Introduction PowerPoint 6/6/2015 7:04:00 PM

Midterm information
- Introduction
 multicellular organism disease
 caused by an accumulation of mutations
o random mutations
 mutated by chance, causing an effect
o inherited predisposition
o viral infections (aka cervical cancer)
 multistep process
o shown by relationship bw age and cancer
 all cancers have a genetic basis
 #2 killer
 Types of cancer:
o Carcinoma- ET cells *most common
 Lung, breast, prostate, colon, bladder
 Adenocarcinomas- ET w/ glandular/mucus secreting
cells
o Sarcoma- connective/mesenchymal tissue
 Fat, bone, muscle, fibroblasts
o Leukemia/Lymphoma
 Bloodstream/lymph nodes
 Hematopoietic cells- blood cells
o Neuroectodermal tumors
 Gliomas, glioblastomas, neruoblastomas, Schwannomas
o Other
 Melanoma, SCLCs
 Most prevalent: prostate/breast
 #1 killer: lung cancer
- What is a definite sign a tumor is malignant? Breaks through the basal
lamina
 If contained, then benign
- Spreading definitions
 Hyperplasia- excess number of cells
o Look normal, just proliferation
 Dysplasia- cytological abnormal; lack of features found in
differentiated; between benign and malignant
  Neoplasia- another word for cancer
 Anaplastic- dedifferentiation, don’t know where it came from
o hard to find tissue of origin
 Invasiveness- cancer cells growing past stromal boundries, but
still enclosed in connective tissue capsule
 Metastatic –spreading to other places
o few cancers can’t: gliomas, basal cell carcinoma (but both
highly invasive)
 -oma- usually benign
 Metaplasia- one type of normal cell layer replaced by another cell
type not found at a particular site in body (Barrett’s esophagous”
- Important people
 Pottchimney = greater skin cancer (occupation and cancer)
 Yamagiwa chemical can directly cause cancer
o Cancer can be studied in lab
Carcinogens- agents that contribute to formation of cancer
 Fats and tobacco = carcinogen
Cancer cells accumulate chromosomal abnormalities, esp late in process
“one renegade cell”
 nature selection
 Darwinian selection
o Tumors work over many many cell divisions
 Mutations occur all the time, but don’t result in disease
o When it fucks up homeostatic control = problem
Less differentiated = worse GX = cannot be determined; G4 –
undifferentiated, high grade
TNM classification
 Tumor, node, metastasis
Cancer Stages
 Stage 0 “in situ” still localized to original tissue
 Stage 1localized to one part of body
 2/3lymph nodes
 4other organs
What makes cancer cells
 Cancer cells are NOT under contact inhibition for proliferation
 Alterations in cell adhesion and cytoskeleton
  Angiogenesis
o Creation of blood vessels
o When starved for O2
o Nutrients and way out to spread
 Immortalized
 Undifferentiated
 Invasive
 Escape apoptosis
Viruses can cause cancer
 Dude named Rous
o Identified that a virus could transmit sarcoma bw chickens
o Rous Sarcoma Virus can transform cells in vitro
o Identified src from virus = oncogene
 Src is a normal cellular gene that chickens and humans
have
 Virus infect cell, takes some host DNA with it when
replicates, then brings an altered version of that host
DNA to a different host cellCANCER
 Led to the thought that viruses were the cause of cancer
o Wrong.
 Ras identified as viral oncogene
Cell Cycle 6/6/2015 7:04:00 PM

