Cell signaling pathways, apoptosis, angiogensis, stem cells, metabolism, senesence Flashcards
Which of the following are split kinases?
PDGFR STAT3 RON CSF-1R VEGFR INSR FGFR KIT/SCFR MET FLK2/FLT3 ERRB2 EGFR TIE1/2 TRK
- PDGFR, CSF-1R (receptor for M-CSF), KIT/SCFR, FLK2/FLT3, VEGFR, FGFR, TIE1/2
- Split kinases have 2 cytoplasmic kinase domains; important autophosphorylation sites are present on a kinase insert within the catalytic domain
Which TKR are homodimers?
TGF-B VEGFR Notch ERRB2 TIE1/2 TRK Integrins PDGFR SCF EGFR
EGFR can form homo- or heterodimers
VEGFR
SCF
Which TKR are heterodimers?
TGF-B Notch ERRB2 EGFR TIE1/2 TRK Integrins PDGFR SCF ERBB2
TGF-B receptors
Integrins
Notch
EGFR
Cytokine receptors lack TK activity, so they noncovalently associate with what receptors?
JAK receptors (cytoplasmic tyrosine kinases)
• Ligand binds –> cytokine receptor dimerization –> JAK transphosphorylates/activates itself –> also phosphorylates/activates the C-terminal tails of their cytokine receptors, creating a docking site for SH2-containing proteins such as STATs
• Type I cytokine receptors of the IL-6 family –> JAK2 –> STAT3
o STAT3 activating mutations in some tumors
How is or what activates and deactivates RAS in its pathway?
• A balance of activity btw GEFs and GAPs determines the activity of normal RAS proteins
Describe the states in which RAS is turned on vs. off.
- GTPase-activating proteins (GAPs) convert GTP to GDP to deactivate RAS
- RAS is activated by guanine nucleotide exchange factors (GEFs), which release RAS-bound GDP to allow for GTP binding
Once RAS proteins are made, they require what process to occur in order to associate with intracellular membranes?
posttranscriptional modification (prenylation)
How does RAS prenylation occur?
Addition of a covalently-linked lipid chain (either farnesyl or geranylgeranyl group) by farnesyl transferase or geranylgeranyl transferase
o H-RAS and N-RAS are additionally modified by the addition of 2 long chain fatty acids to facilitate membrane localization
What treatment would be most effective for a RAF mutation?
MEK 1/2 because is downstream RAF (not RAS)
MEK/ERK inhibition
What are the basic steps of the PI3K pathway?
PI3K –> converts PIP2 to PIP3 –> AKT–>
AKT phosphorylates a million proteins on their serine/threonine residues
Based on the list below determine which of the downstream proteins is inhibited by AKT:
Mdm2 GSK-3B mTOR HIF-1a FOXO4 Tsc2 NF-KB Bad
INHIBITED by AKT
GSK-3B: anti-proliferative protein; phosphorylates B-catenin, cyclin D1, and Myc
FOXO4: anti-proliferative protein; induces expression of p27Kip1, a CDK inhibitor
Tsc2: anti-growth protein; normally forms a complex that inactivates mTOR
Bad: pro-apoptotic protein; belongs to Bcl-2 family
Based on the list below determine which of the downstream proteins is activated by AKT:
Mdm2 GSK-3B mTOR HIF-1a FOXO4 Tsc2 NF-KB Bad
o ACTIVATED by AKT
Mdm2: anti-apoptotic protein; triggers destruction of
p53
HIF-1a: stimulates angiogenesis
mTOR: protein synthesis
NF-KBWhat is the main function of PTEN as part of the PI3K pathway?
Antagonizes PI3K signaling by acting as a lipid phosphatase for PIP3 (converting it back to PIP2)
What would happen if PTEN was downregulated?
Downregulation of PTEN –> increased PIP3 –> hyperactive PKB/AKT signaling
What pathway is the target of stem cells?
NOTCH
Notch plays a critical role in stem cell self-renewal
Describe the process of the Notch pathway?
