Terms and Molecules Flashcards
Type I ovarian cancer
25%, Low-grade
Not spreading
Contained to ovaries
Early diagnosis
Mostly Ras, BRAF, PTEN, and beta-catenin mutations
Type II ovarian cancer
75%, high grade
Highest prevalence and lethality (HGS)
Late diagnosis, rapid, metastatic
P53, operative cytoreduction!
P63
Essential in ectoderm development
P73
Delta Np73 inhibitor of usual p53 fanily function
Anti-apoototic
Important in brain development
STIC
Serous tubular intraepithelial cancer
Stage (in situ carcinoma) of ovarrian cancer
CA125
Tissue marker of peritoneum
Elevated in ovarian carcinoma, pregnancy, cirrhosis, ascites, …
Rituximab
mAb against CD20
On pre and mature B cells
B Lymphomas (NHL)
Fc and Fab region
Regions of antibodys
Fc region interacts with immune system and induces cell lysis
Fab region binds a specific surfaxe marker (e.g. Rituximab binds CD20)
FISH
flourescence in situ hybridization
Flourescent DNA or RNA probes hybridize
Detection of translocations and copy number variations
Her2
Amplification associated with poor prognosis and increased recurrence in breast cancer
Maybe involved in tumorigenesis and therapy resistance to some chemotherapies
Diagnosis importent for therapeutic descicions
Causes of genetic instability
Environmental (lifestyle: smoking, diet and UV and IR exposure, viral/bacterial infections, …)
Genetics: defects in DDR, cellcycle regulation, checkpoints, …
In total causes high mutation rate or chromosomal instability
G1 checkpoint
CDK4/6
Cyclin D
G1/S checkpoint
CDK 2
Cyclin E
S checkpoint
CDK 2
Cyclin A
G2 checkpoint
CDK 1
Cyclin A
M checkpoint
CDK 1
Cyclin B
Checkpoints definition
Monitor and control the completion and irder of major cell cycle events
Types of DNA damage
Loss of bases
Modification of bases
Strand breaks
Blocked DNA replication
Intertumor heterogeneity
Tumors of different patients have different genetic profiles even when stemming from the same tissue or cell type since mutations occur randomly and even driver mutations can vary
Intratumor heterogeneity
Higher mutations rates allow development of driver mutations within one cell and subsequent tumorigenesis
During this process and after many other mutations occur from which some may have replicative benefits, different cells may develop different mutations with reolicative benefits which lads to their increased expansion –> subclones
Initiator caspases
8 (extrinsic)
9 (intrinsic)
10
Activated through dimerization and autoprocessing through adaptors
Executioner caspases
3, 6 and 7
Activation through clevage by initiator caspases
Caspase activation
Executioner through clevage by initiator
Initiator through dimerization through adaptors
Adaptors through apoptosis-inducing signals
–> LOSS of MITOCHONDRIAL INTEGRITY
–> RECEPTOR-LIGAND interaction
–> CELL-CELL CONTACT
Bcl2 family type & role in MOMP
BAX/BAK
pro-apoptotic multidomain
dimerize and release cytochrome c
usually inhibited through anti-apoptotic Bcl2 (Bcl-2, Bcl-XL, …)
Bcl2 family type & role in MOMP
Bcl-2 (Bcl-XL, Mcl-1)
anti-apoptotic
bind Bax/Bak and prevent dimerization
inhibited by BH3 only –> apoptosis
Bcl2 family type & role in MOMP
BIM
pro-apoptotic BH3 only
bind anti-apoptotic Bcl-2 and desinhibit Bax/Bak dimerization
Bcl2 family type & role in MOMP
BID
pro-apoptotic BH3 only
bind anti-apoptotic Bcl-2 and desinhibit Bax/Bak dimerization
Bcl2 family type & role in MOMP
BAD
pro-apoptotic BH3 only
bind anti-apoptotic Bcl-2 and desinhibit Bax/Bak dimerization
Complex I formation
TNF-receptor ligation
Caspase-8 and c-FLIP-L
NFkB signaling, JNK and p38
