Final Review Flashcards
(114 cards)
1
Q
What are EGFs?
A
53 amino acid long (relatively small), when purified works in many cell types, EGF binds to EGFR
2
Q
What are EGFRs?
A
EGF receptor consists of 2 SEPARATED monomers (same chemical composition) when unbound; each monomer consists of
- extracellular binding site for EGF
2.intracellular tyrosine kinase domain
3
Q
How were EGFs discovered
A
- Accidentally discovered as contaminant in salivary gland of male mice when studying NGF (nerve growth factor); lead to premature eyelid opening and tooth eruptions in mice
- V-Src was 1st identified tyrosine kinase, accidentally discovered using Rous Sarcoma Virus probe
C-SRC has 5 parts:
- N-terminal myristoylation site (M)
- SH3 domain
- SH2 domain
- SH1 kinase domain
- C terminal Tail (inhibitory domain)
*V-Src missing part of C-terminal tail leading to constitutive active kinase activity (always on)
*Tyrosine phosphorylation correlates with ability of V-SRC to transform cells
** EGFR cytoplasmic domain has region of homology with SRC SH1 (kinase domain)
- V-Sis discovered to be an autocrine platelet-derived growth factor (PDGF); therefore the cell makes its own receptor and growth factor leading to autonomous cell proliferation
4
Q
What happens when EGF binds to EGFR, what happens when EGF is NOT bound to EGFR?
A
When EGF is NOT PRESENT, EGFR remains monomeric (2 subunits)
When 2 EGF is PRESENT it binds to EGFR, inducing dimerization of receptor leading to the phosphorylation of the carboxyl-terminal tails on the intracellular domain
*cross-phosphorylation creates attachment point for OTHER proteins part of EGF signal transduction pathaway
5
Q
What is the transduction pathway? (8)
A
- 2 EGF’s binds to 2 monomer -extracellular binding domains of EGFR; leads to dimerization and phosphorylation of EGFR
- Phosphorylation of C-terminal tail on intracellular side serves as anchor for Grb-2 protein
- Grb-2 recruits Sos protein
- Sos recruits Ras, a small g protein, which expels GDP and recruits GTP activating it
- Activated Ras then activates Raf protein kinase
- Raf activates MEKs protein kinase
- MEKs activate ERKs (extracellular signal-regulated kinases
- Activated/Phosphorylated ERKs move into nucleus from cytosol and stimulate transcription factors to increase gene expression, this increases rate of protein synthesis leading to enlargement of cytoskeleton and thus cell growth
*Erk phoshorylate ETs (erythroblast transformation specific) and other Transcription Factors proto-oncoprroteins to cause immediate early gene induction t cause cell proliferation
6
Q
What does Viral-ErbB have missing?
A
V-ErbB is missing segment in ectodomain/extracellular EGF binding domain therefore leading to it being constitutively active + activating growth singals without need for ligand binding
7
Q
What are the different mechanisms for constitutive activation of EGFR in cancer?
A
- Over-expression
- Mutations that change structure of receptor
*both lead to ligand-independent firing, no need for ligand to bind to activate growth factor
8
Q
What are the EGFR family members and how are they targeted by anti-cancer drugs
A
- EGFR=HER1=ErbB1
- HER2=ErbB2
- HER3=ErbB3
- HER4=ErbB4
*EGFR and HER2 are most commonly mutated receptors, don’t see as much HER3 or HER4
*ligands (what binds to receptors) can be shared among RTKs
*RTKs can form heterodimers with one another
9
Q
What are the subdomains of EGFR and how do they specifically bind in presence of a ligand (EGF)?
A
There are 4 subdomains of EGF, ligand domain on I and III but unable to be reached by ligand in ABSENCE of EGF since II and IV are folded; when EGF is PRESENT II and IV unfold revealing ligand domain I and III
10
Q
What are the 4 EGFR activating alterations in human cancer and what do they lead to?
A
- Amplification
- Insertion
- Deletion
- Point Mutations
*these alterations lead to either 1) destabilization of inactive conformation or 2) stabilization of active conformation or
3) both
11
Q
what is -nib?
A
kinase inhibitor
12
Q
What is -tinib?
A
tyrosine kinase inhibitor
13
Q
What is -mab
A
monoclonal antibody
14
Q
How are growth factors hijacked in cancer?
A
constitutive activation of RTKs and production of autocrine growth factors are common in human malignancies
15
Q
What is an autocrine growth factor?
A
same cell makes own receptor and growth factor leading to autonomous cell proliferation; an example would be V-sis and how it makes autocrine platelet derived growth factor (PDGF)
16
Q
What are tyrosine kinases?
A
tyrosine kinases are enzymes that selectively phosphorylate tyrosine residues in different substrates
17
Q
What is Grb2? What does it do? How does it lead to Ras activation?
A
-Growth Factor Receptor Bound protein 2
Grb2 is an adaptor protein that serves as the “glue” for other proteins but does not have any enzymatic activity
-Grb2 has SH2 domain (+ some SH3) which is necessary and sufficient to bind pY within EGFR C-terminal, as it brings Grb2 to receptor C-terminal to cause downstream signal to activate proteins
18
Q
What is SH2 domain function?
