cancer genetics since midterm

L#13: Studying cancer-causing mutations: model organism

Outline uses of cell culture models in cancer research

cells in culture

RNAi/CRISPR screens

• library of gRNAs

• lentivirus → deliver DNA to human cells

• each cell deletes a dif gene in genome

• seq to find which gRNA represented (↑,↓, =)

tumor organoids

• 3D culture

• Recapitulate the complex structure/function of the corresponding tissue

• Can be cancer patient derived = can recapitulate genetic complexity

• ↑ success rate, better than mice

• no immune cells

Outline uses of cell culture models in cancer research:

RNAi/CRISPR screens

RNAi

• siRNA complementary to downregulanting the seq you want

• leads to gene silencing (not completely eliminating gene function)

• knock DOWN

CRISPR

• library of gRNAs

• lentivirus → deliver DNA to human cells

• each cell deletes a dif gene in genome

• seq to find which gRNA represented (↑,↓, =)

• positive screen

  • essential for growth → KO → cell wont grow

Outline uses of cell culture models in cancer research:

human organoids

• 3D culture

• Recapitulate the complex structure/function of the corresponding tissue

• Can be cancer patient derived = can recapitulate genetic complexity

• ↑ success rate, better than mice

• no immune cells

L#13: Studying cancer-causing mutations: model organism

Discuss the use of model genetic organisms in cancer research.

Which model organisms have contributed?

• mouse, rat, guinea pig, rabbit

• worm, fly, yeast (genes)

• zebrafish (immune)

Homolog

• ortholog - human + c. elegan a-tubulin

• paralog - within species (human has 10 copies, elephant has many copies of p53)

Forward genetics vs reverse genetics

forward - create random mutations → look for phenotypes

reverse - want to know a gene, what gene does what, seq → why is this mut associated with cancer

Why use mice in cancer research?

99% of genes have human counterpart

Studying gene function in mice

L#13: Studying cancer-causing mutations: model organism

Compare/contrast traditional approaches to making knockout/knock-in mice with use of crispR-cas9 system

CRISPR/Cas9: Genome Editing

What types of changes can be made?

CrispR-cas-9 injection produces

______ results

various genotypic

Controls, controls, controls...

• Potential problems:

  • Mosaicism in founder

  • Off-target effects

  • Upregulation of homologous (unmodified) genes

  • Potential reactivation of gene product via use of alternative start, splicing out of premature stop etc.

  • Suppressor mutation in a second gene

L#13: Studying cancer-causing mutations: model organism

Explain why inducible systems may be used and how they work.

How can we study essential genes?

knockdown

tet off

tet on

Some knockouts aren’t viable

Cre-lox in practice

Diagram the 2 constructs required to make a mammary-specific deletion of BRCA-1 exon11.

conditional KO

• promotor + CRE (control expression in certain tissues)

• loxP sites + GOI

• CRE induces recombination

how to put in loxP sites

CRISPR + repair template

Variations on using the CRE-LOX system

If cre was expressed under control of a liver- specific promoter, what would this system allow?

not liver → no GOI protein

liver → protein of interest

Tet off system

2 components

Use drug to control expression.

• Dox present = off

• Promoter gives spatial control

Tet on system

2 components

Use drug to control expression.

• Dox present = on

• Promoter gives spatial control

Tamoxifen-regulated

Use drug to control activity

• add tamoxifen to ‘switch on’.

• Promoter gives spatial control

Tamoxifen - used to control GOI getting into nucleus

• (-) tam → outside nucleus

• (+) tam → inside nucleus

L#13: Studying cancer-causing mutations: model organism

Design and interpret experiments that use model genetic organisms, including knockouts, knock-ins and conditionals.

L#14: Cancer stem cells

Explain self-renewal of normal stem cells, the role of transit amplifying cells and relate both to tissue homeostasis.

What is a stem cell?

An undifferentiated cell which has the ability to self-renew and to give rise to differentiated cells.

• asymmetric division

• A Stem cell maintains the ability to self-

  renew over many divisions

• daughter cells have different fates

• Stem cells maintain tissue homeostasis

Transit amplifying cells

L#14: Cancer stem cells

Describe methods used to find CSCs and analyze data related to their use.

Are all tumor cells created equal?

Cells within a tumor show functional differences.

Only a subpopulation are capable of initiating tumors after transplant

• = tumor propagating cells

• = only 1% of tumour

_____ can be used to separate cells based on surface markers

fluorescent activated cell signaling (FACS)

flow cytometry

Cell fractionation by FACS separates tumor propagating cells from bulk tumor cells

1. stain → antibody sticks to certain cells, color relate to proteins expressed on surface

2. laser → makes glow

3. detector

4. current → sorts

Cancer stem cell hypothesis

• A subpopulation of cells within the tumour has preferential capacity for driving tumor growth

• These CSCs promote and regrowth at a new site

• The CSCs can recreate tumor heterogeneity.