SURVIVAL
Complex II formation
TNF-receptor ligation
Caspase-8 and Caspase-8
activation of Caspase 3 and BID
APOPTOSIS
Complex III formation
TNF-receptor ligation
Caspase-8 and v-FLIP or c-FLIP-S
NECROPTOSIS
Cell death pathways as target for anti-cancer therapy
BH3-mimetics (= Bcl2 inhibitors)
XIAP inhibitors = Smac mimetics
RTKi target BIM, BAD, BMF
Biogenesis of miRNA
Transcription of pri-miRNA
Processing through DROSHA/PASHA to pre-miRNA
Nuclear export (Exportin 5)
Dicing through TRBP/Dicer-1 to miRNA:miRNA* duplex
RISC loading from pre-RISC to mature RISC
DROSHA/PASHA
processing pri-miRNA to pre-miRNA within the nucleus
Exportin-5
nuclear export of pre-miRNA into the cytosol
TRBP
in complex with Dicer-1 dicing of pre-miRNA to miRNA:miRNA* duplex
Dicer-1
in complex with TRBP dicing of pre-miRNA to miRNA:miRNA* duplex
TRBP/Dicer-1
dicing of pre-miRNA to miRNA:miRNA* duplex
global miRNA reduction
example for reason
consequnces
e.g. through Exportin-5 defect
promotes tumorigenesis
oncomiRNA
examples and consequences
overexpression promotes tumorigenesis (e.g. miR-17-92 inhibits PTEN)
can also induce EMT (e.g. miR-21 is induced through androgens, causes EMT and inhibits pro-apoptotic protein PDCD4)
miR-17-92
oncomiRNA
inhibits PTEN
onco-miRNAs
miR-17-92: inhibits PTEN
miR-21: induces EMT and inhibits PDCD4 (pro-apoptotic), regulated via androgens
miRNA seed region
nts 2-6
require perfect base pairing –> determine selectivity of targets
lncRNAs in gene regulation
mechanisms
guides
decoys
scaffolds
enhancers
ceRNA
competing endogenous RNA
coregulation with mRNA transcript through identical RISC-binding sites
Ras activation and consequence
inactive GDP-bound Ras activated through GEF that exchanges GDP with GTP
shift in swith regions I and II
enables interaction with effectors containing RBD
MAPK cascade with examples
small G protein (Ras)
MAPKKK (BRAF)
MAPKK (MEK)
MAPK (ERK1/2)
effector
Markers of EMT (up and down)
E-cad down
N-cad up
MET up
HGF up
HDGF up
Oct4 and BIM-1 up
TF regulating EMT
Snail & Twist (induced by TGF-beta)
tumor suppressive miRNAs
miR-29b: inhibits metastasis, E-cad up, N-cad down, Snail&Twist down
miR-338-5p & miR-421: reduce EMT markers and stemness, proliferation, growth and metestasis
regulation of EMT through steroid hormones
onco-miR-21 redgulated through androgens
promotes EMT and inhibits PDCD4 (anti-apoptotic)
experimental therapies to target EMT
ZOLEDRONIC ACID: reverses EMT, Snail&Twist, N-cad, Oct4 and BIM-1 down
SD-208: reverses TGF-beta induced EMT, BRachyury, migration and invasion down, chemosensitivity up
PROTEOSOME INHIBITOR: reduces Snail
Wnt INHIBITORS: reduce EMT
targeting EGF, EGFR, and ErbB2 –> reduces EMT
TGF-beta and EMT
causes growth, immunosuppression, angiogenesis and EMT
E-cad down, Fibronectin and Twist&Snail up
induces TF Brachyury –> increases invasiveness
Heat shock proteins and EMT
Hsp27 induces EMT
chaperone of oncogenes
E-cad down
Wnt, mesenchymal proteins and MMP up
Hsp27 essential for IL6 and therefore the IL6/STAT/Twist pathway
Zoledronic acid
experimental therapy to target EMT
reverses EMT
Snail&Twist, N-cad, Oct4 and BIM-1 down
SD-208
experimental therapy to target EMT
reverses TGF-beta induced EMT
Brachyury, migration and invasion down
chemosensitivity up
Proteosome inhibitors
experimental therapy to target EMT
reduce Snail
Wnt inhibitors
experimental therapy to target EMT
reduces EMT
target EGF, EGFR and ErbB2
experimental therapy to target EMT
reduces EMT
Hsp27
heat shock protein
chaperone to oncogenes
induces EMT (E-cad down
Wnt, mesenchymal proteins and MMP up)
