Identification of the genetic alterations in prostate cancer metastases

207 Pages·2017·8.36 MB·English

Identification of the genetic alterations in
prostate cancer metastases

Elzbieta Stankiewicz

Submitted in partial fulfilment of the requirements of the Degree of Doctor of
Philosophy

Supervisor: Dr Yong-Jie Lu and Prof D. Berney
Molecular Oncology and Imaging
Barts Cancer Institute
Queen Mary University of London
John Vane Science Centre
Charterhouse Square
London EC1M 6BQ

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STATEMENT OF ORIGINALITY

I, Elzbieta Stankiewicz, confirm that the research included within this thesis is my own
work or that where it has been carried out in collaboration with, or supported by others,
that this is duly acknowledged below and my contribution indicated.
Mrs Tracy Chaplin provided help with the hybridisation, staining and scanning of
Affymetrix SNP array. Dr. Xueying Mao helped me with SNP data analysis and FISH
methodology. Dr. Marc Yeste-Velasco helped me with generation of stable 22RV1
shFBXL4 cells.
I attest that I have exercised reasonable care to ensure that the work is original, and
does not to the best of my knowledge break any UK law, infringe any third party’s
copyright or other Intellectual Property Right, or contain any confidential material.
I accept that the College has the right to use plagiarism detection software to check the
electronic version of the thesis.
I confirm that this thesis has not been previously submitted for the award of a degree by
this or any other university.
The copyright of this thesis rests with the author and no quotation from it or information
derived from it may be published without the prior written consent of the author.

Signature: Elzbieta Stankewicz
Date: 30.06.16

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ACKNOWLEDGEMENTS
I would like to thank my supervisors, Dr. Yong-Jie Lu and Prof Dan Berney for giving me
the opportunity to do this PhD and for all their support, patience and guidance without
which I would not be able to complete my dissertation.
I would also like to thank all my colleagues in Barts Cancer Institute that advised, helped
and supported me through my PhD.
This work was supported by the ORCHID charity.

