Functional role of dysregulated PI3K/AKT/mTOR signaling in circulating tumor cells:
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| Format: | Abschlussarbeit Buch |
| Sprache: | English |
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2022
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| Beschreibung: | 181 Blätter Illustrationen, Diagramme 30 cm |
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| 100 | 1 | |a Smit, Daniel Jan |d 1996- |e Verfasser |0 (DE-588)1251596630 |4 aut | |
| 245 | 1 | 0 | |a Functional role of dysregulated PI3K/AKT/mTOR signaling in circulating tumor cells |c vorgelegt von: Dr. med. Daniel Jan Smit |
| 264 | 1 | |a Hamburg |c 2022 | |
| 300 | |a 181 Blätter |b Illustrationen, Diagramme |c 30 cm | ||
| 336 | |b txt |2 rdacontent | ||
| 337 | |b n |2 rdamedia | ||
| 338 | |b nc |2 rdacarrier | ||
| 502 | |b Dissertation |c Universität Hamburg |d 2023 | ||
| 655 | 7 | |0 (DE-588)4113937-9 |a Hochschulschrift |2 gnd-content | |
| 856 | 4 | 2 | |m B:DE-101 |q application/pdf |u https://d-nb.info/1297464400/04 |3 Inhaltsverzeichnis |
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| 883 | 2 | |8 2\p |a dnb |d 20230814 |q DE-101 |u https://d-nb.info/provenance/plan#dnb | |
Datensatz im Suchindex
| _version_ | 1804186448225632256 |
|---|---|
| adam_text | TABLE
OF
CONTENTS
TABLE
OF
CONTENTS
........................................................................................................................................................
3
1
INTRODUCTION
..............................................................................................................................................................
7
1.1
C
ANCER
AND
CARCINOGENESIS
.......................................................................................................................................
7
1.1.1
BREAST
CANCER
....................................................................................................................................................
7
1.1.1.1
EPIDEMIOLOGY
.........................................................................................................................................................
7
1.1.1.2
DEVELOPMENT
OF
BREAST
CANCER
..............................................................................................................................
7
1.1.1.3
CLASSIFICATION
OF
INVASIVE
BREAST
CANCER
................................................................................................................
8
1.1.1.4
TREATMENT
OF
BREAST
CANCER
..................................................................................................................................
8
1.1.2
COLORECTAL
CANCER
.............................................................................................................................................
9
1.1.2.1
EPIDEMIOLOGY
........................................................................................................................................................
9
1.1.2.2
DEVELOPMENT
OF
COLORECTAL
CANCER
.......................................................................................................................
9
1.1.2.3
TREATMENT
OF
COLORECTAL
CANCER
..........................................................................................................................
10
1.1.3
METASTATIC
CASCADE
.......................................................................................................................................
10
1.2
C
IRCULATING
TUMOR
CELLS
...............................................................
11
1.2.1
IN
VIVO
EXPANSION
OF
CTCS
.............................................................................................................................
13
1.2.2
CTC
CELL
LINES
...................................................................................................................................................
14
1.2.3
CIRCULATING
TUMOR
CELLS
IN
BREAST
CANCER
......................................................................................................
15
1.2.4
CIRCULATING
TUMOR
CELLS
IN
COLORECTAL
CANCER
................................................................................................
15
1.3
PI3K
/
AKT/
M
TOR
S
IGNALING
..................................................................................................................................
15
1.3.1
PI3K/AKT/MTOR
SIGNALING
IN
CANCER.
...........................................................................................................
19
1.3.2
AKT
ISOFORM
SPECIFIC
SIGNALING
......................................................................................................................
22
1.3.3
INHIBITORS
OF
PI3K/AKT/MTOR
SIGNALING
PATHWAY.
23
1.4
AIM
OF
THE
THESIS
....................................................................................................................................................................................................
25
2
MATERIAL
AND
METHODS
...........................................
27
2.1
M
ATERIAL
.......................................
27
2.1.1
DEVICES
........................................................................................................
,
.................................................
27
2.1.2
CONSUMABLES
.................................................................................................................................................
28
2.1.3
CHEMICALS
.......................................................................................................................................................
28
2.1.4
KITS
..................................................................................................................................................................
28
2.1.5
SOFTWARE
.........................................................................................................................................................
29
2.1.6
VECTORS
...........................................................................................................................................................
