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Copy of cancer biology synthesis

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R. Aaron Ramey

on 4 December 2013

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Transcript of Copy of cancer biology synthesis

Cancer Synthesis: Cancer as a Progressive Process
Hyperplasia
cells appear normal but undergo over-proliferation.
Dysplasia
Many mutations have accumulated, but cells have not yet invaded the basement membrane.
Neoplasia

Tumor cells invade underlying tissue by breaking basement membrane; the tumor has become invasive. considered malignant
Angiogenesis
When tumors have penetrated the basal membrane, blood vessels extend into the tumor to supply the cell mass with nutrients and oxygen.
Accumulation of Mutations leads to Cancer Progression
External Sources
UV Rays, X-rays, carcinogenic chemicals etc.
UV rays
Carcinogenic Chemicals
Viral infection
Mistakes in DNA replication
X-rays
Example:
Higher incidences of leukemia have been detected in humans exposed to x-rays.
Example:
An example of amplification?
3. Small intragenic changes which cause protein structural changes
Example of chromosomal translocation?
Proto-oncogenes to Oncogenes:
- Constitutive activation of genes encoding protein products that promote cell survival and proliferation.
- Dominant mutations (gain of function)
Example of over expression?
Cells sometimes accumulate mutations which cause receptors to become activated and induce cell survival and proliferation even in the absence of growth factors.
Some cells produce their own growth factors which subsequently activate their own receptors and initiate survival and proliferation.
For Example:
Example:
Staining for EGF receptor
TGF-alpha ligand stain
(ligand for EGF receptor)
Overlay:
Yellow represents co-expression of receptor and ligand. Co-expression is an indication of autocrine signaling with in this case is an oncogenic mutation.
Burkitts lymphoma involves a chromosomal translocation between chromosomes 8 and 14.
basement membrane
Cells begin to appear abnormal:
- nucleus
is

bigger than normal
- increased nuclear staining
- increased mitotic activity
Stroma
Tumors require angiogenesis for metastasis to occur!
Metastasis:
The process of tumor cells spreading from the original tumor site to another part of the body.
Don't worry! This will be explained throughout the presentation
What types of genes are mutated to cause carcinogenesis?
???
Now lets get specific...
But wait...
How exactly are proto-onco genes changed to oncogenes?
I'll give you a hint...
it's all in the DNA!
Multiple copies of genes resulting in over production of certain proteins.
Types of Mutations that convert proto-oncogenes to oncogenes
I'll give you a hint...
It's all in the DNA!
Amplification
Gene A
Gene A
Gene A
amplification of myc in human childhood neuroblastoma
Over-expression
Sources include promoter and enhancer mutations, as well as provirus integration.
Gene A
Mutation in Gene A promoter causes it to be constitutively expressed.
Small intragenic changes, or, point mutations
Some types of point mutations
Chromosomal Translocations
Rearrangement of parts between non-homologous chromosomes. Fusion of the gene X promoter with gene Y may result in consitutive expression of gene Y
Benign
Malignant
A tumor which has not broken through the basal membrane. It is confined to one tissue/organ.
A tumor which is broken through the basal membrane and has the ability to invade surrounding tissues and organs.
RSV transforming tumor virus
Introduces and transcribes src, which promotes cell proliferation
Over-proliferation of cells with c-terminal truncation of EGFR.
Probe to HER2 in breast carcinoma cells: clear amplification in RNA levels, indicating increased levels of gene expression.
Gene X
Gene Y
Gene X
Gene Y promoter
3. Other Important Receptors
So that's it, right?
I'm afraid it's not that simple, here are some more potential oncogenes.
E. Wnt Signaling
F. Integrin Induced Signaling
with oncogenic potential
- Ligands include ECM components

- Ligand depends on alpha/beta subunits

- When Ligands bound, integrins cluster to form focal adhesions.
- focal adhesion formation may activate signaling pathways.
Responses downstream include:
Ras activation...