NOTCH matures in Golgi and is cleaved into 2 fragments forms heterodimer on cell surface
binds DELTA-LIKE or JAGGED ligands
ligand binding triggers 2 sequential proteolytic cleavages:
(1) ADAM family MMPs release extracellular domain
(2) Presenilin-protease (y-secretase) complex releases cytoplasmic domain (NIC)
NIC enters nucleus to activate transcription of HES and HEY
Based on the targets of c-MYC listed below, what are the functions of each of these in regards to the development of cancer? (Hint: They are the hallmarks of cancer)
- IL-1B
- Cyclin D1, CDK4, CDC25, GADD45, E2F1, E2F2, E2F3
- TERT
- VEGF
- EZRIN
- MAD2 and BUBR1
- LDHA and GLUT-1
- ICAM-1
- IL-1B : tumor promoting inflammation
- Cyclin D1, CDK4, CDC25, GADD45, E2F1, E2F2, E2F3: self-sufficient growth signals, resistance to growth inhibition
• Inhibits p21CIP1, p27KIP1 - TERT: limitless replication
- VEGF: angiogenesis
- EZRIN: upregulation of metastasis
- MAD2 and BUBR1: genomic instability
- LDHA and GLUT-1: deregulated metabolism
- ICAM-1: avoidance of immune destruction
What is the activated (“on”) canonical WNT signaling pathway?
- WNT binds to Frizzled/LRP5/6 complex
- cytoplasmic recruitment of Dishevelled
- inactivation of glycogen synthase kinase 3 (GSK3)
- β-catenin stabilized
- translocation to nucleus
- β-catenin acts alongside LEF/TFC to enhance transcription of c-myc, cyclin D1, and MMP-7
What is the in-activated (“off”) canonical WNT signaling pathway?
- WNT is absent
- β-catenin is phosphorylated by a destruction complex (APC, GSK3, AXIN)
- targeted for ubiquitin-dependent proteasomal degradation
- TCF acts with coreceptors such as HDACs and Groucho/transducing-like enhancer of split (TLE) to repress gene transcription
The key mediator of canonical WNT signaling is what?
B-catenin
What are the known target genes of B-catenin?
c-myc, cyclin D1, and MMP-7
Which TSG is part of the WNT pathway and has is encoded by the gene responsible for the onset of familial adenomatous polyposis that predisposes patients to colorectal cancer?
APC (part of the destruction complex)
What is the progression of normal epithelium to a carcinoma in a patient that develops colon cancer?
Normal epithelium (loss of APC)--> hyperplastic epithelium (DNA hypomethylation)--> Early adenoma (activation of K-ras)--> intermediate adenoma (loss of 18q TSG)--> late adenoma (loss of p53)--> carcinoma--> invasion and mets
Describe the “off” HH pathway.
- In the absence of HH, patched silences smoothened (the key signal transducer of pathway)
- If smoothened is silenced, there is prevention of disassembly of the Cos2, FU, SU[FU], Gli complex which is bound to microtubules
- Gli cannot be transfered to the nucleus
Describe the “on” HH pathway.
- Binding of HH to patched prevents the inhibition of smoothened
- Activation of smoothened causes dissassembly of the complex, releasing Gli and allowing nuclear translocation of activated Gli transcription factors and HH target gene expression
What are the target genes of HH signaling?
WNT, BMP, Patched
Which pathway can be a tumor suppressor initially and a tumor promoter in at the end?
TGF-B
o Loss of Rb = cells lose responsiveness to cytostatic effects of TGF-B
o Pushes TGF-B to favor cell proliferation
Which members of the TGF-B family of extracellular ligands perform the following:
(1) inhibit stem cell expansion by suppression of Wnt signaling, (2) induce CSC differentiation, (3) increase sensitivity to chemotherapy in vivo – potential therapeutic approach for CSC drug target
BMPs
Answer the following questions regarding TGF-B:
- What are the 2 major branches of TGF-B superfamily?
- Describe the TGF-B receptor pathway.
- Which receptor signals to Smads?
- Under basal conditions Smad receptors are doing what activity?
- TGF-B/activin/nodal branch
BMP branch – diverse and often complementary effects - TGF-β is synthesized as an inactive precursor and proteolytically processed into a mature ligand –>
must dimerize to becomes active –> binds to type I and II cell surface receptors that contain an intracellular serine-threonine kinase –> heterotrimeric complex –>
phosphorylation and activation of type I receptor by type II - Type I receptor phosphorylates R-SMADs R-SMADs interact with SMAD4 nuclear accumulation and associated with cofactors regulation of target genes
o Smad receptors are in constant motion and shuttle between the nucleus and cytoplasm (predominantly cytoplasmic).