29
2.1.7
OLIGONUCLEOTIDES
31
2.1.8
INHIBITORS
........................................................................................................................................................
32
2.1.9
ANTIBODIES
......................................................................................................................................................
32
2.1.10
CELL
LINES
........................................................................................................................................................
34
2.1.11
BACTERIAL
STRAINS
............................................
35
2.1.12
ANIMALS.
35
2.2
M
ETHODS
36
2.2.1
CELL
BIOLOGY
TECHNIQUES
..................................................................................................................................
36
2.2.1.1
CULTIVATION
OF
CIRCULATING
TUMOR
CELLS
..................................................................................................................
36
2.2.1.2
CULTIVATION
OF
ADHERENT
CANCER
CELL
LINES
.............................................................................................................
36
2.2.1.3
FREEZING
AND
THAWING
OF
CELLS
..............................................................................................................................
37
2.2.1.4
CELL
COUNTING
........................................................................................................................................................
37
2.2.1.5
LENTIVIRAL
TRANSDUCTION
.........................................................................................................................................
37
2.2.1.6
TUMOROID
FORMATION
/
3D
CELL
CULTURE
OF
TUMOR
CELLS
.........................................................................................
38
2.2.1.7
PROLIFERATION
ASSAY
...............................................................................................................................................
38
2.2.1.8
MIGRATION
AND
INVASION
ASSAY
..............................................................................................................................
38
2.2.1.9
CHEMOTAXIS
ASSAY............................
39
2.2.1.10
COLLAGEN
IV
ADHESION
ASSAY
...............................................................................................................................
39
2.2.1.11
APOPTOSIS
ASSAY
...................................................................................................
39
2.2.1.12
2D
INHIBITOR
ASSAY
.............................................................................................................................................
40
2.2.1.13
3D
INHIBITOR
ASSAY
.............................................................................................................................................
40
2.2.1.14
FLOW
CYTOMETRIC
ANALYSIS
OF
CANCER
STEM
CELL
MARKERS
.....................................................................................
40
2.2.1.15
IMMUNOFLUORESCENT
STAINING
OF
CELLS
AND
FLUORESCENCE
MICROSCOPY
...............................................................
41
2.2.1.16
CALCEIN
VIABILITY
STAINING
OF
TUMOROIDS
.............................................................................................................
41
2.2.2
MICROBIOLOGY
TECHNIQUES
..............................................................................................................................
41
2.2.2.1
AGAR
PLATES
FOR
BACTERIA
CULTURE
..........................................................................................................................
41
2.2.2.2
PREPARATION
OF
COMPETENT
CELLS
..........................................................................................................................
42
2.2.2.3
TRANSFORMATION
OF
COMPETENT
XL-1
BLUE
............................................................................................................
42
2.2.3
MOLECULAR
BIOLOGY
TECHNIQUES
......................................................................................................................
43
2.2.3.1
PLASMID
DNA
EXTRACTION
........................................................
43
2.2.3.2
DNA
EXTRACTION
FROM
MURINE
TISSUE
...................................................................................................................
43
2.2.3.3
NUCLEIC
ACID
CONCENTRATION
AND
PURITY
DETERMINATION
.......................................................................................
43
2.2.3.4
OLIGONUCLEOTID
DESIGN
...........................................................................................
43
2.2.3.5
SITE
DIRECTED
MUTAGENESIS
USING
THE
QUIKCHANGE
METHOD
.................................................................................
44
2.2.3.6
QUANTITATIVE
REAL-TIME
PCR
.................................................................................................................................
44
2.2.4
PROTEIN
BIOCHEMISTRY
TECHNIQUES
.................................................................................................................
45
2.2.4.1
PREPARATION
OF
WHOLE
CELL
PROTEIN
LYSATES
..........................................................................................................45
2.2.4.2
PROTEIN
CONCENTRATION
DETERMINATION
................................................................................................................45
2.2.4.3
SDS-PAGE
ELECTROPHORESIS
..................................................................................................................................45
2.2.4.4
WESTERN
BLOTTING
..........
.........................................................................................................................
46
2.2.4.5
DENSITOMETRIC
QUANTIFICATION
..............................................................................................................................46
2.2.4.6
SEPARATION
OF
CYTOSOL
AND
MEMBRANE
FRACTIONS
BY
ULTRACENTRIFUGATION
....................................
