Midterm 4 Flashcards
Genomic instability and evolution
there needs to be a certain amount of genomic instability for mutations to occur–this powers evolution. But not so much so that you win dup having a decrease in viability
g2/m checkpoint
last chance to check before mitosis
did we duplicate properly did we correct for errors
compartmentalization of tissues & proliferation
some cells in tissues/organs have different levels of ability to proliferate based on their function
so only some cells we are worried about their genome integrity. we only care about cells that are sticking around in regard to cancer
PROLIFERATIVE CELLS ARE DANGEROUS
Stem cells
divide rarely and are anatomically shielded, preserve genomic integrity
maintain ability to proliferate
so they are the ones we want to protect because of they will always be able to proliferate
stem cells divide _____
asymmetrically
one daughter cell stays in the stem compartment and one goes to eventually differentiate.
in regard to genome integrity we only worry about the one in the stem cell compartment
how do we protect stem cells?
we anatomically shield them (ex. hide them in the crypts in the intestine. crypts also secrete mucins that trap digestive acids)
how do the crypts specifically serve as an example of the anatomical shielding of stem cells?
they physically keep the stem cells away from digestive acids and toxins. they also secrete mucins that capture toxins and digestive acids which makes them unable to wreak havoc
how does pulse chasing work?
you radioactively label cells and cells that have stopped replicating would have a lot of radioactively labelled dna whereas the proliferating cells would have way less become diluted, and it wouldn’t be labelled
how can you tell transit amplifying cells move from the crypts?
radiolabelling pulse chasing
how do tissues protect themselves from mutant stem cells?
apoptosis
pumps
asymmetric dna strand allocation
stem cells and apoptosis (what is different about stem cells when they are damaged in comparison to a normal differentiated cell?)
stem cells undergo apoptosis rather than cell cycle arrest and DNA repair, after DNA damage
stem cells and pumps:
stem cells over express protein pumps (MDR-1) which effectively remove toxins from cells, preventing dna damage
stem cells and asymmetric dna strand allocation:
preserves template strand in stem cell lineage, gives newly synthesized strand to transit cell
basically new strand goes to differentiate
template strand stays in the stem compartment (immortal)
transit amplifying cells
undifferentiated cells that are sort of the transition between stem cells and truly differentiated cells
how can you see the migration of transit amplifying cells in the crypts (lab technique)?
pulse chasing/radio-labelling they keep the radioactive labels because they do not replicate as much as they head toward becoming fully differentiated cells
cell genomes are threatened by ________
errors made during DNA replication
for an error to become permanent during DNA replication what needs to happen?
that cell would need to replicate twice. because if it goes AC in the first mistake it would need to replicate again to GC to become a permanent mutation that could stay in the cell without alerting a problem
DNA polymerase has __________
proof reading exonucleolytic activity
means that as it adds nucleotides on the 3’ end it checks its work. if one is wrong, it undergoes a conformational change to cut out the mistake nucleotide
proof reading function of DNA polymerase detects ________errors
99%
1 in every 10^7 nucleotides will be wrong with just dna polym alone
1 in ________ nucleotide errors happen which corresponds to _____ errors per cell genome replication
10^9
6
mutation in exonuclease activity in DNA polymerase results in ________
tumor phenotype
what nucleotide scenarios give dna polymerase a hard time when its trying to exercise its endonuclease activity?
mono and di nucletoide repeats. super easy for them to misalign and result in insertions and deletions
MMR
repairs the mono and dinucleotide repeats that dna polymerase usually misses
scans genome after dna polymerase is done. looks for nicks and mismatches.
How does MMR work?
MutS scans for mismatch, then determine which strand is the newly synthesized strand, then repair
How does MMR know which strand is the new strand?
methylation or looking for nicks (places where removing the primer and binding together okazaki fragments hasn’t happened yet)
why is the single stranded DNA in the replication fork more susceptible to breakages than double stranded ?
only one phosphodiester bond has to break no ligase to hold together. this can cause deletions inversions etc.
depurination
purine is cleaved off of the sugar phosphate by spontaneous hydrolysis.
endogenous biochemical attack
RESULTS IN SINGLE BASE DELETION
deamination
cytosine –> uracil
which causes c to t
this leads to a change in the base pair
called a transition because both bases are from the same class pyrimidines and purines
why is the deamination of 5 methylcytosine really bad?
turns into thymine which legit belongs in dna so its a really bad mutation. very common endogenous
frameshift mutations usually result in ______
premature stop codons and wrong amino acids which result in nonfunctional proteins
lead to gene loss if it happens early on
transition mutation
stay in the same class of nucleotide
pyramidine –> pyramidine
tranversion mutation
switch nucleotide class
pyramidine –> purine
endogenous oxidation
free radicals bounce around cells and oxidize stuff around it
what can endogenous oxidation lead to?
