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Title: Cancer Cell Biology
Description: Topics covered: Tumorigenesis, Cell immortalization, Maintenance of Genomic integrity and development of Cancer, Rational treatment of cancer. Detailed semester notes from Course MCB 3211 at the University of Connecticut. Aimed at pre-med, molecular cell biology majors.
Description: Topics covered: Tumorigenesis, Cell immortalization, Maintenance of Genomic integrity and development of Cancer, Rational treatment of cancer. Detailed semester notes from Course MCB 3211 at the University of Connecticut. Aimed at pre-med, molecular cell biology majors.
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Chp 10 Eternal Life: Cell Immortalization and Tumorigenesis
12/27/16 8:17 PM
History
Can an animal cell live forever? Question was addressed by growing animal cells in culture
o Early cell culture labs required their workers to dress all in black in darkened rooms to “protect” cells from the
light
1) Approximately how many “doublings” can a normal human cell undergo before stopping?
Hayflick limit
• Human fibroblasts undergo a limited number of doubling when grown in culture
• Leonard Hayflick proposed that 60 doublings was the maximum
• After reaching the “Hayflick limit” cells undergo replicative senescence
• Culture conditions can also limit the replication capability of a cell
Replicative senescence
• A large, flattened morphology with positive staining for senescence –associated B-‐galactocidase
• Senescence: an irreversible state of growth arrest
...
• Telomeres are composed of a TTAGGG repeat (1000s of base pairs) and 3’single strand DNA overhang at the end (makes a
T-‐loop to protect the end of the chromosome)
4) Name two features of the telomere that are likely to play an important role in stabilizing the chromosome end
...
Activated is mediated by p53
o Telomere erosion à DNA damageà p53à p21
Senescent cells in pre-‐malignant tissue
• Telomere shortening can limit the expansion of preneoplastic lesions
• P16 and ki67 expression analysis in serial sections of a pre-‐cancerous adenoma
11) How do cancer cells usually become immortal? Do they usually pass through crisis before becoming immortal?
• Immortal cells have found a way to stabilize their telomeres
o Often activate telomerase to stabilize telomere (extend the telomere)
o Telomerase (TERT) uses an RNA template (TR) to extend the telomere
• Immortal cells/cancer cells that exit crisis typically don’t re-‐establish their full telomere length
o Frequently have telomerase activity (as determined by a TRAP assay)
o Usually acquire this activity by re-‐expressing hTERT protein (at the transcriptional level)
o The hTR RNA is usually expressed in both mortal and immortal cells
§ Cells can be immortalized by expression of hTERT
16) What is mTERT and mTR? What is hTERT and hTR?
•
•
Telomerase activity is associated with poor survival in neuroblastoma and Ewing’s sarcoma
o Demonstrating the critical importance of telomerase and cellular immortalization in cancer
o Oncogenes (accelerator),
o Tumor suppressor genes (the brakes),
o Telomerase (keeps the gas tank full)
Catalytically inactive hTERT mutants that still bind hTERT act as dominant negative mutants
o Dn-‐hTERT mutants expressed in cancer cells induced senescence
o The number of doublings required to reach senescence depends on the length of the telomere in the cell line
GRN163L
• Promising therapeutic that targets telomerase
• An oligonucleotide with a thiophophoramidate backbone that targets hTR/hTERC
15) What is ALT?
Alternative Lengthening of Telomeres mechanism
• ~90% of cancers express telomerase (how do the others maintain telomere length)
o Some cancer cells have an ALT phenotype and use an Alternative Lengthening of Telomeres mechanism
• ALT cancers appear to share info between telomeres
•
•
Potential mechanism of telomere extension by ALT
o 3’overhang invades the telomere on an adjacent chromosome and is extended by a DNA polymerase
o Such lengthening appears to be permitted in cells with incapacitated/mutated mismatch repair proteins
o
13) Do you think telomerase inhibitors would be good anti-‐cancer drugs? Describe an experiment that would allow you to determine
the value of telomerase as a target for therapeutic agents
Is hTERT a good target for cancer therapy?
• Pros
o Most normal cells do not express hTERT and would not be affected by such an inhibitor
o Cancer cells have divided many times and are “old” usually with shortened telomeres that are maintained at a
minimal length
o hTERT has many similarities with viral reverse transcriptase, and drugs specific to these polymerase have been
developed
• Cons
o A cancer cell could adapt and start to utilize ALT
17) Briefly describe the phenotype of an mTR null mouse at breeding generations 1 and 5
...