TGF-B phosphorylates which Smads?
Smads 2 and 3
What are the 3 classes of SMADS?
- Receptor-regulated or R-SMADs (SMADS 1, 2, 3, 5, and 8)
- Common mediator or Co-SMAD4
- Inhibitory SMADS 6 and 7
- The SMADS within the TGF-B pathway can be inhibited by what?
- What inhibits TGF-B?
- Inhibitory SMADs: counteract R-SMADs
Ski and Sno inhibit SMAD 3 and 4
SMURF 1/2: E3 ubiquitin ligases that target R-SMAD for degradation - MYC: normally TGF-B overrides Myc (they have opposing functions), but when Myc oncogenes are present, cells become unresponsive to TGF-B signaling
TGF-β can also signal through SMAD-independent processes. What are they?
activation of RHOA, RAS, and TGF-β activated kinase 1 (Tak1)
BMPs transmits signals intracellularly by phosphorylating what as part of the TGF-B pathway?
Smads 1, 5, and 8
Answer the following questions regarding mTOR:
- What is mTOR?
- Where is it located?
- mTOR complexes with which factors?
- Describe the MOA of TORC 1.
- Describe the MOA of TORC 2.
- mTOR is a serine/threonine kinase that governs cell growth
- upstream of Akt/PKB and activates this pathway
- mTOR complexes with Raptor or Rictor
- TORC1: mTOR + Raptor + GBL
• activates protein synthesis, promotes expression of HIF-1a, and inhibits autophagy
• Promotes protein synthesis via phosphorylation of 4E-BP1 and S6 kinase (S6K aka p70S6 kinase)
• Phosphorylation of 4E-BP1 inactivates this protein; 4E-BP normally inhibits translation
• Phosphorylation of p70S6 kinase activates this protein - TORC2: mTOR + Rictor + GBL
• activates Akt/PKB via phosphorylation
- What does treatment with rapamycin decrease?
2. Which of the TORCs is inhibited by rapamycin?
- TX with rapa decreases mTOR and p70S6 kinase
o Rapamycin binds to FKBP-12 to form a stable complex that inhibits mTOR kinase - TORC1 is inhibited by rapamycin
TORC2 is only affected by rapamycin if treatment is continued for many hours
What are the adverse events reported with rapamycin?
•
skin toxicity, metabolic disorders (hyperglycemia, hyperlipidemia, hypercholesterolemia), GI toxicity, anemia, lymphopenia, rarely hypersensitivity or pneumonitis
• In human cell lines, rapamycin enhances chemosensitivity to TMZ, CCNU, and cisplatin
Transcription factors are classified into families based on what criteria?
their DNA-binding domain: homeodomain (aka helix-turn-helix), zinc-finger, leucine-zipper, and helix-loop-helix (HLH)
•
Which transcription factor family is described below?
contains three helical regions, the 3rd of which directly contacts DNA
o Function as regulators during development
o Ex: oct-1
Homeodomain
Which transcription factor family is described below?
helical areas with leucine at every 7th AA. Leucines stick out to the same side of helix like the teeth of a zipper. Interact hydrophobically with other leucine zipper proteins.
o Activated by proliferative and developmental stimuli (i.e., stress)
o Ex: fos/jun (AP1)
Leucine zipper
Which transcription factor family is described below?
2 paired cysteine or histidine residues bound to zinc, which causes the protein to form into compact domains with alpha helices that insert into DNA
o Mediates differentiation and growth signals
o Ex: glucocorticoid receptor
Zinc finger
Which transcription factor family is described below?
similar to zippers, but have a loop that separates two alpha helices
o Ex: myc
Helix-loop-helix
Identify the TSGs from this list .
IDH2 p16INK4A KIT p53 BCR-ABL APC PTEN ERBB2(HER2) EGFR p110a FBXW7 MET Rb N-Ras p14 ARF K-Ras NF1 B-RAF JAK2 LKB1 BRCA1/2 NF2 MYC IDH1 H-Ras VHL FLT3
p53 VHL PTEN APC p16 INK4A FBXW7 p14 ARF Rb BRCA1/2 NF1 LKB1 NF2
Identify the oncogenes from this list.