Cell Cycle
- Hallmarks of Cancer
 independent from growth signals
 insensitive to death signals
 insensitive to stop-growth signals
 able to invade surrounding tissue
 can grow own blood vessels (angiogenesis)
 able to survive in foreign tissue
 inflammation allow for tumor growth
 avoiding immune system
 deregulated cellular energetics
 genomic instability
 tumor microenvironment
Oncogenes vs. Tumor Suppressors
 Oncogenes constitutively “on”
o Growth factors; receptors; signal transducers, transcription
factors
o Examples: cyclin D1, MDM2 (take away p53), myc, ras
o An oncogene only needs one mutated copy to become
cancerous (aka heterozygotes = cancerous, too)
 Tumor suppressorshut off
o Inhibits cell division
o Examples: Rb, p53, p16, ARF, PTEN
o Tumor suppressors need BOTH alleles mutated to contribute
to cancer
When do cells proliferate
 Development
 Wounds
 Blood, small intestine, immune system
Cell cycle + checkpoints “Guardian Mechanisms” of Genome
 Start aka restriction point
o G1/S phase
 Interphase: G1, S, G2
 Checkpoints
o G1/S
 Point of no return
  Goes into G0 phase if not good enough
 DNA damage? Good conditions for cell division?
Growth factors? Nutrients?
 Restriction/start point
o G2
 DNA damage? DNA replicated properly? Ready for M
phase?
o M
 Are all chromosomes attached to the spindle?
 If chromatids not properly assembled, then anaphase is
blocked
Cancer & proto-oncogenes/tumor suppressors
 Loss of cell cycle control at R point (G1 checkpoint)
Does it matter if a person is homo/heterozygous for a cancer gene?
 Two possible cases:
o Mutant = nonfunctional (aka tumor suppressor based cancer)
o Mutant = hyperactive (aka oncogene based cancer)
Experiments: G1/S and G2/M are important control points
 Fuse M cell + interphase cell = interphase nucleus enters M phase
(G2/M)
 S cell + G1 cell = G1 enters S phase
 S phase + G2 cell = G2 DOESN’T enter S phase
o Can’t ever enter G2 phase from S
CDKs- cyclin-depedent kinases
 Regulate cell cycle; phosphorylate CDK targets and change activity
 Cyclin + CDK  bind now can bind to target
proteinphosphorylates target protein to activate it = cell cycle
function
 Experiment: budding yeast
o Cdc mutatants arrest at the same cell cycle phase
 At low temps: arrest at different phases
 At high temps (mutants): stop before S phase (at G1
phase) *nonpermissive temps
o Size of buds give insight into cell cycle/timing
o Cdc2/Cdc28 needed at R and G2/M checkpoints for:
 Fission
  Budding
 Summary:
o Genetic approach in fission and budding reveals the genes
that are essential in promoting cells through the cell cycle
 Key genes encode a protein kinase called CDK (cyclin-
dependent kinase)
 CDK1 is protein kinase formed from cdc2/cdc28
genes
CDKs are activated by cyclins (Hunt, Hartwell, Nurse)
Cell cycle has a clock, regulated by 1.) promoting factors and 2.)
checkpoints
 After done with step, cyclin is degrades and new cyclin comes in
(ex. G1S phase)
 Cyclin Dependent Kinases regulate Cell Cycle
o Cyclin + CDK = movement past restriction point
 Purpose of Cell Cycle
o Accurate transmission of genetic info
o Maintain ploidy
Humans have 4 CDKs and 4 cyclins
 Different cyclins are up at different times
 Combinations of cyclins/CDKs are specific
 CDK forms heterodimers with cyclins and become active kinases
 Regulation
o G1/S-Cdk complex commit to new cell cycle
o S-Cdk complex promote S phase and prevents replication in
G2 phase
o M-Cdk complex trigger entry into mitosis
 Inactivated before anaphase
CDK regulation
 Abundance of cyclins
o Appear then disappear
o Destruction box targets degradation of cyclin
 Via Ub
 E1, E2, E3 (E3= ligase; specificity)
 If mutation in destruction box, cyclins always there, cell
cycle can’t be completed bc cyclins NEED to be
destroyed in order to go to next step
o Cyclin regulation
 Make the next cyclin for next step
 Degrade the previous one via E1-E3 destruction box
 CDK phosphorylation (aka “activating phosphorylation”)
o CAKs = CDK activating kinases
 CAKs are abundant and not regulated
o Still need presence of cyclin- no cyclin, no activation
 CDK + cyclin = partially active
 CDK/cyclin + CAK (+P) = fully active via an activating
phosphate
 Binding to CKI (ex. P21 attach to cyclin/CDK = inactive now, stops
cell cycle)
o INK4/p16bind to CDK4/6 prevent cyclin D binding (early G1
phase)
 If cyclin can’t bind, then CDK is inactive = no cell cycle
progression
 Commonly mutated in tumors
o Cip/Kip (p21/p27); block active site of MANY Cdks
o CKI normally for entrance into S phase
Cdk/cyclin pairs
 G1CDK4/6 cyclin-D (CKI: p16 or INK4)growth
 G1/SCDK2/cyclin-E (CKI: p21/p27)
 S CDK2/Cyclin-A
 G2/MCDK1/cyclinA/B
o Phosphorylates laminnuclear envelope breakdown in early
mitosis; cyclinB-CDK1 degraded during mitosis before
anaphase to allow formation of new nuclear envelope
breakdown during telophase
Onset of cell cycle requires mitogens
 Mitogens effect
o Activate CDK4/6 cyclin D complex
o Inhibit CKI (p16/INK4)
 Without mitogens
 o Doesn’t meet threshold of enzymesG0 phase
Retinoblastoma
 What does Rb normally do?
o Nuclear protein that undergoes +P and –P in concert with
cell cycle
o G1hypophorylation
o G1/S (R checkpoint)hyperphosphorylation
o S/G2/Mextent of pRb phosphorylation
o End of Mdephosphorylation
 Un/hypo (end of M to G1)
o Inhibits cell from entering new cell cycle
o Upon further phosphorylation at R point, hyperP pRb becomes
inert and cell cycle can proceed
 Rb/E2F complexes act as transcriptional repressors
o pRb + histone deacetylase + E2F = repressed transcription
o if get rid of E2F + histone deacetylase, then transcription is
activated
 NOTE: E2Fs have 100s of target genes, most involved
in DNA replication
o E2F (a transcription factor) activate genes necessary for cell
cycle PROGRESSION
 However, NOT functional when bound to
hypophosphorylated Rb
 When CDK4/6-cyclinD phosphorylate Rb, its causes a
conformational change that releases E2F, which can
activate expression of target genes
 “autoregulation of E2Fs”
E2F target genes
DNA polymerase alpha; CDK1; cyclin E; cyclin A; ARF; securing;
p73DNA synthesis or cell cycle progression/differentiation
When pRb is hyperphosphorylated and inhibited only upon cycE
expression
 Released from its role as guardian
 E-CDK2 can phosphorylate Rb ONLY AFTER Rb is (hypo)
phosphorylated by D-CDK4/6 in mid-G1
 E2F targets: the cycE gene
 o Transcription of cycE starts a positive feedback loop
Rb control of E2F is the restriction point
Rb Summary
 Cell-cycle Off?
o Rb bound to E2F; no transcription of E2F, no entry into S
phase
 Cell-cycle on?
o D-CDK4/6 hypophosphorylates Rb
 E2F and Rb separate bc of conformational change
 E2F transcription = promoting factors
 Without 2 copies of Rb: NO cell cycle arrest
Oncogenes and Tumor Suppressors 6/6/2015 7:04:00 PM

Oncogenes and Tumors Suppressors

- Proliferation
 proliferation = cell division (#) + cell growth (size)
o if you have cell growth but no cell divisionelongated
 example: neuron (NO cell division, can’t be replaced)
o if you have division, but no growthsmall cells
 early development (ex. Tadpole, frog)- still needs to
develop
- Alleles
 tumor suppressors require both alleles to be mutated to be
cancerous
 oncogenes require only one allele to be mutated to be cancerous
 MOST cancers are recessive
- Tumor Suppressor Genes (specifically: Retinoblastoma)
 Retinoblastoma (white eye cancer)
o Can be either sporadic or familial
 sporadic
 more common
 unilateral cancers (affects one eye)
 spontaneous loss of both alleles in one cell
 reason why less common then hereditary
 Must lose both copies of Rb in order to be
expressed (tumor suppressor)