5-‐10kb
§ mouse telomeres are so long, it takes 5 generations before a phenotype can be observed in mTR
knockout animals
§ at generation 5, mTR null mice show signs of premature aging (wasting muscles and hunkced back)
o 2) most somatic cells in mouse express some level of telomerase activity
• since mice have shorter lives and fewer cells, their risk of developing a terminal cancer is much lower than a human’s
• there is not a pressing need to use the telomere to regulate cancer research in a mouse
th
To determine the impact of telomerase expression in a mouse model, 5 generation my mTR knockouts were combined
with a p16INK4A deletion
...
after NIH3T3 cell transformation
Multiple steps of cancer development
• Genetic and epigenetic changes that increase the ability of a cell to expand within the tissue
o Accelerated by genomic instability (caretaker gene mutation)
o Facilitated by tumor promoters
•
21) How does an initiating agent differ from a tumor-‐promoting agent?
• Non-‐mutagenic factors that promote the formation of cancer are called “tumor promoters”
o TPA (also known as PMA) is one of the first identified tumor promoter
o It was identified in a mouse skin cancer model
§ Permutations of the mouse 2-‐step skin cancer model helped define the differences between an initiator
and a promoter
ú DMBA is a genotoxic initiator (which induces a stable genetic change)
ú TPA is a non-‐genotoxic promoter
•
Interaction between initiator and promoter (top right)
o Promoter promote the proliferation and survival of mutated/initiated cells
22)
...
TPA
• TPA stimulates cell proliferation by activating protein kinase C
• PKC normally activated by diacyglycerol (DAG) produced transiently following physiological stimuli – TPA provides a
chronic signal
•
•
Phospholipid cleavage sites by phospholipases: A1, A2, C and D
PLA2 is also involved in tumor promotion through the prostaglandin pathway
•
•
•
One mechanism by which a tumor promoter functions is by directly stimulating cell proliferation
Tumor promoting agents can also function by inducing inflammation
o Recruiting WBC to the tissue)
...
Estrogen
• Is a tumor promoter that stimulates cell proliferation in the mammary tissue
• Fewer menstrual cycles lower the risk of breast cancer
• Removal of ovaries can dramatically reduce breast cancer risk
•
Example of a cancer initiator and promoter in a HUMAN CANCER:
A promoter that induces cell turnover
• Smoking and ethanol consumption combine to increase head and neck cancer risk 100-‐fold
• Cigarette smoke contains many carcinogens, so it can clearly act as an initiator
• Ethanol is not DNA reactive
...
e
...
o The increased rate of cell division to repair the tissue promotes cancer development
Inflammation
• Is also tumor promoting
• Inflammation generally characterized by the infiltration of cells from the blood stream
...
• HBV-‐induced inflammation as a tumor promoter:
o HBV-‐infection à increases liver cancer risk ~7 fold
o Aflatoxin exposure (a DNA damaging carcinogen) à increase liver cancer risk ~3 fold
o Exposure to both increases liver cancer risk by ~60 fold
•
•
27) How do TNF and NFkB interact to promote cancer development?
• Evidence has been obtained that TNF secretion by inflammatory cells and transcription factor NF-‐kB activation in
transformed cells plays an important role in linking inflammation and carcinogenesis
•
NF-‐kB activation involves a phosphorylation-‐dependent degradation of the IkB inhibitor
o Signals include TNF, interleukins and other inflammatory cytokines
o Also oxidative stress, UV radiation and other cell stressors
•
•
Additional evidence for the link between inflammation and cancer comes from epidemiological studies:
Nonsteroidal anti-‐inflammatory drugs like aspirin decrease cancer risk at multiple tissues
One aspirin a day
§ Lung cancer risk: 0
...
7
§ Colorectal cancer: 0
...
Coxibs include Vioxx and Celebrex
29) Why were Cox-‐2 inhibitors anticipated to have fewer side effects than NSAIDs for cancer prevention?
• Coxibs take advantage of the fact that COX2 is only expressed in inflamed or cancerous tissues
• The coxib celecoxib induces apoptosis and reduces proliferation in a mouse cancer model
30) Coxibs are thought to be dangerous because they increase the risk of: ______
...
Stem cells
• 1) Serve as a safe haven for genomic DNA
• 2) They are usually buried within the tissue, shielded from DNA damaging agents
• 3) They replicate their DNA and divide infrequently, so there is a lower chance that they will acquire a mutation
What are the two main lineages derived from hematopoietic stem cells? List two differentiated cell types derived from each lineage
...
