Learning Objectives Lectures 1-6
Lecture 1:
Types of malignancies
85% of all cancers are carcinomas (epithelial cells)
Sarcomas (mesenchymal cells)
Leukemia (blood cancer—originates from bone marrow)
Lymphoma (blood cancer—originates from lymphatic system)
Neuroectodermal tumors (tumors of nervous system)
What are the different steps of malignant progression?
Uncontrolled growth
Benign tumor
Malignant tumor
Metastasis
Why is the basal lamina important for epithelial tissues?
What is the developmental hierarchy in tissue?
Structure of the cervical epithelium
Stem cells are closest to basal lamina
Next layer is progenitor/transient amplifying cells
Then differentiating keratinocytes
Then differentiating squamous layer
Then mature squamous layer
Basically increasing differentiation from the basal lamina
Name the different steps that lead to cancer
In most cancers, the initial cell that acquires proliferative capacity happens to be an undifferentiated cell.
Stem cells are the “longest lived” cells in that tissue, and you need multiple mutations over a lifetime to produce cancer.
If a differentiated cell got that mutation, it would probably die out.
One way cancer can form is if a stem/progenitor cell got a mutation and fails to produce a non-stem cell daughter in each division. This means that it proliferates to form a tumor.
Another way is when a daughter cell fails to differentiate normally and instead proliferates to form a tumor.
Explain the life cycle of a retrovirus
Introduction of nucleocapsid into cell
Association of reverse transcriptase and integrase with viral RNA
Synthesis of viral ssDNA by reverse transcriptase
Removal of viral RNA and synthesis of viral dsDNA by reverse transcriptase
Integration of viral dsDNA into cell chromosome to form provirus
Transcription of provirus by host cell RNA polymerase II
Translation of viral RNA to make new viral proteins
Assembly of viral proteins and viral RNA genomes into progeny virions
Progeny virions leave cell and initiate new infectious cycle
Retroviruses contain a promoter for transcription in a region known as the long terminal repeat. This promoter allows for transcription of the whole viral genome as pre-mRNA
Gag encodes the capsid proteins that surround the whole nucleic acid
Pol encodes the reverse transcriptase
Env encodes the envelope protein found in the plasma membrane surrounding the virus
How does the Rous Sarcoma Virus (RSV) cause cancer in chickens?
RSV has an extra Src domain that it picked up randomly when it integrated into a genome.
V-Src is missing 18 amino acids at its C-terminus that is for tyrosine phosphorylation. This is usually where it is phosphorylated to be inactivated.
Because Src is non-receptor tyrosine kinase, v-Src is constitutively active.
Src is one of the central proteins that sends signals to tell the cell to divide.
This means that v-Src is an oncogene that causes cell proliferation. The more the cell proliferates, the higher chance it’ll accumulate more mutations and alter another gene.
What is an oncogene and a tumor suppressor gene?
How can oncogenes be created?
Lecture 2:
What is the function of Src and why does it cause cancer?
Src is one of the central proteins that sends signals to tell the cell to divide
What are the properties of transformed cells? How are they different from normal cells?
Altered morphology (rounded shape)
Loss of contact inhibition
Ability to grow without attachment to solid substrate (anchorage independence)
Ability to proliferate indefinitely (immortalization)
Reduced requirement for exposure to mitogenic growth factors
High saturation density (ability to accumulate large numbers of cells in culture dish)
Inability to half proliferation in response to deprivation of growth factors
Increased import of glucose
Glycolysis in presence of oxygen
Tumorigenicity
What are the different ways that proto-oncogenes are converted to oncogenes by retroviruses with examples?
Oncogene is mutated: v-src
Oncogene is identical to normal cell protein, bt made from different promoter (LTR) so it is overexpressed: Insertional mutagenesis with Avian Leukosis Virus—80% of the incorporation is near Myc gene where the LTR inserts & overexpresses it
Explain how gene therapy trials lead to the discovery of an oncogene in leukemia
The hypothesis was that if someone is missing a gene, maybe retroviruses can insert that missing gene.