 hereditary/familial
 less common
 75% bilateral
 25% unilateral
 when Rb heterozygotes lose function in the wild
type (WT) allele, tumors arise (via somatic loss of
allele)
 happens in 90% of cases
 if homozygous for loss of Rb, die during embryo
 Must lose both copies of Rb in order to be
expressed (tumor suppressor)
  Familial Rb (bilateral) have high risk of
developing non-retinal tumors
o Why?-->germ-line mutations lead to
predisposition to cancer
Knudson’s Two Hit Hypothesis (LOH)
 Reasoning for the mechanism of inherited RB
 LOH (loss of heterozygoticity) role in tumor suppressors
 Inactivation of both WT alleles of a tumor suppressor gene
PROMOTES tumor
 Explanation
o People have 2 copies of Rb gene (each parent)
o Familial type
 Have one mutant gene already (hetero) Rb+/Rb-
 Easy to cause other mutation (since HAS to lose both,
tumor suppressor)
 Early in life
o Sporadic
 Need to lose both WT alleles- not a very high chance
 Rare; late in life
 Causes of LOH (common in tumor suppressors)
o Missegregation (30%)
o Mitotic recombination at Rb locus
o Methylation of Rb gene (inactives)
o Deletion of Rb gene
o Alteration in chromatin structure
Retinoblastoma genetics
 INHERITED as dominant (familial)
 But really recessive at cellular level
 PREDISPOSITION to cancer is inherited dominantly, but offspring
CANNOT inherit two mutated genes
 Rb is just one example
Tumor Supressors
 Way to lose function
o Mutation within gene
o Chromosome loss
o Methylation of CpG islands
  Near promoter regions of the tumor-suppressor gene
 No transcription
Oncogenes
 Genetic changes
o Missense mutation
 Ras stays GTP-bound
o Gene amplification- more promoters = too much protein
 Myc in leukemia
 EGFR in lung/head
o Chromosome translocation
 BCR (22)-ABL (9) chromosome number
 Promoter from bcr, fused to abl kinase
o Retroviral insertion
 Src
 Viruses take normal oncogene
 A new viral infection inserted an oncogene = cancer
 Put promoter near proto-oncogene
 Without a virus, cells can be transformed
- RAS: Transforming Oncogene = Ras (molecular switch)
 cellular oncogenes = viral oncogene
 Modified by post-translation for going to membrane
o +lipid
o lipid modification targets Ras to the plasma membrane
 Ras binds GTP
o Ras is a enzyme; hydrolyzes GTPGDP (Ras = GTPase)
 Ras = molecular switch
 GTP bound = active; GDP bound = inactive Ras
 Steps:
o Inactivebinds a GDP molecule
o Stimulatory signaltakes out GDP, gets GTP
o Now an “activated signal emitting configuration”
 Cleaves GTP using OWN GTPase activity (hydrolyze)
 Activity of Ras is regulated by GEFs and GAPs
o GAP induce GTPase activity (GTPGDP); turns Ras “off”
 Done by self hydrolysis activity
o GEFactivated by upstream stimulatory signal
  Takes out the GDP and puts in a GTP
 Weak ability to change g proteins
 Weak GDP/GTP exchange
Ras cancer?
 If it loses it’s intrinsic GTPase ability, then constitutively “on”
 Over-proliferation
RTK Signaling Pathway Intro
 Src- non-receptor tyrosine kinase (oncogene)
o Phosphorylates other tyrosine residues
o Has a SH3, SH2, and tyrosine-kinase domain
 RTK = receptor tyrosine kinase
o Transmembrane
 Transmembrane alpha helix
 Extracellular domain
 Cystosolic domain
 Has kinase ability here
 “Activation lip”
o No ligand = inactive, monomer
o w/ ligandactivates RTK via dimerization
 kinase catalytic domain takes two (one for each arm of
RTK) ATPADP, steals phosphates
 “autophosphorylation”own substrates, act on
selves
 SH2 vs SH3
o SH2binds phosphorylated tryosines
 Allows effector proteins to bind to activated (the lips of
RTK)
 SH2 has two binding sites: 1.) phosphotyrosine 2.) AA
side chain
 Effector protein with SH2 domain: Grb2
o SH3binds proline
 Other side of Grb 2 is SH3 domain
 Binds to Sos, made from proline
o Remember Ras?
 Well, with lipid modification attaches onto the
membrane
 GDP-bound, inactive
 Sos binds to Ras
o Sos binding to GDP?
 Sos is a GEF!
 Promotes the flipping of GDP for GTP, now Ras is
active
 Sos disassociates from Ras
 Now forget about RTK, focus on activated Ras
o Ras= molecular switch
 Each activated protein activates the next
o Active RasRaf (MAPKKK)
o Raf uses ATPADP; phosphorylates/activates Mek (MAPKK)
o Mek uses ATPADP; phosphorylates/activates Erk (MAPK)
o Erk enters nucleus
 Uses ATPADP; phosphorylates either a 1.) protein or
2.) gene regulatory protein
 If proteinchanges protein activity
 If gene regulatory proteinchanges gene
expression
 Examples of growth factors
o EGF/PDGF = proliferation
o If you add constitutively “on” Ras into cell, get proliferation
without even adding EGF/PDGF
o RTKs bind specifically to/from soluble polypeptide growth
factors
 Examples: EGF, insulin, NGF, FGF, PDGF
o Fos/jun = targets for Erk
 Increase gene expression SO Fos/jun are both gene
regulatory proteins
 OTHERS: AP-1, Myc/Max (transcription factors)
Ras doesn’t just have to work with RTK
 Also can work with PI3K and RALGDS
 Different pathways = different functions (DOWN STREAM
EFFECTORS)RTK IS A DOWNSTREAMEFFECTOR
o PI3Ksurvival, transcription, translation
o RALGDSvesicle transport, cell-cycle progression
 o RTKcell cycle progression, transcription
o