Cell surface markers ____ for CSC (but don’t purify them)

enrich

Identifying CSCs

colony forming assays

L#14: Cancer stem cells

Discuss CSCs in the context of cell-of-origin.

What is the cancer cell of origin?

Impact of cell-of-origin on cancer can be context-specific

L#14: Cancer stem cells

Describe the features of cancer stem cells. Compare and contrast normal stem cells and cancer stem cells.

normal vs csc

CSC dont reinvent the wheel

• Cell surface markers

• reactivate Signaling pathways

• Asymmetric divisions

• EMT

Normal stem cell signals activated in CSCs

• WNT, NOTCH, HHG signaling pathways active in stem cells AND often mutated (to activate) in cancer cells

  • Colon cancer: APC mutation ->activated WNT

  • Medulloblastoma: SMO, PTCH1, or Gli1 mutation -> Hhg signalling

  • T-ALL: NOTCH mutations

• Signals serve as drivers. i.e. stem cell signals are oncogenic

Dysregulated asymmetric cell divisions

Cell fate determinants are asymmetrically inherited in normal stem cell divisions.

Dysregulation of asymmetric cell divisions can lead to increased self-renewal.

asymmetric localization of proteins can inc SC in tumor

Asymmetric Cell division in oligodendrocytes

Studies in mouse models have shown that oligodendrocyte progenitor cells (OPC) carrying tumor-initiating

mutations are a cell of origin of oligodendroglioma, a progressive primary malignancy of the adult central

nervous system and major glioma class.

Fig. B&C dividing non-cancer cells labelled for the proteoglycan, NG2. E) Asymmetric divisions in WT and a

mouse model of glioma were compared. Modified from Sugiarto et al, 2011.

L#14: Cancer stem cells

Discuss the implications of the existence of cancer stem cells, for tumor initiation, metastasis and therapy resistance.

• Cells that seed metastases are CSCs

• Are all CSCs capable of metastases?

OR

• do they need to evolve new abilities to undergo metastases?

CSCs and treatment.

• Not all cells in a tumor are equivalently sensitive to therapy.

• If CSCs are not eliminated they can reconstitute the tumor

(normal) SC properties acquired by CSCs provide an advantage in surviving chemotherapy

normal SC have properties that make long lived

• drug efflux (dont want to be killed by random toxins)

• good for cancer

• efficient dna repair

• protected niche → buried

Exploiting CSC properties in treatment

• ‘Differentiation therapies’ in development.

• push toward dif = no longer sustain tumor

• Eg SMO antagonists to block Hhg pathway approved for advanced basal cell carcinoma

• Other pathway antagonists in development.

L#14: Cancer stem cells

Describe factors that aid CSCs therapy resistance.

L#15: Insensitivity to anti-growth signals

Discuss cancer-inducing mutations with reference to inheritance (dominant/recessive). How can this be determined experimentally?

insensitivity to antigrowth signals

Two major routes to insensitivity to antigrowth signals

• Pro-growth signals override normal controls

• Loss of antigrowth signaling components (TS)

Tumor suppressor - definition

A gene whose product functions in controlling cell growth

AND

Whose LOF can drive tumorigenesis

Are cancer-inducing mutations dominant or recessive?

normal + cancer → hybrid

~ phenotypes

remains tumoriogenic = dominant

not tumoriogenic = recessive

If a cancer-inducing mutation is recessive what does it mean for a cancer cell genotype?

homozygous (cancer cell genotype of recessive mutation)

• 2 identical (mut) alleles

• 2 different (mut) alleles

hemizygous

• 1 mut allele

• 1 deletion

The first tumor suppressor:

Rb

L#15: Insensitivity to anti-growth signals

Describe knudson’s 2 hit hypothesis.

L#15: Insensitivity to anti-growth signals

Explain what LOH is and interpret data related to detection of LOH.

Direct testing of the LOH theory

L#15: Insensitivity to anti-growth signals

Diagram models of how LOH arises.

1. Mitotic recombination

2. Gene conversion (strand switching during replication)

3. Chromosomal structural changes – hemizygosity

4. Non-disjunction and subsequent chr loss

LOH arises

mitotic recombinaton

dont expect recomb in mitosis

LOH arises

gene conversion

template switching by DNA pol

LOH arises

Non-disjunction followed by chromosome loss

LOH arises

Chromosome Structural Changes

= hemizygosity

L#15: Insensitivity to anti-growth signals

Describe non-mutagenic pathways that can lead to gene inactivation and how they can be monitored experimentally

Non-mutagenic processes -RNA

• Premature polyadenylation.