- oxidize bases themselves
- cleave the bases away from deoxyribose (abasic sites)
- cross link proteins to DNA
oxidation can result from _______
the immune system!!!
it uses oxidants to consume infected cells so we can clear them out. this can damage bystander cells
inflammation can be an initiator and a promoter
most dangerous thing that can happen as a result of oxidation
the oxidation of G
oxo-g if it is not repaired prior to dna replication it will base pair with an A
this would result in a transversion mutation (G GOES TO T)
why are transversion mutations worse than transition mutations
the amino acid changes are more profound because the amino acid class (polar, charged etc) changes. the protein product would then be radically different
MTH1
degrades oxo-gs
mice that do not have MTH1 have a tumor phenotype
cancer genes break down into the following
oncogenes (gas) tumor suppressors (cut breaks) genome maintenance/care-takers (mechanics)
exogenous mutations
come from outside of the cell/the environment
ex. ionizing radiation
ionizing radiation
ionizes water causes base mutations, deletions, and chromosomal translocations
these ions break bonds to get electrons. can cleave off the base, cleave phosphodiester bonds, and can alter the base,.
UV radiation
NOT STRONG ENOUGH TO BREAK PHOSPHODIESTER bonds
but can excite electrons in the bases and can cause pyrimidines
to dimerize. when this happens it distorts the appearance of the DNA double helix
results in transitional mutation.
turns all into TT
CT, TC, CC would then be AA which results in a TT
alkylation
many chemos are alkylating agents
destabilizes linkage between base and deoxyribose so base can be lost
creates mutations
ENU
alkylating agent of guanines.
MGMT (methyl-guanine-DNA transferase) is important in reversing alkylating damage
MGMT (methyl-guanine-DNA transferase)
important in reversing alkylating damage
how do some cancers become resistant to chemo?
upregulating MGMT
how do we fight alkylating agent resistant chemo?
combine it with DNA repair inhibitors that act as a substrate for MGMT and basically if we can keep the bad DNA damaged them we promote a checkpoint and hopefully the cell dies there
MGMT wt mice versus MGMT super physiological mice
Excess mgmt reduces tumor incidence in mice exposed to alkylating agent
some potent mutagens are formed when ________
we ingest or inhale compounds that are altered by cellular metabolic processes
large and nonpolar molecules
cannot be eliminated through the blood and the urine
so detoxification must occur
detoxification
make nonpolar molecules into polar in xenobiotics so that we can eliminate it in the urine
this process can actually make things more carcinogenic
PAHs (polycyclic aromatic hydrocarbons)
products of combustion
they go through detoxification and become carcinogenic by forming adducts
adduct
something covalently attached to nucleotide
DNA transcription hits a wall because it can’t happen
cytochrome p450 enzymes
involved in biosynthesis of steroid hormones, cholesterol, bile acids, and degradation of fatty acids and steroids.
they also oxidize xenobiotics aka pahs
the goal is to detoxify, solubilize and excrete but the result often mutagenic
pahs/cytochrome p450 products result in _______ that result in _______
adducts that result in G to T transversion mutations
G12V mutation
G to T transversion mutation in codon 12 inactivates ras GTPase activity. Ras is GTP bound, with deregulated mitogenic signaling
more than ______ of human tumors have mutated (activated) ras
20%
tp53 mutations
hotspots in lung tumors of smokers coincide with experimentally induced B(A)P adducts
G to T transversion mutations in DNA binding domain region prevent p53 mediated cell cycle arrest, apoptosis, and DNA repair
hupki
human p53 knock in mice–part of the p53 gene in the mouse is human p53 (DNA binding region)
produce huf cells
what do huf cells do?