46
2.2.4.7
KINOME
PROFILING
..................................................................................................................................................
47
2.2.4.8
PEPTIDE
EXTRACTION
...............................................................................................................................................
48
2.2.4.9
LIQUID
CHROMATOGRAPHY/MASS
SPECTROMETRY
.....................................................................................................48
2.2.5
ANIMAL
EXPERIMENTS
...............................................................................
49
2.2.5.1
INTRACARDIAC
INJECTION
OF
CIRCULATING
TUMOR
CELLS
................................................................................................
49
2.2.5.2
ORAL
TREATMENT
OF
MICE
WITH
AKT/MTOR
INHIBITORS
BY
GAVAGE
.........................................................................49
2.2.53
IN
VIVO
OPTICAL
IMAGING
OF
TUMOR
CELL
DISSEMINATION
AND
TUMOR
BURDEN
.....................................
50
2.2.5.4
SACRIFICE
AND
ORGAN
HARVESTING
............................................................................................................................
50
2.2.5.5
EX
VIVO
OPTICAL
IMAGING
OF
HARVESTED
ORGANS
........................................................................................
50
2.2.5.6
HOMOGENIZATION
OF
MURINE
TISSUE
...........................................................................................
50
2.2.5.7
MURINE
BONE
MARROW
PREPARATION
.....................................................................................................................
51
2.2.5.8
CTC
DETECTION
FROM
MURINE
WHOLE-BLOOD
...........................................................................................
51
2.2.5.9
ALU-PCR
...................................................................
51
2.2.5.10
HISTOLOGICAL
ANALYSIS
OF
MURINE
TISSUE
...............................................................................................................
51
2.2.6
BIOINFORMATIC
ANALYSIS
...................................................................................................................................
52
2.2.6.1
LC/MS
DATA
PROCESSING
..............................................................................................................................
52
2.2.6.2
GENE
ONTOLOGY
ENRICHMENT
ANALYSIS
...................................................................................................................
55
2.2.63
DETERMINATION
OF
UNIQUELY
ABUNDANT
AND
COMMON
PROTEINS
..........................................................................
56
2.2.6.4
PROTEIN-PROTEIN
INTERACTION
NETWORKS
FUNCTIONAL
ENRICHMENT
ANALYSIS
............................................................
56
2.2.6.5
KEYWORD-BASED
LITERATURE
SEARCH
USING
OMICSLITMINER
...........................................................................
56
2.2.6.6
STRUCTURAL
MODELING
OF
PIK3CA
MUTATIONS
.........................................................................................................
57
2.2.6.7
DATA
MINING
OF
TCGA
DATASETS
..........................................
57
2.2.6.8
GENE
EXPRESSION
PROFILE
ANALYSIS
.........................................................................................................................
57
2.2.6.9
CALCULATION
OF
COMBINATION
INDEX
.......................................................................................................................
57
2.2.6.10
CORRELATION
OF
DATA
............................................................................................................................................
57
2.2.7
S
TATISTICAL
ANALYSIS
AND
VISUALIZATION
...................................................................................................
58
3
RESULTS
.........................................................................................................................................................................
59
3.1
A
NALYSIS
AND
INHIBITION
OF
THE
PI3K/AKT/
M
TOR
PATHWAY
ACTIVATION
IN
CTC
S
......................................................59
3.1.1
PI3K/AKT/MTOR
SIGNALING
PATHWAY
ACTIVATION
AND
SUSCEPTIBILITY
TO
AKT
AND
MTOR
INHIBITORS
IN
A
2D
MODEL
IN
VITRO
............................................
59
3.1.2
SENSITIVITY
OF
CTC
LINES
TOWARDS
AKT
AND
MTOR
INHIBITORS
IN
A
3D
TUMOR
SPHEROID
MODEL
....................
63
3.1.3
SENSITIVITY
OF
COLORECTAL
CTCS
AGAINST
AKT/MTOR
INHIBITION
IN
A
MURINE
INTRACARDIAC
XENOTRANSPLANTATION
MODEL
IN
VIVO
...............................................................................
:
...........................
67
3.2
C
HARACTERIZATION
OF
PI3K
MUTATIONS
DETECTED
WITHIN
PRIMARY
TUMOR
,
METASTASIS
AND
CTCS
FROM
THE
SAME
PATIENT
.............................................................................................................