A
-different SH2 domains bind to different pY residues (tyrosine phosphorylated residue) and is dictated by the 3 amino acid residues following the pY sites which dictate the specificity of SH2 domain binding
-multiple SH2-containing proteins associate with specific pY residues of activated EGFR terminus (form of signal amplification) some are enzymes like kinases, PCC (phospholipase C), phosphates while others are adaptors like Grb2
19
Q
What is the significance of Ras in cancer and how is it mutated?
A
3 Ras proteins (H, K, N Ras) which are frequently mutated at:
- Glycine 12 (G12)
- Glycine 13 (G13)
- Glutamine 61 (Q61)
*therefore when turned into different amino acid=oncogene
*Grb2 recruits SOS/GEF to activate Ras (small G protein) via stimulating GTP binding + expelling GDP
*Gap inactivates Ras by promoting GTP hydrolysis, i.e. expel GTP and GDP binds to Ras
*Therefore activating Ras point mutations keep Ras proteins in bound, active form thus going through transduction pathway indefinitely
20
Q
What is the Ras cycle?
A
-GTP binding induced by SOS/GEF activates Ras
-GTPase activity stimulated by GAP inactivates Ras via hydrolysis reaction
21
Q
What is the structure of Ras and how does it bind to GTP? GDP?
A
-H-Ras structure has guanine nucleotide binding domain
-G1, G2, G3… contacts GTP/GFP and binding affinity very high so once bound Ras does not let go
-Ras cannot distinguish between GTP/GDP
22
Q
What do mutant Ras do?
A
-K-Ras activation mutants have 1) reduced intrinsic and 2) GAP stimulated GTPase activites
-therefore, change into an other amino acid reesults in partial loss of GDP activity and decrease of both basal rate and GAP
-similar findings found in H-Ras
23
Q
How does Ras hot-spot mutations affect intrinsic GTPase activity?
A
-Q61 (glutamine 61) is involved in catalysis and mutation BLOCKS this
-mutation of G12 sterically hinders Q61-mediated catalysis
24
Q
How does Ras hot-spot mutations affect GAP-induced activity?
A
-G12 and Q61 interacts with GAPs “Arg Finger” which greatly augments GTP hydrolysis
-mutations in G12 and G13 (lies next to G12) cause steric hindrance for GAP and Q61 mediated catalysis resulting in glutamine being unable to function
25
How does Ras induce RTK signaling downstream?
-Raf:a Ser/Thr kinase acts DOWNSTREAM of Ras
-evolutionary conserved "RTK-Grb2-Sos/GEF-Ras-Raf-MEK-ERK" pathaway
26
How does Ras-GTP bind to Raf and activate it?
-Ras GTP binds Raf and activates it via autophosphorylation
switch 1 mediates Ras-GTP interaction with downstream effector proteins
-switch 2 critical for interactions w/ GEF (For Ras-GTP) and GAP (for Ras-GDP)
27
Why can kinase cascade cause signal amplification?
because a single active enzyme can phosphorylate multiple downstream kinase or substrates
ex:ERK which is final kinase transcription factor goes to nucleus and binds DNA turning it ON or OFF
28
How does ERK cause proliferation?
ERKs are the final kinase transcription factor which go directly to the nucleus and activate transcription factors and ETs (erythroblast transformation specific)" a transcription factor that acts downstream of ERK via phosphorylation
29
What do oncoprotein do?
oncoprotein constitutively activate downstream signaling pathways and ignore upstream thereby contributing to uncontrolled cell proliferation
30
What are pathways and oncoprotein importance in human cancer?
- many ways which cancer arises
- oncoprotein aberrantly induce signaling pathways
-specificity of signaling pathways often depend on conditional (or regulated) protein-protein interaction
-oncoprotein can cause multiple downstream effects including deregulation of cell prolif, cell death, etc
-critical steps in oncogenic signaling pathways are targeted by precision cancer therapies
31
If you activate Raf, MEK, or ERK (part of MAPK cascade) is it sufficient to induce proliferation?
Yes, rescue proliferation defect, but other known Ras downstream effectors like P13K could not be rescued
32
What are immediate early genes (IEGs)?
-Include transcription factor (Tf oncoprotein)
-immediate early genes include Fos, Myc, Jun
33
What is multistep tumorigenesis? (Overview 4)
tumorigenesis is driven by series of mutations and epigenetic changes in DNA that affect genes controlling cell proliferation, cell survival, genome stability, and other processes
1. Process of tumor formation is complex involving multiple molecular changes
2. Molecular changes involve the inactivation of tumor suppressor genes and activation of oncogenes
3. # of molecular changes that occur in genome human cancer cells far exceeds the number REQUIRED for tumorigenesis to reach completion, complicating identifying the critical alterations
4. Tumor heterogeneity arises as consequence of hi mutation rates coupled with selection
34
Why do most cancers take decades to form?
age of diagnosis most prevalent in late 60s and early 80s; environmental factors play role, for example incident of lung cancer increased decades AFTER cigarette smoking became popular
35
What is the number of rate-limiting steps needed to drive tumorigenesis?
number of rate-limiting alterations required for cancer to form-6 rate limiting steps
36
What is the crypt in colon cells look like and how does it function?
colon cancer is 2nd most common cancer
-role of colon is to reabsorb water lined with crypts, cells produced in crypt around 200-300 cells
-every 3 to 5 days epithelial lining replaced as protective measure
-composed of 4 cell types + myofibroblast which surround crypt
1. Enterocytes
2. Globlet cells
3. TA cells (transient-amplifying cell)
4. Stem cells
-Stem cells divide asymetrically, some move up some don't
37
What is loss of APC and significance initiating tumorigenesis?