expose huf cells or the mice to carcinogens and see what patters of mutations you see
carcinogen fingerprint
mutation associated with a carcinogen
want to find that a characteristic mutation pattern occurs in exposed individuals vs unexposed individuals
see that suspected carcinogen induces similar mutations in experimental model systems
evidence that mutations occur in early tumor development
uv radiation cause a ________
CC to TT mutation in tp53
aflatoxins cause a ________
G to T mutation in tp53
tobacco smoke causes a _________
G to T mutation in tp53
why is liver cancer elevated in asia?
aflatoxin is an initiator and when liver attempts to detoxify it, it causes mutagenesis in the liver
hepatitis B is the promoter
what are the initiator and promoter in asia liver cancer example?
aflatoxin is the initiator
hepatitis B is the promoter
genome sequencing allows for ____________
scientists to compare normal genomes from a person to the cancerous genomes from the same person to understand the diverse patterns of mutagenesis that can occur
in adults usually from carcinogens
pediatric usually random
G2/M checkpoint
CHECKS FOR DNA DAMAGE
- stop entry into mitosis if damage is detected
- repair damage
how is G2/M checkpoint activated?
CDK1 drives entry into mitosis,
so CDK1 is tightly regulated by cyclins by inhibitory and activating phosphorylations by inhibitors and by subcellular localization
budding yeast experiments to determine what happens in a defective G2 checkpoint
yeast are haploid so no need to worry about dominant or recessive.
expose yeast to radiation:
wt-phenotype rad9, the cell buds but doesn’t go into mitosis
mutant rad9 goes through mitosis no delay
cells with defects in the checkpoint continued into mitosis even though they had DNA damage and died with fragmented chromosomes
what does the cdk at the g2/m checkpoint do when activated?
allow for assembly of mitotic spindle ensure chromosomes are attached to the spindle condenses chromosomes nuclear envelope breakdown actin cytoskeleton rearrangement reorganization of golgi and ER
fission yeast experiments to determine function of G2 check point
identified wee1 kinase and cdc25 phosphatase.
wt- cell expands and divides normally
wee1 deficient- the cell divides prematurely (produced smaller daughter cells WEEEEE DAUGHTER CELLS BRITISH)
cdc25 deficient- the cell does not divide
wee1 deficient cells ______
divide prematurely
cdc25 deficient cells ________
do not divide
wee kinase adds _________
an inhibitory phosphorylation that stops premature cell division and overgrowth
cdc25 ________
removes an inhibitory phosphorylation that allows the cell to continue to grow
How do we activate the cdk in the G2/M checkpoint?
cdk1 associates with M cyclin as M cyclin levels rise.
The complex formed is phosphorylated at the active site by a cdk activating kinase (CAK) and phosphorylated inhibitory sites by Wee1 kinase.
ONLY WAY TO ACTUALLY ACTIVATE IS CDC25 NEEDS TO REMOVE INHIBITORY PHOSPHATE:
The resulting inactive M-cdk complex is then activated at the end of G2 by the phosphatase cdc25.
Cdc25 is further stimulated by active M-CDK resulting in positive feedback. this feedback is enhanced by the ability of M-cdk to inhibit Wee1.
in order to hold a cell at the g2/m checkpoint _______ needs to be inactivated
cdc25
what are cdc25s regulated by?
stress signals (ATM/ATR) that cause stimulatory and inhibitory phosphorylation, sequestration, and ubiquitlation/degradation
ATM and ATR
protein kinases that become activated following specific types of DNA damage. they act through checkpoint signaling pathways that lead to cell cycle arrest
ATM is activated in response to ________
double stranded DNA breaks
ATR is activated in response to ___________
protein coated ssDNA that forms when replication forks become stalled or the DNA is being repaired after various types of damage
DDR kinases _______ CDC25
inactivate
they buy time to do DNA repair
CDK1/cyclin B (mitotic cyclin) are _______ by CDC25
activated
types of DNA repair
reversal of chemical damage nucleotide excision repair base excision repair mismatch repair double strand break
reversal of chemical damage repair is done by ________
alkylating agents. alkyl transferase enzymes remove methyl and ethyl groups (gene is silenced in many cancers). other enzymes oxidize the alkyl groups
restoration of normal base structure by deakylating repair enzymes (like MGMT)
nucleotide excision repair
removes damaged base and adjacent bases, usually during transcription, repaired by pol gamma
used for more profound damage such as when something disrupts the helix shape. happens when there is a covalently attached adduct from carcinogens or UV light.