essential for IL6 and therefore the IL6/STAT/Twist pathway
lymphatic spread
PERMEATION: easy access through lack of thight junctions, BM and astrocytes –> intravasation for hematogenous spread requires proteolytic enzymes
CHEMOTACTIC DIFFUSION: cytokines of lymphatic vessels promote infiltration
LYMPHANGIOGENESIS: tumor cell induced vessel formation (VEGF-C)
MMPs
function
remodelling of ECM components through degradation and clevage
Znc-dependend endopeptidases, activated through clevage by proteinases
MMPs
structure
membrane-bound and secreted MMPs (can interact with cytoskeleton)
metal ion at center (often Znc)
PRO-PEPTIDE: autoinhibition, activation requires its clevage
CATALYTIC DOMAIN: active site, contains Znc2+
C-TERMINUS: protein-protein interactions, e.g. TIMPs
challenges for CTCs
shear forces –> mechanical destruction
immunological clearance
survival mechanisms of CTCs
dynamic regulation of cellular stiffness and contractility
close interaction with blood microenvironment
clusters of CTCs show better survival as single cells
seed & soil hypothesis
Stephan Paget
metastasis formation reuqires the right cell to be in the right environment
for certein tissues of origin certain organs are beneficial (but it also depends on the individual genetics of the cell in question)
anatomical hypothesis
metastasis depends on the blood flow downstream of the primary tumor
CTCs form metastasis in small vessels downstream –> liver or lung
for splanchnic organs = liver, rest = lung
definition CTCs
cells that successfully detached form their primary tumor and intravasated (& survived in the blood)
detection of CTCs
hard, low cell number esp. when comparing to blood cells
MACS: density, CD45 and EpCAM
CellSearch: EpCAM
ScreenCell Filtration: size
ParsortixTM microfluidic system: deformability and size
CTC detection - MACS
depending on density, CD45 and EpCAM
CTC detection - CellSearch
depending on EpCAM (often loss during EMT!)
CTC detection - ScreenCell filtration
depending on size
CTC detection - ParsortixTM microfluidic system
depending on size and deformability
reasons for metabolism alterations in tumors
sustaining proliferative signaling
enabling replicative immortality
evading cell death
evading growth suppressors
activating invasiveness and metestasis
inducing angiogenesis
tumor-specific metabolic adaptations
TCA cycle
lipolysis, proteolysis, glutaminolysis
lipogenesis
redox status
glycolysis –> Warburg and Carbtree effect
Benefits of Glycolysis
indipendent form fluctuations in oxygen tension
generating bicarbonic and lactic acid –> acidification and increased invasiveness
PPP generates NADPH –> antioxidant
intermediates used for macromolecular biogenesis
causes for metabolic adaptations
environment: hypoxia induces HIF –> expression of glycolytic enzymes
cancer genes themselves
tumor specific isoforms: M2 of PK (catalyzes rate limiting step)
Mutations: e.g. IDH (D-2HG)
therapies targeting metabolism
targeting METABOLIC ENZYMES of CARBON metabolism (e.g. LDH, IDH, PDK)
targeting LIPOLYSIS, GLYCOLYSIS, TCA (mitochondrial metabolism)
AEROBIC GLYCOLYSIS INHIBITORS
detection of alterations in ROS levels
reagents that change colour or flourescent emission upon oxidation –> flourescence microscopy or FACS
detection of changes in mitochondrial morphology
microscopy (confocal imaging)
mayby mitochondrial staining
inductors of switch to glycolysis
hexokinase
PI3K/Akt
Myc and MondoA
HIF
p53
Pro and cons of ROS
oxidation of proteins
regulate protein stability
increase/decrease function (constitutive EGFR activation)