-sequences of molecular changes vary but loss of APC/mutation usually early event, leading to tumor progression
-APC is a gatekeeper gene, highly conserved in Mice
-once lost often see formation of adenomas
-loss of APC and activation of KRas leads to metastatic colon cancer= alterations in gatekeepers (APC) and caretakers (Kras) needed for cancers to form
*some molecular changes mutually exclusive but highly variable with number and order of mutations
38
What are the two models for tumorigenesis, and what is the main theory now?
1. Clonal Sweep: accumulation of all mutations had advantage versus some just some mutations; would see sequential mutation; mutations that confer selective advantage overtake bulk of tumor
ex: APC and KRas mutations have advantage over just tumors with APC mutant
2. Big Bang (main theory): lots of mutations occur early on, then coevolve meaning mutations do NOT compete with each other; so final tumor composed of mixture of distinct clonal populations
-see cancers that are heterozygous meaning not composed of just 1 cancer cell but multiple with different mutations/genetic makeup--experimentally validated
39
How was the big bang model studied?
-to determine tumorigenesis process can preform hi-resolution sequencing
-do left and right part of tumor have same or different mutations?
-found tumors OFTEN highly heterogenous meaning big bang (mutations occurred early on) so public mutations (present in entire tumor) and private mutations (happen on other side)
40
What is Wnt signaling and what is the significance?
-Wnt signaling leads to the accumulation of beta-catinin, but if APC present, B-catenin gets phosphorylated and degraded; localized to the cytoplasm
-APC present, normal Wnt is OFF=target gene suppressed
-APC absent/mutated, cancer Wnt ON= target gene activated; localizes to the nucleus
41
What is the difference between drivers (gatekeepers) vs passengers (caretakers)?
driver mutations are mutations present in MOST tumor cells, while passenger mutations are in SOME but not ALL tumor cells
42
What evidence suggests that cancer is a multi-step process?
-*Histopathology (from normal tissue to carcinoma)
- Late onset of lung cancer in smokers
- Tumors can be found in mixtures of adenomas and carcinomas
-* Molecular changes coupled to tumor progression (Loss of APC and activation of KRAS lead to metastatic colon cancer together, but not alone.)
- *Mutations in RAS and p53 Drive Skin Carcinogenesis (from painting example)
-Mutations in PIK3CA or KRAS alone were shown to be not sufficient to cause cancer
43
Multi-step process doesn’t always occur in the same sequence. What are the changes?
-sequences of molecular changes vary, however loss of APC/mutation USUALLY early event leading to tumor progression, but tumors can be initiated by mechanisms other than loss of APC
-some molecular changes mutually exclusive
-molecular changes are numerous
44
What is an initiator? Examples?
mutagenic-like, example:BP, DMBA
45
What is a promoter? Examples?
non-mutagenic, when coupled with an initiation can contribute to tumorigenesis ex:TPA, known skin irritant
46
Explain the rationale and results of the ‘painting’ experiments
When you paint an promoter on top of an initiator on the skin it will cause papilloma development, but when promoter is removed causes tumor regression.
when you paint an initiator, promoter, and then an initiator again it will cause adenoma development
*shows that initiators and promoters work together to contribute to tumorigenesis, but need first a painting with an initiator bc if a promoter is used first there won't be tumor development
Example of RAS (promoter) and P53 (initiator) drive skin carcinogenesis, but if remove promoter-RAS tumor regresses
47
The effect of inflammation on colon tumors
Inflammation is a non-mutagenic/carcinogenic way that can cause tumorigenesis, specifically inflammation in the colon leads to increased COX2 which synthesizes prostaglandins that affect cell adheration and proliferation
48
Role of COX-2 and PGE2 in promotion
specifically inflammation in the colon leads to increased COX2 which synthesizes prostaglandins that affect cell adheration and proliferation
-Cox2 convert arachidonic acid into prostaglandin H2, while PGE2 converts prostaglandin H2 into prostaglandin E2
-inhibition of Cox2 or mediators (receptors) suppresses intestinal tumorigenesis, as reduces inflammation and therefore incidence of tumor formation
-also if lack PGE2 (mediator of Cox2 and dependent on EP2 receptor) or inhibit, leads to suppression of tumorigenesis
-expression of Cox 2 varies among tissues, sometimes stromal cells (intestinal)----controverial
while others epithelial (breast)
49
Considerations to evaluate the usefulness of a mouse model
mouse models helpful for evaluating colon cancer as can induce inflammation (via chemicals, diet, initiator+promoter) increasing risk of colon cancer and can easily monitor this inflammation and tumorigenesis in the colon via colonoscopy, so need very few animals/patients bc can see tumors directly
50
What is Haploinsufficiency?
