Oncogene & Tumor Suppressor Control Pathways in Oral Squamous Cell Cancer I & II Flashcards
How many new cases of oral cavity/oropharyngeal cancer occur in the US?
About 58,450 new cases (1 in 6000)
How many deaths occur in the US from oral cavity or oropharyngeal cancer?
About 12,230 deaths
Oral cavity & oropharyngeal cancers occur most often in which sites?
The tongue
The tonsils & oropharynx (parts of the throat behind the mouth)
The gums, floor of the mouth, & other parts of the mouth
Almost all cancers in oral cavity & oropharynx are…
squamous cell carcinomas or squamous cell cancers
Infections with certain high-risk types of ___ cause most of the squamous cells cancers of the oropharynx
HPV
HPV is rarely associated with what?
oral cavity cancer
HPV + cancers are seen more often in who?
young people with no history of tobacco or alcohol use
HPV + cancers tend to have what kind of prognosis than squamous cell cancers not related to an HPV infection?
better prognosis
What are the 2 main classes of genes involved in tumorigenesis?
oncogenes & tumor suppressor genes
Describe oncogenes
Encode proteins that actively promote aberrant cellular proliferation
Activity is up-regulated in tumor cells (K-Ras mutations in pancreatic cancer), gene amplification (MDM2 in soft tissue sarcomas), chromosomal translocations (BCR-ABL in CML). Present within genomes of tumor promoting viruses
Describe tumor suppressor genes
Encode proteins that negatively regulate cell proliferation
Activity down-regulated in tumor cells (loss of function mutations, gene deletion, promoter methylation)
Malignancy is a ___ ____ process
multi-step
Cancer is a ___ ___ process involving both the activation of ______ and the inactivation of ____ _____ ____
multi-step; oncogenes; tumor suppressor genes
What have given us important info on the nature of mutations in human cancers?
Large scale exome sequencing projects sequences DNA from thousands of different tumors
Oncogenes are recurrently mutated at which AA position?
The same AA position
Tumor suppressor genes are mutated through what? 9
Protein-truncations alterations (nonsence, missense mutations throughout their length)
3 molecular processes relevant to cancer
Cell fate
Cell survival
Genome maintenance
Cell fate 11*
Many of the genetic alterations in cancer abrogate the balance between differentiation & division, favoring the latter
Cell survival 12*
Even though cells divide abnormally bc of cell-autonomous alterations like those controlling their fate, their surrounding stromal cell don’t keep pace
This leads to abnormal vasculature of the growing mass & severe growth factor & nutrient limitations
So a cancer cells getting a mutation in growth factor receptor or downstream signaling molecule that lets it proliferate under limiting nutrient concentrations will have a selective growth advantage
Genome maintenance
Mutation in DNA repair factors can also act earlier in the transformation process by accelerating the acquisition of mutations that function through the process of cell fate/survival
13* The Epidermal Growth Factor Receptor tyrosine kinase is frequently found…
Over-expressed in epithelial tissues. Gene amplification common mechanism
What is the most common Ras mutation from COSMIC?
G12V is the most common
For RAS mutations from COSMIC, what is the type of classic recurring mutation at particular residues in the protein?
Classic recurring missense mutation
What are 5 of the top 20 mutated genes by tissue?
TP53 (45%)
CDKN2A (11%)
FAT1 (24%)
CASP8 (15%)
BRCA2 (6%)
What type of gene is FAT1?
Tumor suppressor gene
What overall type of mutation does FAT1 undergo?
Loss of function
What are two types of specific mutations scene in FAT1? 17
Nonsense substitution (40.57%)
Missense substitutions (19.81%)
What type of mutation is seen with the CDKN2A gene (p16)?
Nonsense substitution (56.82%)
Most cells in our bodies aren’t in cell cycle. In absense of mitogenic stimulation they exit the cell cycle into ____
G0 (quiescent state)
Which cells enter G0 reversibly
stem cells, hepatocytes, etc.
Which cells irreversibly enter G0? *in most cases leads to terminal differentiation
RBCs & epithelial cells
Eukaryotic cell cycle progression is triggered by what?
Cyclin Dependent Kinases (CDKs)`
What do CDKs do?
Phosphorylate other regulatory and structural proteins thereby controlling expression and activities of replication enzymes and cell cycle factors
What is CDKN2A gene (p16) induced by?
Signals that promote cell cycle exit
What do loss of function mutations promote?
Aberrant cell proliferation
CASP8 & apoptosis is a classic _______ example
loss of function
What are 2 mutations for CASP8?