75
3.2.1
FREQUENCY,
LOCATION,
AND
CLINICAL
OUTCOME
OF
PI3K
MUTATIONS
IN
BREAST
CANCER
PATIENTS
......................
76
3.2.2
PREDICTED
EFFECT
OFC2
DOMAIN
MUTATIONS
ON
THE
PROTEIN
STRUCTURE
OFPLLOAAND
THE
INTERACTION
WITH
P85A.
............................................................................................................................................................
77
3.2.3
ESTABLISHMENT
OF
PIK3CA
C2
DOMAIN
MUTANT
E418K
AND
E453K
OVEREXPRESSING
BREAST
CANCER
CELLS
..
80
3.2.4
ANALYSIS
OF
DOWNSTREAM
SIGNALING
OF
PIK3CA
MUTANTS
..............................................................................
80
3.2.5
ANALYSIS
OF
THE
FUNCTIONAL
ROLE
OF
PIK3CA
E418K
AND
E453K
IN
BREAST
CANCER
........................................
84
3.2.6
SUSCEPTIBILITY
OFE418KAND
E453K
MUTANT
TO
PI3K
INHIBITION
..................................................................
88
3.2.7
ANALYSIS
OF
THE
PI3K
MEMBRANE
LOCALIZATION
IN
E418K
AND
E453K
MUTANT
............................................
89
3.3
T
HE
ROLE
OF
AKT
ISOFORM
SPECIFIC
KNOCKDOWN
IN
COLORECTAL
CANCER
CTCS
.............................................................
91
3.3.1
ESTABLISHMENT
OFAKTI,
AKT2
AND
AKT3
ISOFORM
SPECIFIC
KNOCKDOWNS
IN
CTC-MCC-41
........................
91
3.3.2
FUNCTIONAL
ROLE
OF
AKT
ISOFORMS
IN
COLORECTAL
CANCER
CTCS
.......................................................................91
3.3.3
PROTEOMIC
CHANGES
IN
CTC-MCC-41
CELLS
AFTER
AKT
ISOFORM
SPECIFIC
KNOCKDOWN
....................................
92
3.4
A
NALYSIS
OF
SUSPENSION
AND
ADHERENT
FRACTIONS
WITHIN
BREAST
CANCER
AND
COLORECTAL
CANCER
CTCS
...............102
3.4.1
GROWTH
PATTERN
OF
CTCS
IN
CULTURE
.............................................................................................................
102
3.4.2
SIGNALING
PATHWAY
ANALYSIS
OF
SUSPENSION
AND
ADHERENT
CELLS
...............................................................
106
3.4.3
KINOME
ANALYSIS
OF
SUSPENSION
AND
ADHERENT
CTCS
.................................................................................
107
3.4.4
ANALYSIS
OFCADHERIN
AND
EMT
MARKER
EXPRESSION
IN
SUSPENSION
AND
ADHERENT
CTCS
..........................
119
3.4.5
EXPRESSION
OF
CANCER
STEM
CELL
MARKERS
IN
COLORECTAL
CANCER
CTCS
....................................................
120
4
DISCUSSION
.................................................................................................................................................................123
4.1
AKT
AND
M
TOR
INHIBITORS
IN
THE
PRECLINICAL
AND
CLINICAL
SETTING...............................................................................................
123
4.2
I
NHIBITION
OF
CTCS
AS
A
TARGET
FORMETASTASIS
PREVENTION
...................................................................................
125
4.3
THE
FUNCTIONAL
ROLE
OF
AKT
ISOFORM
SPECIFIC
SIGNALING
IN
CANCER
..............................................................................................
125
4.4
PI3K
MUTATIONS
AND
THE
FUNCTIONAL
ROLE
OF
C2
DOMAIN
MUTATIONS
IN
BREAST
CANCER......................................................
127
4.5
S
USPENSION
AND
ADHERENT
CTCS
AND
THE
EPITHELIAL
-
MESENCHYMAL
TRANSITION
......................................................129
5
SUMMARY
...................................................................................................................................................................
134
6
APPENDIX
.....................................................................................................................................................................
135
7
LIST
OF
ABBREVIATIONS...........................................................................................................................................
157
8
LIST
OF
TABLES
...........................................................................................................................................................
162
9
LIST
OF
FIGURES.........................................................................................................................................................