alter subcellular localization (translocation)
altered prot-prot interaction
low levels physiological, intermediate is tumorigenic through adaptive proteins and mutagenesis), high levels is cytotoxic
Carbtree effect
increased glucose levels and uptake do not increase oxygen tension instead oxygen uptake is reduced in presence of glucose
this can be found in tumor cells and other rapidly dividing cells (thymocytes, spermatozoae, mucosal cells, ESCs)
Warburg effect
switch from oxidative phosphorylation to aerobic glycolysis
dependend on glucose
Warbur effect
advantages
switch from oxidative phosphorylation to aerobic glycolysis
less dependency on oxygen
ACIDIFICATION of tumor environment –> immune evasion and DDR inactivation (mutagenesis)
INVASIVENESS and METASTASIS –> elevated MMP, lactate increases HYALURONAN (single cells) and CD44 (motility)
HIF1-alpha stabilization
induces glycolytic enzymes
degraded by Von Hippel Lindau (VHL, E3 ubiquitin ligase)
VHL MUTATIONS
ONCOGENE SIGNALING (Ras, Src, PKB, …)
TCA CYCLE ENZYMES
ROS (stabalizes HIF)
HIF1-alpha
inductor of switch to glycolysis
induces glycolytic enzymes
degraded by Von Hippel Lindau (VHL, E3 ubiquitin ligase)
Hexokinase II
inductor of switch to glycolysis
anti-apoptotic properties
catalyzes the rate-limiting step during glycolysis
regulated by Myc (elevated –> elevated)
PI3K/Akt
inductor of switch to glycolysis
Myc
with MondoA inductor to switch to glycolysis
in combination with MAX: increase of glycolytic enzymes
- Hexokinase II
- GAPDH
- Enolase-1
- Pyruvate kinase
- Lactate dehydrogenase
p53
inductor of switch to glycolysis
loss increases glycolysis
TIGAR: induced by p53, reduces glycolysis
SCO2: induced by p53, necessary for COX complex assembly (important for respiratory chain)
PGM: repressed by p53, catalyzes step in glycolysis
drug-unspecific resistance mechanisms
drug efflux
cell death inhibition
EMT
epigenetics
drug-specific resistance mechanisms
drug metabolism –> inhibition or prohibition of activation
mutations in drug target
DDR
activation of alternative signaling pathways
therapeutic strategies to reduce expression of MDR protein transporters
polypeptide transcriptional repressor
siRNAs
ribozyms
glycosylation inhibitor (important for folding)
extrinsic cell death inhibition
loss of caspase 8
increased expression of FLIP (caspase 8 inhibitory protein)
overexpression of IAPs
DNA methylation in cancer and therapy resistance
hypomethylation of oncogenes (e.g. proliferative proteins)
hypermethylation of tumorsuppressors (e.g. pro-apoptotic proteins)
DRUG RESISTANCE: changes in methylation pattern for better DDR, drug efflux, changes in drug metabolism, EMT, …
example: hypomethylation of UGT1A1 –> resistance to irinotecans active metabolite SN38
preventing hypermethylation of anti-tumor sequences through cytidine-analogs that cannot be methlyated
evasion of cell death by DNA damaging drugs
drug efflux
cell death inhibition
increased DDR
epigenetics
drug inactivation/metabolisation
lactate in tumor survival and metastasis
increases HYALURONAN –> less adhesion, more single cells
increases CD44 –> cytoskelletal interaction -> motility
acidification of tumor environment –> immune evasion and mutagenic (nactivation of DDR)
glycolysis inhibitors
2-deoxyglucose
3-bromopyruvate
DCA (dichloroacetat)
siRNAs inhibiting LDH (lactate dehydrogenase)
Somatostatin and derivate TT-232
2-deoxyglucose
glycolysis inhibitor
3-bromopyruvate
glycolysis inhibitor
DCA (dichloroacetat)
glycolysis inhibitor
Somatostatin
glycolysis inhibitor
derivate is TT-232
TT-232
derivate of Somatostatin