Downstream effectors transcription factors

Apoptosis 6/6/2015 7:04:00 PM

Apoptosis
 p53 hypothesis: oncogene vs tumor suppressor
o DISCOVERY
 P53 was first discovered from the precipitation of 1.)
SV40 Large T Antigen and 2.) cellular protein of 53 kDa
 From hamster fibroblastsaddition of SV40 =
transformed cells
 Works for both mice and monkeys: results in
monkey kidney cells to turn into cancerous cells
After precipitation, got out: Large T antigen and p53
 Large T = oncogenic protein
 Both Large T and p53 are ONLY expressed upon
viral expression
 Shown by a western blot, both active when virus
(darkened lines)
o TUMOR SUPPRESSOR OR ONCOGENE? (answer: tumor
suppressor)
 When thought oncogene?
 Fucked up tumors have high p53 levels
 Half-life of p53 was longer in tumor cells than
normal cells
 THUS: overexpression/stabilized when
transformed, so oncogene makes sense
 Original hypothesis: low p53 levels in normal
cells, T-antigenoverexpression of p53 to act like
an oncogene
 How did they prove that it was a tumor suppressor
 Cloned p53 and saw effects = less tumors =
tumor suppressor
 Autosomal Dominant Li-Fraumeni
 Germ-line mutated (inherited) in p53 cause
predispositions for DISTINCT cancers
o Examples:
 Adrenal , soft/bone sarcoma,
brain, breast, lung
 Mice experiment
 o P53+/+survived
o P53+/-some died
o P53-/-a lot died
o Proved that, without p53 activity, then
tumors go crazy; if an oncogene, then
would survive longer if it was p53-/-
 Some p53 cDNA could immortalize cells in culture
and enhance transformation by activated Ras
 When wild type/regular p53 was transfected
into cells
o Blocked G1 cell cycle progression
o INTERFERED with this oncogene
transformation
 Found that those with tumors had both
alleles of p53 fucked up
 T-antigen
 Oncogenesequesters p53 and pRb
 P53 responsible for both apoptosis and
stopping cell cycle
 pRb is responsible for the negative
regulation of the cell cycle
 p53 characteristics/functions
o transcription factor- protein that binds to specific DNA
sequences, controlling the rate of transcription from
DNAmRNA
o homotetramer- protein complex with 4 identical subunits
associated, but not covalently bonded
 “loss of function copy”- homotetramers will still
assemble into p53 and they will bind to partners, but
WON’T be able do do the job; they will just prevent
other normal p53 from binding to cause apoptosis
 even 1/16 of p53 molecules have some activity
 as such, you need both copies of negative p53-/-
to result in tumor progression
o Possible mutations:
 Nonsenseresults in a STOP codon
  Frameshiftaddition or deletion, major problems
 Missensepoint mutation, results change in AA
 Some missense can still assemble, but function is
lost; not homotetramer
 Null mutations- loss of function but but doesn’t
really form the tetramer
 So can’t bind to self and take up a real p53
spot
 More specific common mutations
 Point mutations (85%)
 Deletions (9%)
 Splice mutations (3.5%)
 frameshift
 insertions (2%)
 frameshift

 Categorized Mutations
 Transcription-activation mutations
 MDM2 binding
 the addition of P
 Sequence-specific DNA binding domain
 Most common
 SV40T comes into play here
 Non-specific DNA interaction domain
 Problem with tetramerization
 The addition of P
 Regulation of p53 expression
o Inputs (what would normally increase p53 expression)
 Direct causes
 genotoxic stress (aka DNA damage)
ex. UV damage
 DNA damageA
 proliferative stress (aka oncogenes)
 SV40T
o If p53, itself, wasn’t regulated itself, then is ALSO a problem
 Would just go and start causing apoptosis
  P53 is constantly made at all times, but is
degraded via ubiquitin proteolysis
 Thus, in normal cells, p53 concentration is
low
 Keeping p53 in check- 3 mechanisms
 Protein stability via MDM2
 Regulatory proteins E1-E3 control p53 via
ubiquitin (inactivation once attached) Ub-
76-aa tag
o E1activates Ub and binds to it;
passes on Ub to E2
o E2conjugating enzyme: 2 options
 Transfer Ub to E3 ligase
 OR can transfer directly to the
target (in this case, p53)
o E3Ub ligase
 Ligases glue together things
 Functions in conjunction with
E2; bind to/transfers Ub to the
lysine residue
 Specificity in E3!
 Forms a poly-Ub chain to target
p53 for degradation by
proteasomes, 26S via E4,
ubiquitin chain assembly factor
 Specific E3 ligase
 MDM2
 MDM2 ligase (an E3)
o Genetic evidence that MDM2 INHIBITS
p53: mice
 P53-/-
 Mice lives, but with cancer
 P53-/- AND MDM2 -/- inactive
ligase
 Mice lives
 MDM2-/-
  Mice dies
 Normal copy of p53, but
no way to regulate to
OVER-APOPTOSIS; mouse
dies
 Really shows that
regulation of p53 is also
important; too much is
bad
o Works via a negative feedback loop
(auto-regulatory loop)
 In presence of the inputs of
apoptosis (genotoxic and
oncogenes):
 P53 is needed; destroys
and breaks down the
MDM2 ligase so can
continue working
 If everything is dandy (normal
cell)
 These broken MDM2 ligase
pieces are taken and put
together to form the E3
ligase to Ub p53 and stop
if from over-apoptosis
 Some p53 mutants show the overexpression of
inert p53 (no function)
 This takes up the stops the
normal/functional p53 act to destroy bad
shit
 Thus, MDM2 transcription can Ub and take
these bad copies of p53 out