163
10
REFERENCES
..............................................................................................................................................................
165
11
ACKNOWLEDGMENT
................................................................................................................................................
176
12
CURRICULUM
VITAE
................................................................................................................................................
178
13
PREVIOUS
PUBLICATIONS
.......................................................................................................................................
179
14
STATUTORY
DECLARATION
....................................................................................................................................
181
|
| adam_txt |
TABLE
OF
CONTENTS
TABLE
OF
CONTENTS
.
3
1
INTRODUCTION
.
7
1.1
C
ANCER
AND
CARCINOGENESIS
.
7
1.1.1
BREAST
CANCER
.
7
1.1.1.1
EPIDEMIOLOGY
.
7
1.1.1.2
DEVELOPMENT
OF
BREAST
CANCER
.
7
1.1.1.3
CLASSIFICATION
OF
INVASIVE
BREAST
CANCER
.
8
1.1.1.4
TREATMENT
OF
BREAST
CANCER
.
8
1.1.2
COLORECTAL
CANCER
.
9
1.1.2.1
EPIDEMIOLOGY
.
9
1.1.2.2
DEVELOPMENT
OF
COLORECTAL
CANCER
.
9
1.1.2.3
TREATMENT
OF
COLORECTAL
CANCER
.
10
1.1.3
METASTATIC
CASCADE
.
10
1.2
C
IRCULATING
TUMOR
CELLS
.
11
1.2.1
IN
VIVO
EXPANSION
OF
CTCS
.
13
1.2.2
CTC
CELL
LINES
.
14
1.2.3
CIRCULATING
TUMOR
CELLS
IN
BREAST
CANCER
.
15
1.2.4
CIRCULATING
TUMOR
CELLS
IN
COLORECTAL
CANCER
.
15
1.3
PI3K
/
AKT/
M
TOR
S
IGNALING
.
15
1.3.1
PI3K/AKT/MTOR
SIGNALING
IN
CANCER.
.
19
1.3.2
AKT
ISOFORM
SPECIFIC
SIGNALING
.
22
1.3.3
INHIBITORS
OF
PI3K/AKT/MTOR
SIGNALING
PATHWAY.
23
1.4
AIM
OF
THE
THESIS
.
25
2
MATERIAL
AND
METHODS
.
27
2.1
M
ATERIAL
.
27
2.1.1
DEVICES
.
,
.
27
2.1.2
CONSUMABLES
.
28
2.1.3
CHEMICALS
.
28
2.1.4
KITS
.
28
2.1.5
SOFTWARE
.
29
2.1.6
VECTORS
.
29
2.1.7
OLIGONUCLEOTIDES
31
2.1.8
INHIBITORS
.
32
2.1.9
ANTIBODIES
.
32
2.1.10
CELL
LINES
.
34
2.1.11
BACTERIAL
STRAINS
.
35
2.1.12
ANIMALS.
35
2.2
M
ETHODS
36
2.2.1
CELL
BIOLOGY
TECHNIQUES
.
36
2.2.1.1
CULTIVATION
OF
CIRCULATING
TUMOR
CELLS
.
36
2.2.1.2
CULTIVATION
OF
ADHERENT
CANCER
CELL
LINES
.
36
2.2.1.3
FREEZING
AND
THAWING
OF
CELLS
.
37
2.2.1.4
CELL
COUNTING
.
37
2.2.1.5
LENTIVIRAL
TRANSDUCTION
.
37
2.2.1.6
TUMOROID
FORMATION
/
3D
CELL
CULTURE
OF
TUMOR
CELLS
.
38
2.2.1.7
PROLIFERATION
ASSAY
.
38
2.2.1.8
MIGRATION
AND
INVASION
ASSAY
.
38
2.2.1.9
CHEMOTAXIS
ASSAY.
39
2.2.1.10
COLLAGEN
IV
ADHESION
ASSAY
.
39
2.2.1.11
APOPTOSIS
ASSAY
.
39
2.2.1.12
2D
INHIBITOR
ASSAY
.
40
2.2.1.13
3D
INHIBITOR
ASSAY
.
40
2.2.1.14
FLOW
CYTOMETRIC
ANALYSIS
OF
CANCER
STEM
CELL
MARKERS
.
40
2.2.1.15
IMMUNOFLUORESCENT
STAINING
OF
CELLS
AND
FLUORESCENCE
MICROSCOPY
.