glycolysis inhibitor
inhibition of LDH
with siRNAs
inhibition of glycolysis
donor of methyl groups
methionine
mostly as SAM (S-adenosylmethionine) = Methionine + ATP
metabolic cycling of methionine
uptake through diet
+ ATP –> SAM
SAM = SAH + CH3
SAH (S-adenosylhomocystein) –> Hyc (Homocystein)
Hyc remethylated (Vit. B12 and 5-methyl-THF) to methionine or catabolized (Vit. B6)
epigenetically regulated metabolic enzymes
CYP1A1: metabolization of PAHs (polycycling aromatic hydrocarbons) to carcinogenic intermediates
UGT1A1: drug resistance to irinotecan by inactivating active metabolite SN38
NAT 1&2: inactivation of carcinogens
CYP1A1
metabolic enzyme
metabolization of PAHs (polycycling aromatic hydrocarbons) to carcinogenic intermediates
hypomethylated in cancer
UGT1A1
metabolic enzyme
metabolization of small lipids to excretable water-soluble molecules
drug resistance to irinotecans active metabolite SN38
hypomethylated in cancer
Irinotecan
cancer therapeutic
active metabolite = SN38
inactivated through metabolization by UGT1A1
SN38
active metabolite of irinotecan
inactivated by metabolization through UGT1A1
NAT2
N-acetyltransferase
can inactivate carcinogens by metabolization
hypermethylated in cancer
epigenetic effects of D-2HG
D-2 hydroxygluterate
oncogenic driver via epigenetic reprogramming in mIDH associated cancers
produced by mIDH1/2
TET2 INHIBITION: leads to CpG hypermethylation
KDMs INHIBITION: leads to histone hypermethylation
promotes BM disruption
TET2
prevents CpG methylation
inhibited by D-2HG (oncometabolite of mIDH1/2)
KDMs
lysine demethylases (e.g. JHDM)
prevent histone methylation
inhibited by D-2HG (oncometabolite of mIDH1/2
metabolite of mutated IDH1/2
D-2HG
D-2 hydroxygluterate
oncogenic driver via pigenetic reprogramming in mIDH1/2 associated cancers
SIRT6
HDAC (histone deacetylase)
reduces the expression of glycolytic genes
may act as tumorsupressor by preventing switch to aerobic glycolysis
repress Myc transcriptional activity
deficiancy promotes Myc activity and ribosome biogenesis
interplay of epigenetics and metabolism in glucose metabolism
GlnAc: produced from glucose entering the hexosamine biosynthetic pathway –> GlnAc of histones
Sirtuins: HDACs, regulated through NAD+ levels
TCA intermediates
- Citrate: conversion to Acetyl-CoA –> HAT
- alpha-KG: cofactor for DE-METHYLATION of histones and DNA
- Methionine: donor for HMT and DNMT
- low ATP/AMP ratio: activation of AMPK –> histone phosphorylation
GlnAc
produced from glucose entering the hexosamine biosynthetic pathway
GlnAcylation of histones
Citrate and epigenetics
converted to Acetyl-CoA
donor for HAT (histone acetylation)
alpha-KG and epigenetics
cofactor for DE-METHYLATION of histone and DNA
methionine and epienetics
donor for HMT and DNMT
mostly as SAM
ATP/AMP ratio
low ratio activates AMPK
causes histone phosphorylation
FRET
flourescence resonance energy transfer
energy transferred from a donor flourophore to an acceptor without emission
acceptor emission is changed/induced, …
enzyme assays: e.g. protease inhibitors
protein conformation
protein-protein interaction
application of FRET
enzyme assays: e.g. protease inhibitors
protein conformation
protein-protein interaction
main questions in drug discovery project planning
SCIENTIFIC & TECHNICAL: hypothesis, target, assays, animal models
STRATEGIC: unmet medical need, market predictions, …
OPERATIONAL: staff, facilities, cost, timescale
HTS
high throughput screening
analysis of a large number of compounds in microtiter plates
HTS-Assays
ENZYMATIC: chromogenic or flourescent substrate