Nonsense substitution (26.44%)
Missense substitution (55.17%)
In 1842, Karl Vogt…
noticed cells in notochord “disappeared” and were replaced by cells of vertebrae
1972: Kerr, Wylie, Currie
Apoptosis suggested an active, inherently programmed phenomenon
1973: 3 classes of cell death
I: Cell shrinkage, membrane blebbing, DNA fragmentation, chromatin condensation (apoptosis-regulated cell death)
II: Formation of large scale autophagic vacuolization containing cytosolic materials and organelles (autophagy-regulated cell death)
III: Loss of membrane integrity and swelling of subcellular organelles (accidental and regulated cell death-necrosis)
1990s, Robert Horvitz got 2002 Nobel prize for…
Identification of key apoptosis genes in C. Elegans
Apoptosis 6 points
Outer membrane blebbing
Shrinking
Condensation of the nucleus
Fragmentation of chromosomes
Formation of cytoplasmic vacuoles
Cell fragments generated by apoptosis are taken up and eliminated by neighbouring cells and phagocytes
Describe apoptotic signaling via extrinsic pathway in 5 points
25*
Mediated by death receptors (e.g. CD95, TNFR1, TRAIL)
Formation of DISC (death-inducing signaling complex)
Recruitment of pro-caspase 8 (cysteine dependent aspartic specific proteases) and self-cleavage
Activation of effector caspases 3,6, and 7. Destruction of essential cellular proteins
Can also feed into the intrinsic pathway by cleavage of the BH3 only protein BID
Describe apoptotic signaling via intrinsic pathway in 5 points
Marked by one central event: mitochondrial outer membrane permeabilization (MOMP)
Results in release of cytochrome C from mitochondrial intermembrane space
Cytochrome C triggers assembly of a caspase-activating complex between caspase 9 and APAF1 (apoptotic protease activating factor 1) via Caspase Recruiting Domain (CARAD) of APAF1
Also leads to release of AIF & Endonuclease G, which degrades DNA
Caspase 9 activates effector caspases 3, 6, and 7, which destroy essential cellular proteins
Main mutations involved in BRCA2 gene and genomic instability
Missense substitution (46.67%)
Frameshift insertion (36.67%)
Recombination is a mechanism with ___ distinct functions
2
Describe 2 functions of recombination
- In all cells, repair of double strand damage to chromosomes
- In germ line cells undergoing meiosis, shuffling genetic info between homologous chromosomes (allows redistribution of parental genetic traits)
Further explain repair of ds damage to chromosomes
When both strands are damaged, use the info from homologous chromosome to replace lost info
What enzymatic activities are required for homologous recombination? 30*
5’-3’ exonuclease
Recombinase
DNA polymerase
Ligase
Resolvase
BRCA2 loss promotes what?
DNA loss
Genomic instability
In response to DNA damage, what does p53 tumor suppressor do?
Halts cell cycle to allow DNA repair
p53 is…
The most frequently mutated gene in oral cancer
In a tumor cell lacking p53…
Cell division continuous with damaged chromosomes (genetic instability) and this can result in:
Massive mitotic failure and cell death where tumor regresses
or
Continued mutation, selection, and tumor evolution (cancer)
The p53 tumor suppressor is also a transcription factor that does what?
Up-regulated genes that negatively regulate proliferation in response to various stress signals
List types of treatment for oral and oropharyngeal cancer
Surgery
Radiation therapy
Chemotherapy
Targeted therapy
Immunotherapy
Surgery
Goal is complete removal of tumor with “negative margins” meaning no trace of cancer in margin’s healthy tissue
Radiation therapy
Use of high-energy X-rays or other particles to destroy cancer cells. Specific # of treatments over given period. More precise
Chemotherapy
Use of drugs to destroy cancer cells, usually by keeping cancer cells from growing, dividing, and making more cells. Certain drugs block DNA replication, induce DNA damage
Targeted therapy
Targets cancer’s specific genes, proteins, or tissue environment that contributes to cancer growth & survival
Blocks growth & spread of cancer cells & limits damage to healthy cells
Not all tumors have same targets
Currently Abs directed against epidermal growth factor receptor are being used in combo with radiation therapy for head and neck cancers
Cetuximad (Erbitux) is targeted therapy approved by FDA for use in combo with rad therapy
Immunotherapy
Uses body’s natural defenses to fight cancer by improving immune system’s ability to attack cancer cells
Pembrolizumab (Keytruda) & nivolumab (Opdivo) are 2 immunotherapy drugs approved by FDA for treatment
What do Pembrolizumab (Keytruda) & nivolumab (Opdivo) treat?