41
2.2.1.16
CALCEIN
VIABILITY
STAINING
OF
TUMOROIDS
.
41
2.2.2
MICROBIOLOGY
TECHNIQUES
.
41
2.2.2.1
AGAR
PLATES
FOR
BACTERIA
CULTURE
.
41
2.2.2.2
PREPARATION
OF
COMPETENT
CELLS
.
42
2.2.2.3
TRANSFORMATION
OF
COMPETENT
XL-1
BLUE
.
42
2.2.3
MOLECULAR
BIOLOGY
TECHNIQUES
.
43
2.2.3.1
PLASMID
DNA
EXTRACTION
.
43
2.2.3.2
DNA
EXTRACTION
FROM
MURINE
TISSUE
.
43
2.2.3.3
NUCLEIC
ACID
CONCENTRATION
AND
PURITY
DETERMINATION
.
43
2.2.3.4
OLIGONUCLEOTID
DESIGN
.
43
2.2.3.5
SITE
DIRECTED
MUTAGENESIS
USING
THE
QUIKCHANGE
METHOD
.
44
2.2.3.6
QUANTITATIVE
REAL-TIME
PCR
.
44
2.2.4
PROTEIN
BIOCHEMISTRY
TECHNIQUES
.
45
2.2.4.1
PREPARATION
OF
WHOLE
CELL
PROTEIN
LYSATES
.45
2.2.4.2
PROTEIN
CONCENTRATION
DETERMINATION
.45
2.2.4.3
SDS-PAGE
ELECTROPHORESIS
.45
2.2.4.4
WESTERN
BLOTTING
.
.
46
2.2.4.5
DENSITOMETRIC
QUANTIFICATION
.46
2.2.4.6
SEPARATION
OF
CYTOSOL
AND
MEMBRANE
FRACTIONS
BY
ULTRACENTRIFUGATION
.
46
2.2.4.7
KINOME
PROFILING
.
47
2.2.4.8
PEPTIDE
EXTRACTION
.
48
2.2.4.9
LIQUID
CHROMATOGRAPHY/MASS
SPECTROMETRY
.48
2.2.5
ANIMAL
EXPERIMENTS
.
49
2.2.5.1
INTRACARDIAC
INJECTION
OF
CIRCULATING
TUMOR
CELLS
.
49
2.2.5.2
ORAL
TREATMENT
OF
MICE
WITH
AKT/MTOR
INHIBITORS
BY
GAVAGE
.49
2.2.53
IN
VIVO
OPTICAL
IMAGING
OF
TUMOR
CELL
DISSEMINATION
AND
TUMOR
BURDEN
.
50
2.2.5.4
SACRIFICE
AND
ORGAN
HARVESTING
.
50
2.2.5.5
EX
VIVO
OPTICAL
IMAGING
OF
HARVESTED
ORGANS
.
50
2.2.5.6
HOMOGENIZATION
OF
MURINE
TISSUE
.
50
2.2.5.7
MURINE
BONE
MARROW
PREPARATION
.
51
2.2.5.8
CTC
DETECTION
FROM
MURINE
WHOLE-BLOOD
.
51
2.2.5.9
ALU-PCR
.
51
2.2.5.10
HISTOLOGICAL
ANALYSIS
OF
MURINE
TISSUE
.
51
2.2.6
BIOINFORMATIC
ANALYSIS
.
52
2.2.6.1
LC/MS
DATA
PROCESSING
.
52
2.2.6.2
GENE
ONTOLOGY
ENRICHMENT
ANALYSIS
.
55
2.2.63
DETERMINATION
OF
UNIQUELY
ABUNDANT
AND
COMMON
PROTEINS
.
56
2.2.6.4
PROTEIN-PROTEIN
INTERACTION
NETWORKS
FUNCTIONAL
ENRICHMENT
ANALYSIS
.
56
2.2.6.5
KEYWORD-BASED
LITERATURE
SEARCH
USING
OMICSLITMINER
.
56
2.2.6.6
STRUCTURAL
MODELING
OF
PIK3CA
MUTATIONS
.
57
2.2.6.7
DATA
MINING
OF
TCGA
DATASETS
.
57
2.2.6.8
GENE
EXPRESSION
PROFILE
ANALYSIS
.
57
2.2.6.9
CALCULATION
OF
COMBINATION
INDEX
.
