Watson Lectures Flashcards
Pre-Molecular Era (Important Concepts)
- Cancer is a disease of the cells
- Cancer cells have become immortalized
- Properties of cancer cells are transmitted from parental to daughter cells.
- Cancer involves changes in DNA (the vehicle of heredity)
How can you detect Oncogenes in Human Tumours?
1) DNA Transfections.
To detect oncogenes in human tumors, DNA from tumor cells is prepared and co-precipitated using a phosphate buffer and calcium ions, forming a calcium phosphate-DNA complex. This DNA is then introduced into NIH/3T3 cells, a line of mouse fibroblasts, which are cultured for two weeks. Transformed NIH/3T3 cells, which grow distinctively as foci among untransformed cells, indicate the presence of oncogenic sequences in the tumor DNA.
How does Ras oncogenes get activated? Where are they found?
- They get activated by Point Mutations.
- Ras mutations in tumours usually involve coodns 12, 13, 59, and 61.
- Mutated ras is found in 50% of colorectal cancers and 95% of pancreatic cancers.
- plays a major role in tumour maintenance.
Ras GTPase
1) Exists in 2 states.
- Active: GTP bound.
- Inactive: GDP bound.
2) Ras is a crucial regulator of:
- cell shape
- motility
- growth
What are some different characteristics found in normal cells and tumour cells?
1) Density-dependent inhibition of growth:
normal = present
tumour = absent
2) growth factor requirements
normal = high
tumour = low
3) anchorage dependence
normal = present
tumour = absent
4) proliferative life span
normal = finite
tumour = indefinite
5) contact inhibition of movement
normal = present
tumour = absent
6) adhesiveness
normal = high
tumour = low
7) morphology
normal = flat
tumour = rounded
What is cancer caused by? And what are the 2 categories of Cancer Research?
- Cancer is caused by the accumulation of mutations that result in the activation of oncogenes and the inactivation of tumour suppressors.
2 categories:
- tumour suppressor genes (inactivated)
- proto-oncogene becomes an oncogene (activated)
Onocogenes
- frequently amplified in human tumours
- can be identified as alterations in chromosome structure
- take the karyotype (all chromosomes of cancers) and visualize them.
- oncogene amplification can be identified as alterations in chromosome structure.
Proto-oncogene amplification
Multiple copies of proto-oncogenes have been found in various tumours.
1) homogeneous staining regions (HSRs): Areas in chromosomes where amplified genes are integrated, resulting in uniform staining during karyotyping.
2) double minute chromosomes (DMs): Circular extrachromosomal DNA fragments that carry amplified oncogenes and segregate with cell division.
In studies with 3T3 fibroblasts, DMs were analyzed, revealing three genes—MDM1, MDM2, and MDM3—on these structures. The MDM2 gene was identified as responsible for the transformation phenotype, demonstrating its role as an oncogene.
function of p53 & its negative regulator
- MDM2 is a negative regulator of p53. (promote cancer growth)
- p53 act as checkpoint for entry into S phase.
lack of nucleotides, UV radiation, ionizing radiation, oncogene signalling, hypoxia, blockage of transcription, all lead to activation of p53, which will activate cell cycle arrest/senescence/return to proliferation, DNA repair, block of angionensis, apoptosis. (inhibit cancer growth - tumour suppressor gene)
What is the main oncogene present in breast cancer?
- 20-25% of breast cancer have amplification of the HER2 RTK (EGFR family)
- HER2 is a prognostic factor and therapeutic target
- a prognostic factor provides information regarding patient outcome at time of diagnosis and provides information on the likelihood of response to a therapeutic.
- HER2 is important for breast cancer growth and is accessible as a cell surface receptor.
One thing that is important for HER2 and other receptor tyrosine kinases, they are on the cell surface of cells, can identify therapies such as monoclonal antibodies that target signal
What is the personalized medicine that will target HER2 (breast cancer oncogene)?
Herceptin or Pertuzumab (monoclonal antibody 2C4, similar strategy to herceptin)
will block HER2.
HER2 monoclonal antibodies:
- inhibited proliferation of HER2 over expressing tumour cells.
- inhibited in vivo growth of HER2 tumours in nude mice.