• Promoter methylation can inactivate ts genes – crosstalk with chromatin modifications.

EVIDENCE:

• CpG islands in promoters – increased me in cancer

• CIMP – CpG island methylator phenotype can be associated with increased DNMT3B expression.

• Me promoters show LOH

Monitoring methylations status

BS seq

nanopore seq - direct detection

L#15: Insensitivity to anti-growth signals

Discuss the network of interactions that underlies cell cycle entry.

Functions of tumor suppressors

• Unifying feature:

  • Operate to decrease likelihood of cancer development

  • Regulate circuits that govern cell proliferation and survival

Tumor suppressors often involved in negative feedback:

NF1

switches off at end

Ras → small GTPase, downstream signaling

ACTIVE - Ras+GTP

• signals grow + divide

Ras GAP - GTPase activating protein NF1 mut = Ras stuck in active form

INACTIVE - Ras+GDP

Ras GEF - GTP exchange factor

Focus on cell cycle entry

Rb regulates cell cycle entry

mitogenic - pro prolif signals

R point - point of no return

Mitogenic signal activates CDK

• Mitogen signaling activates Myc

• Myc is a TF

• Myc activates cyclinD-CK4/6

Cyclin D/CDK initiates Rb phosphorylation,

CyclinE/CDK continues

mittogenic signaling → myc (TF) → cyclinD+E → (P) Rb

Rb - binds E2F → no transcription

↓ cyclin D+E

Rb (P) - does not bind E2F → active transc

RB regulates progression through G1 phase

Rb regulates E2F activity

E2F = transcription factor of S phase proteins

bond to Rb = inactive

unbound = active transcription

hyperphosphorylation of Rb = dissociation from E2F = transcription activation

Rb, the retinoblastoma protein regulates the cell cycle

L#15: Insensitivity to anti-growth signals

Use information on gene function in the cell cycle to hypothesize the outcome of a given experiment.

investigating a genetic interaction between Drosophila Rb and E2F

conditional KO - cre lox

overexpress E2F = ↑ cell cycle = rough eye

overexpress Rb = gets better

L#15: Insensitivity to anti-growth signals

Diagram methods used to study tumor suppressor function and interpret data stemming from their use.

Does knockout of RB cause retinoblastoma in mice?

—nesprin eye prom—cre——————-lox-Rb-lox—

Does knockout of RB cause retinoblastoma in

mice?

L#16: Increased growth signaling

Contrast proto-oncogenes and oncogenes.

Oncogene - definition

• A gene whose expression contributes to cancer. eg. pro-proliferation signal, Ras

protooncogene → oncogene

how?

The path to oncogene

protonco → onco

1. change in coding seq - hyperactive protein (normal amounts, abnormal protein)

2. gene amplification - more copies of protein (normal protein, abnormal amount)

3. chr rearrangement - bring together seqs

Normal protein – more of it:

• Gene amplification

• Altered expression

Disruption of gene function

• Gene fusion – novel hybrid protein

• Deregulated activity – ligand independent signaling

• mutation = increased activity

L#16: Increased growth signaling

Explain with examples how oncogene creation happens without changing protein function and diagram how it may come about.

gene amplification

• homogeneously staining regions (tandom duplications in chr)

vs

double minutes

• extra chr arrays - not under normal regulation

detecting amplifications - how

direct

• nanopore - chr changes (LRS)

• short read seq

• fish

• rna seq

• array cgh - imbalances

indirect

• western blot

• rna seq- dont involve amplification

altered expression - how

1. viral integration (random)

2. promoter fustion

3. loss of miRNA binding sire from a transcript - loss of reg of that transcript

miRNA - genome encoded

ds miRNA processed → ss miRNA → binds mRNA → degradation, chromatin changes

How to detect altered expression?

western blot

RNA seq

RT pcr

• ^^^^ not looking at DNA

northern blot

microarrays

L#16: Increased growth signaling

Explain with examples how altered function can create an oncogene and diagram how it may come about.

altered gene function

novel hybrid protein - BcrAbl (kinase)

How could you test whether a novel hybrid gene is driving tumor?

TEST

• sufficient - express novel hybrid → cancer

• necessary - down regulate novel hybrid (RNAi, CRISPR) in tumor cells

Deregulated activity eg. Ligand-independent

signaling

truncation = growth without signal

L#16: Increased growth signaling

Explain with examples how dysfunction of signaling pathways may contribute to cancer.

• Explain how deregulated receptor function contributes to cancer.

• Explain with examples how monomeric G proteins are activated and inactivated and how they function in signaling pathways.