36) Some people believe that cancers form from mutated stem cells
...
What are they?
• They have 5’à3’ polymerase activity, and 3’ à 5’ exonucleases activity to correct mistakes
•
38) What is the phenotype of a mouse in which the proofreading activity of DNA polymerase d has been eliminated through
mutation?
• A mutation of DNA pol sigma that eliminates its proofreading activity and increases cancer development in mice (top right)
39) When a mistake occurs during DNA replication, it is important for the cell to identify, which base is correct and which is incorrect
...
How does this instability usually arise?
Patients with HNPCC have mutated MMR proteins
• Their tumors will show contractions or expansions of microsatellites – called microsatellite instability (MSI)
• BAT25 is a microsatellite on chromosome 4
•
•
•
•
DNA damage can also result from
• The chemical reactivity of the DNA base
• The physiological generation of reactive molecules
• For a mutation to occur, the damaged base must lead to a base-‐pair change, either through base conversion or base mis-‐
reading by the DNA polymerase
Purines bases
• Prone to depurination leaving an basic site that will require repair
• This reaction is promoted at lower pH
• Pyrimidine are more stable
42) Many bases undergo deamination, but deamination of 5-‐methylcytosine is very frequent
...
Reactive oxygen species are generated by
• Mitochondria
• Peroxisomes
• Macrophages and neutrophils during an inflammatory response
ROS can damage DNA in many ways, including strand breakage and base oxidation
45) Would you expect a mouse with a deleted 8-‐oxo-‐deoxyguanosine glycosylase to have a higher or lower cancer risk?
• Oxidation of guanosine can lead to the formation of 8-‐oxo-‐guanosine
• If un-‐repaired, 8-‐oxo-‐guanosine can be read as a “T” by DNA polymerase leading to a G to A transition
o (G to T to C would be a transversion)
• 8-‐oXo-‐guanosine is a potentially carcinogenic lesion
o If you prevent its repair in mice, their risk of cancer will increase
• Loss of expression of enzymes involved in repairing 8-‐oxodeoxyguanosine reduces breast cancer survival (evidence that 8-‐
oxo-‐dG accelerates progression)
46) Compare how X rays and UV damage DNA
• Chemical and physical damage of DNA by environmental agents is responsible for most cancers
o X-‐radiation is ionizing radiation
...
o A direct hit to DNA will cause strand breakage
o UV radiation is a more common source of DNA damage, particularly to skin cells
47) What are the two major photoproducts formed by UV radiation?
• Common DNA photoproducts include: cyclobutane dimers and 6-‐4 photoproducts between adjacent pyrimidine’s
•
48) Many environmental agents can alkylate DNA
...
How do they become carcinogens?
Pro-‐carcinogens
• Most carcinogens enter the body as non-‐relative molecules known as pro-‐carcinogens
49) Why do cytochrome P450 enzymes attach oxygen molecules to pro-‐carcinogens?
• Cytochrome P450 performs this catalysis
...
1) Benzoapyrene (Gà T transversion)
• Benzo [a] pyrene from tobacco smoke is metabolized to BPDE in the lung
• BPDE binds to guanine, causes it to be read as a “T during DN replicatioin
• This lead to a G to T transverison
2) Aflatoxin B
• Is formed by molds that grow on improperly stored grains
• It is activated in the liver, where detoxifying pathways compete with DNA reactivity
• DNA reactivity consistent with the AGG to AGT mutations found in the p53 gene in individuals exposed to alfatoxin B
• Cytochrome P-‐450s performed the phase I metabolism
...
3) Heterocyclic amines (HCAs)
• Like PhIP are derived from broiled and charred meats
• PhIP and other HCAs may contribute to the elevated risk of western cancers, such as prostate cancer
Cytochrome P450 enzymes are present on the smooth ER
52) Many dietary pro-‐carcinogens are absorbed by the small intestine
...
Inducers such as sulforaphane modify cysteines of Keap1, which leads to stabilization and translocation of Nrf2 to the
nucleus, and its binding to ARE and stimulation of phase 2 gene transcription
Compare base excision DNA repair and nucleotide excision repair
...
What are they called and which is more error prone?
