Ras and other oncogenes, Rb and Tumor Suppressors

Genetic Basis of Cancer ppts 3 and 4

Ras was founded by

Robert Weinberg

What did Robert Weinberg do?

Determine if cancer-causing genes could be transferred directly from cancer cells to normal cells

Cancer cell DNA

should make normal cells proliferate

What was Weinberg’ process?

DNA bind to calcium phosphate to form tiny white particles. The particles are ingested by cells, and would therefore ingest DNA bound to it

Chiaho Shih

a Grad student in Weinberg’s lab transferred mouse cancer cell DNA to normal cells- grew in foci → cancer. Next it was confirmed in human cells

Contact inhibition

cell process where cell movement and cell division stop when cells come in contact with other cells

Normal cells have _____; Cancer cells _______

contact inhibition; no contact inhibitionC

Cancer cells

no contact inhibition, continue to grow, can grow in low serum, adopt round morphology, rather than flat extended, can grow without attaching to a surface- “anchorage independence”

3 groups isolated Ras

Weinberg (MIT), Mariano Barbacid (NCI), and Michael Wigler (Cold Spring Harbor HY)

Has also been discovered in virus before

“Ras”- rat sarcoma

Ras in normal cells

tightly regulated

Mutated Ras

hyperactive and always on

Ras was

first “native” human oncogene discovered

Myc

originally discovered as v-myc oncogene

Myc is a

transcription factor, binds to regions of the DNA to promote transcription

Human myc

consistently altered by chromosomal translocation on Burkitt lymphoma and multiple myeloma

Myc is one of the most

highly amplified oncogenes in several human cancers (colon, lung, stomach, cervix)

Mouse genetics

could introduce exogenous genes into mouse embryo- “transgenic mice”

OncoMouse

first one attempted- c-myc, overexpressed Myc only in breast mammary cells, to specifically study overexpressed of myc and if it resulted in breast cancer; first animal to be patented

OncoMouse (c-Myc)

only developed small breast tumors, and not in every mouse

2nd OncoMouse

Leder created another OncoMouse- activated 2 oncogenes: Ras and Myc, multiple tumors sprouted within months and cancer has been artificially created in an animal

Neu

Lakshmi Charan Padhy, isolated oncogene from rat tumor- neuroblastoma, (neu)

Her2

Researchers discovered the human homolog of the neu gene

• Human EGF Receptor (HER)- Her2

Her2 member

member of EGFR family of receptors

Her2 has

an extracellular domain (large fragment hangs outside) while most oncogenes encoded proteins that were inside the cell

Her2 dimer

if overexpressed it dimerized not needing ligandsH

Her2 doesn’t

have it’s own ligand

Reason why HER2 cancers are aggressive

HER2 does not have it’s own ligand

Dennis Slamon

found that Her2 was increases in ~20% breast cancer samples

Her2 positive

more aggressive than Her2 negative

Genentech

developed antibody drug to target outside domain of Her2

• Trastuzumab-Herceptin

Herceptin

Her2 intercept and inhibitor

Most commonly altered oncogenes in cancer

• PIK3CA

  • KRAS
    EGFR

What does PI3K stand for?

Phosphoinositide 3-kinase

How many classes of PI3ks exist?

Several classes, including Class 1

What are the subunits of Class 1 PI3k?

catalytic subunit (110 alpha) and regulatory subunit (85)

What gene encodes the catalytic subunit 110 alpha of PI3K?

PIK3CA

Why is PIK3CA clinically significant?

It is highly mutated in cancers

What reaction does PI3K catalyze?

PI3K converts PIP2 into PIP3

What are PIPs?

Lipid signals in the cell membrane

What does PIP3 activate?

PIP3 activates PDK1, leading to ATK phosphorylation and mTOR activity

What is the cellular effect of PI3k signaling?

Promotes cell proliferation

What is the function of PTEN in the PI3K pathway?

PTEN converts PIP3 back to PIP2 removing a phosphate group, reversing PI3K signaling

Where are phosphoinositides (PIPs) located?

In the plasma membrane

What does PIP2 stand for?

Phosphatidylinositol (4,5)-bisphosphate

What does PIP3 stand for?

Phosphatidylinositol (3,4,5)-trisphosphate

What is the role of PI3K in PIP signaling?

PI3K phosphorylates PIP2 to form PIP3

What role does PIP3 play in the cell?

PIP3 is a substrate for kinases that promote proliferation

What is PIP in general?

Phosphatidylinositol phosphate, a phospholipid signaling molecule in membranes

E542K, E545K, H1047R

3 of the most common mutation in PIK3A

PIK3CA mutations

very common in breast and colon cancer

What is the H1047R mutation in PIK3CA?

A mutation near the hinge region of the activation loop that may affect its mobility

What is the overall effect of mutations in PIK3CA?