Haploinsufficiency: The situation that occurs when one copy of a gene is inactivated or deleted and the remaining functional copy of the gene is not adequate to produce the needed gene product to preserve normal function. (i.e 1 copy of good is NOT enough to rescue good phenotype
Haplosufficiency: 1 copy of good IS ENOUGH to rescue good phenotype
51
Cell autonomous vs. non-autonomous
-Cell autonomous gene action suggests that the product is involved in signal reception, signal transduction, or does not participate in a process involving cell-cell interactions.
-Cell non-autonomous gene action suggests that the product is a signaling molecule or participates in the synthesis of a signaling molecule.
*Prostaglandin E2 can act in an autocrine (cell targets itself, releasing signal molecule like ligand which binds to own receptor) or paracrine manner (cell targets neighboring cells)
52
Examples of how inflammation is a promoter
- Why smokers who drink alcohol 100x more likely yo develop head and neck cancer than nonsmokers, cigarette smoke very carcinogenic (initiator) but while alcohol (promoter) is weak mutagen, it is toxic to epithelial lining, stripping lining so it has to be repaired
- patients with ulcerative colitis (inflammation in GI tract) have significant risk of developing colon cancer
- Gut Flora and bacteria like heliobacteria pylori or chemicals like (DSS) can cause inflammation
53
What is a monoclonal tumor?
tumor derived from cells from a single progenitor; homogenous
54
What is a polyclonal tumor?
tumor derived from cells from multiple progenitors; heterogenous
55
What is Lyon’s hypothesis?
-inactivation of X chromosome occur randomly in somatic cells early on during embryonic development
- because females have 2 X chromosomes, one from mom and one from dad, they have 2 copies of clone
-therefore, monoclonal tumors should only be comprised of cells with the SAME X-inactivation
*but patches of X-inactivation are large, as pattern of mosaicism introduces a strong bias as overtly polyclonal only form on borders-i.e. not enough cells on boarder to determine if tumor is polyclonal
56
What early experiment(s) demonstrated that tumors were always monoclonal?
1. one of first paper stated colorectal tumors had monoclonal origin, used x-inactivation to determine if it was monoclonal or polyclonal
2. another looked at adenomas and cancers, observed only a single band in tumors (southern blot) when DNA cleaved using methylation-sensitive restriction enzyme, indicating tumors monoclonal (all cells in tumor had either paternal/maternal X chromosome inactivated)
57
What evidence demonstrates that polyclonal tumors exist?
tumors can be derived from multiple progenitors, some studies have indicated mosaicism in detected tumors
-ex: examined colon tumors from FAP patients who was mosaic (some cells lost Y chromosome XY/X0) 13/263 tumors composed of XY cells and X0
-ex 2: A study found intestinal tumors had polyclonal origin (mix of blue and white staining); used APC min/+ (white cell stain) and APC min/+ (blue cell stain) Rosa26; if mix=polyclonal and if 1 color=monoclonal
58
What is an aggregation chimera?
an aggregation chimer (2 mice in 1): take 2 embryos and remove fertilization envelope so embryos float and fuse together, insert into pseudopregnant mother who then birth the chimeric mouse (phenotype: see black and white fur)
59
60
Mouse models used to study clonality
1. need an animal model that develops intestinal tumors, or tumor of interest
2. need to be able to distinguish cells lineages, ideally, each lineage would be uniquely marked
-therefore use aggregation chimeras, which develop intestinal tumors that can be detected and are easily identifiable, coat color black and white
61
How did they distinguish recruitment and cooperation experimentally?
-in order to distinguish between cooperation and recruitment experiment using APC min/+ (white) and APC +/+ w/ Rosa26 (blue)
-if cooperation ALL tumors should be white bc need to lose BOTH copies for tumor to form (meaning APC needs too lose both good copies for tumor to form and this is very unlikely)
-if recruitment, expect to see tumors mixed with white (mutant) and blue (wild-type APC)
-result: found recruitment was best explanation (25% of tumors were mixed)
*mutated min has a dominant-negative effect, meaning one bad copy is enough to have bad phenotype, i.e. faulty allele affects wild type allele
-another experiment used VAP1 activation, found pancreatic tumors formed following induction of activated KRAS but if KRAs expression stopped tumors regressed; but found that even when KRAS inactivated, the % of survival decreased significantly, indicating that VAP1 cells were recruited, so patients dying from tumors via recruitment
62
Recruitment and cooperation
polyclonal tumors form via 2 interactions:
1. Cooperation:2 or more cells become independently initiated within close physical and temporal proximity and neoplastic descendants of cells found together in single tumor
2. Recruitment: possibly through wnt signaling, an initiated progenitor transforms nearby cells and neoplastic descendants of these cells found together in a single tumor
--recruits neighboring cells to tumors
63
Do Wnt ligands mediate signaling and how does this lead to cancer development?
-loss of Wild-type Wnt leads to accumulation of beta-catenin which leads to tumor development
-tumor cells with loss of APC/Wnt can also RECRUIT cells that have Wild-type APC/Wnt so even though no mutation, these cells look like tumor because receiving signals from tumor cells
-several Wnt-signaling molecules are transforming factors in-vitro and in-vivo
-once beta-catenin has translocated into the nucleus can stimulate the expression of numerous genes including Wnt3A
64
What is Wnt signaling and why is it important?