base excision repair
DNA glycolase cuts the base from the sugar, endonuclease removes the base free sugar, DNA pol beta repairs.
typically endogenous DNA damage. used for when depurination occurs, and for bases that have been oxidized, reduced, alkylated, or deaminated
MMR = mismatch repair
uses poly gamma as well and repairs any mismatched based we went over this
Double stranded break repair
Nonhomologous end joining in G1 and by homology dependent repair in S and F2
MGMT has been shown to be _______ by a promoter Ch3’n in a variety of tumor types
silenced
tumors that express high levels of MGMT have _____ response to chemotherapy and a ____ outcome
poor; worse
it helps dealkylate the DNA during chemo
DNA glycosylase
cleaves covalent bond between base and deoxyribose.
ex. uracil glycosylase removes uracil created by deaminating cytosine; t:g glycosylases remove the ts that are opposite gs
genetic disorders that impact NER repair:
XP - xeroderma pigmentosum: 1000x increase in skin cancer. cant repair uv light induced pyramidine dimers. onset is significantly earlier age.
CS: Cockayne’s syndrome: short stature, premature aging, photosensitivity
PIBIDS: photosensitivity, ichtyosis, brittle hair, impaired intelligence, decreased fertility, short stature
HNPCC is due to:
mutations in MMR enzymes
HNPCC: accerlerated progression from adenoma to carcinoma due to germline mutations in mismatch repair enzymes
homo-polymeric sequences (stretches of the samenucleotide) are susceptible to ____________
mismatches. will need MMR to fix the mutations
a mutation in tgf-beta results in _________
a defect in MMR. the frameshift mutation means a loss of the anti-mitogenic signaling.
TGF-beta normally is responsible for anti-mitotic signalling. it turns on CDK inihibitors
BRCA risks
50% familial breast cancer have this mutation
70-80% familial ovarian cancer have this mutation
BRCA – what does it do?
plays a role in fixing double stranded breaks
protein transits to the site of the double stranded break.
how did we discover that BRCA transits to the double strand breaks?
In the experiment HU inhibits the production of dNTPs which causes the replication fork to stall which leads to breaks and you see it transit to the breaks
PCNA is always at replication fork
BRCA is only at the fork when there is DNA damage
H2AX
variant histone H2A. it gets phosphorylated by ATM and ATR kinases and then becomes yH2QX which recruits BRCA1 and other repair proteins
two kinds of double strand break repairs
non homologous end-joining (less accurate) homologous recombination (more accurate)
NHEJ
looks for ends and sticks them together. could be a problem when some of the DNA is not cut back. can lead to translocations, deletions, inversions etc.
HR
same as HDR. happens in G2 the damaged DNA cheats of sister chromtid that isn’t damaged as a template to fix itself.
BRCA1 enables the entire complex that does this to form. it is the park bench it comes to sit on.
homologous recombination how it works
broken dna is in proximity to unbroken homologous dna which serves as a template in G2
they do a strand dance where the damaged strand uses the complementary strand as a template for repair
resection, strand exchange, extension, displacement, ligation
BRCA’s role in double strand breaks and forming complexes
it sits on where the break is and recruits the complex. rad50 can sit on it and initiate HR. then it gets phosphorylated which is key to its ability to function. a lot of mutations in brca cause a frame shift there to make it not function
BRCT
c terminal domains common to proteins with genome maintenance functions. interacts with Rb, HDAC
it supports chromatin remodeling and transcriptional regulation
BRCA1 controls ____ and promotes _____
expression, phosphorylation, and localization of cdc35 and cdk1/cyclin b kinase
BASICALLY REGULATES KEY EFFECTORS OF THE G2 CHECKPOINT
promotes mono-ub of histone H2A which is associated with constitutive heterochromatin formation (gene-poor regions such as centromeres)
THEREFORE LOSS OF BRCA 1 PROMOTES WIDESPREAD GENETIC DESTABILIZATION
PARP inhibitors
kill cells that have defects in BRCA1 or BRCA2
parp is a pathway to repair DNA – so if it is inhibited you can’t repair DNA with double stranded breaks if you also dont have BRCA so that cell will die.