COUPLED ENZYME ASSAY + PROMOTOR: e.g. effect of protein of interest on a promotor inducing luciferase –> measurment of luciferase activity
MEMBRANE PREPARATIONS
PHENOTYPIC ASSAY: e.g. whole living cells and GFP-labeled protein, detection of translocation into the nucleus
SPR
surface plasmon resonance
measurment of direct molecule-molecule interactions in realtime
polarized light on a gold-film causes them to resonate –> measurment of resonance and reflection
on the gold film a protein/fragment/DNA etc. can be bound, if it interacts with its binding partner then there is a change in mass and therefore resonance and reflection (measured)
presentation in sensogramm (resonance over time) –> association, dissociation ans regeneration
ideal drug target
no IP or competition
3D structure known or clear active/catalytic domain
disease causing/modifying
analyzation with HTS possible
bimarker to observe effects in organisms
whole animal screening
C. elegans, D. melanogaster, D. rerio
cultivation of animals in microtiter plates
THERAPEUTICAL SCREEN: reversion to wildtype, control = phenotype
CHEMICAL GENETICS SCREEN: induction of phenotype, control = wildtype
therapeutical screen
(whole animal screening)
compound should induce a reversion to wildtype
control = phenotype
chemical genetics screen
(whole animal screening)
compound should induce a phenotype
control = wildtype
innate immune cells involved in tumor immunity
NK cells
DCs
macrophages (TAM2 pro-tumorigeneic)
adaptive immune cells involved in tumor immunity
B cells
CD4+ T cells
CD8+ T cells
T regs
tumor immune escape mechanisms
- upregulation of immune-inhibitory proteins (CTLA4, PDL-1, PD-1)
- loss of antigen and MHC-I
- acidification of tumor environment
- exclusion of immune cells through ECM modification
- cytokines and molecules inhibiting DC maturation and differentiation (e.g. IL-6, IL-10, VEGF)
- chemokines recruiting tumor-promoting immuen cells
immunotherapeutical approches
TUMOR-Ag VACCINATION: isolation and injection + adjuvant
ADOPTIVE T CELL THERAPY:
- CAR-T cells: selection or transfection
- NK cells: isolate, purify, expand & activate
- DC therapy: load precursors with Ag –> immune response and boost
ANTIBODY THERAPY: desinhibition of T cells (a-CTLA4, a-PD(L)-1)
adoptive T cell therapy
- CAR-T cells: selection or transfection
- NK cells: isolate, purify, expand & activate
- DC therapy: load precursors with Ag –> immune response and boost
tumor-Ag vaccination
isolation of Ag
injection i.v. or i.d. with adjuvant
antibody therapy
e.g. desinhibition of T cells (a-CTLA4, a-PD-1, a-PDL-1)
DCs in tumor immunity
initiators of anti-tumor immunity
incorporation of tumor cells, degradation, transport and presentation of Ag
in tumors numbers reduced and functionally impaired
impairment promotes tumorigenesis and progression
key steps of autophagy
initiation
expansion of phagofore an formation of autophagosome
transport to lysosome and fusion
autophagic breakdown and recycling
kinase-complex essential for supression of autophagy
mTORC1
sensor of AA and GF
if enough is present then inhibition of autophagy by phosphorylation of ULK1-complex
kinase-complex essential for autophagy
ULK1-complex
consits of ULK1, ATG13 and FIP200
continously phosphorylated and inhibited through mTORC1
mTORC-1 inhibition allows dephosphorylation and activation
types of selective autophagy
mitophagy
pexophagy
reticulophagy
ribophagy
xenophagy
aggrephagy
inductor of bulk autophagy
starvation
starvation causes …
bulk autophagy
cell types in stroma
edothelial cells
immune cells
smooth muscle cells
