Molecular Bio of Cancer Exam 3

Is cancer dominant or recessive?

recessive

Is hereditary cancer common?

rare - relative to all cancers observed

• rare diseases are rare, but rare disease patients are numerous

what is a familial cancer

prostate cancer and retinoblastoma

20% of prostate cancers are familial

40% in retinoblastoma 

Why don’t adults get retinoblastoma

mutations are precursor cells so cells stop growing after younger years  

What is bilateral in retinoblastoma

both eyes are affected—they are prone to tumors later in life in a variety of tissues, but especially sarcoma

what happened in the Hybridoma experiments?

cancer is not dominant; something in the good cell is “suppressing” the cancer phenotype

How does Knudson’s two-hit hypothesis relate to cancer?

have two copies of every gene, so maybe we need to get rid of both copies of a gene, because it “suppresses” tumor growth

what cancer causing gene remains present?

oncogene

what cancer causing gene disappears?

tumor suppressor gene

best method for detecting loss of chromosome/regions?

• Fluorescence in situ hybridization (FISH) & Spectral Karyotyping (SKY)

• Western or Northern Blot analysis

• Array Comparative Genomic Hybridization (aCGH) & SNP arrays

• Southern blot analysis

• Next Generation Sequencing (NGS)

5 ways loss of heterozygosity can happen

1. homologous mitotic recombination 

2. chromosome loss (e.g. non-disjunction)

3. Chromosome loss and replication 

4. deletion

5. point mutation, small deletion, or gene conversion
   

When would recombination happen?

meiosis, but on occasion (rarely) can ocur in somatic cells during mitosis

what is karyotype analysis

identifying chromosome abnormalities in metaphase

• can see the region of chromosome that is gone

• retinoblastoma cells have deletions in 13q12 - 13q14 region

What is linkage analysis

method used to find disease-related genes by studying high-risk families

• looks for genetic markers that are passed down along with disease

two parts of linkage mapping

genetic linkage map and genetic marker

genetic linkage map

position where a gene is using recombination effects

• shows relative locations of specific DNA markers on a chromosome

genetic marker

any inherited trait that varies between people and can be easily tested in the lab

• needs to have different forms (polymorphism) so they can be detected in family studies 

  • Ex: SSLPs, SNPs, RFLPs

positional cloning

sequence overlapping pieces of DNA until you find the culprit

linkage analysis used for…

1. enzymatic activity as a genetic marker

2. restriction fragment length polymorphism (RFLP) analysis

requirements for linkage analysis

1. sequence probe

2. nearby restriction site

3. restriction site is heterozygous

other methods of tumor suppressor gene detection

1. array comparative genomic hybridization (aCGH)

2. single nucleotide polymorphism array (SNP)

3. high throughout next generation sequencing (NGS)

SNP probes

50 million SNPs in human genome

array has spots for up to about 1 million SNPs

a SNP is about 12 nucleotides long

ability to discriminate btwn its target and a highly related mismatch sequence

probes can be allele or non-allele sequencing (NGS)

High throughput next-generation sequencing

chop up DNA, add linkers on both ends, stick both ends to slide, sequence by synthesis, take a picture after adding single nucleotide, copy number variation analysis based on number of reads

what experiment could one do to test if a gene was a bona fide tumor suppressor?

mouse double knockout?

• look for the knockout gene of interest in cell culture and look for transformation (ex: loss of contact inhibition, indefinite cell proliferation, reduced growth factor requirements, etc)

• look to see if gene is missing/disrupted in patients with the disease

compared to normal cells, cancer cells are…

1. hyper-methylated

2. hypo-methylated 

3. generally hyper-methylated, with some genes hypo-methylated

4. generally hypo-methylated, with some genes hyper-methylated

where does DNA methylation occur

CpGs

• 40% of genes have CpG islands in their promoter

• - promoters are not normally regulated

description of methylated regions

compact DNA

less accessible to transcription factors

Connection between TSGs and methylation

• TSG can be silences (inactivated) by promoter methylation

• TSG expression silencing by methylation is variable between cancer types

NF1

Neurofibromatosis

NF1 gene product normally functions as a GAP (GTPase activating protein)

tumor suppresor gene

How does NF1 interact with Ras cycle

NF1 inactivates Ras by promoting Ras-mediated GTP hydrolysis. - takes over GAP not allowing RasGTP to be hydrolyzed into Ras GDP

FAP

Familial Adenomatous Polyposis

APC (adenomatous polyposis coli) gene product promotes B-catenin degradation

intestinal polyps early in life which progress to adenocarcinoma

B-catenin is stabilized and leads to ligand-independent Wnt signaling (proliferation/cytoskeletal changes) when APC is defective

most observed APC mutations result in truncated protien that cannot bind B-catenin

What therapeutic approaches could mitigate loss of a tumor suppressor gene?

1. gene therapy (reintroduce)

2. engineer fix by CRISPR

3. modulate downstream effects

Phases of Mitosis

prophase, prometaphase, metaphase, anaphase, telophase 

Phases in cell cycle

Go, G1, S, G2, M

what happens at Go

quiescent phase

What happens at G1

growth phase 1 (12-15 hr)

What happens at S

Dna synthesis (6-8hr)

What happens at G2?