-Wnt is a key effector in the transduction signal to the nucleus and the transcription of wnt-specific genes that control the cell and its development in different types of cells and tissues
-Wnt important to cell mobility, growth, and differentiation during embryonic development
-Wnt is regulated by APC, as APC inhibits Wnt signaling; therefore if 2 bad copies of APC=embryonic lethal
-Wnt "On": Wnt is able to bind to receptor Frizzled and therfore APC is UNABLE to interact with beta-catenin resulting in the accumulation of beta-catenin in the cytoplasm
-Wnt "Off: Wnt is UNABLE to bind to the Frizzled receptor, allowing APC to interact with beta-catenin and destroy it
65
What is the evidence that supports tumorigenic recruited cells
-recruited cells are present in human tumors, 1 study looked at individual crypts to see if there was APC mutations, 2/12 crypts APC WT but LOOKED cancerous indicating possible recruitment
-additionally, when recruited cells were isolated and injected into model organisms lead to tumor/cancer development
-as add additional mutations, cancers have more recruited cells, as accumulation of mutations may lead to negative health consequences soo they recruit cells to get needed nutrients and hormones
66
What is the evidence that supports presence of recruited cells may depend on stage
-presence of recruited cells may depend on the stage, as they found that in later cancers like adenocarcinomas there was no WT APC crypts but in early tumors found WT crypts (i.e. early tumors see WT crypts but not later/advanced tumors)
-
67
What is field cancerization?
-large area of cells affected by carcinogenic alterations; saw larger fields of mutant cells led to more tumors
-experiment used APC mutation that was CRE dependent (snips at loxp sites and turned ON my adding tomooxifin)
-absence of CRE=normal APC; Presence of CRE=loss of APC
-@ lo dose tomoxifin (absence of CRE) APC present, @ hi dose tomoxifin (CRE present) APC deficient=see larger fields of mutant cells with more tumors
68
How does field size affect tumor formation efficiency?
-bigger fields=more tumors
69
Why do fields of mutant cells appear in normal-looking tissues?
- fields affected by mutations and environmental factors
-1 study looked at health "normal" esophagus crypts and found LOTS of mutations which depended on age (so as get older=increase in mutations)
-many of these mutations (51%) occur PRIOR to tumorigenesis
70
What is the evidence that supports tumors arising from fields of mutant cells
tumors do arise from fields of mutant cells as tumors share molecular features ex:benign tumors related to malignant cancers
-looks like colon has field of mutant cells where have different polyps and carry disease
71
How does polyclonality affect tumor biology?
-different clones affect tumor biology, as usually polyclonal tumors are more aggressive than monoclonals
-different mutant clones talk to each other forming more aggressive cancers
-cooperation of cells affects metastasis, for example IL11 (tumors pale, not good vasculature but big); FIGF-small tumors but when combine both IL11 and FIGF tumoros for aggressive polyclonal tumor
72
What is immortalization?
-process where cell population normally has limited replicative potential acquires ability to multiply indefintetly meaning overcame senescence and crisis
-Henrietta Lacks developed cervical cancer, died but cells from 1951 still cultured today=first immortalized cell line which is still used today
73
What is senescence?
-cell is alive but stops dividing and permanently withdraws from cell cycle
-senescence is provoked by physiological stresses and is mediated by p16 and p21 (both tumor suppressors)
-cancer cells can escape senescence if tumor suppressor genes are lost
-senescent cells can be both anti-tumorigenic and pro-tumorigenic
74
How can senescent cells promote tumorigenesis?
example senescence fibroblast promote cell proliferation, migration, and invasion; growth and division more rapid when cultured with senescent fibroblasts
75
How did they observe pattern of cell replication?
Observed in C. elegans that cell replication was invariable during development
-saw cells had programmed cell death genes
76
What is crisis?
-crisis triggered by erosion of telomeres/loss of telomeres (ends of chromosome) normally resulting in cell death (apoptosis), chromosomal fusions, karyotypic chaos
-telomeres composed of a hexanucleotide sequence that is repeated 1000x
-telomeres can be regenerated via telomerase (enzyme)
-cancer cells escape crisis by activating telomerase or an alternative mechanism to lengthen chromosomes
77
What is telomerase?
-composed of hTERT catalytic subunit and associated HTR RNA subunit
-activation of telomerase allows escape from crisis, so in mortal cells lack telomerase activity (highly regulated) but in immortal cells always present
-therefore the suppression of telomerase activity results in loss of neoplastic growth; cells stop dividing so stops telomerase activity
-dominant-negative hTERT: outcompetes WT allele for binding, stops/reduces telomerase activity so in these cells see a pleateu being reached quickly, meaning cells failed to double/proliferate
78
Why is telomerase activity a prognostic indicator of survivability/outcome?
TA positive: patient low survivability, neuroblastoma and telomerase activity so less likely to senescence
TA negative: patients hi survivability
79
What is the main difference between crisis and senescence?
Senescence represents a halt in cell proliferation with retention to cell viability/being alive over extended amounts of time while crisis involves cell death via apoptosis
80
What is a min mouse?
