Lecture 2, 4 - Cancer Evolution Raych. Flashcards
2011 - In what ways are cancer cells different from normal cells? How do the differences arise
a. avoid apoptosis
- override cell cycle checkpoints
- become independent at GF signalling
- change biosynthesis
- acquire ability to migrate
- induce angiogenesis
- avoid immune system
- spread to distal sites
- no limit to cell division
b. these differences arise due to mutations that result from genomic instability. Mutations build up over time to result in cancer phenotypes
Properties of Tumor Cells + Emerging Hallmarks
- sustaining PROLIFERATIVE signalling
- evading GROWTH SUPPRESSORS
- resisting CELL DEATH
- inducing ANGIOGENESIS
- activating INVASION and METASTASIS
- Enabling replicative IMMORTALITY.
Emerging Hallmarks
- avoid IMMUNE destruction
- tumor promoting INFLAMMATION
- Genome INSTABILITY and MUTATION
- Disregulating cellular ENERGETICS
Warburg Effect
- Cancer cells undergo increased rates of lactate production under aerobic conditions, aerobic glycolysis
- Oncogenic changes (ex +Ras or -p53) + Hypoxia -> increased HIF-1a -> increased glycolysis.
- HIF-1a is induced by hypoxia. Encites other METABOLIC changes such as increase in glycolytic enzymes, and ^ in glutamate uptake.
How does a tumor utilize immune cells”?
- macophages: TAM1: is anti-tumor, but is induced to TAM2: which is pro-tumor and is associated with wound healing and promotes tumor development.
- Tumor secretes signals to suppress the immune system:
1. TGF-B: secreted by tumor and TAMS - promotes M2 development, inhibits NKs and dendritic cells, suppresses CD8 cells.
2. IL10: Secreted by TAMS. anti-inflammatory cytokine that favors tumor escape from surveillance.
3. Arginase 1: marker for m2 TAM. Generates polyamines = disregulation of the TCR = unresponsive CD8
PD1L
PD1 is found on the T cell.
PD1-L is found on the tumor cell. It binds to PD1 and silences the T cell.
This is a target of immunotherapy (try to block this binding)
Mutations in these pathways lead to immunosuppression
Hyperactivation of following pathways
- EGFR
- PI3K (ko PTEN)
- MAPK (ko BRAF)
*What epigenetic changes in a cancer cell should you look for”?
- CpG island in the promoter region of TSG leads to inactivation and allows tumor to develop
- De-acetylated histones at site of TSG -> inactive chromatin.
- Look for “inactive” chromatin markers at: H3K9me, H3K27me
- How do you look for epigenetic changes?
- Bisulfate sequencing to analyze changes in DNA methylation.
- compare normal vs cancer genome for CpG methylation and look at where there are differences. - Chromatin-IP using antibody against modified histones, and then deep sequencing to ID modification sites
- compare chromatin markers at “inactive” sites H3K9 and H3K27 and look for methylation.
- this can be done using ChIP-Seq.
*How do you detect chromosome copy number changes in cancer”?
aCGH: Array Comparative Genomic Hybridization
- probes are made from normal DNA and tumor DNA, each with a different color
- If both anneal, you will get a yellow signal.
- If tumor anneals more, there is a duplication. get a green signal
- if normal anneals more, there is a deletion. get a red signal. Look for the green and red signals.
- this has been largely replaced by WGS or WES.
RNA expression Analysis for genetic and epi changes
compare RNA levels of tumor vs control. ID where there are abnormal expression levels.
- for example, can look at amplification of HER2/Neu.
- expression signature may indicate prognosis and five year survival rate.
- based on if a gene is over expressed or underexpressed.
- can predict THERAPY FAILURE.
*How does genomic instability contribute to microevolution of cancer cells? / Discuss how mutations and epigenetic changes synergize in the development of a tumor
cell, referring to the hallmarks of cancer cells. (2017)
- genomic instability not only contributes, but is ESSENTIAL to the dev of cancer
- different types of cancer cells (stem and progenitor) must acquire the right amount of mutations over time in order to allow for (6 essential functions).
- these mutations must not be established too early or the tumor will die. So genomic instability grows over time as the cell gets new mutations
- genomic instability PROVIDES the microevolution needed by tumor cells to become established and then gain new functions such as angiogenesis and metastasis.
2013*How does expression profiling allow determination of the prognosis of a cancer patient? Give example.
- RNA expression profiling looks at the transcriptome to identify genes that have changes in expression. These changes may correlate with cancer development.
- Certain genes are more highly expressed in certain cancers.
- EX: RNA expression profiling may give a breast cancer patient information about what genes have changes in expression. This may help determine prognosis and can also predict therapy failure. An example is the upregulation of HER2 as a prognostic indicator.
2013* You have IDed a new protein that has similar seq homology as MDC1. You want to see if it has any role in the DNA damage response pathway. Design expts to determine:
a. is it modified by ATM?
b. involved in recruiting BRCA1?
c. required for the activation of cell cycle checkpoints?
a. phos of MDC1 by ATM
b. Focus formative assay to check if BRCA1 is recruited by MDC1. If we KO MDC1 in control, there will be NO recruitment to (IC5) if MDC1 is involved in recruitment.
c.
2012* How do gene expression arrays help diagnosis of cancer types?
Gene expression arrays allow you to see what genes are amplified or deleted in cancer types, as compared to a normal control. They can also tell you the timely manner in which cancer causing genes are being expressed.
- can also show comparative gene expression of different SUBTYPES of the same cancer
- this info can give info on prognosis and what genes to target for treatment.
2012* You have isolated a ssDNA binding protein. What expts would you design to show it participates in DNA response?
How would you show it is required for activation of ATR following DNA damage?
colocalize fluro
coIP
KO