IDH2 p16INK4A KIT p53 BCR-ABL APC PTEN ERBB2(HER2) EGFR p110a FBXW7 MET Rb N-Ras p14 ARF K-Ras NF1 B-RAF JAK2 LKB1 BRCA1/2 NF2 MYC IDH1 H-Ras VHL FLT3
p110a BCR-ABL EGFR IDH1 ERBB2(HER2) IDH2 B-RAF JAK2 K-Ras KIT H-Ras MET N-Ras FLT3 MYC
The intrinsic apoptotic cascade is activated by what?
Activated by p53 and non-p53 dependent mechanisms (excess intracellular Ca2+, excess oxidants, certain DNA-damaging agents, microtubule disruptors)
What is the entire intrinsic (mitochondrial) pathway?
- P53 increases transcription of Bax and Bak which permeabilize the outer mitochondrial membrane
- release cytochrome c, SMAC, and OMI into cytosol
- As apoptosis proceeds, Bax and Bak aggregate on mitochondrial surface to increase its fragmentation
- In the cytosol, cytochrome c complexes with APAF-1 and forms apoptosome
- Activates procaspase9 to form Caspase 9
- activates executioner caspases (3, 6 and 7)
- Executioner caspases cleave Caspase 8
- activates Bid to link to extrinsic pathway
Which BCL2 family members are proapoptotic?
BAX, BAK, BOK, BID, BAD
Which BCL2 family members are anti-apoptotic?
BCL2, BCL-XL, MCL1
Which BCL2 family members contain BH3 domains only?
BIM, BAD, BMF, NOXA, HRK, PUMA, BIK
Which BH3 only Bcl-2 family member is important for death receptor-induced necrosis?
BMF
Name 2 ways in which the extrinsic pathway is blocked and apoptosis is prevented.
- Bcl-2 family members keep mitochondrial channels closed (anti-apoptotic)
- Akt/PKB phosphorylates/inactivates Bad to oppose apoptosis
What are the executioner caspases ?
3, 6, and 7
The intrinsic or mitochondrial pathway is inhibited by what things?
- Inhibitors of apoptosis (IAPs)
(a) bind to caspases to prevent proteolytic activity and (b) tag caspases for ubiquitination
What inactivates IAPs to allow for apoptosis?
SMAC
• SMAC antagonizes IAPs; IAPS normally ubiquitinate caspases to target them for degradation – so SMAC allows apoptotic cascade to continue
Where do the intrinsic and extrinsic pathways interact?
CASP8 cleaves Bid
Bid + Bax forms holes in mitochondrial membrane
What is the entire extrinsic (death receptor) pathway?
Governed by death receptors (TNF family) binding to their ligands (n=6):
- Fas binds FasL
- DR4 or DR5 binds APO2L/TRAIL
- Ligand binding
- recruitment of Fas-associated death domain (FADD)
- recruitment of procaspase 8
- formation of death-inducing signaling complex (DISC) 8. releases caspase 8 to cytosol
- activates executioner caspases + BID
- DR4/5 recruit Cullin-3
- polyubiquitinates CASP8 to cause aggregates
- increases pathway activation
What are the endogenous angiogenic factors?
o PlGF – binds VEGFR1, stimulates angiogenesis
o FGF-1, 2, 3, 4 – stimulates EC mitogenesis, survival, and angiogenesis
o IL-8 – binds CXCR1 to stimulate ECs and inflammatory cells
o IL-6 – binds IL-6R to stimulate inflammatory angiogenesis
o TNFa – stimulates EC and induces VEGF
o Bv8 – stimulates endocrine and tumor ECs
o MMP9 – releases GFs from ECM
What are the endogenous angiogenic inhibitors?
TSP-1 Endostatin Agiostatin Tumstatin SFlt-1/sVEGFR1 VEGF165b PEX INKa/B
What are the stimulators of angiogenesis that act directly on the endothelial cells?
VEGF, PIGF, FGF 1/2, HGF, IL-8
Which receptor/ligand interaction is involved in the regulation of endothelial cell survival, vascular permeability, and recruitment of mural cells?