fibroblasts
neurons
upregulated markers in CAFs (3)
FAP
alpha-SMA
S100A4 (= FSP1)
PDGF-receptors
CD90/Thy1
Tenascin C
tumor-promotig effects of CAFs
DIRECT: secretion of mitogenic factors (paracrine), direct cell-cell-contacts
INDIRECT: modulation of immune cells, alterations of ECM, angiogenesis, metabolic reprogramming
THERAPY RESISTANCE: reduce efficacy of chemotherapy, andocrine/target resistance
factors remodelling ECM in tumor-associated stroma
IL-1b, Wnt and TGF-beta drive CAF-subsets
Rho-ROCK: modulates cytoskelletal organisation –> collagen-track formation
CAVEOLIN-1: activates Rho-ROCK, elevated stromal Cav-1 correlates with increased metastasis
YAP & TAZ: promote proliferation and motility, translocation to nucleus induced by mechnical stimuli
FAP
upregulated marker in CAFs
alpha-SMA
upregulated marker in CAFs
FSP1
= S100A4
upregulated marker in CAFs
S100A4
= FSP1
upregulated marker in CAFs
PDGF-receptor
upregulated in CAFs
Rho-ROCK
factor remodelling ECM in tumor-associated stroma
modulates cytoskelletal organisation –> collagen-track formation
Caveolin-1
factor remodelling ECM in tumor-associated stroma
activates Rho-ROCK
elevated stromal Cav-1 correlates with increased metastasis
YAP
with TAZ
factor remodelling ECM in tumor-associated stroma
promote proliferation and motility, translocation to nucleus induced by mechnical stimuli
TAZ
with YAP
factor remodelling ECM in tumor-associated stroma
promote proliferation and motility, translocation to nucleus induced by mechnical stimuli
immune suppressive mechanisms of tumor-associated ECM
- reduced T cell proliferation (stiff)
- reduced T cell activation (impaired Ag presentation, activation)
- limited motility (stiff and dense)
- TAM2 promoton (collagen-rich and rigid ECM favor polarization to M2 phanotype)
infiltrating cell types from which CAF can originate
endothelial cells
adipocytes
marrow-derived stromal cells
pericytes
(tumor cells)
consequences of increased mechanical tension in tumor-associated microenvironment (3)
- induces fibroblast activation to SMA+ myCAFs
- activates latent MMPs
- collagen production elevated
- local release of ECM-stored GFs (TGF-beta, IGF, HGF)
- reveals cryptic sites in ECM molecules
- modified cellulare responses via integrin/FAK association
consequences of increased mechanical tension in tumor-associated microenvironment (3)
- induces fibroblast activation to SMA+ myCAFs
- activates latent MMPs
- collagen production elevated
- local release of ECM-stored GFs (TGF-beta, IGF, HGF)
- reveals cryptic sites in ECM molecules
- modified cellulare responses via integrin/FAK association
CAF regulating tumor immune response
RECRUITMENT OF INNATE & ADAPTIVE IMMUNE CELLS: e.g. via CXCL12 for macrophages or CCL2 for monocytes
MODULATE THEIR ACTIVITY:
- FAP+ CAFs are immuno suppressive
- TGF-beta: released by CAFs, reduces T, NK and CD8+ cells, increases recruitment of tumor-promoting cells
- TSLP: secreted, polarizes T cells to Th2 immunity (tumor- promoting)
TGF-beta
RELEASED BY ECM AND CAFs:
reduces numbers of T cells, NK cells and CD8+ T cells
increses recruitment of tumor-promoting immune cells
IN EMT:
causes growth, immunosuppression, angiogenesis and EMT
E-cad down, Fibronectin and Twist&Snail up
induces TF Brachyury –> increases invasiveness
safety features of oncolytic viruses
non-human pathogenic viruses
dependency of replication on tumor-associated features (active cell cycle, signaling pahways, deficiency of IFN or p53)
safety concerns of oncolytic viruses
specifity for tumor tissue
cytokine storm (virus progeny!)