- Genentech developed a humanized monoclonal antibody (Herceptin)
- HER2 amplification in breast cancer - Chromosome 17q. (can visualize using DNA - southern blot, RNA - northern blot, protein (immunoprecipitation)).
Identifications of Oncogenes at Chromosomal Breakpoints
- Creation of the Philadelphia chromosome. Found in >95% of CML patients.
- chromosome 9 and 22 contain the genes c-abl and bcr at their breakpoint, respectively.
- formation of bcr-abl chimeric gene.
- formation of bcr-abl chimeric RNA.
- formation of bcr-abl chimeric protein.
- Bcr-Abl rearrangement present in 98% of CML.
How do we target Bcr-Abl (CML)?
- Abl is a tyrosine kinase (cytosolic - not transmembrane - hence not suitable to target mAb type therapies). Instead, develop small molecule inhibitors to the ATP binding pocket of the tyrosine kinase domain.
- the small molecule, Gleevec, is defined to fit into that ATP binding pocket of the Bcr-Abl tyrosine kinase, this competes with access to the activate site.
- Gleevec “personalized medicine” for CML.
How can we test the efficacy of potential small molecule inhibitors?
- must develop in vitro screens.
In normal BaF3 pre-B cells, growth requires IL-3, and they die when IL-3 is removed. However, BaF3 cells transduced with Bcr-abl continue to grow without IL-3, showing that Bcr-abl acts as an oncogene; Gleevec is not toxic to normal cells but specifically inhibits cells with Bcr-abl.
Patients in the acute blast crisis phase of CML often develop resistance to Gleevec due to mutations in the ATP-binding pocket of the Abl-kinase domain. These mutations occur in critical amino acids for Gleevec binding, leading to the need for alternative inhibitors targeting these resistant mutants.
Why did alternative inhibitors, for example for CML, need to start being developed?
- Patients in acute blast crisis of CMLoften develop resistance to Gleevec
caused by mutations in the ATP
binding pocket of Abl-kinase domain. - Many of the mutations in patients are
found within the same amino acids in
Abl kinase-those critical for binding
Gleevec.
Hence can start to develop
alternate inhibitors that would target
the mutants!
What are some strengths and weaknesses of anti-receptor antibodies versus low-molecular-weight tyrosine kinase inhibitors as anti-cancer agents?
1) Target:
Small molecule: tyrosine kinase domain
Antibody: receptor ectodomain
2) Specificity:
Small molecule: +++
Antibody: ++++++
3) Binding:
Small molecule: most are rapidly reversible
Antibody: receptor internalized, only slowly regenerated
4) Dosing:
Small molecule: oral daily
Antibody: intravenous, weekly
5) Distribution in tissues:
Small molecule: more complete
Antibody: less complete
6) Toxicity:
Small molecule: rash, diarrhea, pulmonary
Antibody: rash, allergy
7)Antibody-dependent cellular cytotoxicity:
Small molecule: no
Antibody: possibly
Some translocations don’t lead to a new chimeric protein, but uncouples the gene from its regulating elements. What is an example?
- The Myc gene can become deregulated through various mechanisms, disrupting its normal function.
- Myc is a type of protein in the basic helix-loop-helix family.
- It forms heterodimers (pairs) with other proteins to bind DNA and regulate genes.
- Myc-Max: Promotes transcription of genes containing an “E box” in their promoters.
- Mad-Max: Represses the same genes.
- As cells differentiate, Mad protein levels increase.
- This displaces Myc from the complex, reducing Myc-Max activity.
- The decrease in Myc-Max activity promotes differentiation and slows proliferation.
- Myc regulates genes that control the cell cycle and proliferation, including:
- Cyclin D2 and CDK4: Promote cell cycle progression.
- Cul1 and E2F: Important for DNA replication and S phase entry.
- Myc-Max complexes promote progression through the S phase of the cell cycle.
Identification & Isolation of Tumour Suppressor Genes
- Usually multiple genetic changes in any single tumor.
- tumor suppressors are usually recessive.
Retinoblastoma (Rb)
1) Role of Rb Gene:
Rb gene prevents uncontrolled cell division (tumor suppressor).