-mice carrying min allele of the adenomatous polyposis coli gene develop tumors along entire length of colon tract but tumor multiplicity dependent on genetic background
--------Example: AKR min mice < 1 (tumor) vs BTBR min > 500 (tumors)
-loss of heterozygosity
-loss of telomerase activity suppresses the development of intestinal tumors in min mice --by 4th gen see min mice develop fewer tumors
81
What is Hayflicks limit?
-cells become senescent eventually during lifespan, meaning cells alive but don't divide
-age vs proliferative capacity: doesn't matter which tissue there is limited amount of doubling, age only SLIGHTLY matters
-when combine 2 tumor suppressors (like p16 and p21) expression increases when cells cultured and witness population doubling but at some point cells will reach senescence
82
Contribution of p16 and p21 to tumorigenesis in ApcMin/+ mouse
-loss of p16 affects intestinal tumorigenesis, senescence mediated by p16 and blocks progression of tumors via angiogenesis which are smaller and lack vascularization,
-loss of p21 affects intestinal tumorigenesis, as senescence mediated by p21 blocking tumorigenesis in intestine, therefore less tumors form in WT p21 vs loss of p21/mutant
83
The effect of telomerase function on neoplastic growth
therefore the suppression of telomerase activity results in loss of neoplastic growth; cells stop dividing so stops telomerase activity
84
What is the effect of telomere length on mouse vitality?
erosion of telomeres in mice decreases vitality
-1st gen mice: don't live very long so not enough time for telomeres to erode
-by 5th gen: mice start showing signs of different phenotypes like age quicker, slow wound healing, wasting--telomeres of mTR-deficient mice erode
85
What are heterotypic interactions in cancers?
cancer is a disease of complex tissues interacting among a variety of cell types
86
What kinds of different cell types can be recruited in a cancer?
-stromal cells can be recruited to primary and metastatic tumors, present in many cancers; sometimes more stromal cells than tumor cells
-4 types of stromal cells with different functions
1. Immune Cells
2. Cancer-Associated Fibroblastic Cells
3. Endothelial Cells
4. Pericytes
87
What is the role of stromal cells and what do they interact with ?
-epithelial cells create growth factors for neighboring stromal cells and vice versa;
- ex:mesenchymal cell (stromal cell, fibroblast) creates TGF-alpha which binds to EGF-R of epithelial cells while epithelial cell/tumor cells release PDGF which binds to PDGF-R on mesenchymal/stromal cell
-paracrine signaling is like co-dependent neighbors
88
What is paracrine signaling? Give an example
cells create growth factors that stimulate neighboring cells
ex:Wnt is a paracrine signal. Fibroblast (stromal cells) signal stem cells to proliferate via Wnt signaling
-in normal intestinal crypt, Wnt is OFF once cells terminally differentiated
-in APC -/- Crypt, Wnt is ON and NOT regulated allowing cell proliferation to continue leading to build-up of beta-catenin where accumulations @ site of future polyp formation
89
What is autocrine signaling? Give an example
cells create own growth factor and stimulate own proliferation, self-sufficient neighbors, common in cancer
ex:activate GF gene in cytoplasm---pass through plasma membrane---go into ECF--GF stimulates own receptor---activate GF gene in cytoplasm (again, repeat)
90
What is a xenograft? Patient derived xenograft?
-xenografts that are derived from cancer cell lines fail to recruit stromal cells
-take surgical specimen (human) and extract stromal cells + tumor
-engraft patient specimen (model organism)--some stromal cells but not as many as human
-engrafted cell line-grown in culture for decades so they have LEARNED to grow without stromal cells, so don't need or know how to recruit stromal cells
-therefore cancer cell lines selected in-vitro for their ability to grow INDEPENDENTLY of stromal cells
91
Explain the similarities and differences between cancer and wound healing
-mediators of heterotypic signaling is NOT unique to cancers, wound healing involves many of the SAME signaling pathways which are tightly regulated
-cancers resemble wound that does not heal
Cancer: SEIMEIR
1. sustained proliferative signaling
2. evasion of growth suppressors
3. invasion and metastatsis
4. enabling replicative immortality
5. inducing angiogenesis
6. resisting cell death
-wound healing:
1. transient (controlled) proliferate signaling
2. transient evasion of growth suppression
3. activation of cell migration withoout invasion or metastasis; basement membrane repair
4. No enabling replicative immortality
5. inducing angioogenesis
6. transient decrease in terminal differentiation; transient increase in cell death
92
Describe the effect of fibroblasts on tumorigenesis
-stromal cells are active contributors to tumorigenesis
-if cancer associated fibroblast exhibit an expression pattern similar to that observed in cultured fibroblast responding to serum, the prognosis for the patient is WORSE
-versus cancer-associated fibroblast that do not align with signature=better prognosis
-addition of normal fibroblasts facilitates tumor growth, tumors formed earlier and larger vs control
-carcinoma-associated fibroblasts better fascilitators vs normal fibroblast, as carcinoma-associated fibroblasts recruit more endothelial cells than normal fibroblast, increase presence of red blood cells
93
What is angiogenesis?