 Nuclear localization
 Post-translational Modification (PTM)
 impact on p53a
 o INCREASE in p53 expression (tumor suppressor)
 Normal cells have low levels of p53- don’t need to kill
them off
 More exposure to UV = more expression of p53, ex.
genotoxic effects
 Outputs
o Cell-cycle arrest
o Apoptosis
 Pathways of Inputs
o I. Genotoxic (DNA) damage (kinases)
 GAD45 activated by DNA damage; binds to nuclear
proteins involved in DNA nucleotide excision pair and
can arrest cell cycle
 DNA damage response
 Involves 2 protein kinases: 1.) ATM 2.) ATR
 phosphorylates downstream effectors
 Involves 2 protein kinases: 1.) ATM 2.) ATR
 JOB: phosphorylate p53
 ATR/Chk1
o cases:
 DNA damage (single)
 Stalled replication fork
 ATM/Chk2
o Case
 Double strand break
 Phosphorylation of p53 ACTIVATES P53
 How does this make sense?
 The phosphate group prevents MDM2 from
Ub p53 and lysing it; the phosphorylation of
MDM2 inactivates it (inhibitory
phosphorylation)
o Stabilizes p53 so can act as a
transcription factor and do it’s job of
apoptosis
 Pathway
 DNA DAMAGE PATHWAY
 o Single
StrandATRChk1p53p21 no
mitosis
o Double strand
breakATMChk2p53p21no
mitosis
o P21 use- cyclin/CDK inhibitor, arrest
cell cycle at G1/S and G2/M
checkpoints
 NORMAL PATHWAY (low p53 level, normal
DNA)
o DNA
damageATR/ATMChk1/2Cdc25
mitosis
o If mitosis than activates Cyclin-CDK to
allow the progression through cell
cycle
o II. Hyperproliferative Stress Response (aka high E2F activity)
 Results in high p53 activity
 Controlled by ARF, a regulative protein
 P19ARFtumor suppressor, increasing p53
expression by INHIBITING MDM2
 E2Fs induce transcription of ARF gene, bind to MDM2
and send it to nucleolus, stabilize p53
 So ARF sends MDM2 upon need for apoptosis in
hyperproliferative stress.
 Pathway
 Myc/Ras/E1A= oncogenes
 ARF (Alterantive Reading Frame) found in same locus as
p16 gene
 Together, p53 (nucleotide subst.)/INK4 (deletion,
sub, etc) = most common mutated genes in
human tumors
 Uses Alternative promoter
 P16- tumor suppressor protein
  Responsible for decelerating cell progression
from G1 phase to S phase aka tumor
suppressor like pRb
 2 reactions for the price of one stopping of cell
division AND cell death upon excess
mitogens/profliferative agents
 Two possible outputs
o Cell cycle arrest
 P16 aka INK4 = tumor suppressor; responsible for
inhibiting the entrance into S/replication phase
 Deals with CDK4/6-cyclin-D
 P21 (made by/activated by p53)
 A CKI (inhibitor)
 Deals with CDK2-Cyclin-E
 G1/S phase!
 P27helps P21 arrest at CDK2/E
o Apoptosis (2nd possible “cell fate”/output)
 Random facts
 Syndactylyl
 “impaired apoptosis”results in joined
fingers
 10^11 cells die/day via apoptosis
 only 1/3 of neutrons generated during
embryogenesis survive at birth
 other 2/3 undergo apoptosis, important for
sculpting tissue (ear, digits, etc)
 T-cell development in thymus (chest region)
 Greater than 90% of developing T-cells die
o T-cellimmune cell, responsible for
immune cells
 Apoptosis of “extra” 90% of T-
cells prevent autoreactive ones
 Cell-division + Apoptosis
 Proliferation of a group of cells
 Apoptosis kills the inside cells
  Now left with a hollow lumen with a
polarized/growth arrested ET
 Apoptosis requires ATP and is highly ordered
 Via proteases/nucleases = break down DNA,
protein, etc
 DNA fragments = DNA oligonucleosomes
 membrane blebbingproduces apoptotic bodies
that contain debris that the cell wants to get rid of
 apoptotic bodies engulfed by macrophages
 C-elegans—worm (model organism)
 1910 cells, 131 die during development
 in worms, Bcl-2 is actually CED9
 CANCEROUS PATHWAYS:
 Death signal Ced 9Ced-4—ced-3cell
death
o Ced4 activates Ced-3
 Ced-3 is a caspase (protease)
responsible for killing
o Ced-9 = inhibitor
 Eg1-1 = promoter
o Ced-4 = activator
o Ced-3 = protease
 Without death cignal C-9 is inactive, so no cell
death/apoptosis
 Intro
 Bcl-2 is an anti-apoptotic protein
 If mutated results in apoptosis
 Bcl-2 = b-cell lymphoma (translocation)
 Mutations causes “gain of function” to bcl-2,
resulting in cancer
 Same process in apoptosis as programmed cell
death
 Similar to humans
 BAD + Bcl2 = Inhibitor
o BAD = Bcl-2 Associated Death
Promoter
  Death signal induces BAD
 Apaf1 = activator
 Caspase-8/9 = protease
 Pathway (intrinsic pathway)
 Anti-apoptotic (aka survival) signals
o Closed channel to mitochondria
o Bcl-2 helps with this; helps maintain
mitochondrial membrane integrity
o Keeps cyt c pores closed
 Pro-apoptotic (aka death) signals
o Open channel to mitochondria
o Cytochrome C exits the mitochondria
 Releases VERY potent inducer of
apoptosisinducer binds to Apaf
(around the flower kind of
figure)caspase 9/3
activated/recruited to make
apoptosome
 Initiator caspase
 Caspase 8/9, first, forms
apoptosome
 Also has Apaf-1
 Executioner procaspase 3, 6, 7
 Break down death
substrates into cleavage
 Intrinsic Pathway Summary
 OMM (outer mitochondrial membrane) intact
 BH3-only proteins bind to Bcl-2 (death signals)
 Tells cell to recruit Bax/Bak
 Bax (a proapoptotic gene) recruited to OMM
 Bax/Bak (bak is always present, BAX needs to be
recruited oligomers form (another Proapoptotic
gene)
 Oligomers/heterodimers allow the opening of cyt c
pores, caspase activation
 Originally, only BAK (always present) and Bcl-2
present on normal cells
 When need apoptosis (remember inputs),
Bcl-2 ripped off via BH3 protein, BAX
attaches to OMM, forms oligomers (both
pro-apoptosis), opens channel, allows cyt c
to exit, releases a very strong apop factor;
Apaf1casp-8/9casp3,6,7
 IAPs = inhibitors of apoptosis
 Helps to maintain balance so apoptosis is
not in appropriately triggered
Invasion and Metastasis 6/6/2015 7:04:00 PM