• Double strand DNA breaks can be repaired by homology-‐directed repair (HR)
o HDR is a high fidelity repair pathway of double strand breaks that are caused by mutagens (like X-‐rays) and during
normal replication (at replication forks) requires sister chromatid
•
Nonhomologous end-‐joining (NHEJ) is a low-‐fidelity DNA repair mechanism: deletions occur frequently (top right)
o In the absence of Brca1/2, homology-‐directed double strand break repair is not possible
...
o Brca1 and 2 are in a complex with other DNA repair proteins
Li-‐Fraumeni
• TP53 (multiple cancers), DNA damage alarm protein
• CHK2 (colon, breast), Kinase signaling DNA damage
What is the difference between chromosomal instability and microsatellite instability? How do these states arise?
• Microsatellite instability vs
...
non-‐hypermutated colon cancers compare with regards to: anatomical location, DNA mis-‐match repair,
microsatellite instability, and chromosome stability?
• Whole genome sequencing confirms at least two distinct types of colorectal cancer: hypermutated (MIN) and non-‐
hypermutated (CIN)
o There are distinct (but overlapping) mutations in hypermuated and non-‐hypermutated cancers
o MSI/MIN: has MMR proteins, CIN don’t
• Distribution of MSI/MIN and CIN tumors in the human colon shows an anatomical preference
o Right colon (ascending): Hyper-‐mutated (MSI) BRAF mutations, DNA methylation
o Left colon (descending): non-‐hypermutated (CIN)
What type of colon cancer is responsive to 5-‐fluorouracil? What is the molecular basis for this differential sensitivity?
• impact of mutations on colon cancer therapy:
o Standard chemotherapy is 5-‐fluorouracil (5FU), 5FU promotes uracil incorporation into DNA
o MMR proteins detect uracil and trigger cell death, No MMR proteins, no 5-‐FU benefit
o Tumor typing is not always done
In a normal cell, mitosis will not occur until the spindle assembly checkpoint in passed
...
o All batches will be identical and specific to just one epitope
List the general steps for making a mouse monoclonal antibody? (This question may be on the exam
...
In the short term, this can sometimes cause an immune response
...
How was a mouse monoclonal antibody altered for to become Herceptin?
• Monoclonal antibodies that bind HER2 were isolated
• The gene encoding one antibody was humanized (so that it doesn’t provoke an immune response in humans)
List three general types of breast cancer and indicate which one can be treated with Herceptin
...
• Trastuzumab (Herceptin) à ERBB
• Bevecitzumab (Avestin)àVEGF
What do gene expression arrays measure? How might they be used for cancer diagnosis and treatment?
Cancers (particularly some breast cancers) that over-‐express the HER2/neu growth factor receptor are responsive to Herceptin
Herceptin reduces HER2 expression and downstream pro-‐survival pathways
It also makes cells sensitive to apoptosis (the example shows X-‐ray-‐induced apoptosis
Cetuximab and panitumumab are EGFR inhibitors
...
Metastatic colorectal
carcinoma
• Cetuximab –(chimeric human-‐murine IgG1) n combination with radiation therapy for the initial treatment of locally or
regionally advance SCCHN; as a single agent for patients with SCCHN for whom prior platinum based therapy has failed ;
and palliative treatment of pretreated metastatic EGFR-‐positive colorectal cancer
Ofatumumab and Arzerra and the same molecule
...
fully human monoclonal antibody (for the CD20 protein), which appears to inhibit
early-‐stage B lymphocyte activation
...
What is the 10-‐year survival rate for women with a “good”
gene expression signature? What is the 10-‐year survival rate for women with a “poor” signature?
• Survival curves for breast cancer patients stratified by the expression of 70 prognostic genes
•
Diffuse large B cell lymphoma was originally thought to be a single disease
...
Despite this complexity, their growth and survival can
often be impaired by the inactivation of a single oncogene
...
What vitamin metabolite was found to cure for acute promyelocytic leukemia (APL) when used at pharmacological doses?
• All-‐trans retinoic acid (ATRA) can induce the differentiation of APL promyelocytes into post-‐mitotic neutrophils
•
• What fusion gene is frequently found in patients with APL?
• Retinoic appears to destabilize the PML/RAR fusion oncoprotein
Small molecules can be designed to inhibit oncogenic proteins, but since cancers normally have multiple mutations, can we be sure
that inhibiting a single oncoprotein will be sufficient to induce growth arrest/apoptosis in a cancer?
•
Experiments in animal models indicates that shutting off the initiating oncogene can sometimes, but not always, lead to
tumor regression
•
What type of pre-‐clinical models can be used to determine whether an oncogene is a good target for therapeutic agent
development?
• Preclinical studies define the mechanism of action and demonstrate specificity to cancer cells
...