• Explain how dysregulation of cell cycle effectors contributes to cancer.

• Explain how deregulated receptor function contributes to cancer.

What types of genes are proto-oncogenes?

function in signal tranduction

Ras, myc

• Explain with examples how monomeric G proteins are activated and inactivated and how they function in signaling pathways.

Receptor tyrosine kinase - monomers → ligand binding → dimerize → P eachother

RTK →receive prolif singal → pass to Ras → G proteins, phosphinstol

How are signals conveyed downstream of RTKs

• Activate:

• monomeric G protein signaling eg ras

• Phosphoinositol signaling eg PI3K

Ras

L#16: Increased growth signaling

Use knowledge of signaling pathways to predict the consequence of dysfunction of particular components.

Oncogene addiction

Is the continued action of the oncogene required to sustain the cancer phenotype once a tumor is formed?

once oncogene arises, is it required for initiation or maitance?

L#17 Paper 5

Chin et al 1999

https://docs.google.com/document/d/1H059zRHq81ifEUV4Ik_DlpgZAfJPMwcsnVe6J4iUX2Y/edit

L#18: apoptosis/programmed cell death

Diagram techniques used to detect apoptosis and interpret data stemming from their use.

tunel assay - detects breaks during apoptosis

Tdt - terminal deoxynucleotide transferase, adds onto end

Brd UTP - nucelotide analog, recognized by Ab

add Rb to Brdu, see staining = undergoing apoptosis

apoptosis

L#18: apoptosis/programmed cell death

Describe/diagram apoptotic pathways. Predict consequences of mutations.

BCL2 - regulator of apoptosis, pro survival = ↑ tumor

Bcl-2 and Bax are members of gene families

The balance of bcl2 and bax leads to ______ decision

life or death

L#18: apoptosis/programmed cell death

Describe the role of the mitochondria in apoptosis.

mitochondria – the home of apoptosis

anti- and pro- apoptotic signals regulate release of cytochrome c from the mitochondria

Cytochrome c release triggers apoptosis

Cytochrome c triggers caspase activation

procaspase 9 → (cut) → caspace 9

proproteins - translated in inactive form

apoptosome binds + cuts other procaspaces

activates ICAD - fragments DNA

Smac/DIABLO are also released from the mt

INHIBITORS of apoptosis

L#18: apoptosis/programmed cell death

Compare and contrast intrinsic and extrinsic mechanisms of apoptosis

Where do the death ligands come from?

intrinsic -Cells can secrete the ligand to induce their own death

extrinsic - immune cells secrete death ligand

L#18: apoptosis/programmed cell death

Explain how we know that many p53 mutations are dominant negative.

most frequently mutated gene in cancer

need all 4 subunits to function

Is p53 a tumor suppressor or an oncogene?

L#18: apoptosis/programmed cell death

Describe the role of p53 and how p53 mutations lead to impaired apoptosis and cancer.

p53 - not required for development, required for longevity

p53 required for apop after exposed to radiation

Why is it worse to have a p53 point mutation than a p53 deletion mutation?

What is the function of p53?

P53 is a transcription factor

p53 modulates response to cellular stress

p53 is required for induction of cell death after radiation exposure

How does p53 induce apoptosis?

Bax - opens gate to mitochondria, lets out cytochrome c - induces apoptosis, apoptosome, inhibitor/executioner

FAS - cell surface receptor, receives death ligands

↑ # receptor = ↑ sensitivity to apop

* inhibits pro-survival factors

induce pro - apoptosis

L#18: apoptosis/programmed cell death

Describe the regulation of p53 and use this information to hypothesize how perturbations might affect a cell

p53 levels increase in response to radiation

How are p53 levels regulated?

no stress - constantly made + degraded

mdm2 (e3 upiq ligase) →→ proteosome

= low p53 levels

stress - p53(P)

cant be degraded, blocks mdm2 binding

= high p53 levels

promote apop, repair, etc

p53+mdm2 promoter = transcription of mdm2 and more degradation

L#19: Why don’t elephants get cancer

C: Elephants can evade cancer through enhanced TP53 signaling.

2 models

P53 - tetramer Found at low levels in cell that is healthy growing, Levels regulated at protein level

• Normal cell - p53 bound by mdm2 (e3 ubiquitin ligase, send for degradation), constantly making p53 and tagging it for degredation

• DNA Damage - p53 gets phosphorylated - no longer binds to mdm, stabilized in cell

Decoy

• Some of mdm binding to retrogene, leaving more p53 available under normal circumstances

Guardian

• Retrogene binding to tp53 , prevents it from being degraded by mdm2

• Found by immunoprecipitation that rtg bound to tp53 and NOT mgm2

• Computational modeling - dimers more stable