myriad genetics
story of a researcher. he thought he could get exclusive rights to sequence the brca1 gene and patent it and refuse to license it to anyone else
ACLU sued and won in the supreme court
why is BRCA related cancer limited to certain tissues?
its also involved in sex hormone signaling so if it goes to shit that means that we won’t have it and its why you see this related to breast and ovarian cancers
Folkman
THE MANS who was convinced that angiogenesis, the ability of cancers to promote their own vascularization, was the key to treating it
during WW2 was asked to develop a way to freeze dry blood and preserve it.
Folkman experiment set up
normal thyroid from a rabbit was grown in glass chamber with a cancer cell. they grew to a certain size and then stopped every single time. but if you took those cancer cells and put them into an animal they grew like crazy
REASON FOR THIS in the small thyroid gland they didnt have enough blood vessels to link up to so they didn’t grow!
Folkman eye experiment set up
tumor cells transplated into the anterior chamber of the eye of a rabbit form tiny tumors that soon stop growing. BUT when they are transplanted into the iris area, which has a blood supply the tumors grow CRAZILY because they are infiltrated by blood vessels
angiogenesis
cancer can tell the organism to make new blood vessels. it normally happens in us when we are developing and growing. it continues throughout our life, pregnancy also involved! but its all tightly regulated
angiogenesis activators
vascular endothelial growth factor VEGF
fibroblast growth factor (FGF)
when cells are ________ vegf goes crazy
oxygen deficient
how does vegf and fgf have a role in cancer?
cancer cells release vegf and fgf into surrounding tissue. they bind receptors on endothelial cells lining the blood vessels. this binding activates a signaling pathway. in response the endothelial cells divide and secrete matrix metalloproteinases (MMPs). the MMPs break down the extracellular matrix allowing endothelial cells to migrate into the surrounding tissues. they organize into hollow tubes and develop into new blood vessels.
t or f: angiogenesis can occur prior to invasion
true
what is the association between angiogenesis and disease outcome
the more vascularized the tumor the worse the outcome because it has progressed further
t or f: there are natural activators and inhibitors of angiogenesis
true
anti-angiogenesis therapies
angiostatin & endostatin- naturally occurring. effective because it only goes after neoangiogenesis. difficult and expensive to produce.
interferons- suppress bFGF and IL-8 (both angiogenic) which leads to tumor regression
drugs-
neutralizing anti VEGF monoclonal antibody (avastin) it blocked growth of human sarcoma and glioblastoma in mice. used in combo with conventional chemo
also some tyrosine kinase inhibitor for VEGF and PDGF
do angiogenesis inhibitors have side effects?
cancer doesn’t advance. but are there side effects? people would need to be on these for decades
can cause problems with bleeding, clots in the arteries, hypertension, and protein in the urine.
likely possible complications are still unknown
HIF-1alpha pathway
turns on in hypoxia conditions. recent studies have found a strong correlation between elevated levels of HIF-1 and tumor metastasis, angiogenesis, poor patient prognosis, and tumor resistance to therapy.
tumors turn on survival pathways to adapt to hypoxic stress
HIF is a transcription factor so theres many ways to attack it like sequestering it in cytoplasm or targeting it for destruction
metastasis
first cancer cells invade surrounding tissues and gain access to bloodstream
then they get transported throughout the body
finally they leave the bloodstream and establish new metastatic tumors in various organs
angiogenesis happens ______ metastasis
before
what changes make cancer cells able to metastasize?
cell-cell adhesion proteins that cause cells to adhere to one another are often missing or defective.
E-cadherin is an important one.
cancer cells can also produce proteases (plasminogen, mmps) that degrade protein containing structures such as the basal lamina and the ECM
they also get increased motility from stimulating signaling molecules from the surrounding tissues
EMT
epithelial mesenchymal transition.
in order to acquire motility and invasiveness, epithelial (carcinoma) cells change their characteristic phenotype, morphology, and pattern of gene expression, and become more invasive and motile. happens naturally during developmental morphogenesis (like gastrulation neuralation etc)
MET
mesenchymal epithelial transition. revert back to epithelial once you get to where you were going so that you can proliferate
MMPs
extracellular proteases that all cancer cells to remodel their tissue environment by creating a passageway through the EXM.
