Cyclic AMP Modulation and its Effects on Chemo-resistant Colon Cancer Cell Proliferation and

316 Pages·2008·9.97 MB·English

Cyclic AMP Modulation and its Effects on Chemo-resistant
Colon Cancer Cell Proliferation and Survival.

by

David George McEwan

This thesis is submitted to the University of Glasgow as part fulfilment of the
requirements for the degree of Doctor of Philosophy.

The Faculty of Medicine The Beatson Institute for Cancer Research
University of Glasgow Cancer Research UK Laboratories
Glasgow Bearsden
Glasgow

© David G McEwan October 2007
i
Abstract
One of the major problems associated with colorectal cancer is resistance to cytotoxic
chemotherapeutic agents. New strategies are therefore required to inhibit colon cancer
proliferation and survival. Here I use modulators of cAMP pathways, including inhibitors
of phosphodiesterase 4 (PDE4) enzymes, which are under clinical development for other
disease states, to inhibit the breakdown of cAMP and to assess the effects of raising
intracellular cAMP on colon cancer proliferation and survival. I found that some chemo-
resistant cancer cells are addicted to keeping low cAMP in PDE4 regulated compartments,
and modulation of this pool causes G1/S-phase arrest and apoptosis. I also show that
PDE4 controlled cAMP negatively regulates the PI 3-Kinase/Akt pathway, which some
cells are addicted to for survival. Furthermore, I investigated the expression and role of
PDE4 enzymes in metastatic colon cancer cells and assessed the effects of modulating their
expression on survival. Also, I used a clinically relevant analogue of forskolin, an agonist
of adenylyl cyclase, to examine the general effect on growth of epithelial cancer cell lines.
This work might provide new strategies for the treatment of advanced colon cancer.
ii
Acknowledgements
First and foremost, I would like to thank my supervisors, Margaret Frame and Miles
Houlsay for their continuous help and support during my PhD and without whom this
thesis would not have completed. Also, I would like to thank George Baillie for his input,
help and supply of reagents, again without which my project would have been that little bit
more difficult.
Thanks to both the R1 and Gardiner lab members past and present.
Also I would like to that Prof David Gillespie and Dr. Owen Sansom for their help
throughout my PhD.
A big thank you to my partner Alison for her continued love and support.
Finally, I also wish to thank the Beatson Institute and Cancer Research U.K. for funding
this project.
iii
Table of Contents

Abstract i
Acknowledgements ii
Table of contents iii
List of tables vii
List of figures viii
Abbreviations ix
Declaration xv