cell migration proliferation survival
When receptors dimerize, JAK1 and JAK2 transphosphorylate to activate each other.
once activated both kinases phosphorylate c-terminal tail of cytokine receptor.
JAK1 and JAK2(tyrosine kinsases for this system) are non-covalently linked to cytosolic side of the receptor.
Wnt binds and activates Frizzled cell membrane receptor
With Wnt
2. Ras
and downstream signaling proteins
First of all, ras is a small g-protein which is mutated in 30% of cancers. Ras is activated by GEF, which "kicks off" GDP and binds GTP. GAP induces GTP hydrolysis and de-activates Ras
Let's take a closer look at the events leading up to the activation of ras...
Upon activation receptor molecules like RTK can bind SH2 domains at phosphotryosine sites (in this case, the adaptor protein Grb2).
Grb 2 has SH3 domains which can then bind sos, a GEF which is then able to activate ras!
Once ras is activated, it can signal downstream proteins in multiple pathways. Let's focus on three of them:
Ras
PI3K
MAP
kinase pathway
Ral-GEF
Integrins can also activate ras!
Erk (a kinase) is activated, so what?
PIP3 can then activate proteins with PH domains like Akt*
PIP3 is dephosphorylated (and deactivated) by PTEN
stimulates growth
By activating the mTOR pathway
Ral-GEF activation promotes cell motility
Downstream effect? Called the Jak/STAT pathway!
Most Important downstream effects:
transcription of proliferation and cell survival genes like...
myc
cyclin D2
cyclin D3
1
2
anti-apoptotic proteins like...
Bcl-X
How???
*BUT ALSO- can activate PI3K/Akt and MAPkinase pathways.
Okay, but how?
phosphorylation of FAK creates a phosphotyrosine for SH2 domains to bind
multiple receptors can activate IKK

- myc (cell division)
- c-cyclins (cell division)
-Bcl-2 (anti-apoptotic)
NF-kB can now go the nucleus and transcribe...
Wow, so I only need one dominant mutation on any one of those proteins and I'll have cancer? That's actually not as complicated as I thought it would be.
Not quite, cancer only really develops after the accumulation of multiple mutations. In addition to oncogenes, these mutations actually often include recessive mutations in what we call tumor-suppressor genes. It's all quite complex.
learning is hard.
Tumor Suppressor Genes
- Typically encode growth-limiting proteins
-Recessive or epigenetic events contribute to cancer development.
- loss of both functional copies of tumor suppressor gene is required for cell transformation
1. First mutation of tumor suppressor gene is a rare event.
mutant tumor suppressor gene allele
* still functional copy of tumor suppressor gene available
(heterozygous)
2. Loss of heterozygosity (LOH) is not as rare...
2 copies of mutant allele=mutant phenotype
Why?
A.) Mitotic Recombination
the exchange of DNA between homologous chromosomes
B. Gene Conversion
STAT SH2 domains can bind phosphotyrosine, facilitating phosphorylation by JAKs
phosphorylated STATs can dissociate from docking site...
...dimerize
...Act as a transcription factor
Grb2/Sos complex is also formed by activated RTKs! (to be discussed)
loss of function mutations
"unidirectional transfer of genetic information"
C. Epigenetic changes
methylation of tumor suppressor gene promoter
TS-gene
TS-gene
promoter
promoter
(Methylated)
(unmethylated)
transcription
no transcription
NF-kB pathway
Cytokine Receptors
Hmmmm... well I guess PTEN because it counteracts PI3K by dephosphorylating PIP3 to PIP2.
I bet you can already identify some tumor suppressor genes just with that little bit of information! Think about it, what protein products inhibit some of the oncogenic pathways we have talked about so far?
TSC2, Bad, TE-BP-1... hmm, those all seem so specific though, are there any tumor suppressor genes that have more widespread affects?
Right! Can you think of any other proteins that counteract some of those pathways?