MT-1 MMP on the cancer cell membrane cleaves cell surface adhesion molecules such as cadherin and integrins
how do cancer cells become more motile?
cell locomotion requires changes in the actin cytoskeleton and the making and breaking of contacts with the underlying substrate. Lamellopodia extend the leading edge, stress fibers contract the trailing edge. these are controlled by rho family gtpases, downstream targets of ras
how does EMT happen?
programmed by transcription factors that orchestrate key steps in embryogenesis.
- loss of e-cadherin
- stromal proteases cleave off ecm domain of e-cadherin
- mmps chew through the basal lamina
twist
twist (transcription factor) turns genes on and off, if you introduce it into the epithelial cells you will see less epithelial markers and more mesenchymal markers
association of EMT-inducing transcription factors with cancer
more tf more cancer
t or f: proliferation is required for metastasis
f
you could metastastize and not be crazy proliferative. converse also true.
how does EMT impact drug sensitivity in the treatment of cancer?
when epithelial tumor cells undergo emt they become mesenchymal cells in order to metastasize. in the process we lose sensitivity to many drugs that target epithelial tumors.
t or f: most cancer cells can survive in the blood
f
not hospitable for cancer cells. flow usually kills them. but some get stuck to the capillary beds and thats how they metastasize
blood flow model
first capillary bed cancer cell lands is where it will go. so lung or liver
seed and soil model
cancer cells grow where there is optimal growth for its kind.
Coley
Could an infection prompt the immune system to attack tumors?
Saw 25% of his patients cured
is cancer foreign or self?
based on failed implants of tumors in mice we think foreign
humoral versus cell mediated response in adaptive immunity
humoral - antibodies
cell mediated - t cells
helper t cells
activate both humoral and cell mediated responses
antigen presenting cell hits up helper t cell they release cytokines. then they hit up b cells to make antibodies and and t cytotoxic cells
cytotoxic t cells
put peforin into the bad cell and then it causes apoptosis
cancer immunoediting
3 phases:
elimination
equilibrium
escape
how cancer still winds up developing in bodies even though it is seen as foreign
elimination
tldr; immune system takes care of the lowest hanging fruit – the cells that it has easy access to / look the most foreign
recognition of tumor cells by innate immune cells and their limited killing.
migration of antigen presenting cells and priming of t lymphocytes
generation of tumor-antigen-specific t lymphocytes and activation of cytotoxic mechanisms
homing of tumor-antigen-specific t lymphocytes to the tumor site and elimination of tumor cells
equilibrium
cancer evolves and the immune system is selecting against the ones it can kill.
continuous sculpting of tumor cells and selection of those with reduced immunogenicity. promoting production of resistant variants.
can go latent until a mutation comes that increases resistance to immune pressure
escape
tumor stops expressing antigens, or they are resistant to cell death, or the cancer cells can keep t cells from killing them
checkpoint inhibitors
target receptor-ligand interactions that negatively regulate immune response.
drugs inhibit the cancer cells ability to inhibit the t cells.
PD-1 receptor
PD-1 programmed cell death checkpoint.
long term exposure to antigen causes t cells to express the pd-1 receptor which prevent chronic, prolonged, excessive activation. most cancer cells express ligands that bind to it.
this protects against auto immunity but is an immune suppressive pathway that makes it difficult to mount and sustain anti tumor t cell responses
What does the MAb to PD-1 do to bring back t cell immunity to cancer cells?
it blocks the interaction between PDL1 ligand on the tumor cells and PD-1 receptor on the t cells, which reactivates them
CTLA-4
stimulatory signals on t helper cells become replaced by inhibitory signals normally. CTLA4 suppresses T cell activation
drugs can be made to inhibit CTLA4
just keeps your own t cells active for longer.