1. Introduction…………………………………………………………………….......1
1.1 Colorectal cancer……………………………………………………………......2
1.2 Oncogene addiction…………………………………………………………......4
1.3 Treatment of colorectal cancer……………………………………………….....8
1.4 Chemoresistance……………………………………………………………….10
1.5 Cell-based models of colon cancer…………………………………………….11
1.6 Animal-based models of colon cancer………………………………………....12
1.7 Fidler model of human colorectal metastasis…………………………………..15
1.8 Previous work using the Fidler model of colorectal cancer cells……………....16
1.9 New strategies: Molecular targeted therapies………………………………….21
1.10 Cyclic nucleotide signalling………………………………………………….....25
1.11 cAMP 2nd messenger signalling………………………………………………...25
1.12 Compartmentalisation of cAMP signalling……………………………………..26
1.13 GPCR signalling and cancer…………………………………………………….28
1.14 cAMP effectors: PKA…………………………………………………………...30
1.15 cAMP effectors: Epac…………………………………………………………...33
1.16 Down stream effects of Epac activation………………………………………...34
1.17 PKA and cancer………………………………………………………………....37
1.18 Epac/Rap1 and cancer…………………………………………………………...38
1.19 cAMP degradation………………………………………………………………39
1.20 PDE3 enzymes…………………………………………………………………..41
1.21 PDE3 enzymes and their role in cancer…………………………………………42
1.22 PDE4 enzymes…………………………………………………………………..45
1.23 PDE4 isoforms…………………………………………………………………..45
1.24 PDE4s, cAMP and cross-talk with other signalling pathways………………….49
ERK regulation of PDE4 activity…………………………………………….....49
cAMP-mediated modulation of ERK activity…………………………………...50
PI 3-kinase/Akt pathway.......................................................................................51
cAMP, PDE4s, and modulation of the PI 3-kinase/Akt pathway……………….57
1.25 cAMP, PDE4s and the cell cycle………………………………………………..57
1.26 PDE4s and cancer……………………………………………………………….62
1.27 Summary………………………………………………………………………...64
2. Materials and methods……………………………………………………………..65
Materials………………………………..……………………………………………....66
2.1 Cell culture reagents…………………………………………………………….66
2.2 Cell culture plasticware…………………………………………………………67
2.3 Treatments………………………………………………………………………67
iv
2.4 MTT assay……………………………………………………………………….67
2.5 PDE assay………………………………………………………………………..68
2.6 Rap1 activity assay……………………………………………………………....68
2.7 Cell cycle analysis……………………………………………………………….68
2.8 Annexin-V staining……………………………………………………………...69
2.9 Immunofluorescence…………………………………………………………….69
2.10 Western blotting………………………………………………………………....70
2.11 Reverse-transcription (RT)-PCR…………………………………………….......72
2.12 Stock solutions and buffers……………………………………………………...72
2.13 Cells and plasmids……………………………………………………………….76
Methods…………………………………………………………………………………78
2.14 Routine cell culture…………………………….………………………………...78
2.15 Treatment of cells……………………………………………………………..…78
2.16 MTT proliferation assay…………………………………………………………78
2.17 PDE assay………………………………………………………………………..79
2.18 Preparation of protein extracts…………………………………………………...79
2.19 Western blot analysis…………………………………………………………….80
2.20 Rap1 activation assay…………………………………………………………….81
2.21 Cell cycle analysis………………………………………………………………..81
2.22 Annexin-V detection of apoptosis………………………………………………..82
2.23 Transient transfection.……………………………………………………………83
2.24 Immunofluourescence. ..…………………………………………………………83
2.25 Sub-cellular fractionation………………………………………………………...84
2.26 Immunoprecipitation (IP)………………………………………………………...84
2.27 RT-PCR…………………………………………………………………………..85
2.28 Preparation of DNA……………………………………………………………....86
2.29 Retroviral infection……………………………………………………………….87
2.30 Nucleofection……………………………………………………………………..87
2.31 Stable knockdown of PDE4Din KM12L4A cells………………………………...87
2.32 Statistical analysis………………………………………………………………...88
2.33 Densitometry……………………………………………………………………...88
3. cAMP effects of KM12C proliferation…………………………………….………..89
3.1 Aims……………………………………………………………………………....90
3.2 cAMP inhibits the proliferation of KM12C cells………………………………....90
3.3 PDE enzymes can regulate anti-proliferative effects of cAMP…………………...94
3.4 Epac did not regulate anti-proliferative effects of cAMP…………………………98
3.5 Fsk/rolipram induces a partial G1/S-phase arrest………………………………..103
3.6 Fsk/rolipram induces specific G1/S-phase CKIs………………………………...106