These experiments
employ cell culture and animal models
Why are kinases usually good targets for drug development? Even though kinases are druggable, why is their targeting sometimes
problematic with regard to toxicity?
• Kinases are very druggable, they contain clefts that bind ATP and substrate molecules
• Drugs can be designed that dock in these clefts and inhibit enzyme activity
• Drugs obtained through small molecule screening and then refined by molecular docking
Define the terms pharmacodynamics and pharmacokinetics
...
• Iressa and Tarceva were developed to inhibit EGF-‐R tyrosine kinase activity
• Phase I trials showed that the drug’s IC90 could be achieved with acceptable toxicity
• Initial studies showed that Iressa blocked EGF-‐R and MAP kinase phosphorylation in skin cells
• Although the target was not the skin, this data showed that the compound worked in humans
•
What is a goal of a phase III clinical trial? What type of patient is usually recruited for a phase III trial and what type of outcomes
would be deemed a success?
• Phase III trials: A large patient base is enrolled and “real” endpoints measured (patient survival, tumor shrinkage, improved
quality of life, etc
...
Where do the Gleevac resistant cells come
from?
• Usually there are additional mutations in Bcr-‐Abl that decrease Gleevec binding and inhibition
• These mutations arise spontaneously in the tumor, and then out-‐compete the other cancer cells in the presence of Gleevec
• Blast cells in blood
Describe two experiments that could be performed to determine if Gleevec therapy was working on a CML patient?
• molecular response to Gleevac/Imatinib predicts long-‐term survival
• mapping drug specificity on the human kinome
What is the target of Iressa, and why did only ~10% of lung cancer patients respond favorably to this drug in a phase III trial?
• Iressa treatments showed dramatic improvements in some patients with lung cancer (which are difficult to treat)
• However, the drug was only found to work in ~10% of patients in the US (27% in a Japanese study)
• When the lung cancers in responding patients were analyzed, only those with specific EGF-‐R mutations were found
• These were small deletions and point mutations near the catalytic domain
• Even though Iressa was designed as a general EGF-‐R inhibitor, it is only effective in a subset of cancers (for reasons that are
not entirely clear)
•
After a few years, patients developed resistance to Iressa
...
• 1) cancer vaccine – direct injection of killed cancer cells (injecting cancer antigens into patients to stimulate the immune
response
2)Dendritic cells (DCs) are most efficient antigen presenting cells (APCs)
o Immune cell transfer can be used to “force” a stronger immune response
...
o Remove antigen presenting dendritic cells from patient, load them up with tumor antigen and re-‐inject
...
• 3) immune checkpoint block
o Treatments can be used to block the immune checkpoint
...
These checkpoints can be disrupted to reactivate the immune response in cancers
Name 2 immune checkpoint targets that have been used to treat cancer
...
Blocking this interaction with an antibody activates CTLs
in the tumor
The PD-‐1 immune check point – cancer cells can express a PD-‐1 ligand (B7-‐H1) which binds PD-‐1 and kills CTLs
Antibodies that block PD-‐1 or PD-‐L can increase CTL activity in the cancer
o
o
•
•
•
What is presently considered the most promising approach to cancer treatment?
• Immune therapy
o Works best on neoplasms with a high mutation rate
...
o Might be best when combined with a complementary targeted therapy
o Immune checkpoint block (ICB) and complementary treatment (TX)
Student presentation
12/27/16 8:17 PM
To what part of Mdm2 does Nutlin-‐3 bind? What is the consequence of this binding? What type of cancer might be responsive to
Mdm2 inhibition?
•
P53 binding sites on MDM2 block interaction of MDM2 with p53,lymphoma
Is reactivation of mutant p53 possible? What result might be anticipated after reactivation of mutant p53 in a cancer cell?
•
Yes
Where in the cell are proteins degraded during autophagy? What is the role of mTOR in regulating autophagy? Do oncogenes usually
activate or repress mTOR activity?
•
Lysosome, inhibition of mTOR à autophagy
Title: Cancer Cell Biology
Description: Topics covered: Tumorigenesis, Cell immortalization, Maintenance of Genomic integrity and development of Cancer, Rational treatment of cancer. Detailed semester notes from Course MCB 3211 at the University of Connecticut. Aimed at pre-med, molecular cell biology majors.
Description: Topics covered: Tumorigenesis, Cell immortalization, Maintenance of Genomic integrity and development of Cancer, Rational treatment of cancer. Detailed semester notes from Course MCB 3211 at the University of Connecticut. Aimed at pre-med, molecular cell biology majors.