They make PI3K constitutively active (always on)

How do PIK3CA mutations affect PIP signaling?

Increase PIP2 → PIP3 conversion and keep downstream signaling active

What are the three family members of RAS?

KRAS, HRAS and NRAS

What does KRAS stand for?

Kristen Ras, named after the Kristen murine sarcoma virus

What does HRAS stand for?

Harvey Ras, named after the Harvey murine sarcoma virus

What does NRAS stand for?

Neuroblastoma Ras

What is the function of RAS proteins?

They act as GTPase

RAS proteins are similar to what common energy molecule?

ATP

GTP bound to Ras

on

Acts as molecular switch depending on if it’s

GTP or GDP

Where does a common KRAS mutation occur?

In the P-loop

What is the effect of a KRAS P-loop mutation?

Prevents conformational change back to the inactive state

Mutated KRAS is especially common in which cancer>

Pancreatic cancer

When is KRAS active?

When bound to GTP

What does GTP stand for, and what is it similar to?

Guanosine 5’-triphosphate; similar to ATP as an energy molecule

What is the most common oncogene mutated in cancer?

KRAS

What do KRAS mutations G12C and G12D represent?

Substitution of glycine (G) with cysteine (C) or aspartic acid (D)

What percentage of tumors have KRAS mutations?

~25–30% of all tumors

What cancers are strongly associated with KRAS driver mutations?

Pancreatic (90%), lung (32%), and colon (40%)

Which KRAS mutations are most common in specific cancers?

G12C in lung cancer; G12D in pancreatic cancer

In which structural region are KRAS mutant residues often found?

The switch II pocket

What mnemonic helps remember KRAS mutation associations?

C = cigarette (lung), D = diabetes (pancreas)

How do most KRAS mutations affect KRAS activity?

Lock it in an active state, enabling downstream signaling without GTP activation

What is EGFR commonly associated with in cancer?

EGFR is often mutated or amplified in cancers

Which cancers most frequently have EGFR alterations?

Lung cancer and brain cancer (glioblastoma)

How do EGFR mutations differ between cancers?

Lung cancer: mutations in the kinase domain

Brain cancer: mutations in the extracellular/ligand-binding domain

What is the function of tumor suppressor genes?

Encode proteins that inhibit tumor formation by regulating cell cycle and repairing DNA

What happens when tumor suppressor genes are lost or mutated?

Cells can freely enter mitosis, DNA damage goes uncorrected and tumors may form

How do germline mutations in tumor suppressor genes affect cancer risk?

Predispose individuals to hereditary cancers

What processes do tumor suppressor proteins regulate?

Cell cycle checkpoints, DNA repair, and correction of replication errors

What is Loss of Heterozygosity (LOH)?

Loss of one parent’s copy of a gene, leaving only the other copy

How does LOH affect heterozygous tumor suppressor cells?

Converts a heterozygous cell (1 normal + 1 mutated) into homozygous for the remaining allele

What impact does LOH have on cancer development?

Loss of the last functional tumor suppressor gene copy gives the cell a growth advantage

What are common mechanisms causing LOH?

Gene/chromosome deletion, gene conversion, mitotic recombination, whole chromosome loss, chromosomal instability

Who proposed the Two-Hit Hypothesis and what cancer did he study?

Alfred Knudson; retinoblastoma

What is the “Two-Hit Hypothesis”?

Both copies of a tumor suppressor gene must be mutated for cancer to develop

Why do oncogenes like Src only need one mutation, but Rb needs two?

Oncogenes activate division (dominant); tumor suppressors like Rb inhibit division (recessive)

Analogy for tumor suppressor vs oncogene mutations?

Two gas pedals (oncogenes) and two brakes (tumor suppressors); losing one brake still allows control, but both lost = uncontrolled division

What is the main function of Rb protein?

Gatekeeper for cell division; sequesters E2F to prevent premature G1→S progression

How is Rb inactivated during normal proliferation?

Phosphorylation releases E2F, allowing cell cycle gene transcription

What happens if Rb is mutated or lost?

E2F is free, uncontrolled proliferation occurs

Which cancers commonly have Rb mutations?

Lung, bone, esophageal, breast, and bladder cancers

What is TP53 and p53?

TP53 = gene; p53 = protein (“Guardian of the Genome”)

How is p53 normally regulated?

Expressed at low levels; degraded by MDM2/MDM4 until cell stress occurs

How does p53 respond to cellular stress?

Phosphorylation → tetramer formation → DNA binding → transcription of genes for cell cycle arrest, DNA repair, apoptosis

Where do most p53 mutations occur?

DNA binding domain; usually missense mutations

How does mutant p53 affect the cell?

Accumulates in the cell, prevents normal p53 function, and disrupts protective pathways

How can p53 be targeted therapeutically?

Drugs either prevent wild-type p53 degradation or attempt to restore mutant p53 function