding is
not known. It was hypothesized that the opposing findings describing the role of TMEFF2
in prostate cancer result from proteolytic processing of TMEFF2 by different proteases
which are co-expressed with TMEFF2 in prostate cancer cells, such as the type II
transmembrane serine proteases (TTSPs), prostasin and ADAMs. To support this
hypothesis co-expression of TMEFF2 and serine proteases was analyzed in prostate
cancer cell lines and clinical samples. The shedding of TMEFF2 by ADAMs and serine
proteases was investigated using HEK293 cells expressing AP/V5 TMEFF2 or shedding
resistant AP/V5 ∆ TMEFF2 mutant (Ali and Knäuper 2007). The data obtained from
303-320
AP activity assay and Western blot analysis of cell lysates showed that TMEFF2 is
cleaved by serine proteases (matriptase and hepsin) and ADAMs (ADAM9, ADAM12).
Moreover, serine proteases and ADAMs cleave TMEFF2 in different positions, generating
several soluble TMEFF2 fragments. To establish the biological role of TMEFF2
processing, N-terminal TMEFF2 fragments predicted to be generated by TTSPs and
ADAMs were expressed in E. coli and mammalian cells. Preliminary experiments using
HEK293 and PNT2-C2 cells indicated that soluble TMEFF2 does not signal through ErbB
receptors and suggested several signaling pathways that might be regulated by TMEFF2.
The fate of TMEFF2 C-terminus following ectodomain shedding was examined by
confocal microscopy and Western blotting, indicating that TMEFF2 cytoplasmic domain is
likely degraded following the release of TMEFF2-ECD.

V
Table of content
Chapter 1 : Introduction 1
1.1 Prostate cancer 2
1.2 TMEFF2 – a novel protein expressed in prostate cancer 8
1.2.1 TMEFF2 protein structure. 8
1.2.2 Expression of TMEFF2 9
1.2.3 Regulation of Tmeff2 gene expression 9
1.2.4 TMEFF2 null mice 9
1.2.5 The role of TMEFF2 in central nervous system 10
1.2.6 Controversial role of TMEFF2 in normal prostate and prostate cancer 10
1.2.7 Soluble TMEFF2 fragments 11
1.2.8 The role of TMEFF2 cytoplasmic domain 12
1.3 Differential processing of TMEFF2 – possible explanation of TMEFF2 13
controversial role in prostate cancer
1.3.1 Proteolysis on the cell surface – an important regulatory mechanism of 13
protein function
1.3.1.1 Ectodomain shedding 14
1.3.1.2 Regulated intramembrane proteolysis (RIP) 15
1.3.1.2.1 RIP-dependent release of transcription factors 18
1.3.1.2.2 Regulation of cytoplasmic proteins by RIP products 19
1.3.1.2.3 Degradation of RIP products 19
1.3.2 Biological consequences of dysregulated cell surface proteolysis 20
1.3.3 The role of proteolysis in modulating TMEFF2 function 22
1.4 Membrane-anchored proteases implicated in prostate cancer 23
1.4.1 ADAMs 23
1.4.1.1 Expression of ADAMs 23
1.4.1.2 ADAM domain structure 23
1.4.1.3 ADAMs proteolytic activity 26
1.4.1.4 ADAM9 27
1.4.1.4.1 Expression of ADAM9 27
VI
1.4.1.4.2 ADAM9 substrates 27
1.4.1.4.3 ADAM9 as an adhesion molecule 27
1.4.1.4.4 Cytoplasmic domain of ADAM9 27
1.4.1.4.5 ADAM9 null mice 28
1.4.1.4.6 ADAM9 in cancer 28
1.4.1.5 ADAM12 30
1.4.1.5.1 Expression of ADAM12 30
1.4.1.5.2 ADAM12 synthesis and activation 30
1.4.1.5.3 ADAM12 substrates 31
1.4.1.5.4 The role of ADAM12 in cell adhesion 31
1.4.1.5.5 Cytoplasmic domain of ADAM12 32
1.4.1.5.6 ADAM12 deficiency in mice 32
1.4.1.5.7 ADAM12 in cancer 33
1.4.1.6 ADAM15 35
1.4.1.6.1 ADAM15 expression 35
1.4.1.6.2 The role of ADAM15 in adhesion 35
1.4.1.6.3 ADAM15 proteolytic activity 36
1.4.1.6.4 ADAM15 cytoplasmic domain 36
1.4.1.6.5 ADAM15 null mice 37
1.4.1.6.6 ADAM15 in cancer 37
1.4.2 Type II transmembrane serine proteases (TTSPs) 40
1.4.2.1 The structure of TTSPs 40
1.4.2.2 Classification of TTSPs 41
1.4.2.3 Activation of TTSPs 44
1.4.2.4 Expression of TTSPs 44
1.4.2.5 Regulation of TTSPs activity 45
1.4.2.6 TTSPs implicated in prostate cancer 45
1.4.2.6.1 Matriptase 45
1.4.2.6.2 Matriptase-2 50
VII
1.4.2.6.3 Hepsin 52
1.4.3 Prostasin – a GPI-anchored serine protease 54
1.4 Aims of the thesis 58
Chapter 2: Materials and methods 59
2.1 Cell culture 60