Invasion and Metastasis

- What’s the main difference bw Invasion and Metastatic
 malignancy (tumor that can harm) are usually BOTH metastatic and
invasive (compared to benign)
 tumor progression = slow/gradual
o Order:
 normalhyperplasia, dysplasia, carcinoma in situ,
invasive
 malignant is the last two steps
o tumor- mass/growth of cell that have no useful purpose
o first invasive THEN metastatic
 Main difference
o Invasive is the actual growth of the tumor; it’s spreading
within the same body part
o Metastasis- actual movement of tumor cells to different body
parts
- Steps to Metastasis
 Actual Steps
o Primary tumor formation
 “progression"
o Localized invasion
 “progression”
o Intravasation- interaction with platelets, lymphocytes, and
other blood components
 Remember: in order to metastasize, needs to get into
the blood!
 Intravasation is the invasion of cancer cells through
the basal membrane into a blood or lymphatic vessel
o Transport through circulation
o Arrest in microvessels of various organs
o Extravasation- formation of micrometastasis
 Leakage of fluid out of container; lead from blood
(capillaries) to tissue
 Movement of the cancer cells to a new place
o Formation of micrometastasis
o Colonization- formation of macrometastis
- Cancer cells frequently metastasize to common places
 possible “targets”, “metastatic trophism”
o brain, lung, liver, bone marrow
o makes sense bc supply of blood, vessels, etc
 cancers cells need to lose adhesion/ET properties to break through
and metastasize
o same method as regular cells
- It is possible to have micrometasteses that are “dormant”
 the chance for survival is usually higher if dormant
 IntravasationLatencyColonizatino
- How do cells become invasive?
 Cells need to regulate movements via:
o Structure/organization (to self-Bcl-2, take care of this)
 ECM
o Detach and reattach
 ECM
 Other cells
o Actual movement
- For cells to move, they need to be able to stick and un-stick
 Cell-cell attachements
 tight junctions
o Claudin
o Occluding
 adherens junctions
o E cadherin- connect adjacent actin filament bundles bw cells
o Beta-catenin-
 Junctions bw ECM and cell

-EMT (Epithelial to Mesenchymal Transition)

 process by which Epithelial cell loses adhesion and polarity, has
ability to move now with mesenchymal cells
 important in development
o cells need to generate new compartments, get new places
o more EMT = more invasive/metastatic
 important in cancer
o most common cancers= carcinoma = ET
 o in order for ET cell to become malignant, needs to become
mesenchymal cell **not normal behavior
 Process
o Necessary transcription factor Twist = good central regulator
 Twist fucks with adherens
 Twist inhibits E-cadherin, so cell-cell interactions
are affected
 Stopped expression
 Gains b-catenin
 transcriptional factor
 structural protein
 correlates with genes critical for
proliferation/differentiation AKA GROWTH
 beta-catenin leaves adherens junction and
accumulates in the nucleus
 now can activate genes (transcription
factor!) like c-myc, resulting in proliferation
 Analysis of changes typical to EMT
 Process can actually be seen
 ET marker = cytokeratin
 Mesenchymal marker = vimentin
o gain mesenchymal proteins
o Cell Movement
 Leading edge projects
 Lamellipodia that attach to ECM while
simultaneously breaking disrupting connections at
trailing edge
 Actin polymerization on leading edge (lamellipodium)
 Stretches out the actin cortex, resulting in
movement of unpolymerized actin, too
 Myosin II aids in the contraction of this filament
 Release of focal adhesion on back
 Leading Edge
o What happens at the leading edge?
 Actin polymerization
 Projection of lamellipodium
  Localized decreases in cell-membrane tension
o What’s there: (all interact with integrins)
 Clustered integrins
 Receptors
 Required to form and react to new adhesive
contacts
 Cytoskeletal protein (aka actin)
o Focal adhesions
 Integrins (at leading edge) binding ECM become linked
to cytoskeleton to form new focal adhesion
 Read End
o for cell to move:
 adhesive contacts at rear are released
 proteolysis of integrins at the rear end
 reduction of integrin affinity for ECM
 ECM no longer wants to bind to integrins
o Integrins
 Cell-surface, transmembrane receptor glycoproteins
 Has 1 alpha and 1 beta subunit
 18 different alpha types
 8 different beta type to choose from
 each pairing of alpha/beta binds a different ECM protein
ligand
 vary in specificity
 aVb3 binds fibronectin, fibrinogen, VWF,
vitronectin, collagen, laminin
 a5b1 only binds fibronectin