3.7 Fsk/rolipram inhibits Rb/E2F regulated cell cycle proteins……………………...106
3.8 Fsk/rolipram induces a cell death morphology…………………………………..113
3.9 Fsk/rolipram induces DNA fragmentation………………………………….........114
3.10 Fsk/rolipram induces apoptosis…………………………………………………..114
Discussion………………………………………………………………………………..122
3.11 Inhibition of chemo-resistant colon cancer cells by cAMP……………………...122
3.12 Is Epac or PKA regulating KM12C proliferation?................................................124
3.13 PDE3 vs PDE4 induced inhibition of proliferation……………………...............125
3.14 PDE4/cAMP controlled cell cycle arrest………………………………………...127
3.15 Rolipram mediated apoptosis................................................................................128
v
3.16 Summary………………………………………………………………………..129
4. cAMP interference with oncogene addiction……………………………….…….130
4.1 Aims…………………………………………………………………………….131
4.2 Loss of pAkt (Ser473) is an early event in PDE4/cAMP inhibition of
proliferation……………………………………………………………………..131
4.3 Fsk/rolipram perturbs PtdIns(3,4,5)P localisation……………………………..134
3
4.4 Fsk/rolipram displaces PI 3-kinase p85α subunit from the cell periphery..........135
4.5 PDE4/cAMP inhibits downstream effectors of the PI 3-kinase pathway……...141
4.6 LY294002 induces similar effects to Fsk/rolipram…………………………….144
4.7 LY294002 induces apoptosis and inhibits proliferation…………………..........145
4.8 Exogenous expression of PTEN inhibits Akt phosphorylation and
sensitises KM12C cells to Fsk……………………………………….................150
Discussion.......................................................................................................................155
4.9 PI 3-kinase localisation……………………………………………....................155
4.10 PDE4/cAMP induced loss of Akt/PKB phosphorylation....................................156
4.11 PI 3-kinase regulation of proliferation................................................................158
4.12 Effects of PTEN expression................................................................................160
4.13 Oncogene addiction and its inhibition.................................................................161
4.14 Summary………………………………………………………………………..162
5. PDE4 expression and activity is altered in metastatic cells – consequences for
Fsk sensitivity……………………………………………………………………..163
5.1 Aims………………………………………………………………....................164
5.2 Metastatic cells have increased resistance to Fsk mediated inhibition of
proliferation…………………………………………………………………….164
5.3 Metastatic cells have increased PDE4 activity and expression...........................168
5.4 PDE4D RNAi sensitises metastatic cells to Fsk………………………………..172
5.5 PDE4D3 does not regulate the apoptosis in KM12C cells……………………..176
5.6 PDE4D5 may regulate apoptotic response to cAMP in KM12C cells...………...177
Discussion………………………………………………………………………………182
5.7 Metastatic resistance to Fsk inhibition of proliferation……….………………...182
5.8 Increased PDE4 expression and activity in metastatic cell lines………………..183
5.9 PDE4D3 versus PDE4D5 regulation of proliferation and cell death…………....185
5.10 Summary………………………………………………………………………...186
6. NKH477: A potentially clinically relevant Fsk analogue.....................................188
6.1 Aims………………………………………………………………......................189
6.2 Sensitivity to growth inhibition by cAMP modulation is not restricted to
KM12C cells…………………………………………………………………….189
6.3 NKH477 induces p27Kip1 and loss of pAkt in KM12C cells…………………....194
6.4 NKH477 inhibits the proliferation of numerous cancer cell lines……………....195
Discussion………………………………………………………………………………199
6.5 Cell lines sensitivity to Fsk/rolipram……………………………………………199
6.6 NKH477 as an anti-cancer therapy……………………………………………...200
6.7 Summary………………………………………………………………………...201
vi
7. Concluding remarks and future perspectives…………………………………..202
7.1 cAMP, PDE4s and their therapeutic potential in cancer……………………….203
7.2 Does PDE4s elevation correlate with disease stage?..........................................204
7.3 Can PDE4s be exploited as therapeutic targets for cancer?................................206
7.4 Future work…………………………………………………………………….210