57
2.2.6.10
CORRELATION
OF
DATA
.
57
2.2.7
S
TATISTICAL
ANALYSIS
AND
VISUALIZATION
.
58
3
RESULTS
.
59
3.1
A
NALYSIS
AND
INHIBITION
OF
THE
PI3K/AKT/
M
TOR
PATHWAY
ACTIVATION
IN
CTC
S
.59
3.1.1
PI3K/AKT/MTOR
SIGNALING
PATHWAY
ACTIVATION
AND
SUSCEPTIBILITY
TO
AKT
AND
MTOR
INHIBITORS
IN
A
2D
MODEL
IN
VITRO
.
59
3.1.2
SENSITIVITY
OF
CTC
LINES
TOWARDS
AKT
AND
MTOR
INHIBITORS
IN
A
3D
TUMOR
SPHEROID
MODEL
.
63
3.1.3
SENSITIVITY
OF
COLORECTAL
CTCS
AGAINST
AKT/MTOR
INHIBITION
IN
A
MURINE
INTRACARDIAC
XENOTRANSPLANTATION
MODEL
IN
VIVO
.
:
.
67
3.2
C
HARACTERIZATION
OF
PI3K
MUTATIONS
DETECTED
WITHIN
PRIMARY
TUMOR
,
METASTASIS
AND
CTCS
FROM
THE
SAME
PATIENT
.
75
3.2.1
FREQUENCY,
LOCATION,
AND
CLINICAL
OUTCOME
OF
PI3K
MUTATIONS
IN
BREAST
CANCER
PATIENTS
.
76
3.2.2
PREDICTED
EFFECT
OFC2
DOMAIN
MUTATIONS
ON
THE
PROTEIN
STRUCTURE
OFPLLOAAND
THE
INTERACTION
WITH
P85A.
.
77
3.2.3
ESTABLISHMENT
OF
PIK3CA
C2
DOMAIN
MUTANT
E418K
AND
E453K
OVEREXPRESSING
BREAST
CANCER
CELLS
.
80
3.2.4
ANALYSIS
OF
DOWNSTREAM
SIGNALING
OF
PIK3CA
MUTANTS
.
80
3.2.5
ANALYSIS
OF
THE
FUNCTIONAL
ROLE
OF
PIK3CA
E418K
AND
E453K
IN
BREAST
CANCER
.
84
3.2.6
SUSCEPTIBILITY
OFE418KAND
E453K
MUTANT
TO
PI3K
INHIBITION
.
88
3.2.7
ANALYSIS
OF
THE
PI3K
MEMBRANE
LOCALIZATION
IN
E418K
AND
E453K
MUTANT
.
89
3.3
T
HE
ROLE
OF
AKT
ISOFORM
SPECIFIC
KNOCKDOWN
IN
COLORECTAL
CANCER
CTCS
.
91
3.3.1
ESTABLISHMENT
OFAKTI,
AKT2
AND
AKT3
ISOFORM
SPECIFIC
KNOCKDOWNS
IN
CTC-MCC-41
.
91
3.3.2
FUNCTIONAL
ROLE
OF
AKT
ISOFORMS
IN
COLORECTAL
CANCER
CTCS
.91
3.3.3
PROTEOMIC
CHANGES
IN
CTC-MCC-41
CELLS
AFTER
AKT
ISOFORM
SPECIFIC
KNOCKDOWN
.
92
3.4
A
NALYSIS
OF
SUSPENSION
AND
ADHERENT
FRACTIONS
WITHIN
BREAST
CANCER
AND
COLORECTAL
CANCER
CTCS
.102
3.4.1
GROWTH
PATTERN
OF
CTCS
IN
CULTURE
.
102
3.4.2
SIGNALING
PATHWAY
ANALYSIS
OF
SUSPENSION
AND
ADHERENT
CELLS
.
106
3.4.3
KINOME
ANALYSIS
OF
SUSPENSION
AND
ADHERENT
CTCS
.
107
3.4.4
ANALYSIS
OFCADHERIN
AND
EMT
MARKER
EXPRESSION
IN
SUSPENSION
AND
ADHERENT
CTCS
.
119
3.4.5
EXPRESSION
OF
CANCER
STEM
CELL
MARKERS
IN
COLORECTAL
CANCER
CTCS
.