CTLA-4 Antibody and Tumor growth
tumors don’t grow with CTLA-4
Adoptive Cell Transfer (ACT)
try to find something specific to the tumor and prime our t cells with it and then put them back into the human
problems with this: you can have too much at once. tumor lysis syndrome can occur.
there has been an overall ______ in cancer mortality
decrease
due to better food storage, testing, etc.
what does it mean for a cancer to be refractory?
they don’t respond to conventional therapy or respond and then come back and progress to a highly malignant metastatic stage
usually in cancers that the first symptoms are vague like abdominal pain
incidence vs detection
is there actually increased incidence or do we just have better detection or are more people going to get more testing ?
can compare to global populations where there is less detection but then lifestyle factors confound
indolent
slow growing cancer
do we treat it or do we watch it
aggressive cancers
should we treat them because it will ruin quality of life :/
conventional treatments
surgey/radiation/chemo
rational therapies
new precision medicines that are all in pre-clinical and clinical trials
radiation
ionizing radiation removes electrons from water and other molecules producing free radicals that damage DNA
these cells cant undergo apoptosis because they lost p53. radiation kills cells through chromosomal damage so severe that they wouldn’t survive mitosis
rapidly growing cancers respond better to radiation than slower growing ones
why do you want multiple small doses of radiation rather than a few big ones?
want to minimize damage to normal cells. the time gives the normal cells a chance to repair damage prior to replication
chemotherapy
kills all dividing cells and are therefore not tumor specific
more likely to make apoptosis go down
are mutagens and therefore are carcinogens so there is risk of second-site cancers when under chemo
classes of chemo
alkylating agents:
platinum compounds:
anti-metabolites
topoisomerase inhibitors
mitotic inhibitors
anti-metabolites:
interfere with metabolic pathways. methotrexate inhibits dihydrofolate reductase which is important to making bases in DNA. it could slow cancer growth
also analogs of purine and pyramidine. can competitively inhibit some of the enzymes in dna synth
platinum compounds
discovered by accident. can form covalent bonds with nitrogens in protein so when you cross link to them it disrupts ability to DNA to be transcribed and synthesized
alkylating agents:
bind directly to DNA and alkylate it.
topoisomerase inhibitors
prevent religation of single strand dna breaks during replication. so the replication fork will fall apart and there will be no replication.
these drugs irreversibly bind dna so transcription is disrupted as well.
mitotic inhibitors
mess with the mictrobules in spindle assembly so that mitosis can’t happen properly.
prevents spindle disassembly and promotes abnormal MT bundles
resistance to conventional therapies
- increased drug efflux (MDR pumps out wide range of molecules)
- decreased drug uptake
- increasing target molecules
- alterations in drug metabolism
- increased DNA damage response efficiency
- drugs are not killing the cancer stem cells which eventually regrow the tumro
problem with single drug vs multi drug treatment
single drug - resistance
multi drug- MUY toxic could hurt people / kill them
prevention
decrease initiators and promoters in our lives
ex. diet!
obeseogens (chemicals that alter metabolism and promote adipogenesis)
prevention in diet
fruits and vegetables contain vitamins minerals and dietary fibers that protect against cancer
SFN
inhibits histone deacetylase. derepresses epigenetically silenced genes in cancer cells and activates genes in normal cells
vitamin D
dominant negative ligand for EGFR and it upregulates BRCA1 and p21, downregulates Bcl-2 and IAPs
tldr vitamin D blocks ras signaling and supports p53 mediated arrest, repair, and apoptosis
hormones as ______
promoters
lifetime estrogen exposure contributes to risk
oral contraception, hormone replacement therapy, and obesity increase exposure while pregnancy, lactation, and physical activity decrease exposure
hormone therapy
some cancers are estrogen or testosterone dependent. looked for molecules that can antagonize hormones in their role in cancer (usually competes to bind to receptor)
functional genomics
classify biopsies in microarrays of 300 tumor samples to see which genes were upregulated or downregulated in breast cancer
created prognosis genes
signature genes
genes associated with proliferation
took the prognosis genes from the previous experiment to predict the outxome of 150 women on 10 year follow up
targets
enzymes with well defined catalytic cleft and easily assayed
drugs ______ biological function
inhibit.
such as remove tumor suppressors or gate keepers or shut off oncogenes
____ are the most attractive targets for drug development
oncoproteins
valid target for drug k-ras example
if a mutation early on (say in kras) is what causes tumor progression but then subsequent things happen such as loss of p53 or p16, could the tumor still survive if you inhibit kras? that would mean it wouldnt be a good target for the drug
oncogene addiction
if you need the oncogene to be on for the tumor to survie
how do we find a drug?
compound libraries- collection of compounds
high throughput screening- test the function of a drug on the target
how are high throughput screens done?