Loss of Rb function predisposes cells to tumor development.
2) Familial Retinoblastoma:
Zygote carries 1 defective Rb copy; all cells have only 1 functional copy.
A somatic mutation in a retinal cell eliminates the functional Rb copy, leading to tumor formation.
Patients are prone to bilateral tumors (both eyes).
Higher risk of second tumors later in life.
3) Sporadic Retinoblastoma:
Both Rb copies must be inactivated by 2 sporadic mutations in the same retinal cell.
Rare; typically causes a single tumor in one eye.
4) Mechanisms of Rb Loss:
Mitotic recombination: Exchange of genetic material during cell division.
Gene conversion: One allele is replaced by another, eliminating the functional Rb copy.
5) History of Rb Research:
1914–1984: Rb patients studied; bilateral cases showed higher second tumor risks.
1986: Rb gene cloned using loss of heterozygosity (LOH) in chromosome 13 from tumor tissue.
Gene walking mapped deletions to pinpoint Rb gene location.
6) Key Facts:
Familial Rb: Germline mutation + somatic mutation.
Sporadic Rb: Two somatic mutations required.
Role of cyclin D1, Cdk4 and Rb in cell cycle progression
- Rb controls cell cycle progression by switching between active and inactive states through phosphorylation.
- In M/G1 phase, Rb is dephosphorylated (active) and stops cells from progressing.
- In early G1 phase, Cyclin D-CDK4/6 partially phosphorylates Rb (hypophosphorylation), starting to inactivate it.
- At the restriction point (R), Cyclin E-CDK fully phosphorylates Rb (hyperphosphorylation), fully inactivating it and allowing entry into S phase.
- Rb and p53 act as checkpoints to prevent uncontrolled cell division.
- If Rb is lost, cells bypass control, leading to continuous cell cycle progression.
What are different hallmarks of cancer and why are they useful?
- they are useful because by understanding the biology oncogenes and tumour suppressors the field has found common hallmarks that are frequently found in cancer.
1) Sustaining proliferative signalling.
2) Evading growth.
3) Activating invasion and metastasis.
4) Enabling replicative immortality.
5) Inducing angiogenesis.
6) Resisting cell death.
What is the definition of Genome and what is meant by Genomics?
Genome: fusion of the words gene and chromosome.
Genomics involves the quantitative analysis of DNA, RNA, and epigenetic using technologies such as microarray and sequencing.
Cytogenetic Tools/Genomic Methods and Technologies (DNA)
- Cytogenetic methods were the first tools that enabled genomic analysis of chromosomes.
- First genomic maps were constructed by staining chromosomes with Giemsa (G-banding).
- Dark regions have low GC content and are gene-poor, while light regions are gene-rich and have high GC content.
1) Can see on a karyogram.
2) Spectra karyotyping (SKY) uses fluroescently labeled DNA probes to colour chromosomes allowing detection of translocations, deletions and amplifications.
3) Array comparative genomic hybridization (CGH):
- can measure genome-wide copy number aberrations at high-resolution to detect amplifications and deletions.
- Use probes to look at different regions of the genome and compare the DNA we get from tumours vs DNA we get from normal blood, can look at all the copy number alterations by comparing to a reference set (which would be normal DNA).
4) Affymetrix SNP arrays (for copy number)
(what was used in the cancer research atlast project)
What are some problems with Genomic Methods and Technologies (RNA)?
1) Northern analysis of gene expression.
- Amount.
- Limited to few known genes/scalability.
2) PCR based analysis of gene expression.
- limited to known genes/scalability.
3) Microarray of gene expression.
- thousands of known and unknown genes analyzed at one time.
- nanoscale technology.
The use of expression profiling for discovery in breast cancer.
Problem:
- cancer development is a complex process of selection for multiple genetic events that provide phenotypic advantages to tumour cells for growth, survival, metastasis and drug resistance.
- any one of numerous combinations of genetic defects can lead to similar cancer phenotypes.
- many oncogenic changes result in altered gene expression.
- personalized medicine!!!
- one direction-do gene expression changes reflect common genetic events:
-high-throughput analysis of many thousands of genes in numerous cancers to identify prevalent targets for drug discovery and diagnostic development. - development of a more global understanding of gene expression profiles.