120
4
DISCUSSION
.123
4.1
AKT
AND
M
TOR
INHIBITORS
IN
THE
PRECLINICAL
AND
CLINICAL
SETTING.
123
4.2
I
NHIBITION
OF
CTCS
AS
A
TARGET
FORMETASTASIS
PREVENTION
.
125
4.3
THE
FUNCTIONAL
ROLE
OF
AKT
ISOFORM
SPECIFIC
SIGNALING
IN
CANCER
.
125
4.4
PI3K
MUTATIONS
AND
THE
FUNCTIONAL
ROLE
OF
C2
DOMAIN
MUTATIONS
IN
BREAST
CANCER.
127
4.5
S
USPENSION
AND
ADHERENT
CTCS
AND
THE
EPITHELIAL
-
MESENCHYMAL
TRANSITION
.129
5
SUMMARY
.
134
6
APPENDIX
.
135
7
LIST
OF
ABBREVIATIONS.
157
8
LIST
OF
TABLES
.
162
9
LIST
OF
FIGURES.
163
10
REFERENCES
.
165
11
ACKNOWLEDGMENT
.
176
12
CURRICULUM
VITAE
.
178
13
PREVIOUS
PUBLICATIONS
.
179
14
STATUTORY
DECLARATION
.
181 |
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| author | Smit, Daniel Jan 1996- |
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| id | DE-604.BV049582184 |
| illustrated | Illustrated |
| index_date | 2024-07-03T23:32:09Z |
| indexdate | 2024-07-10T10:11:17Z |
| institution | BVB |
| language | English |
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| physical | 181 Blätter Illustrationen, Diagramme 30 cm |
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| spelling | Smit, Daniel Jan 1996- Verfasser (DE-588)1251596630 aut Functional role of dysregulated PI3K/AKT/mTOR signaling in circulating tumor cells vorgelegt von: Dr. med. Daniel Jan Smit Hamburg 2022 181 Blätter Illustrationen, Diagramme 30 cm txt rdacontent n rdamedia nc rdacarrier Dissertation Universität Hamburg 2023 (DE-588)4113937-9 Hochschulschrift gnd-content B:DE-101 application/pdf https://d-nb.info/1297464400/04 Inhaltsverzeichnis DNB Datenaustausch application/pdf http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=034927100&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA Inhaltsverzeichnis 1\p dnb 20230814 DE-101 https://d-nb.info/provenance/plan#dnb 2\p dnb 20230814 DE-101 https://d-nb.info/provenance/plan#dnb |
| spellingShingle | Smit, Daniel Jan 1996- Functional role of dysregulated PI3K/AKT/mTOR signaling in circulating tumor cells |
| subject_GND | (DE-588)4113937-9 |
| title | Functional role of dysregulated PI3K/AKT/mTOR signaling in circulating tumor cells |
| title_auth | Functional role of dysregulated PI3K/AKT/mTOR signaling in circulating tumor cells |
| title_exact_search | Functional role of dysregulated PI3K/AKT/mTOR signaling in circulating tumor cells |
| title_exact_search_txtP | Functional role of dysregulated PI3K/AKT/mTOR signaling in circulating tumor cells |
| title_full | Functional role of dysregulated PI3K/AKT/mTOR signaling in circulating tumor cells vorgelegt von: Dr. med. Daniel Jan Smit |
| title_fullStr | Functional role of dysregulated PI3K/AKT/mTOR signaling in circulating tumor cells vorgelegt von: Dr. med. Daniel Jan Smit |
| title_full_unstemmed | Functional role of dysregulated PI3K/AKT/mTOR signaling in circulating tumor cells vorgelegt von: Dr. med. Daniel Jan Smit |
| title_short | Functional role of dysregulated PI3K/AKT/mTOR signaling in circulating tumor cells |
| title_sort | functional role of dysregulated pi3k akt mtor signaling in circulating tumor cells |
| topic_facet | Hochschulschrift |
| url | https://d-nb.info/1297464400/04 http://bvbr.bib-bvb.de:8991/F?func=service&doc_library=BVB01&local_base=BVB01&doc_number=034927100&sequence=000001&line_number=0001&func_code=DB_RECORDS&service_type=MEDIA |
| work_keys_str_mv | AT smitdanieljan functionalroleofdysregulatedpi3kaktmtorsignalingincirculatingtumorcells |
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