FRET- fluorescence resonance energy transfer. interaction between two proteins is detected by FRET, and HTS looks to see what compounds disrupt the interaction
how do we turn a hit into a drug?
preclinical testing- tissue culture and animals! test there and see if it works in physiology.
THEN you submit an IND to the FDA.
THEN you move into clinical trials in humans
THEN you apply for an NDA and when it is accepted it is cleared as a drug
phase 1 clinical trial
toxicity trial. what is the maximum tolerated dose?
small group of people not statisically significant. monitor side effects
also pharmacokinetics and pharmacodynamics
therapeutic window
minimum tolerated dose to maximum tolerated dose
phase 2 clinical trial
efficacy
test in a larger groups, come up with indications for the drug and define efficacy. does it work in patients
phase 3 clinical trial
is the clinical response statistically significant? is the therapeutic benefit greater than the current standard of care
very expensive
phospholipase D
lipid signaling enzyme superfamily that has been studied for its roles in cell communication
PLD1&2 converts phosphatidylcholine to phosphatidic acid (PA) which can also become diacylglycerol etc.
____ & _____ activate PLD1&2
GPCR & RTK
what is the main role of PLD in cancer?
progression including growth, metabolism, angiogenesis, and mobility
matrigel plug assay
what happened to mice pld knock out
allows you to study vascularization
double knock out pld mice showed no vascularization
wt mice showed vascularization dependent on VEGF
platelets and pld1
found absence of pld1 reduces tumor cell and platelet interaction
pld knockout mice have _______ tumors than regular
less
FIPI
pld inhibitor in humans
showed decreased platelet interaction and decreased metastasis
current research ta1
is there a biological pathway being altered by pdl1&2 that we could narrow to focus in on
working on mouse breast cancer
MAMPs
microbial associated molecular patterns
immune checkpoint inhibitors
basically the body has this downreg t cell after a while so tumor cells take advantage of this machinery to program t cells to not hurt it. but gut microbiome can prevent this
gut microbiome can ________
impact response to different therapies that are meant to stim immune cells
herceptin
targets EGFR receptor and did well in all clinical trials
tarceva & iressa
tyrosine kinase inhibitor egfr
lead to discovery of egfr addicted cancers in certain people who responded even better to treatment
nutlins
keep mdm2 from binding to p53
if p53 is _____ it can’t be ______
acetylated; ubiquitylated
onyx 015
mutated adenovirus that kills only cells with mutant p53. E1B (normally shuts down p53 to keep the virus host alive) is defective in it so that only cells with wt p53 can fight it off
velcade
proteosome degradation
why did scientists want to improve from velcade and what did they do ?
velcade was non-specific would impact all ubiquitin mediated proteasomal degradations
so they began to target E3 ubiquitin ligases that were specific to their substrate
kras targeting one ad10
basically this plus the pd-1 immunotherapy showed crazy results
Sulforaphane (SFN)
a. n isothiocyanate found in cruciferous vegetables such as broccoli, is a potent anticarcinogen that may act through epigenetic mechanisms.
b. SFN has been shown to inhibit histone deacetylase (HDAC) activity in human colon and prostate cancer lines, with an increase in global and local histone acetylation status, such as on the promoter regions of p21 and bax genes.
i. So, SFN can inhibit HDAC and allow suppressor genes to be expressed
deamination: biggest issue
either form u which isnt usually in dna
i. Biggest issue is last one – doesn’t raise red flags
• C to T transition (deamination of 5-me-cytosine)
⇒ Transition = staying in same class of base (purinepurine, v/v/)
⇒ Transversion: purinepyrid or v/v
oxidation of G causes
G to T tranversion
DNA adducts cause
a. When DNA poly comes along, it’s like slamming into a wall – adducts are huge molecules
b. Causes collapse of replication fork
c. Introduces GT transversion mutations, as well as others
BRCA enables ______ to happen
HR