This was tested on kids born with X-linked Severe Combined Immunodeficiency (X-SCID).
These kids cannot survive in a normal environment.
The idea was to extract stem cells in the bone marrow, infect them with the retrovirus, and put back into the patient.
9 out of 10 boys were cured, however 4/10 developed leukemias.
3 had activation of the LMO2 gene, which created pre-leukemic cells.
Because retroviruses have random insertion, it happened to be in front of LMO2.
This was not successful because there is no way to control where the retrovirus inserts.
Why does expression of LMO2 in T-cells lead to a pre-leukemic state?
Expression of LMO2 in T-cells prevents differentiation.
This means that cells keep accumulating in the pre-differentiated state
Lecture 3:
Why do NIH mouse 3t3 cells transform with one oncogene when cancer needs 5-7 mutations?
NIH-3T3 cells were cultured extensively until a spontaneous immortal cell line was identified.
This cell line has a mutation in the tumor suppressor gene called p16.
This means that these cells are already “on the way” to forming cancer, so just adding 1 oncogene is sufficient to turn them into cancer.
What is the function of Ras in the cell?
Ras is a weak GTPase.
In its active form, Ras-GTP signals the MAP kinase cascade eventually leading to entry into the cell cycle.
Why does the mutation G12V cause Ras to be in a constitutively active form?
Glycine 12 is at the protein active site. Glycine is also the smallest amino acid.
While Ras can still bind to GTP, when glycine is replaced by valine, a bigger amino acid, static hindrance blocks GAP so the GTP can’t be hydrolyzed.
This leaves Ras constitutively active.
Why are the mutations in Ras dominant in the cell?
Ras mutations are dominant because only one copy of Ras needs to be mutated in order to affect downstream signaling pathways.
Even if the other copy is normal, the “gain of function” mutation in one Ras overpowers the normal copy.
What are receptor tyrosine kinases?
RTKs are catalytic receptors.
Each receptor is a single pass transmembrane protein.
RTKs are activated upon binding of the extracellular ligand.
This causes dimerization and transphosphorylation.
What are the different ways RTKs can be altered so that they are constitutively active—with one example of each?
Growth factors: When simian sarcoma virus infects a cell, the oncogene causes the cell to release copious amounts of PDGF-like Sis proteins into the extracellular space.
PDGF is a growth factor that stimulates proliferation of mesenchymal cells.
Because the Sis oncoprotein is similar to PDGF, it can then bind to PDGF receptors (RTKs) that are on the same cell.
This results in strong stimulation of the PDGF receptors and a large amount of growth-stimulating signals.
This is an autocrine signaling loop.
Growth factor receptors: HER2/ERBB2 is an epidermal growth factor receptor. It is amplified/overexpressed in about 15% of all breast cancers.
When the receptor is overexpressed, there are too many receptors on the cell surface. The chance that they will dimerize without the presence of a growth factor is high.
Another way Her2 can be altered is with a point mutation that deletes the ectodomain of the receptor. This allows for dimerization in the absence of a ligand.
Downstream signaling proteins:
How is the signal from the outside transmitted inside the cells?
Once a signal has bound and RTKs have dimerized, the RTKs will phosphorylate each other.
There are many different RTKs, so the pattern of phosphorylation will be different on to cytoplasmic side.
An SH2 domain binds to specific phosphorylated tyrosines on the RTK.
What is the relationship between growth factors and RTKs? How can these pathways be activated in cancer cells?
Growth factors activate RTKs which activate downstream signaling cascades.
These pathways can be activated in cancer when there is an excess of growth signal, or when the RTKs can activate with little growth signal around.
This will cause over signaling and will force the cell to proliferate.
What is the function of SH2 domain containing proteins in the cell?
SH2 domains are adaptor proteins.
How does the MAPK pathway function? What is the downstream effect of activation of the MAPK pathway?