Growth Phase 2 (3-5hr)

mTOR pathway

controls cell metabolism and physiology (regulates cell growth)

restriction point

represents a point at which the cell commits itself, irreversibly, to complete the remainder of the cell cycle (major decision maker)

Where is the R point located

near the end of G1 but before the break betwene G1 and S

What is interphase

Go, G1, S, G2 - period at which cells are responsive to mitogenic growth factors and TGF-B 

What/where are the checkpoint controls in the cell cycle?

1. DNA damage checkpoint: entrance into S is blocked if the genome is damaged

2. DNA damage checkpoint: DNA replication halted if the genome is damaged

3. entrance into M is blocked if DNA replication is not completed

4. anaphase is blocked if chromosomes are not properly attached to the mitotic

What is different in cell cycles of cancer cells?

one or more checkpoint controls are inactivated in most cancer cells.

difference between ATM ad ATR

ATM = responds to double-stranded DNA breaks

ATR= responds to broader things, such as single-stranded breaks or single-stranded DNA from stalled replication forks

ATM & ATR

help monitor if DNA is damaged and arrest the cell cycle until the damage is fixed

serine/threonine kinase 

related to Rad3

loss of ATR results in breaks in DNA at fragile sites 

What controls the cell cycle clock?

cyclins and CDKs

CDK

cyclin-dependent kinases are serine/threonine protien kinases

cyclin-CKS complexes

CDKs are active when complexed with their cognate cyclin

What happens during the cell cycle with CDK an cyclins?

CDK levels remain constant while cyclin levels change during the cell cycle

CDKs phosphorylate substrates that drive steps in the cell cycle

Cyclin functions

1. activate CDKs (partially*)

2. ]direct CDK to substrates

why can cyclins only activate CDKs partially?

CDKs must be phosphorylated by CAK before they can phosphorylate their substrates

Types of Cyclins in cell cycle

A,B,D,E

Types of CDKs in cell cycle

CDK1, CDK4/6, CDK2

What does cyclin A bind to 

CDK1 and CDK2

What does cyclin B bind to

CDK1

What does cyclin D bind to

CDK4/6

What does cyclin E bind to 

CDK2

Where is Cyclin B in cell cycle

Mitosis

Where is cyclin D in cell cycle

middle of G1

Where is cyclin E in cell cycle

after R point in G1

Where is cyclin A in cell cycle

A-CDK2 is beginning of S

A-CDK1 is bridge between S and G2

How does the binding of cyclin to CDK work?

PSTAIRE helix on CDK binds to cyclin

A threonine residue in CDK complex activation loop becomes exposed

The activation loop is phosphorylated by CAK that increases CDK activity 40,000-fold.

CAK

CDK activation kinase (complex between cyclin H-CDK7)

what do all pathways lead to

cyclin D

what is cyclin D controlled by

growth factors

what are cyclin E,B,A controlled by

are on autopilot

what happens when the R point is passed 

the cell cycle clock only continues to move forward

What needs to happen for something to be mitogenic

Cyclin D1 needs to be turned on

Which cyclins are rapidly destroyed

Cyclin E, A, and B gradually accumulate, and are then rapidly destroyed via ubiquitylation

Cyclin CDKs activate the next cyclin-CDK and inhibit the previous cyclin-CDK

CDKI

cyclin-dependent kinase inhibitors 

• inhibitors block the actions of CDKs at various points in the cell cycle 

INK4

inhibitors of CDK4 by binding to them and bending them, which distorts the ATP binding site

• reduces CDK affinity for cyclin and compromise catalytic activity of CDK

Kip

kinase interacting protein

p21

(CIP/KIP) inhibit CDKs by obstructing the ATP-binding site in the catalytic cleft of the CDK

YIN

TGF-B promotes CDKI expression, which in turn, inhibits cell cycle via P15INK4B and p21CIP1

YANG

mitogens can counteract TGF-B activity by blocking CDKIs

mechanism of action for inhibitors

AKT-mediated phosphorylation prevents p21CIP1 entry into the nucleus, sequestering the CDKI in the cytoplasm

What is the effect of AKT signaling in breast cancer

p27 sequestering in the cytoplasm is bad news for breast cancer patients

• activated AKT suppresses p27KIP1 in breast cancer

what does p21 and p27 do

• stimulates cyclin D-CDK4/6 complexes 

• inhibits cyclin E-CDK2 complexes

  • Cyclin D-CDK46 eventually sequesters enough p21 and p27 to free cyclin E-CDK2 from inhibition to trigger transition through the R-point

what proteins are phosphorylated by cyclin-CDKs/important substrates of cyclin-CDK?

pRb: retinoblastoma

what are the controls of the pRb protein?

controlled by phosphorylation, not expression 

controls the restriction point 

extent of pRb phosphorylation?

start of G1: pRb dephosphorylation by phosphates

mid G1/R point: pRb hypophosphorylation/monophosphorylation (low)

R point: hyperphosphorylation (becomes inactive)

high throughout proteomics

a new way to find CDK4/6 substrates (generally considered targets)