Recurrent or metastatic head and neck squamous cell carcinoma (HNSCC) not stopped by platinum-based chemo
Both Pembrolizumab (Keytruda) & nivolumab (Opdivo) are…
37*
Immune checkpoint inhibitors that are also approved for treatment of some people with advanced cancers of other kinds
DNA serves as a template to…
Copy itself
DNA also serves as template for…
Synthesis of RNA
RNA serves as a template for…
Synthesis of protein
Reverse transcriptase
Converts RNA genome of retroviruses to DNA which is then used to make new RNA molecules
RNA dependent RNA polymerase
copies RNA genome of RNA viruses to new molecules of RNA
Nucleoside
purine/pyrimidine nitogenous base
pentose ribose/deoxyribose sugar
Nucleotide
nitrogenous base
pentose sugar
phosphate
DNA is a polymer of…
4 deoxyribonucleotides
RNA is a polymer of…
4 ribonucleotides
What are the precursors for DNA or RNA synthesis?
Triphosphates (dNTPs or NTP)
What are incorporated into DNA or RNA where PPi is released?
Monophosphates (dNMP oro NMP)
DNA is ___ ___ than RNA
more stable (less susceptible to degradation by nuclease)
DNA and RNA are formed by linking nucleotides by ______ bonds
phosphodiester
In a phosphodiester bond…
5’ phosphate group on one nucleotide joined to 3’ hydroxyl group of sugar moiety of next nucleotide with release of PPi
RNA is hydrolyzed rapidly under what conditions?
Alkaline conditions due to 2’ OH group on ribose sugar
DNA is ___ in alkaline conditions
stable
How many H bonds between A and T?
2
How many H bonds between C and G?
3
2 strands in DNA are ______
antiparallel
DNA is composed of 2 ____ -______ backbones that wind around each other to form a right-hand double helix
sugar-phosphate
Each turn in DNA is made of ____ nucleotides
10.4
What does the coiling of DNA create?
2 grooves in DNA, major & minor groove
DNA replication is ____-______ and _______
Semi-conservative; discontinuous
DNA replication rate in bacteria
1000 nucleotides/second
DNA replication error rate
1 in 10^9 nucleotides
DNA is read in the ________ direction
3’-5’
DNA is synthesized in the _______
5’-3’
Replisome
Assembly of enzymes that replicate DNA
20-25 proteins are involved in bacterial replisome
3 basic enzymatic activities required:
synthesis of nascent DNA by DNA polymerase
unwinding of DNA by helicase
priming of Okazaki fragments on lagging strand by
DNA primase
All DNA polymerases do/require what?
50*
synthesize in 5’-3’ direction
need a template
need a primer
need 3’ OH on primer
use dNTPs as energy source
incorporate dNMPs into DNA
DNA on lagging strand must be synthesized as short DNA molecules known as…
Okazaki fragments
Is the replication fork symmetric?
No because synthesis of leading strand slightly precedes lagging strand
Steps of DNA replication
DNA double helix unwound ahead of replication fork
Leading strand made in 5’-3’ direction by DNA polymerase
RNA primers made on lagging strand by DNA primase
RNA primers elongated by DNA polymerase in 5’-3’ direction to give Okazaki fragments
RNA primer removed by 5’-3’ exonuclease (RNAse H)
Another molecule of DNA polymerase fills gap
Nick between Okazaki fragment joined with DNA ligase
DNA topoisomerase prevent DNA tangling
What unwinds DNA?
DNA helicase
What straightens out DNA template and facilitates activity of DNA polymerase?
Single-strand DNA-binding (SSB) protein
At the start of replication, how many primers are needed for leading strand?
1
How long are RNA primers on lagging strand?
100-200 nucleotides
DNA primase doesn’t require a _____ and can join 2 _____ _____
primer; nucleoside triphosphates
What is the position at which DNA helix is first opened?
Origin of Replication
Two replication forks form and proceed in opposite directions forming what?
Replication Bubble
In prokaryotes, there are how many origins of replication?
1
In prokaryotes, how many replication forks are there?
2
In eukaryotes, how many origins of replication are there?
A large number to allow for synthesis of complete genome in timely manner
In eukaryotes, replication origins are activated in clusters called what?
Replication units
New replication units in eukaryotes are activated at different times during what phase?
S phase
How many bp apart are unit origins within a replication placed?