Growth factor binds
RTK dimerizes and transphorphorylates
Grb2 adaptor protein (SH2) domain binds to the phosphorylated tyrosines on the RTK
The SH3 domain of Grb2 binds to Sos (GEF) which exchanges GDP for GTP in Ras.
Ras then activates MAP kinase kinase kinase (Raf)
Raf activates MAP kinase kinase (Mek)
Mek activates MAP kinase (Erk)
Erk then activates transcription factors Fos and Jun.
Fos and Jun move to the nucleus where they bind to DNA and expresses many genes like growth factors and cyclins.
This transitions the cell from G1 to S phase.
Lecture 4:
Why are mutations in Ras more common compared to other genes in the MAPK pathway?
Oncogenes downstream of Ras are rare than those upstream of Ras.
This is because the further upstream, the more pathways and greater overall affect the oncogene will have.
The further downstream, the less can be affected.
BRAF mutations occur in a site adjacent (V600E) to the phosphorylation site (602) where a valine is converted to a glutamic acid - why does that lead to constitutive action of this protein?
The substitution from valine to glutamic acid causes BRAF to think that it’s active.
The change in charge leads to the a “phosphomimic” that acts as a phosphorylated serine.
How does PI3K activate AKT/PKB to turn on downstream signaling and inhibit apoptosis?
Phosphatidylinositol (PI) is a phospholipid found on the cytoplasmic side of the membrane.
PI kinase leads to phosphatidylinositol-(4,5) diphosphate (PIP2). It added 2 phosphate groups, and is now a substrate for phospholipase C and PI3K.
Phospholipase C cleaves the head group off of PIP2 leading to inositon 1,4,5 triphosphate (IPC) & diacylglycerol (DAG).
IP3 changes intracellular Ca2+ by opening channels.
DAG activates the serine/threonine kinase protein kinase C.
PI3K has an SH2 domain that binds to a phosphorylated tyrosine on an RTK. This activates PI3K which can turn PIP2 to PIP3.
PIP3 is still anchored in membrane, and is a substrate for binding proteins that contain a PH domain.
When PIP3 is created, PH domain containing proteins gather to bind.
A phosphatase called PTEN removes the phosphates from PIP3 and converts it back to PIP2, reversing the product of PI3K activity.
In many cancers, PTEN has been lost. When this happens, other kinases activate the PH domain proteins like Akt, which activate downstream pathways.
More PIP3 means more recruitment of Akt, which is activated and will turn on genes.
Akt can inhibit apoptosis by binding to Bax, which stops pores from opening on the mitochondria. It is pro-survival.
What is the role of p53 in the cell?
p53 is called “the guardian of the genome”
It arrests the cell cycle and blocks proliferation if DNA damage occurs.
IT also stimulates DNA repair, promotes apoptosis, and inhibits the expression of certain oncogenes that suppress apoptosis.
Why was p53 initially thought to be an oncogene?
p53 was originally thought to be an oncogene because mutations in p53 were acting in a dominant way and leading to cancer.
Role of p53 in cell cycle regulation
When DNA damage occurs, a kinase phosphorylates p53 and causes it to dissociate from Mdm2.
p53 can then enter the nucleus and act as a transcription factor for p21, which inhibits a G1/S cyclin-cdk.
What is the relationship between p53 and Mdm2? p53 and Mdm2 regulation
Msm 2 is an E3 type ubiquitin ligase enzyme. Normally, it adds a ubiquitin to p53, marking it for degradation by proteasome.
It keeps the cell cycle going by repressing p53 when there is no cell damage.
In many tumors, Mdm2 is amplified and overexpressed, thus acting to keep p53 lower.
p53 is always being made in the cell. p53 actually makes Mdm2, so it self regulates.
Why are p53 mutations dominant? Is the dominant mutation a gain of function or loss of function mutation?
p53 is a tetramer that binds to DNA.
If any of the subunits are mutated, the entire tetramer is unable to bind.
This is why it acts in a dominant way—mutations inhibit the function of the non-mutated copy of p53 by forming inactive tetramers.