Yes! You're becoming quite intuitive Mr. Triangle.
Rb
A TSG that plays a critical role in cell cycle progression, but how?
The
Cell Cycle
To start, know that cell cycle progression depends on cyclin-CDK complexes formed
at different stages
in the cell cycle.
D-CDK 4/6 complex forms during G1 and controls whether or not the cell passes the R point, this is when the role of Rb is most important.
But why?...
D-CDK 4/6 complex phosphorylates Rb, which makes it a good substrate for E-CDK2 to hyperphosphorylate.
And when Rb is hyperphosphorylated...
hypo- or unphosphorylated Rb binds and blocks E2F transcription factors
It releases E2F transcription factors, which recruit HATs
Genes transcribed in response to Rb hyperphosphorylation are responsible for cell entrance into S-phase!
CKIs
[the other major tumor suppressor gene]
So what are its targets?
14-3-3 sigma
B-CDK complex
[[G2 Arrest]]
p21
[[G1 Arrest]]
Fas
Bax
p53AIP
APOPTOSIS
GADD45
DNA repair
MDM2
MDM2 is an ubiquitin ligase that maintains low p53 levels under normal cell conditions
DNA damage
ATM and ATR
Over proliferation
Arf
epithelial
tissue
stroma-mesenchymal tissue
(note, this is just one example)
sources?
lets take a look at some carcinoma cell DNA and find out!
1 Receptor Tyrosine Kinases
(some)
D-CDK 4/6
Arf, ATM, and ATR all block MDM2 under certain cellular stresses
[allows for p53
to accumulate]
Let's take a step back. Normal cells may proliferate, but they have limitations to their replicative capacity. Cancer cells are able to escape these limits.
Right, cell immortalization
Cells stop replicating as a
Senescence
many cancer cells exhibit immortality
accumulation of enough mutations, cells may surpass senescence
result of accumulation of physical stress
But what if cells surpass senescence?
After a limited number of replications, cells will undergo
Cells enter
CRISIS
APOPTOSIS
HOW DOES CRISIS HAPPEN?
it is induced due to telomere shortening
"T-loop"
telomeres have 3' overhangs which protect genes from degradation
telomeres shorten with each generation, until they are two short to protect chromosomal ends
Result? Breakage fusion bridge cycle
[and eventually apoptosis]
How are cancer cells able to escape senescence AND crisis though?
Escaping senescence...
LT=SV40 large T antigen
Suggests...?
T- antigen sequesters Rb and p53
p53 and Rb are essential for senescence!
Escaping Crisis...
expression of telomerase
- hTERT activation
- alternative mechanisms
I'm still having a hard time understanding how all of this fits together. Please Mr. Circle, make me understand!
I think it's time to talk about multistep tumorigenesis
The disruption of a combination of ___ different pathways appears to be commonly required to change normal cells to cancerous cells.
5
let's take a look!
carcinoma
So what are these 5 pathways?
ras
pRb
p53
telomeres
PP2A
*the mechanism is not always the same though
*This is the
most common
combination of pathways, not the
only
combination
For example, one study found that many colon cancers followed a specific sequence of pathway interruptions at different stages of cancer development.
Even though common pathways are disrupted, tumors are
heterogeneous
*A stem cell population gives rise to transit amplifying cells that can proliferate a limited number of times.

* If there is a second mutation, gives rise to new.
Transient amplifying cells can also accumulate mutations, which contributes to
heterogeneity.
Now we need to discuss cell interactions with their environment, and how that effects cancer development. Lets start with inflammation
?
inflammation
Multiple studies have shown that chronic inflammation is associated with cancer development
for example:
*initiator

*promoter
mutagen
pro-inflammatory factor
Only initiator or promoter? NO CHANGE
pro-inflammatory factor added after mutagen?
initiator and promoter added together?
*
*
inflammatory factors promote pro-tumorigenesis cellular behaviors
How Exactly? Look at the pathway!
Carcinomas are dependent of support from stromal cells
tumor epithelium
tumor
associated
stroma
regular
(liver)
tissue
These heterotypic interactions resemble
the epithelial-stromal interactions found at wound sites
In wound healing, cells undergo
EMT
in order to migrate to wound space, then revert to epithelial state in MET.
Relevance to cancer? Metastasis!
MAP kinase Pathway
* ERK
activated
cell proliferation
cell motility
chromatin
compaction
cell proliferation
Translational Control
EMT stands for

epithelial mesenchymal transition
.