8. References…………………………………….…………………………………..214

Published material......…………………………………….………………………….252
vii

List of tables

Table 1 The Dukes’ classification of colorectal cancer and 5 year survival rates……….3
Table 2 The TNM classification of colorectal cancer…………………………...……….3
Table 3 Correlation between TNM and Dukes staging of colorectal cancer.…...……….3
viii
List of Figures

Figure number Description Page
Figure 1 Cancer development models 6
Figure 2 5-Fluorouracil mechanism of action 9
Figure 3 APC/β-catenin pathway 14
Figure 4 Fidler mouse model of human colorectal metastasis 17
Figure 5 cAMP generation 27
Figure 6 cAMP effectors 32
Figure 7 Epac domain structure and regulation 35
Figure 8 Cyclic AMP hydrolysis 40
Figure 9 PDE3 domain structure and gene organisation 43
Figure 10 PDE4 domain structure 48
Figure 11 PI 3-Kinase/Akt pathway 55
Figure 12 PTEN domain structure 56
Figure 13 Cell cycle regulation by p21Cip1/Waf1/p27Kip1 61
Figure 14 cAMP inhibits the proliferation of KM12C cells 93
Figure 15 PDE enzymes regulate the anti-proliferative pool of cAMP 97
Figure 16 Epac/Rap1 did not mediate the anti-proliferative effects
of cAMP 102
Figure 17 Fsk/rolipram induces partial G1/S arrest 105
Figure 18 Fsk/rolipram induces a specific G1/S-phase CKI 110
Figure 19 Fsk/rolipram treatment inhibits expression pRb/E2F
regulated cell cycle proteins 112
Figure 20 Fsk/rolipram induces a cell-death like morphology 117
Figure 21 Fsk/rolipram induces DNA fragmentation 119
Figure 22 Fsk/rolipram induces apoptosis 121
Figure 23 Loss of pAkt (Ser473) is an early event in PDE4/cAMP
inhibition of proliferation 133
Figure 24 Fsk/rolipram perturbs PtdIns(3,4,5)P localisation 138
3
Figure 25 Fsk/rolipram displaces PI3-Kinase p85α from
the cell periphery 140
Figure 26 PDE4/cAMP inhibits downstream effectors
of the PI 3-kinase pathway 143
Figure 27 LY294002 induces similar effects to Fsk/rolipram 147
Figure 28 LY294002 induces apoptosis and inhibits proliferation 149
Figure 29. Exogenous expression of PTEN inhibits Akt
phosphorylation and sensitises KM12C cells to Fsk 154
Figure 30 Metastatic cells have increased resistance to Fsk 167
Figure 31 Metastatic cells have altered PDE4 expression and activity 171
Figure 32 PDE4D RNAi sensitises KM12L4A cells to Fsk 175
Figure 33 PDE4D3 does not regulate apoptosis in KM12C cells 179
Figure 34 PDE4D5 may regulate cAMP that controls KM12C
apoptosis 181
Figure 35 Fsk/rolipram inhibits a number of cancer cell lines 193
Figure 36 NKH477 suppresses pAkt, pERK and proliferation
of KM12C cells 197
Figure 37 NKH477 inhibits the proliferation of all cancer
cell lines tested 198
Figure 38 NKH477 treatment of APCflox/PTENflox mice 213
ix
Abbreviations

5-FU 5-Fluorouracil
8-Br-cAMP 8-Bromo-cAMP
8-CPT 8-pCPT-2`OMe-cAMP
8-pMeOPT 8-pMeOPT-2´-O-Me-cAMP
AC Adenylyl cyclase
AKAP A-kinase anchoring protein
AMP 5´-adenosine monophosphate
APC Adenomatous polyposis coli
Arg Argenine (R)
ATP adenosine 5’triphosphate
B-CLL B-cell chronic lymphocytic leukaemia
BrdU Bromodeoxyuridine
C- Carboxy terminal
Ca2+ Calcium
cAMP 3´5´-cyclic adenosine monophosphate
CDK Cyclin dependent kinase
CFP cyan fluorescent protein
cGMP 3´5´-cyclic guanosine monophosphate
CKI Cyclin dependent kinase inhibitor
CLL Chronic lymphocytic leukaemia
CML Chronic myeloid leukaemia
CNB Cyclic nucleotide binding domain
COPD Chronic obtrusive pulmonary disease
CREB Cyclic AMP responsive element binding protein