tumor biology: cellular and molecular aspects of the transformed cell growth factors, receptors, and...

Tumor Biology: Cellular and MolecularAspects of the Transformed Cell

Growth Factors, Receptors,and Signal Transduction II

Rebecca Riggins202.687.7451

[email protected]

Overview

o Intracellular signaling “downstream” of receptors- key pathways: Ras/MAPK, Src, PI3K

o Intracellular signaling defects in cancero Targeted therapies to intracellular signaling moleculeso TNF and TRAIL

Brief Review – Growth Factors and Receptorso most growth factors are secreted o receptors are transmembrane proteinso 3 major features: o extracellular domaino transmembrane regiono intracellular domaino where, when, and how they are expressed determines their biological function

Brief Review – Receptor Activity

o intracellular, or catalytic, domain has kinase activityo kinases add phosphate groups to (phosphorylate) specific amino acids

ATP-binding Phospho-transferase

ATP

ADP

PAmino Acid P

RASP

RTK

PGrb2SOS

PAkt

PI3K

p85

p110

RAS

Raf

MEK

P

P

ERK

GROWTH FACTOR

PIP3 PIP3

PDK1

PROLIFERATION

MDM2BAD

P

NF-ĸB

P

FKHR

P

CELL SURVIVAL

p70S6K

P

GSK3

P P

PROTEIN SYNTHESIS

Consequences of RTK activation

. Aaronson, Growth factor and receptor tyrosine kinases. Sci. STKE 2005, tr6 (2005).

Intracellular Signalingo begins with autophosphorylated or transphosphorylatedamino acids on the receptoro Phosphorylation recruits other proteins to the receptoro Amino acids surrounding the phosphorylation site determine which proteins are bound…

Membrane

Kin

ase

Dom

ain

-Tyr

-Tyr

P

P

4 major protein interaction domains:SH2, PTB, SH3, PH

Basics of Amino Acid Structure

Basics of Protein Structure

o Primary Structure = “beads on a string”

o Quaternary Structure = specific folding creates domains, or “units” of the protein

SH2 domains

o SH2 = src homology 2o was first identified as a 100 amino acid region ofhomology (“sameness”) in the src tyrosine kinaseo specifically recognizes phosphorylated Tyrosineo 2 classes of SH2 domain-containing proteins…

- have enzymatic activity (like Src)- don’t have enzymatic activity

o Those with no enzymatic activity bind otherproteins to the receptor…adaptor molecules

SH2 domain specificity

Science, Vol 278, Issue 5346, 2075-2080 , 19 December 1997

o specificity is determined by the amino acids C-terminalto the phospho-Tyro in most cases, it is the amino acid at position +3 thatdetermines specificity

Hydrophobic amino acid

http://0-www.sciencemag.org.library.lausys.georgetown.edu/content/vol278/issue5346/images/large/se5276134002.jpeg

PTB domains


o phosphotyrosine binding domains recognize aminoacids N-terminal to the phospho-Tyro PTB-containing proteins also participate in hormonesignaling

any amino acid

SH3 domains


o src homology 3 domains recognize amino acidsequences rich in Proline (Pro-rich)o Is a more general protein-protein interaction motif…many cytoskeletal proteins contain it

any amino acid

PH domains

o pleckstrin homology domains recognize phospholipids(components of the plasma membrane)


Protein-Protein Interactions and Receptorso Proteins with many different interaction domainscan bind to growth factor receptor family members

Figure 6.9 The Biology of Cancer (© Garland Science 2007)

o Protein-proteininteractions connectreceptors to theirintracellular signalingnetworks

Receptors bind other Kinases,and Adaptor Proteins

Figure 6.10a The Biology of Cancer (© Garland Science 2007)

EGFR and the Ras Pathway

P

EGFR

PGrb2SOS

RAS

Raf

MEK

P

P

ERK

EGF

PROLIFERATIONCELL SURVIVAL


Grb2: adaptor protein that binds to phosphorylated Tyr on EGFR usingits SH2 domain

Grb2 has multiple protein interaction domains

Cell. 2004 Jan 23;116(2):191-203.

EGFR and the Ras pathway, cont’d.