Also, notice the role of
angiogenesis
in wound healing. Remember when we mentioned it is required for cancer development earlier?
*
*
*
*
Evidence?
Fibroblasts
promote tumor growth and survival
(mesenchymal cell type)
Macrophages
promote angiogenesis
suggests macrophages (involved in wound healing) promote angiogenesis, which is required for tumor growth and survival
I just realized something Mr. Circle, I get what
metastasis
is, but I really have no idea how it works
Mr. Triangle, you picked a perfect time to bring this up, let's take a closer look at metastasis, and what processes are involved in it!
Let's start at the
primary tumor
in epithelial tissue (a carcinoma)

Metastasis required a number of complex steps.
Primary Tumor
Primary Tumor:
- hypoxia induces
HIF-1
activation
*
PDGF, VEGF, TGF-beta expression
- Stromal cells recruited to tumor, also contribute to
ANGIOGENESIS
stromal cells
...and, release MMPs

...promotes EMT...
Cancer cells undergo EMT to increase motility and invasivness.
Here are some cellular behaviors associated with EMT.
...and here are some protein expression changes associated with EMT
EMT
Stromal cells release signals that induce EMT
*they also induce carcinoma cell expression of transcription factors that are critical for EMT such as
Snail
,
Twist
, and
Slug
, which also appear to be required for metastasis
[induces ECM degradation]
EMT and ECM degradation allows for
Localized Invasion
Intravasation
Transport through circulation
arrest in microvessels of various organs
formation of micrometastasis
macrometastasis established in new site;
*colonization
successful
*Akt*
Promotes cell survival
regulate cell cycle progression by inhibiting the formation of cyclin-CDK complexes necessary for cell cycle progression.
A transcription factor when activated can induce cell cycle arrest, promote DNA repair, and induce apoptosis in extreme conditions.
That's pretty cool Mr. Circle! I want to learn more about these 'heterotypic interactions'.
Okay Mr. Triangle, now that you have better grasp on heterotypic interactions, lets take a look at some recent discoveries which have been made in this field of cancer research.
Heterotypic interactions between mesenchymal cells, specifically
cancer-associated fibroblasts
, and cancer cells leads to
EMT
and gain of
cancer stem cell properties
.
These changes lead to increased aggressiveness of the cancer and a higher occurrence of
metastasis
Recall:
We saw
MMPs
in the context of wound healing, but here they act in cancer cells to cleave the ECM and release cytokines which promote both
angiogenesis
and
EMT
. Both changes are necessary for carcinoma growth and metastasis. Part of the reason carcinomas require stromal cells is for access to proteins such as MMP
MMPs are released by CAFs. In order to ensure cancer cells receive MMP, they induce regular fibroblast transition to CAFs by releasing
IL-6.
In some cases, cancer cells have the ability to transform normal cells using microvesicles (MVs).
researchers found that tTG and FN were responsible for the transformative ability of cancer cell associated MVs
I think it's safe to say I am an expert on everything there is to know about cancer.
So naive.
CAFs reprogram to
Warburg metabolism
in result of contact with carcinoma cells.
Warburg Metabolism is the usage of anaerobic glycolysis in place of oxidative phosphorylation.

- glucose transporters
- glycolytic enzymes
- pyruvate dehydrogenase kinase 1 (prevents pyruvate from entering kreb cycle)
HIF-1 plays a major role in reprogramming by up-regulating :
Cancer cells up-regulate lactate importers (MCT1) and down-regulate glucose importers.

Now cancer cells may undergo aerobic respiration using lactate from CAFs
result?
increased cell survival and tumor growth
Cancer associated fibroblasts (CAFs) and carcinoma cells are capable of reciprocal metabolic reprogramming
Full transcript