30,000 to 250,000 bp apart
Error rate in 5’ to 3’ polymerization
1 error/10^5
Errpr rate in +3’ to 5’ exonucleolytic proofreading
1 error/10^7
Error rate in + strand-directed mismatch repair
1 error/10^9
How to analyze specific molecules
DNA
Sequencing
PCR/qPCR
RNA
Reverse transcriptase-PCR/RT-qPCR
Protein
Western blotting
Immunohistochemistry
Global analysis
DNA
SNP arrays
Genome sequencing
RNA
RNA seq
Protein
Proteomics
DNA sequencing
Sanger DNA sequencing involves enzymatic DNA synthesis using base specific chain terminators & radiolabeled primer
Size DNA products on large denaturing polyacrylamide gels that can resolve sizes of products by one nucleotide
Automatic DNA sequencing with fluorescent primers
Concept is same except 4 rxns contain same primer with a different fluorescent label. Rxns are then combined prior to separation and signal is detected by laser
PCR is…
A major technical advance for DNA analysis. A way of amplifying specific DNA elements in a test tube
Temperatures in PCR:
Heat to separate strands - 95 deg C
Cool and add primers - 60 deg C
Add heat-stable DNA polymerase - 70 deg C
In PCR the number of copies of defined product _____ every cycle
doubles
In PCR, there are about ___ copies of defined product (“amplification”)
10^6
You need how many copies of each primer per template in PCR to amplify target DNA sequence?
millions of copies of each primer
What is automation of PCR called?
Thermocycling
Thermocycling requires what?
A DNA polymerase to withstand high temps
Application for PCR
Restriction fragment length polymoriphism (RFLP) analysis
Restriction Fragment Length Polymorphism Analysis
64*
Identify individuals harboring disease genes that have polymorphic restriction site. E.g. of VCP mutations in ALS (autosomal dominant disorder)
Applications of Southern Blotting
Variable Number Tandem Repeat (VNTR) Analysis
Variable Number Tandem Repeat (VNTR) Analysis
Identify individuals based on differing size of DNA repeats (forensics, paternity ;) Arrows denote primers used for PCR
5 applications of PCR in medicine
RFLP & VNTR analysis using primers that span the polymorphic restriction site or VNTR
Looking for mutations in known genes by PCR, followed by DNA seq analysis of the PCR product
Embryo typing and genetic counseling for Huntington’s disease: IVF of mutiple embryos, grow embryos to 8 or 16 cell stage, isolate one cell and DNA for PCR
Remission test cancer patients
Virus detection
qPCR
Machine based quant PCR that measures amount of PCR product after each cycle
qRT-PCR can be used to measure what?
RNA levels of specific transcripts, giving an idea of expression changes in disease or experimental conditions
Real time PCR relies on what?
Specialized probes that bind sequence between PCR primers
Western blotting
Prep of protein extract
SDS-polyacrylamide gel electrophoresis
Transfer to nitrocellulose
Incubation with monoclonal antibody that recognizes specific protein and washing
Incubation with secondary antibody that recognizes primary and also contains an enzyme that when exposed to substrate, emits a signal
Washing, expose to chemical that activates enzyme on secondary and reading of this signal
Immunohistochemistry
70
Assesses protein abundance in cells or tissue, including disease tissue
Very little material is needed
Need highly specific monoclonal antibodies for this analysis
Global analysis at DNA level-Detecting Single nucleotide polymorphisms
SNP is a variation at a single site in DNA that may alter the expression or function of a gene/gene product. Represent over 80% of genetic variation between individuals
10,000,000 in human genome
Dictates phenotypic differences between individuals, including sensitive and resistance to human disease
SNP-based genetic linkage analysis can be used to map disease loci
Future appls of SNP analysis (DNA seq/microarrays) may determine disease susceptibility and in pharmacogenomics, the concept of assessing efficacy of drug therapies for an individuals
SNP genotyping using microarrays and allele specific oligonucleotides
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RNA-seq applications
Allele specific expression: prevalence of transcribed SNPs
Identification & quantitation of alternatively spliced RNAs, novel transcripts, & edited RNA
*Gene expression profiling, i.e. determining gene activity of all genes in cells under different conditions
SIngle cell RNA seq: to identify heterogenous cell pos in a given sample (e.g. tumor heterogeneity in a single biopsy)
Global analysis at protein level mass spec based proteomics methods
Determine relative expression profiles of proteins in and out of cells
Also can determine differences in protein modification
Sensitively, quantitation (in patient samples) and coverage still not there yet. WILL NOT DETECT ALL LOW ABUNDANCE PROTEINS
Requires a lot of cells/protein