ANG/TIE2
Fill in the blank with either sprouting or regression for the following statement:
In the presence of VEGF, the exposure of ANG2 promotes vascular ____a______, but when VEGF levels are low, ANG2 promotes vascular ______b________.
a. sprouting
b. regression
Which angiogenesis process is explained below?
occurs when nonendothelial cells adopt endothelial-like phenotypes and line vascular channels
Normally occurs in the placenta (epithelium transforms to endothelium)
Also occurs when embryonic stem cells are injected SQ (leads to teratoma formation) and in the bone marrow of certain cancers
Vasculogenic mimicry
Which angiogenesis process is explained below?
cancer cells exploit preexisting tissue vasculature by growing around and enveloping the vessels
Reported in highly vascular organs (lung, brain)
Interaction can trigger blood vessel regression and thrombosis
Eventually followed by onset of angiogenic reaction
Vascular cooption
What regulators have been implicated in the process of vascular mimicry?
tissue factor, TF pathway inhibitor 2, PI3K, FAK, MMPs, Ephrins, and Laminin chains
These markers are used to identify what cells?
CD31, CD144, integrin αvβ3
endothelial cell markers
These markers are used to identify what cells?
Prospero homeobox transcription factor (PROX-1)
Podoplanin
Lymphatic vessel hyaluronan receptor-1 (LYVE-1)
lymphatic endothelial cells
This explanation below is in regards to what metabolic process created by cancer cells?
Increase in glycolysis
Glucose is converted to lactate and secreted, rather than being oxidized to CO2 in mitochondria
Similar to Pasteur effect (switch to glycolytic ATP production in hypoxia), but occurs under normoxic conditions as well
The Warburg effect
Cancer cells must generate enough ATP for normal processes and further cell division
Must also evade normal checkpoint controls during metabolic stress
T/F: ATP production via glycolysis is more rapid than by oxidative phosphorylation, but it’s much less efficient in terms of # of ATP per molecule of glucose consumed; therefore tumors need much more glucose to meet energy requirements?
True
What is this a definition of?
after a certain number of population doublings, cells undergo senescence and cease dividing although generally retaining viability
Hayflick limit
Choose which of the following are markers of senesence.
NF1 JAK2 LKB1 SAHFs PROX-1 integrin αvβ3 y-H2AX acidic B-galactosidase
- acidic B-galactosidase (may also stain some apoptotic cells)
- senescence-associated heterochromatic foci (SAHFs) arising from covalent modification of histones (can ddx senescent cells from G0)
- y-H2AX (IDs dsDNA breaks not necessarily senescent cells)
Answer the following questions regarding senescent cells:
- What happens to the metabolism of a cell in senesence?
- Can they re-enter the cell cycle?
- What happens when these cells express GFs?
3.
- remain metabolically active
- have irreversibly lost the ability to re-enter the cell cycle
- Express growth factor receptors but downstream signaling pathways have been inactivated through unknown mechanisms
What is meant by the senescence-associated secretory phenotype (SASP)?
secrete pro-inflammatory cytokines such as interleukins, IGFBPs, TGF-B
chronic inflammation may lead to tumor formation
According to Harlet et al, VCO, 2018, what CSCs were identified in Canine LSA?
B-cell = CD34, CD90, CD117, Oct3/4 T-cell = Oct3/4
What are the markers for cancer stem cells?
CD133 CD44 CD34 CD117 ALDH1
What is the most common surface protein in CSC detection – used for epithelial tumors?
EpCAM (epithelial cell adhesion molecule)
What is MAGE an example of?
Cancer/testis antigens: proteins expressed in gametes and trophoblasts and in many types of cancers, but are not present in normal somatic tissue
Which TFs are associated with progression of EMT?
TWIST NF-κB STAT Snail Slug SMAC TGF-B Oct-1
TWIST, Snail and Slug
Others:
o TGF-β via 2 downstream pathways: Smad and p38/Rho
o HGF, which activates c-Met, EGF and PDGF
o ZEB 1 and 2
Repression of E-cadherin mediated by TFs?
Yes this is kind of trick question!
TWIST, Snail and Slug
- Snail and Slug are zinc finger TFs
- TGF-β induces expression of Snail, Slug, Twist, Goosecoid
- TFs can also induce expression of one another; Twist binds to promoters of Twist, Snail, Slug, and Zeb1
- Snail has unique “futile cycle” like p53; quickly made/degraded
What are the suppressors of metastasis?
NME, MKK4, KAI, KiSS, BrMS
What process is a post-translational lipid modification essential for localization of Ras to the plasma membrane?
Prenylation