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Abstract
Prostate cancer (PCa) is the most common cancer among men in Western developed
countries. While the majority of PCa diagnosed by PSA screening are indolent, advanced
and metastatic disease has a significant mortality and morbidity. Bone metastases are
extremely common in PCa and identification of bone metastasis associated genes may
provide insights into PCa progression and assist in finding new drug targets. However,
the genetic study of bone metastases is very limited due to the difficulty of sampling.
We performed genome-wide analysis of six fresh frozen PCa bone metastases. We
found several alterations commonly present in advanced PCa, including gains at: 1q32.1,
broad gains of 8q (MYC, NCOA2), 9q33.2-34.3, 11q13.1-14.1 (CCND1), 12q24.23-
24.31, 16p13.3, 16p12.1-11.2 and Xq12-13.1 (AR) as well as losses at: 5q11.1-22.1,
5q14.3-23.1, 6q14.1-22, 8p23.2-p21, 13q13.2-31.1 (RB1), 17p13.1-12 (TP53) and
18q11.1-22.3. Two cases also showed PTEN loss and one sample had deletion
indicative of TMPRSS2-ERG fusion.
For downstream analysis we concentrated on CCND1 oncogene at 11q13 and FBXL4
at 6q16 as potential drivers of these genomic changes. Using fluorescence in situ
hybridisation we found common CCND1 gain and FBXL4 loss in PCa bone metastases
(54.5%, 12/22 and 47.8%, 11/23, respectively), much less frequent in primary tumours
(7%, 10/142 and 13.8%, 20/145, respectively) and absent in BPH cases (0/55). The
expression levels of cyclin D1 protein, coded by CCND1 correlated with CCND1 copy
number gain (p < 0.0001) and were higher in metastatic tumours than in primary PCa (p
= 0.015), confirming cyclin D1 involvement in advanced PCa.
Presence of FBXL4 loss in early stage primary PCa strongly correlated with current PCa
prognostic markers and with worse patient survival. Therefore, we propose that FBXL4
may be a tumour suppressor gene in prostate, whose loss in early PCa could be
indicative of more aggressive disease. Using in vitro experiments we demonstrated that
FBXL4 regulates cells motility and invasion. We confirmed that ERLEC1, an ER lectin
involved in ER stress response pathway is a degradation target of FBXL4. As activation
of ER stress response pathway is linked to enhanced cell migration and invasion, loss of
FBXL4 could be one of the mechanisms by which cancer cells increase their efficiency
to respond to stress and to escalate their metastatic potential through stabilisation of
ERLEC1. Further studies of FBXL4 – ERLEC1 axis are necessary to establish how they
contribute to PCa progression. This knowledge can potentially help to develop novel
targeted therapies for aggressive disease harbouring FBXL4 abnormalities.
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ABBREVIATIONS
ADT Androgen deprivation therapy
AR Androgen receptor
BAC Bacterial artificial chromosomes
bp Base pair
BPH Benign prostatic hyperplasia
BSA Bovine serum albumin
cDNA Complementary deoxyribonucleic acid
CGH Comparative genomic hybridisation
CIN Chromosome instability
CAN Copy number alteration
CNV Copy number variation
CRL Cullin-RING E3 ubiquitin ligases
CRPC Castration resistant prostate cancer
DAPI 4,6-diamidino-2-phenylindole dihydrochloride
DEPC Diethylpyrocarbonate, inactivates RNase enzymes
DHT Dihydrotestosterone
DIG Digoxigenin
DMEM Eagle's minimal essential medium
DMSO Dimethyl sulphoxide
DNA Deoxyribonucleic acid
dNTP Deoxyribonucleotide triphosphate
DRE Digital rectal examination
DPX Di-n-butyle phthalate in xylene
DTT Dithiothreitol
EB Tris/HCl
EMT Epithelial-mesenchymal transition
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ER Endoplasmic reticulum
ERAD Endoplasmic reticulum associated degradation
FAM Carboxyfluorescein
FCS Foetal Calf Serum
FFPE Formalin-Fixed, Paraffin-Embedded
FISH Fluorescence in situ hybridisation
GOLF Genome Oriented Laboratory File system
GWAS Genome-wide association study
H&E Hematoxylin and eosin
HGPIN High grade prostatic intraepithelial neoplasia
IF Immunofluorescence
IHC Immunohistochemistry
Kb Kilobase
LB Lysogeny broth
LNM Lymph node metastasis
LOH Loss of heterozygosity
MAD Mitochondria associated degradation
mCRPC Metastatic castration resistant prostate cancer
MIN/MSI Microsatellite instability
mRNA Messenger RNA
MOR Minimal overlapping region
NaCl Sodium chloride
PBS Phosphate-buffered saline
PCa Prostate cancer
PCR Polymerase chain reaction
PIN Prostatic intraepithelial neoplasia
PSA Prostate-specific antigen
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qPCR Quantitative PCR
RNA Ribonucleic acid
rpm Revolutions per minutes
RPMI Roswell Park Memorial Institute medium
SCF Skp, Cullin, F-box containing ubiquitin ligases
shRNA Small hairpin Ribonucleic Acid
siRNA Small interfering Ribonucleic Acid
SNP Single nucleotide polymorphism
SSC Sodium chloride sodium citrate
TAPG Transatlantic Prostate Group
TBE Tris/Borate/EDTA
TE Tris/EDTA
TMA Tissue microarray
TNM Staging System: T-primary tumour, N- number of involved lymph nodes,
M-metastasis
TSG Tumour suppressor gene
TURP Transurethral resection of the prostate
UPR Unfolded protein response
UTP Uridine 5'-triphosphate
V Volt
WB Western blot