- diagnostic and prognostic and biomarkers of response.
- identification and validation of new targets.
- predicting function and efficacy of drugs.
Cancer functional genomics
- global (genome-wide) experimental appraoch to assess gene function in normal physiological and pathological processes.
experimental approach:
1) Tumour/normal tissue banking
2) analysis of gene expression (microarray analysis - glass slides coated with thousands of oligonucleotides probes)
3) correlation of microarray results with pathology and follow-up.
4) early detection/prognosis/tailored therapy
can lead to construction of a gene expression matrix, the GEM is in a computer readable format. The expression profile is generally visually represented by colour bars-each colour representing a level of expression of a gene in a particular sample.
- affymetric microarray
- gene expression heat map
(rows correspond to each gene on the chip, colour represents the expression level of the gene, columns represent each sample.)
How to analyze microarray data?
1) Class Discovery
“is there a natural classification of the data?”
Choose genes in an unbiased manner.
“unsupervised clustering”
-> identifies distinct molecular subtypes of breast cancer (whole tumour expression profiling) -> then can make an immunohistochemical profile, and get personalized recommended treatment.
mRNA gene expression analysis allows categorization of heterogenous tumour subtypes that can inform patient outcome and therapeutic decision-making
2) Class Distinction
“what are the genes which distinguish between known classes?”
Choose genes biased for the sample classes.
“supervised clustering”
First Generation Sequencing - Sanger Sequencing
- Fred Sanger developed first DNA sequencing
method in the 1970’s using dideoxy termination chemistry - Initial method used radioactive isotopes (P32) and poly acrylamide gels to detect nucleotide sequences
- Modern capillary sequencers use fluorophores and can sequence 700 bases per reaction
- Mix 1% dideoxy nucleotide (ddNTP), which causes termination of the strand during
polymerization, with the other dNTPS - Perform 4 reactions, each using a different ddNTP
- Run the sequence on a polyacrylamide gel (no longer used)
- Now: detect fluorescence on a modern capillary seqeuncer (ABI) to produce a trace
file
Melanoma: Poster Child for Targeted therapy
Focused sequencing of the MAPK pathway.
Mutations of the BRAF gene in human cancer.
Around 20% NRAS mutated.
Around 50% BRAF mutated.
Biological investigation of BRAF V600E-induced melanogenesis, then could develop Plexxikon/Vemurafenib as targeted therapy.
Developed BRAF V600 inhibitor to treat patients with late-stage (metastatic) or unresectable melanoma.
what are three promising targeted therapy by elucidating oncogenes?
1) Herceptin (breast)
2) Gleevec (CML)
3) BRAF inhibitors (melanoma)
Next is comprehensive sequencing of all cancers.
What are two Early large-scale genomic studies?
1) Johns Hopkins (Vogelstein) group.
An Integrated Genomic Analysis of Human Glioblastoma Multiforme.
- Screened 2043 genes and found 2325 somatic mutations.
- Discovered Hotspot mutations in a gene called IDH1.
- could make tables and see which genes/pathways were mutated, amplified, deleted, or altered.
2) TCGA
Cancer Genome Atlas Research Network
Cancers:
- Glioblastoma multiforme
- Squamous cell lung cancer
- Ovarian cancer
Goal:
- assemble high-quality samples of each cell type
- characterize tumour genome by various approaches
- rapidly share data with scientific community
- compare and improve technologies
- integrate and analyze data to illuminate genetic basis of cancers
Cross-platform data integration, comparison
- TCGA: focused sequencing and copy number analysis: frequent genetic alterations in three critical signalling pathways:
1) RTK/RAS/PI(3)K signalling altered in 88%
2) p53 signalling altered in 87%
3) RB signalling altered in 78%
Needed tissue samples, sent to centralized tissue repository, and did QC of tissue, isolated DNA and RNA and sent to various centres to do sequencing and epigenetics etc. Data was made available in real time.
You have 100 fresh frozen tumor tissue of a previously unstudied cancer type, and you performed a microarray experiment to elucidate mRNA expression levels. What information can you obtain by performing a class discovery analysis (i.e., unsupervised clustering)? What variables would you input for a class distinction analysis (i.e., supervised clustering)?