2.2 Immunolocalisation of TMEFF2 and ∆ TMEFF2 in HEK293 61
303-320
2.3 Transient transfection of HEK293 cells 61
2.3.1 Transfection of HE293 cells for shedding experiments 61
2.3.2 Transfection of HEK293 cells with EGFP-TMEFF2 and TMEFF2-YFP 61
2.4 Assay for alkaline phosphatase activity (AP assay) 62
2.5 Analysis of cell lysates by Western blotting 62
2.5.1 Cell lysis 62
2.5.2 Protein concentration assay 62
2.5.3 SDS-PAGE 63
2.5.4 Western blotting 63
2.5.5 Stripping for re-probing Western blots 63
2.6 Coomassie Blue R-250 staining 65
2.7 Silver staining 65
2.8 Construction of HREGF TMEFF2 MBP-fusion expression vector 65
2.8.1 Overlap extension polymerase chain reaction (PCR) 65
2.8.2 Restriction digest 68
2.8.3 Ligation 68
2.8.4 H-REGF TMEFF2 MAL pRSET amplification 68
2.9 Expression and purification of MBP-fusion proteins in E.coli 68
2.9.1 Transformation of bacteria 68
2.9.2 Expression of recombinant proteins 69
2.9.3 Analysis of protein expression following IPTG induction 69
2.9.4 First step purification: affinity chromatography on amylose resin 69
2.9.5 Second step purification: gel filtration 70
VIII
2.9.6 Calculation of protein concentration using extinction coefficient factor 70
2.10 Generation of N-protein A TMEFF2 expression plasmids 70
2.10.1 Amplification of TMEFF2 ectodomain fragments by PCR 70
2.10.2 Restriction digest 72
2.10.3 Ligation and plasmid amplification 72
2.11 Generation of Fc-TMEFF2-ECD pcDNA5/FRT vector 73
2.11.1 Restriction digest 73
2.11.2 Ligation and plasmid amplification 73
2.12 Generation of CHO cell lines stably expressing Fc- and N-protein A 73
fusion TMEFF2 fragments
2.13 Collection of conditioned medium 74
2.14 Purification of N-protein A TMEFF2 fragments 74
2.14.1 Packing and equilibration of IgG Sepharose column 74
2.14.2 Binding and elution of protein A fusion protein 74
2.14.3 Re-equilibration and storage of IgG Sepharose 75
2.15 Purification of Fc-TMEFF2 ectodomain 75
2.15.1 First step purification: affinity chromatography using protein G 75
Sepharose
2.15.2 Second step purification: HIS-Select HF Nickel Affinity Gel 75
2.16 Analysis of ERK1/2 phosphorylation 76
2.16.1 Cell stimulation and sample analysis 76
2.16.2 Quantification of the pERK1/2 to total ERK1/2 ratio using ImageJ 76
2.17 Cell proliferation assay 77
2.17.1 Proliferation of CHO cells the presence of N-protein A TMEFF2 78
medium
2.17.2 PNT2-C2 proliferation in the presence of Fc-TMEFF2-ECD 78
2.18 Preparation of nuclear and cytoplasmic extract 78
2.19 Statistical analysis 78
Chapter 3: Expression of TMEFF2 and serine proteases in prostate cancer 79
3.1 Introduction 80
3.1.1 Expression of TMEFF2 in prostate cancer 80
IX
3.1.2 Dysregulated expression of potential TMEFF2 sheddases in prostate 80
cancer
3.1.3 Prostate cancer stem cells 82
3.2 Aims 82
3.3 Results 83
3.3.1 Expression of TMEFF2 in prostate cancer stem cells and cell lines 83
3.3.2 Expression of serine proteases in prostate cancer cells 88
3.4 Chapter summary 91
Chapter 4: Shedding of TMEFF2 by serine proteases and ADAMs over- 94
expressed in prostate cancer
4.1 Introduction 95
4.1.1 ADAM-mediated ectodomain shedding 95
4.1.2 Cleavage of transmembrane proteins by serine proteases 96
4.1.3 TMEFF2 as a potential substrate for serine proteases 97
4.2 Aims 97
4.3 Results 99
4.3.1 Characterization of the cellular model to study TMEFF2 shedding 99
4.3.2 Optimisation of TMEFF2 shedding assay 109
4.3.3 Shedding of TMEFF2 by membrane serine proteases expressed in 113
prostate cancer
4.3.4 Analysis of AP/V5 TMEFF2 shedding by matriptase-2 119
4.3.5 Shedding of TMEFF2 in the presence of matriptase-activated 124
prostasin
4.3.6 Shedding of TMEFF2 by ADAMs involved in prostate cancer 127
progression
4.3.7 Shedding of TMEFF2 in the presence of PDGF-AA. 132
4.4 Chapter summary 137
Chapter 5: Expression and purification of TMEFF2 ectodomain fragments 143
in E. coli
5.1 Introduction 144
5.1.1 The characterization of an EGF-like domain 144
5.1.2 Signaling of EGF-like proteins through ErbB receptors 144
X