P

EGFR

PGrb2SOS

RAS

Raf

MEK

P

P

ERK

EGF



SOS binds to the Grb2 SH3 domain

SOS activates Ras

DH PH CDC25

Proline-rich

Dbl-Homology

Membrane-targeting

Catalytic Domain SH3-Binding

Rho GTPases (Rac)

Grb2

EGFR

o SOS is an exchange factor (it exchanges onenucleotide for another)

A. Chan, Ras-MAPK Pathways. Sci. STKE 2005, tr5 (2005).

What is Ras?o Ras is an oncogeneo Ras is a small GTP-binding protein…it bindsguanine triphosphateo Ras bound to GTP is active…Ras bound to GDPis inactiveo Ras mutation is implicated in many kinds of cancer…

Incidence of RAS mutations in human cancer

Lung (30% ki-ras)Breast

Brain

Colon (50% Ki-ras)

OvaryProstate

Pancreas (90% Ki-ras)

Bladder (10% Ha-ras)

Thyroid (50% ras)

Liver (30% N-ras)

Skin (14% N-ras)

Head & Neck

Leukemia (30% N-ras)

Kidney (10% Ha-ras)



Lung carcinoma K-Ras codon 12 (GGTgly) >AGTser

Pancreatic Carcinoma K-Ras codon 12 (GGTgly) >GTTval

Bladder Carcinoma H-Ras codon 12 (GGCgly) >GTCval

Melanoma N-Ras codon 61 (CAAgln)>CGAarg

Ras Mutations display Tumor Specificity

How is Ras activated?


What does active Ras do?


Autocrine growth factor signaling

What else does active Ras do?

P

EGFR

PGrb2SOS

RAS

Raf

MEK

P

P

ERK

EGF



Ras activates Raf and the Erk/MAPK pathway

The Erk/MAPK pathwayo Erk = extracellular signal regulated kinaseo MAPK = mitogen activated protein kinaseo MAPK promotes cell growth and survival byphosphorylating other proteinso An immediate consequence of MAPK activationis transcription (DNA → RNA)o MAPK activation is a major event following EGFstimulation…

Nature Reviews Cancer 4, 937-947 (2004)

Inhibitors of the MAPK Pathway

Nature Reviews Cancer 4, 937-947 (2004)

Mechanisms of Ras/MAPK inhibitorso inhibit Ras binding to the plasma membraneo inhibit Rafo inhibit MEKo some of these have entered clinical studies…

PPGrb2SOS

RAS

Raf

MEK

P

P

ERK

EGFR

EGF


P

RTK

P PAkt

PI3K

p85

p110

GROWTH FACTOR

PIP3 PIP3

PDK1

MDM2BAD

P

NF-ĸB

P

FKHR

P

CELL SURVIVAL

P

GSK3

P

EGFR and the PI3K pathway


What is PI3K?o PI3K = phosphatidyl inositol 3-kinaseo phosphatidyl inositol is a lipid, part of the plasmamembraneo so, PI3K phosphorylates lipids (fats) instead ofother proteinso There are 2 subunits of PI3K…

PI3K

p85

p110

p85 = regulatory subunitp85 has an SH2 domain that binds phospho-Tyrosine on the EGFR

p110 = catalytic subunit that phosphorylates lipidsK

inase

Dom

ain

-Tyr

-Tyr

P

P

PI3K Target(s)o PI3K phosphorylates PIP2 to make PIP3o PIP3 is now a binding site for Akt…


Akt – the real master regulator

P

RTK

P PAkt

PI3K

p85

p110

GROWTH FACTOR

PIP3

MDM2BAD

P

NF-ĸB

P

FKHR

P P

GSK3

P

CELL SURVIVAL


o Akt is a Serine/Threoninekinaseo Akt has targets in the cytoplasm as well as the nucleuso Akt inhibits the cell cycle inhibitorso inhibitor + inhibitor = GROWTH,and SURVIVAL

S

M

G1G2 Cyclins,Cyclin-dependentkinases, andinhibitors

Table 6.3 The Biology of Cancer (© Garland Science 2007)

PI3K/Akt Defects in Cancer

PI3-K

GF RTK

PIP3

Akt1/2

TSC1

RheB

mTOR

S6K 4EBP-1

Protein synthesis

Cell growth/size/survival

p

(Tuberous Sclerosis Complex)

TSC2TuberinHamartin

Perelman (2004) Dematology Online Journal 10: 17.Kovich & Cohen (2004) Dematology Online Journal 10: 3.