- Unsupervised – look at tumours, do clustering, look at natural grouping of tumours.
- In supervised, you are putting in what YOU want to look at. (example is class distinction example)
In a study of class discovery, researchers performed unsupervised clustering of the 1500 most variable genes in melanomas. This revealed subgroups based on elevated epithelial/skin markers, immune infiltration (associated with better outcomes), and low MITF expression (linked to de-differentiated melanomas). These subgroups correlated with patient outcomes, where, for instance, high keratin levels were associated with the poorest outcomes.
For class distinction, uveal melanoma patients were categorized into two groups based on gene expression and outcomes: one group with no metastasis and another with liver metastasis, the latter having shorter survival. Further analysis focused on identifying specific genes predicting melanoma metastasis and poor outcomes. Narrowing down genes that can be used that predict melanoma metastasis.
What are mutations doing to Ras?
The mutations are present in the kinase active site, and it is preventing the hydrolysis of GTP to GDP.
Where would Gleevac target?
ATP binding pocket of the bcr-abl tyrosine kinase,
competes with active site.
This allows for personalized therapy for CML.
What are four types of cytogenetic tools?
1) G-banding (Giesma) – dark regions have low GC content and are gene-poor, while light regions are gene-rich and have high GC content.
2) Spectra karyotyping (SKY) – uses fluorescence to colour chromosomes, allowing the detection of translocations, deletions and amplifications.
3) Array comparative genomic hybridization (CGH) – measure genome-wide copy number aberrations at high resolution to detect amplification and deletions.
4) Affymetrix SNP arrays – analyze SNPs (single
nucleotide polymorphisms) across the genome, used to detect genetic variations at specific locations in a genome.
many numerous combinations of genetic defects can lead to similar cancer phenotypes and many oncogenic changes result in
altered gene expression. Therefore, how can we can get around this issue to ensure that a patient is receiving the proper treatment?
designing personalized medicine through expression profiling to reflect different genetic events.
Examples: microarrays, gene expression matrix, Affymetrix microarrays, gene expression heat maps.
analyzing mRNA gene expression analysis can allow us
to make informed therapeutic decisions… why?
1) allows distinct profiling of subtypes of different cancers.
Different cancer subtypes might require different therapies
depending on their profiles. These tools allow us to identify
differences in subtypes to determine appropriate modes of action.
Synthetic Lethality, What are they especially great for targeting?
loss of function tumour suppressors
example from slides – EZH2.
Next Generation Sequencing (NGS)
Sequence capture for whole exome sequencing.
Can do sequence alignment.
Single nucleotide variant detection.
Visualize NGS data (Integrative Genome Browser - IGV)
Can find:
- point mutations
- coding
- non-coding
- indel
- copy number alterations (homozygous deletion, hemizygous deletion, gain)
- translocation breakpoint
- pathogen
46% of ovarian clear-cell carcinomas with ARID1A mutations.
84% of metastatic uveal melanoma tumours.
Frequent mutation of BAP1 in metastasizing uveal melanomas.
Recurrent promoter mutations identified: TERT promoter.
New hallmarks of cancer identified from NGS: epigenetic and mRNA splicing factors.
Cutaneous Melanoma
- Melanoma is the most deadly form of skin cancer that begins in melanocytes, which arise from neural crest cells and are the melaninproducing cells located in bottom layer of the skin’s epidermis.
- Melanocytes provide photo-protection to the skin.
Secondary method to validate recurrent mutation:
mass-spectrometric genotyping.
validation and extension set analysis, example looking in melanoma cell lines.
Lessons in validation
- manual review sequencing data
- validate mutations with an additional sequencing method
- validate mutation presence in an extension set
- functional studies
Transferring Human Cancer Cells Into Mouse Models
- Human cancer cells can be transferred into mice to study hereditary cancer.
- Cancer cells are prepared for transfer.
- Must be transferred into two lines of mice.
- A healthy mouse can develop cancer through this process.
Applications:
- research cancer pathways and contributing factors.
- test treatments in mouse models.
- clone transforming genes and transfected human oncogenes.
- detect oncogenes in human tumours.