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TABLE OF CONTENT
STATEMENT OF ORIGINALITY ........................................................................ 2
ABBREVIATIONS .............................................................................................. 5
LIST OF TABLES ............................................................................................. 16
1 INTRODUCTION AND AIMS ................................................................. 20
1.1 PROSTATE CANCER .................................................................................... 20
1.1.1 The prostate anatomy and origin of prostate cancer ..................................... 20
1.1.2 Prostate cancer incidence, mortality and survival ......................................... 21
1.1.3 Risk factors ................................................................................................... 22
1.1.4 Role of androgen in PCa .............................................................................. 22
1.1.5 Prostate cancer screening using PSA ........................................................... 23
1.1.6 Gleason grading system ............................................................................... 24
1.1.7 Staging of prostate cancer ............................................................................ 26
1.1.8 PCa treatment .............................................................................................. 27
1.1.8.1 Localised low-risk indolent prostate cancer .................................................................... 28
1.1.8.2 High-risk prostate cancer ................................................................................................ 28
1.2 GENETIC ALTERATION IN PROSTATE CANCER ........................................ 29
1.2.1 Genomic instability and cancer ..................................................................... 29
1.2.2 Prostate cancer susceptibility genes ............................................................. 31
1.2.3 Somatic genetic changes in prostate cancer ................................................. 33
1.3 METASTATIC PROSTATE CANCER ............................................................. 38
1.3.1 Mechanism of metastasis ............................................................................. 38
1.3.2 Bone metastases .......................................................................................... 41
1.3.2.1 Normal bone physiology.................................................................................................. 42
1.3.2.2 Types of bone metastases .............................................................................................. 43
1.3.2.3 Model of osteolytic bone metastases .............................................................................. 44
1.3.2.4 Model of osteoblastic bone metastases in prostate cancer ............................................ 45
1.4 AIMS ............................................................................................................... 48
2 MATERIAL AND METHODS ................................................................. 50
2.1 CLINICAL SAMPLES ..................................................................................... 50
2.2 CELL LINES AND CELL CULTURE .............................................................. 51
2.3 HEMATOXYLIN-EOSIN (H&E) STAINING ..................................................... 51
2.4 DNA EXTRACTION ........................................................................................ 51
2.4.1 Fresh frozen samples and cell lines .............................................................. 51
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2.4.2 Simultaneous DNA and RNA extraction ........................................................ 52
2.4.3 FFPE samples .............................................................................................. 53
2.5 SAMPLE PREPARATION FOR SNP 6.0 ARRAY ANALYSIS ....................... 53
2.5.1 Restriction enzyme digestion ........................................................................ 54
2.5.2 Ligation ......................................................................................................... 54
2.5.3 PCR .............................................................................................................. 55
2.5.4 PCR product purification ............................................................................... 55
2.5.5 Fragmentation .............................................................................................. 56
2.5.6 Labelling ....................................................................................................... 56
2.5.7 Hybridisation ................................................................................................. 57
2.5.8 Washing and Staining ................................................................................... 58
2.5.9 Scanning ...................................................................................................... 58
2.5.10 SNP array data analysis ............................................................................... 59
2.6 FLUORESCENT IN SITU HYBRIDISATION (FISH) ....................................... 59
2.6.1 FISH probe preparation ................................................................................ 59
2.6.1.1 Bacterial artificial chromosome (BAC) DNA preparation ................................................ 59
2.6.1.2 BAC DNA amplification, cleaning and labelling .............................................................. 60
2.6.2 Cell and tissue pre-treatment ........................................................................ 61
2.6.3 Hybridisation and post-hybridisation washes ................................................ 62