In heterozygotes, only 1/16 tetramers will be functional due to the mutated and wt subunits forming tetramers.
It is the only exception of a dominant tumor suppressor.
Lecture 5:
Homework questions
Why does the knockout of the p53 gene in embryonic stem cells depend on a double-recombination event?
The researchers designed a plan based on homologous recombination to disrupt p53 in mouse embryonic stem cells. They designed a DNA targeting construct that, if homologous recombination were to occur between it and the genomic p53 gene, would remove most of the highly conserved p53 exon 5 and replace it with a neo cassette. Double homologous recombination is required so that this can occur; a crossover in between the normal p53 gene and the targeting construct between exons 2 and 5, and another crossover in between exons 6 and 10 are required to replace exon 5 with the neo cassette. The presence of the neo cassette can be detected with a drug selection.
To make p53 knockout mice, embryonic stem cells heterozygous for the p53
knockout allele are injected into 3.5-day old embryo blastocysts from mice that have a different coat color than the mice from which the embryonic stem cells were derived. The mice are implanted into the uterus of pseudopregnant mice. The mice that are born are referred to as chimaeric. What are chimeric mice and how are they used to generate homozygous p53 null mice?
Chimeric animals have a mixture of two types of genomes among the cells in their body yet they come together to form a whole organism - this is evidenced by mixed patches of different fur color. These chimeric mice are then tested for the ability to carry the mutation in their germ line, and if it is present there, then half of the sperm or eggs produced (haploid cells) will have the p53 mutation. Breeding these heterozygous mice together can then generate homozygous null p53 mice. The key idea here is that once the embryonic stem cells become part of the mouse germ line, then it is straightforward to use genetic crosses to generate homozygous mutant mice for testing.
When generating a null mutation in this type of experiment, it is important to demonstrate that the mice are truly incapable of making p53 protein (or a functional portion of it). How did the authors fully conclude that they had eliminated p53 activity in their mice and why was it necessary to do so for this p53 knockout?
The authors performed multiple tests to ensure that there was no p53 activity in the homozygous mutant mice. They tested the mouse DNA for the desired mutation and then they tested the mouse mRNA, and protein products. They showed that the homozygous p53 knockout animals lacked the highly conserved exon 5, that no mRNA that spanned the site of insertion was produced, and that no protein-containing parts of p53 could be found in the homozygous knockout mice. An alternative hypothesis to explain the viability of the mutation in p53 is that the parts of the p53 gene that remain (for example exons 1 - 4) may still be capable of producing part of the protein that still might have the p53 activity. Thus, it was important to prove that the mutation is a true null mutation that resulted in no p53 protein fragments being made.
p53 is highly conserved in evolution, yet this paper demonstrates that p53 is not essential for proper mouse development. p53 clearly has a relationship to cancer prevention. What are the implications from this study for the role of p53 in animal biology?
p53 null mice are viable and have no developmental defects. This suggests that p53 is not essential, yet the high evolutionary conservation of the protein would suggest that it has an important function. When p53 is lost, the animals have a strikingly high rate of cancer when they are young. This indicates that p53 has an essential role in preventing cancer growth and that there is evolutionary pressure to maintain the p53 function. The correlation between strong evolutionary conservation of protein sequence and "tumor suppression" indicates that mammals depend on p53 anti-tumor activity. The importance of p53 appears to be related to its role in preventing cancer.
How was the role of p53 determined in the cell?
Functions of p53 in apoptosis
Apoptotic cascade—what proteins are pro and anti-apoptotic
Intrinsic and extrinsic signals for apoptosis
Lecture 6
What does the loss of heterozygosity (LOH) mean in a cancer cell? What are the different ways LOH can occur?
Why is it necessary to lose the second copy of Rb to cause retinoblastoma
What is the function of Rb in the cell and why does loss of Rb lead to cancer?
How does Rb control the cell cycle?
What is the restriction point in the cell cycle and why is it necessary?
Explain the different components in cell cycle progression—what is the role of cyclin/cdks and CIPs and INKs?