-formation of blood vessels
-implanted tumors recruit more blood vessels
-recruitment of blood vessels dependent on VEGF, can block using inhibitor
-tumor-associated vasculature is disorganizes + not uniform in size or shape
-therefore if increase blood vessels, increase likelihood of tumor formation
94
Why do tumors need angiogenesis?
-tumors need source of blood, so when good blood flow=tumor formation/cells hyperplastic
- tumor associated vasculature is leaky though, allowing tumor cells to penetrate into the circulation and metastasize to other parts of the body.
tumor cells are heterogeneous w/ respect to ability to recruit blood vessels, as tumors varied in size +micro-vessel density depending on cancer colony
-generally: more blood vessels=bigger tumors
95
What are characteristics of the angiogenic switch?
a. Role of immune cells, MMP9, VEGF
-angiogenic switch is necessary for tumor expansion
-angiogenic switch involves recruitment of inflammatory cells
---production of metalloproteinaises (MMP9) from immune cells (Mast cells and macrophages) cleaves and releases VEGF away from extracellular matrix making it soluble and provoking angiogenesis
---VEGF normally inactivated when sequestered in the extracellular matrix
-in Rip-tag transgenic mice, viral oncoprotein expressed in beta cells in pancreas, eventually some cells acquire ability to provoke angiogenesis
96
What are metastases?
-formed by cancer cells that have left the primary tumor mass and traveled through the blood vessels and lymphatics to form new colonies in sites throughout the body
-cancers that spread are difficult to treat
97
What fundamental changes enable cancer cells to become mobile?
-Epithelial-mesenchymal transition (EMT) is a complex biological process that enables cancers cells to invade and move
- where epithelila cells start looking like stromal cells
-expression of Twist (protein) induces EMT
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Steps necessary for metastasis (i.e. Invasion-Metastasis Cascade)
1. Primary tumor formation-carcinoma
2. Localized invasion-invasive carcinoma
3. Intravasation (into blood vessels + lymphatics)
4. Transport through circulation
5. Arrest in micro-vessels of various organs
6. Extravasation
7. Formation of a micrometastasis
8. Colonization-formation of macrometastasis (rate limiting step)
*cancer Co-opt developmental programs like embryo development
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Role of blood vessels and lymphatics in metastasis
metastasis formed by cancer cells that have left the primary tumor mass and traveled through the blood vessels and lymphatics (intravasation) to form new colonies in sites throughout the body
100
Characteristics of epithelial mesenchymal transition (EMT)
-Epithelial-mesenchymal transition (EMT) is a complex biological process that enables cancers cells to invade and move
- where epithelila cells start looking like stromal cells
-expression of Twist (protein) induces EMT
-intravasation depends on proteases that breakdown tissue and epithelial-mesenchymal transition (EMT) that causes cancer cells to become mobile and go into blood vessels/vascularture
-EMT necessary for development and wound healing' this normal process is reversed AFTER extravasation (Mesenchymal-epithelial transition), as change necessary for colonization-rate limiting step
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Role of Twist with EMT
-expression of Twist protein induces EMT, as epithelial cells look like stromal and express stromal markers (fibronectin and vimentin) and less epithelial markers (E-cadherin, Beta-catenin)
-inhibiting Twist suppresses metastasis
-transcription factors orchestrate EMT during development and tumorigenesis ex:Twist, Snail, Slug
--ex: 4TI is a mouse mammary cancer cell line where cells metastasize from grafted sites in lungs, inhibition RNA prevents twist and metastatic lesions but does not totally decrease it as some modules/tumors formed due to twist proteins not being fully inhibited or redundancy (dif proteins responsible for tumor formation)
102
Seed and soil hypothesis
-different cancers metastasize to different sites
-seed (cancer) and soil (dif organ) hypothesis: cancer cell (seeds) find a new home in certain compatible tissues (soil)
-metatstasis cannot be explained by random scattering of cancer cells
103
What is the rate limiting step of metastasis? Why?
-the rate limiting step appears to be colonization as micrometastasis persists for weeks BEFORE becoming macrometastasis
-primary tumors and metastasize (other organs/parts) have SIMILAR appearance
-small tumors can metastize, individual cancer cells in small and large tumors might be equally capable of metastizing but large tumors simply "dispatch" a greater # therefore we see trend that as tumors grow=more likely to form metastasis
104
Where do cancers spread?