2.7 RNA EXPRESSION ANALYSIS ..................................................................... 63
2.7.1 RNA extraction ............................................................................................. 63
2.7.2 Complementary DNA (cDNA) synthesis ........................................................ 64
2.7.3 Quantitative Polymerase Chain Reaction (qPCR) ......................................... 64
2.8 PROTEIN ANALYSIS ..................................................................................... 66
2.8.1 Western blot (WB) ........................................................................................ 66
2.8.1.1 Protein extraction ............................................................................................................ 66
2.8.1.2 Bradford assay ................................................................................................................ 66
2.8.1.3 Sodium Dodecyl Sulphate-PolyacrylAmide Gel Electrophoresis (SDS-PAGE) and PVDF
membrane transfer .......................................................................................................... 67
2.8.1.4 Antibody incubation and protein detection ...................................................................... 68
2.8.2 Immunochistochemistry (IHC) ....................................................................... 71
2.8.2.1 Cell lines .......................................................................................................................... 71
2.8.2.2 FFPE tissue sections ...................................................................................................... 72
2.8.3 Immunofluorescence (IF) .............................................................................. 73
2.9 FBXL4 GENE KNOCKDOWN ........................................................................ 74
2.9.1 Gene knockdown with small interfering RNA (siRNA) ................................... 74
2.9.2 Gene knockdown with short hairpin RNA (shRNA) constructs ...................... 75
2.10 FBXL4 CLONING ........................................................................................... 78
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2.10.1 C-terminus Flag tag FBXL4 construct preparation ........................................ 78
2.10.2 DNA purification using Minelute Gel extraction Kit ........................................ 80
2.10.3 Cloning of FBXL4 construct into TOPO® TA vector ...................................... 81
2.10.4 Transformation of Chemically Competent Cells ............................................ 82
2.10.5 Plasmid purification with The Wizard® Plus Minipreps DNA Purification System
83
2.10.6 Restriction digestion of C-Flag FBXL4 pCR™4-TOPO® TA vector and
subcloning into pcDNA4/TO expression vector ............................................. 83
2.10.7 Subcloning C-Flag FBXL4 fragment into pcDNA4/TO expression vector ...... 84
2.10.8 Large scale pcDNA4/TO expression vector with C-terminus Flag tag FBXL4
construct amplification .................................................................................. 86
2.11 TRANSIENT FBXL4 OVEREXPRESSION ..................................................... 87
2.12 PRODUCTION OF STABLY TRANSFECTED CELLS WITH C-FLAG FBXL4
PLASMID DNA ............................................................................................... 88
2.12.1 Production of cells constitutively overexpressing FBXL4 .............................. 88
2.12.2 Tetracycline - inducible system for FBXL4 overexpression ........................... 88
2.13 CELL VIABILITY ASSAY ............................................................................... 89
2.14 CELL MIGRATION ASSAY ............................................................................ 90
2.15 CELL INVASION ............................................................................................ 91
2.15.1 Matrigel invasion ........................................................................................... 91
2.15.2 Collagen invasion ......................................................................................... 91
2.15.2.1 Single cell collagen invasion assay through native collagen type-I matrice ............ 91
2.15.2.2 Single cell invasion assay through collagen type-I/Matrigel matrix .......................... 92
3 GENOME-WIDE ALTERATIONS IN PCA BONE METASTASES ........ 95
3.1 INTRODUCTION ............................................................................................. 95
3.2 RESULTS ....................................................................................................... 95
3.2.1 Sample and SNP 6.0 array data quality control ............................................. 95
3.2.2 Genomic copy number changes detected in PCa bone metastases ............. 98
3.2.3 Confirmation of copy number changes in six fresh frozen bone metastases 108
3.3 DISCUSSION ................................................................................................ 109
3.3.1 The common regions with copy number changes with same chromosomal
breakpoints ................................................................................................. 109
3.3.2 The common regions with copy number changes with different chromosomal
breakpoints ................................................................................................. 111
3.3.3 Copy number confirmation at CCND1 and FBXL4 locus ............................. 113
4 ANALYSIS OF 11Q13 GAIN/ CCND1 GENE IN PCA ......................... 116
4.1 INTRODUCTION ........................................................................................... 116
4.1.1 The 11q13-14 gain and potential driver genes ............................................ 116
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