tumor targeting mechanisms of oncolytic viruses
EXTRACELLULAR: inf. of tumor vasculature, increased potency through tumor-associated e.g. proteases in ECM
CELL SURFACE: natural tropism or engineered retargeting towards tumor-associated markers or neoantigens
CYTOSOLIC: dependency on oncogenic signaling apthways (Ras, EGFR), dependency on IFN deficiency
NUCLEAR: active cell cycle, p53 deficiency, active RB pathway
extracellular tumor targeting mechanisms of oncolytic viruses
infection of tumor vasculature
increased potency through e.g. tumor-associated proteases in ECM
cell surface tumor targeting mechanisms of oncolytic viruses
natura tropism or engineered retargeting towards tumor-associated surface markers or neoantigens
cytosolic tumor targeting mechanisms of oncolytic viruses
dependency on oncogenic signaling pathways (EGFR, Ras)
dependency on IFN deficiency
nuclear tumor targeting mechanisms of oncolytic viruses
dependency on active cell cycle
dependency on deficient p53
dependency on RB pathway
heterologous cancer vaccines
prime & boost principle
combination of oncolytic virus with non-oncolytic vaccine
oncolytic virus induces anti-viral and anti-tumor response, release of neoantigens
vaccine boosts the existing anti-tumor response selectively
transient oncolytic effect and long-lasting, more robust and more effective anti-tumor response
challenges of oncolytic-viruses-based therapy
granting tumor specifity and no infection of helathy cells
generating anti-tumor and not only anti-viral responses
overactivation of immune system (cytokine storm)
changes in ECM prevent viral infiltration
pre-existing immunity to virus (e.g. adenovirus)
structural element of the protein kinase domain
N-lobe
C-lobe
activtation loop
catalytic cleft
classification of protein kinases
Serin/threonin kinases
Tyrosin kinases
role of protein kinases in regulating protein function
SIGNAL RECEPTION/TRANSDUCTION: receptors have PK activity or are associated with PK
ACTIVITY: conformational shifts through phosphorylation can change activity status, reveal binding sites
INTERACTION: phosphorylation itself as binding partner (SH domains)
FUNCTION: posttranslational modifications for correct function
regulation of substrate recognition in protein kinases
physical interactions
CATALYTIC CLEFT: accomodates site of phosphorylation and several neighbouring AA –> dependend on size and charge
DOCKING SITE & DOMAIN INTERACTIONS: increase specifity and affinity, can induce conformational shifts (activation)
ADAPTORS & SCAFFOLDS: increase proximity and speed, can cause conformational shifts to promote phosphorylation
structure of intracellular signaling pathways
signal
reception
processing
output
signal propagation
a defined cascade of protein/factor activation and inactivation
elevated/accelerated through scaffolds
additional regulation, inhibition or increase through feedback loops
role of UPR in tumorigenesis
involved in nearly all hallmarks of cancer
- evading growth supressors, cell death and immune destruction
- inducing tumor promotig inflammation, angiogenesis, invasion and metastasis, genome instability and mutagenesis
- sustainming prolifertive signaling
- deregulation of cellular energetics
examples/pathways of UPR in tumorigenesis
inflammation, mutagenesis, stemness, growth and angiogenesis
ER stress in macrophages –> PERK, IRE1, ATF6 –> cytokines –> inflammation –> elevated ROS –> mutagenesis
ER stress –> IRE1 –> XBP1s –> increased stemness and tumor growth
ER-stress –> PERK –> ATF4 –> VEGF –> angiogenesis
elevated XBP1s in tumors
increases stemness and tumor growth
ATF4 in tumors
VEGF release –> angiogenesis
ER stress in macrophages
induction of PERK, ATF6 and IRE1
cytokine production
inflammation
elevated ROS
muatgenesis
sensors of ER stress + TF
PERK –> ATF4
IRE1 –> XBP1s
ATF6 –> intracellular fragment (ATF6p50)
enzymatic domains in IRE1 and function
KINASE domain –> trans-autophosphorylation upon oligomerization
ENDONUCLEASE domain –> splicing of one intron in XPB1 –> active TF XBP1s