o
Cancer Migration Part II 6/6/2015 7:04:00 PM

- Src
 Src is post-translationally modified
 Kinases vs. Phosphatase
o Kinases- add phosphate groups
o phosphatase-removed phosphate groups
 Src is a tyrosine kinase NOT receptor
o Experiment: Src + presence of labeled Src showed that
substrate will be phosphorylated
 Aka the P from ATP is stolen from Src and transfers it to
the substrate
o When phosphorylatesactivates substrate
o Src activity is usually pretty low NORMALLY
 CANCER: high in cancer
o C-src vs v-src
 C-Src = normal
 structure
 [N-side] Myr + SH3 + SH2 + Kinase [C-
side with tryrosine group]
 V-Src = fucked up one
 structure
 [N-side] Myr + SH3 + SH2 + Kinase
o Difference: NO carboxy terminus with
a tyrosine group; just ends with
kinase
 Remember:
 SH3 for proline
 SH2 for tyrosine
 Kinase for +P
 3-D of Src known!
o SH2 + SH3 + Kinase + tyrosine tail connecting back to SH2
 Kinase spot has a distorted kinase active site
 Src is normally inactive due to intramolecular (inside)
inhibition
o 2 different conformations of Src
 Closed conformation
 Kinase inactive (normally)
  SH2 and SH3 domains bound
 Resides in endosome
 Tail is “hidden”
 Open conformation (ACTIVE)
 How got here?  moved around SH2/SH3,
 Kinase active/open
 SH2 and SH3 are NOT bound; available for
attachment
 Location: plasma membrane
 Via focal adhesions
Where is Src in cells?
 Myr = Myristylated tail
o Hidden in inactive form
o Lipid covalently bonded
 Inactive to active?
o Closed in the endosome
o Then the myr tail is not hiddenallowed to attach onto the
membrane
o Get rid of the C-terminal region
 = activation of v-Src
 use ATP to pull that baby off: ATPADP
 via Csk (tyrosine kinase): pulls the C-terminal
region right off
 Src = Tyrosine Kinase
o Experiment
 Western blotting
 Antiphosphotyrosine antibodies
 V = vSrc
 2A/V = nonmyristylated v-Src
o Targets of Src
 P120 cateninmodulates cell-cell adhesion
 CadherinCa2+ dependent adhesion
 Hemophilic interaction
 Cortactin A regulates actin polymerization
 Focal Adhesion Kinase (FAK)cell-matrix
interactions
  Integrin A3B1
 Fibronectinpart of matrix, binds to integrins
 Heterophilic interactions
 Focal adhesions are linked to the actin
cytoskeleton
 Fibronectin of matrix linked to
transmembrane protein of integrin
transmembrane receptors
 Linked to actin in a very organized manner
How the focal adhesion (integrin +
fibronectin) links to actin AND is able to
regulate its polymerization
o VERY Complex network of proteins
o Focal adhesions = GREAT target for protein tyrosine
phosphorylation
o Src function example: WOUNDS
 Normal cells are tightly adherent to ECM
 Wounded cellsplatelet recruitmentcell
migration/proliferation
 Platletsclotting blood, in RBC
 Phosphorylated, so focal adhesions disattach from
actin upon PDGF wound signal
Process for focal adhesion and migration
 FAK is recruited by integrins to membrane
o Autophosphorylates using ATP (recall: RTK)
 Src binds to (auto)phosphorylated FAK (on cytoplasm side)
o Src can now change conformation (closedopen) and now
active!
 SRC phosphorylates FAK (even more)
o Now you have a Src-FAK power team to phosphorylate other
proteins
o Remember: Src is the one with the Mry-CH3-CH2-Kinase-
Src-FAK acts together to regulate OTHER focal adhesion proteins
 Now that active = less adhesion, MORE migration
Without Src and close relatives (fyn and yes)- cells can adhere, but not
move
  Without these, only a “modest” phenotype is observed
o Why? Redundancy as Src is similar to yes and fyn
 Different receptor = different Src family kinase
 T-cell = Fyn
 B-cell = lyn
 Why? Well, if Src isn’t present, then can’t phosphorylate the FAK,
so stuck there
Triple mutants
 Don’t have Src, Fyn, or Yes (SFY)can’t move, can only make focal
adhesions
 Compared to c-Src, which CAN move, SFY triple mutant can’t move
The structure of Src is known (3D)
 Active site between the small and large lobe
 Bc structure is known, you add a Src inhibitor to this active site
 In Leukemia adding Src inhibition to either Src or it’s family Abl,
improves prognosis in phase II trials
o Src inhibitor: dasatinib
o Why use this instead of other drugs?-->prevents drugs
resistance in CML leukemia
o Src inhibition in trial for various cancers
o sp. dasatinib)
 Ex. Breast cancer, sarcoma,
o AZD0630
 Other Src inhibitor
 Used for:
 Pancreatic, breast, ovarian, prostate
Relatedness of Ras/Src/RTK signaling pathways
 Densely connected to other networksaka crossover effects of
therapies, more bang for your buck
NEDD9
 Member of Cas family scaffolding proteins
o Scaffolding protein- regulators of many signaling pathways;
tethers multiple members of a pathway into complexes
 Regulate signal transduction
 Help localize pathway components (into complexes) to
places like membrane, nucleus, mitochondria, etc
  May be implicated in cancer phenotypesputs together FAK and Src
o Motility/invasion
o Proliferation
o Survival
 NEDD9 = HEF1 + Cas-L
 Cas proteins are regulated by many upstream signals
o Toxins
o Chemokines/hormones
o GFRMS
o TGF-B
o Integrin-mediated attachment/adhesion
NEDD9 is very complicated
 High NEDD9 in a lot of tumor cell lines
o Migration/invasion in: breast, melanoma, glioblastoma, lung,
etc
 NEDD9 RNA increases migration of MCF-cells
o NEDD0 mRNA is downregulated in lung metasis
NEDD9 loss inhibits initiation of MMTV-Neu (HER2/ErbB2)-induced tumors
 MMTV-neu; NEDD9 +/+a lot of cancer
 MMTV-neu; NEDD9 -/- little cancer
o Tumor cell lines grow slower as metastasize less
 Mammary tumor inhibition of NEDD9-/- is at the initiation stage
o Need to investigate NEDD9 dependent biology before tumors
appear
Other factors that promote tumor progression
 Cancer cells have altered metabolism (via genetic alterations)
o Formation of fatty acids = upregulated in tumor cells
 More glycolysis
 More fatty acid synthase
o Mychelps promote altered metabolism
 Myc = transcription factor *Myc/Max
 Helix-loop-helix
 leucine zipper domain
 Controls many things
 Cell progression: G1S phase
 Differentiation
  Apoptosis
 Myc binds numerous promoters, but only regulates a
fraction of corresponding genes
 c-Myc
 viral oncogene
 1.)translocations
 blood cancers
 2.) amplifications
 solid cancers
o Myc family
 C-Mycviral homolog
 Cancers: carcinoma, melanoma, myelcytomatosis
 L-MYCDNA amp
 Lung carcinoma
 N-MYCDNA amp
 Neuroblastoma, lung carcinoma,
rhabdomyosarcoma
o
 Angiogenesis
 Tumor microenvironment
o Examples: hypoxia, pH, nutrients, autophagy
o Myc/Max
If apoptosis is inactivated via Bcl-2, for example, then sufficieint for MYC to
trigger immediate carcinogenic progression
 Increase angiogenesis
 Metastasis
 Drug resistance
 Decreased survival
o …all bc of Myc
Hypoxia can also cause tumor progression
 Angiogenesis
 Increased metastasis
 Resistance to chemo
 Decreased patient survival
o Patients with hypoxic tumors have worse overall survival than
those with more oxygenated tumors
  Other:
o Apoptosis of p53 cells
o Alteration of gene expression
o Cell-cycle arrest
o Inhibition of global translation
Angiogenesis
 Formation of new blood vessels from existing ones
 Dormant tumorproliferation balanced out by apopotosis
o Angiogenic switchgoing from dormant, perfectly balanced
tumor to a proliferative tumor due to angiogenesis growth
 VEGFvascular endothelial growth factor
o Receptor: VEGFR
o Pro-angiogenic molecule
 Angiogenic inhibitorsnaturally expressed molecules that
counteract pro-angiogenic factors
o Maybe a solution to cancer?
Tumor Vasculature highly anomalous
 Chaotic flow
 Blood lakes
 Localized avascular regions
 DISORDEReD signaling events