other parts of body/organ/tissue that the tumor/cancer is compatible with
105
Role of climate in metastasis
-not just soil or seed but CLIMATE important in development of metastasis
-climate is the genetic background/mutations and the mitochondria (more from mother)--10 genes identified that control whether or not metastasis form
-mitochondria important; mitochondria influence metastasis as FVB nucleus is in each cell but transplant different mitochondrial DNA, some mitochondria like BL6 took longer to form metastasis while BALB mitochondria promoted metastasis/ shorter period of time to develop metastasis
-MMTV-PyVT trans genetic mice express the Polyoma virus middle T-antigen under direction of mouse mammary tumor virus promoter/enhancer--females develop mammary tumors which metastasize in lung while males develop at later age of onset
106
What cell type gives rise to cancers
-stem cells appear to be targets of cancer
-tumors often have differentiated cell types that are present in tissue of origin as found dif markers present in tumors via staining ex: markers for enterocytes, goblet cells, and paneth cells present in tumors from min mice
-problem: most stem dont have definitive markers or could lose expression of marker when transformed into cancer cell
-distinct hematopoietic cell types within a patient with chronic myelogenous leukemia (CML) carry philadelphia chromosome- same translocation of chrom 9 and 22 forming BCR-ABL gene/protein complex
-hematopoietic cells produced by myeloid stem cell have translocation
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How are these cells protected from damage at the molecular level? How are cells protected from damage at the physical level
Molecular: stem cells divide infrequently, relatively slowly, and present in very lo numbers (2 to 3 in each crypt)
-surrounding goblet cells filled with mucin which protects from mutagens, therefore if lacking mucin (Mucin2)=mice develop inflammation+intestinal tumors, mucin2 haploid sufficient/not haploid insufficient (1 copy is enough to rescue bad phenotype)
Physical: stem cells shielded from damage by location and protective elements, stem cells in base of crypt in intestine
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Characteristics of stem cells:
- Frequency of division
- Source of differentiated cells
- Why are stem cells often not killed by chemotherapy?
- Symmetric vs. Asymmetric cell division
1. Frequency of division: very slow + infrequent, protects stem cells from damage
2. Source of differentiated cells: stem cells differentiate into transit-amplifying cekks which divide rapidly, transit-amplifying cells turn into differentiated cells which shed off into lumen of intestine
**therefore damage often occurs in post-mitotic cells/differentiated cells=less likely to form tumor vs if stem cell damaged bc cell damage would persist during division
3. Why are stem cells often not killed by chemotherapy?
-bc can self-renew and differentiate into heterogenous lineages of cancer cells
4. Symmetric vs. Asymmetric cell division: asymmetric stem cell division, 1 daughter cell remains in stem cell crypt and 1 cell moves up
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Mechanisms that a stem cell uses to protect itself against damage: (6)
a) Location of stem cells: deep in the crypt/at the pit so hard for mutagens/damage to occur
b) Conserved strand hypothesis/Immortal DNA:
protecting from accumulation of mutations in the DNA
c) Apoptosis: programmed cell death
d) Drug pumps : if damage, will pump drug/mutagen out of the cell
e) DNA repair mechanisms: errors can occur during replication like spontaneous changes, incorporation of altered nucleotides, mutagenic insult
-proofreading activity like 3' to 5' exonuclease activity: removing misincorporated nucleotide replacing w/ correct 1 and matching with template
f) Mucus (mucin expression): from goblet cells surround stem cells in crypt which protects from possible mutag
110
What is conserved DNA strand hypothesis?
-conserved strand is less likely to have DNA damage vs nonconserved strand/newly synthesized strand can accumulate mutations
-but if conserved strand theory correct then why do cancers develop?
-bc no biological system perfect, mechanisms affect protection @ replication level but not from other exogenous sources
-due to plasticity (cancer look like derived from stem cells)- some transient ampligying cells can revert BACK into stem cells under certain biological context which could CAUSE MUTATIONS
111
How and why do germline mutations of DNA repair mechanisms increase tumor and cancer incidence?
-# of DNA repair mechanisms repair damage resulting from endogenous and exogenous sources
-mistmatch repair activity: can repair errors as protein recognizes mismatch, bulge occurs, strand is removed and new DNA synthesizes
-human homolog of MutL (E. coli) is MLH1, and MutS is MSH2
- mutations to these repair mechanisms can then cause mutations
-mice that lack proofreading have increased incidence of cancer
112
What is Lynch syndrome/Hereditary Nonpolyposis Colon Cancer (HNPCC)?
-afflicted individuals @ increased risk of developing cancers of colon, stomach, gall bladder, urinary tract, endometrium, ovaries
-carry germline mutations in MLH1, MSH2 (predominately H1 and H2), MSH3, MSH6, and PMS2--these genes important is mismatch repair activity, lacking mismatch repair activity
-many ppl with lynch undiagnosed, each patient has usually 3 relatives with mutation
-tumors lacking mismatch repair activity exhibit microsatelite instability
113
Importance of below genes in regard to Lynch syndrome
a) MLH1 and MSH2
b) PMS2 and MSH6
a) usually MLH1 pairs with MSH2, so partner becomes less table (usually heterodimer)
b) PMS2 pairs with MSH6
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What is microsatellite instability (MSI)? How can MSI be detected?
-tumors lacking mismatch repair activity exhibit micro-satellite instability
-look @ DNA repeat sequences since susceptible to damage due to faulty mismatch as DNA poly may lose track/add extra nucleotides causing extension of repeats
-micro-satellite instability can lead to insertions and deletions, but severity of damage depends on place and what nucleotides coding for
-micro-satellite instability is detectable, peaks that are NOT normal (more than 2) can see bp deletions
-MSI as a biomarker for immunotherapy: proteins have neoantigens detected by immune system, tumor inactivates protection=blockst-cell activation
-however can take antibody attached like PD-1 and bind antagonists, blocking the interaction with tumor and allowing detection for T-cells to kill tumor cells
*immunotherapy does NOT work if patient has mismatch-repair proficient, works if deficient in mismatch repair