(Ras-homology enriched in brain)

(Target of rapamycin)

Lipid Kinase

Ser/Thr Kinase

GI, Br, Ov

Cancer Syndromes

PANC

Inhibitors to PI3K and/orAkt are being developedfor patient use

pp60 c-Srco “normal,” cellular Src is another protooncogeneo viral Src (v-src) is the transforming gene of theavian Rous sarcoma viruso Src is a tyrosine kinase, but NOT a receptoro Cooperation/synergy with the EGFR in promoting proliferation and tumorigenesis in breast cancer cells

Kin

ase

Dom

ain

-Tyr

-Tyr

P

P

Src

Cell Growth

Phosphorylation of transcriptionfactors

MAPK and PI3K pathways

c-Src phosphorylates many cellular proteins


Isolate protein andload onto poly-acrylamide gel;detect phospho-Tyrosine with

specific antibodies

c-Src and Cancer

o 30-70% of breast cancers overexpress Src, show elevated activity…many of these also overexpress

EGFRo Other cancers Src may contribute to are: colon,

lung, skin, endometrial, head/necko Src is usually not mutated…how is it activated?

Regulation of Src Activity

“Inactive”N

C

pY527

SH2

Kinase

Kinase

SH3Kinase domain has noaccess to targetproteins

YX

“Active”N

C

KinaseKinase (p)Y527

SH2SH3

Kinase domain is nowaccessible

Viral Src has lost thisTyrosine

How does Src become activated?

o Src can bind to EGFR or PDGFR, and the active configuration is stabilized

o However, are other proteins that can bind to and activate Src besides RTKs…

“Active”N

C

KinaseKinase (p)Y527

SH2SH3

YX

Adaptor proteins with the rightbinding sites for SH2 and SH3 domains could activate Src

The adaptor protein Cas can activate Src

RP640LPSPP pY668DYV

N C

“Active”KinaseKinase (p)Y527

SH2SH3

Cas

o Cas is important for cell proliferation, cell migration,

and transformation by oncogenes (including viral Src)

Cas, Src, and Breast Cancero Cas, like Src, can also be overexpressed in breast

cancero Cas and Src bind to each other in breast cancer

cellso Cas and Src are localized to the same areas of

breast cancer cellso Cas overexpression in breast cancer is associated

with resistance to a particular kind of drug (Tamoxifen)…this involves Src activation

Cas Src Cas + Src

Inhibitors of Src in Cancer Therapyo Inhibit protein-protein interactions (like those with

Cas)o Inhibit kinase activity (preclinical/phase I trials)o Inhibit protein stability (accelerate Src

degradation)o These will likely be used in combination with other

chemotherapy drugs, EGFR inhibitors, etc.

Adhesion Receptors

Figure 6.24b The Biology of Cancer (© Garland Science 2007)

Tyrosine Kinases and Adhesion Receptors:Overlap in Cytoplasmic Signaling


G protein-coupled receptors:More overlap

Tumor Necrosis Factor (TNF) Reviewo in some cells, TNF is mitogenic, but in most it promotes cell death, or apoptosiso this growth factor is not soluble, but is inserted intothe plasma membraneo TNF receptors have no catalytic domain…rely on intracellular proteins to signal

Juxtacrine

Intracrine

TNF family of growth factors

Schulze-Osthoff, Trends Cell Biol. 1994 Dec;4(12):421-426.

TNFR Signaling to Death

DD = death domain, another protein interaction motif

DED = death effector domain

DD and DED formdimers

CELL DEATH

Sci STKE. 2004 Jun 22;2004(239)

TNFR Signaling to Survival

Otherproteins

CELL SURVIVAL

Sci STKE. 2004 Jun 22;2004(239)

TNF Signaling and cancer therapy:Will this work?

TRAIL Signaling to Survival and Death

TRAIL = TNF-related apoptosis-inducing ligand

tumor biology: cellular and molecular aspects of the transformed cell growth factors, receptors, and...

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