Therapeutics 6/6/2015 7:04:00 PM

Therapeutics
 Cancer Drug Targets
o 5-Fluorouracil (DNA replication)
 pyrimidine
 blocks synthesis of thymidine
o AraC (DNA replication)
 Comp inhibitor of DNA Poly III
o Etoposide (DNA replication)
 Blocks topoisomerase
o Adrimycin (Stops transcription)
 Antibiotic
 Inhibits topoisomerase by intercalating (becomes part
of) into DNA
o Vincristine/Taxol (post translational)
 Blocks microtubule assembly/disassembly
 Targeted Therapies
o What makes a good target
 Tumor specific
 Druggable
 Low toxicity, high efficacy
 Required for tumor maintainance
 “oncogenic addiction”
 central player in tumor signaling networks
 Personalized Medicine
o Get molecular def of tumor and fuck around with it using
pathways
 Imatinib
 CML (BCL-ABL)
 GIST (KIT)
 Monosomy 14, 22, partial deletion of 1
 Gefitinib
 NSCLC (EGF receptor)
CML
 Nowell/Hungerfordfind one copy of chromosome 22 in CML
o Leukemia
 Chromosomal translocation
 o Chromosome 9 (ABL), chromosome 22 (BCR)
 22 is smaller
 translocation results in both genes on the 22nd
chromosome; “Philadelphia Chromsome” = Leukemia
o N terminus of BCR + Abl = fusion
 Different variations of BCR/ABL (Philly chrom.) causes
different cancers
ALL (acute lymphoblastic leukemia)
 Affects B/T cells
 Ph+ chromosome, mainly in adults
o Prognosis is worse in adults than childrenrelapse)
o 40% cure rate; cure: bone marrow transplant
Abelson
 ABL gene on chromosome 9
o Oncogene carried by virus
o Causes B cell fuck up
Diagnosis
 FISH (fluorescence)good for diagnosing CML
o PCR good technique
ABL KINASE
 Fatty-acid modified
o Myr-SH3-SH2-Kinase-Actin binding
o Similar to Src
 Actin-binding non-receptor tyrosine kinase
 Fucked up types:
o Bcr-Abl replaces Myr on N terminus
 NORMAL ABL
o Phosphorylates Focal Adhesion proteins
 and regulators of actin cytoskeleton
 Fucks up every way and causes cancer
CML
 Stages3 phases, characterized by increasing refractoriness to
therapy; IS possible to go straight from chronic to blast crisis
o Chronic Phase
 5-6 years
 less than 10% blasts in periphereal blood/marrow
  well differentiated
 overproduction of normal looking
granulocytes/neutrophils
o Accelerated Phase
 6-9 MONTHS
 10-29%
 changes in morphology
 too much blast cells (myeloid, lymphoid
o Blast Crisis
 3-6 MONTHS of survival
 >30%
 rapidly fatal
 Definition
o Too much myeloid cells
 Neutrophils- fight infection by phagocytosis
 Monocytes- release immune modulators/histamines
 Platelets- cell fragments of megakaryocytes
 Different Leukemia = different affected stem cell types/distinct
stages in development of stem cells
o CML in stem cell that is granulocyte precursor AND THUS
gives rise to neutrophils, basophils, and megakaryocytes
(platelets)
Therapy for CML
 Determining if a drug is working
 First goal: reduce WBC count
o Hematologic response VS Cytogenetic response
 Hematologic response (Complete)
 Normal peripheral blood count
 No immature cells
 Lowering symptoms
 Must be maintained for a month
 Cytogenetic Responsereducing unwanted cells
 Complete: 0% Ph+
 Partially working: 1-35% Ph+
 Minor working: 36-95% Ph+
  Via FISH and PCR find out number of myeloid
dividing cells
 If can find cytogenetic response:
 Improve survival for IFN-alpha patients!
Treatments (Major)
 Allogenic bone marrow transplant
 Interferon alphase (IFN-alpha)tells body what can go into/out of
cell
o Better survival rates by a little compared to CHT
(chemotherapy)
o Mechanism not known
 STI-571 (imatinib/Gleevec)SPECIFIC for BCR/ABL
o Treat the BCR/ABL as a Src/FAK combo
o Block active site and now tumor cells can’t proliferate
o 17% get resistance to this = relapse
 BCR/ABL have different mutations to combat =
resistance
o If combine with stem cells, come right back; NOT depleted by
therapy
 ONLY progenitors and differentiated cells appear
sensitive to Gleevec
 Single agent therapy is useless EVOLUTION
 Dastinib
o Secondary treatment after relapse, FDA approved
o Gets around drug resistance in CML
Cocktailsused to overcome resistance
 Gleevec = BCR/ABC
 Iressa = EGFR receptor
 PLX = BRAF protein
Limits of targeted therapies
 Preclinical vs clinical
o Not many make it to clinical trials for many reason
 Delivery issues
 Toxicity
 Side-effects
 Low efficacy if only use one drug
  Cost
 Too specific, companies not gonna make $
HER2Herceptin
 RTK
 Breast cancer
 HER2 = gene, AMPLIFICATION bc its solid
 Combos:
o Herceptin + chemo = early breast cancer treatment
 ½ likely for relapse
 different EGFR amplified in some glioblastomas/lung cancer
o Metlung cancer
o HER2breast/bladder
o KitGI tumors
Iressa/Gefetinib, Tarceva, Eroltinib
 Blocks ATP binding site of tyrosine kinase domain EGFR
 EGFR TK domain fuck ups = responsive to gefitinib
 EGFR inhibitors not very effective
 Iressa replaced by Erlotinib/Tarceva (EGRF inhibitors)
o 2nd line treatment, lung cancer
o not very effective
o
Questions 6/6/2015 7:04:00 PM

Questions
- Do we need to know all the different kinds of integrin alpha/beta pairs and
what the do?
- How detailed do we need to know the Metastasis/Invasion PowerPoint?
End become very detailed
Is E-cadherin is tight or adherin shit?

be able to understand this: