HER1/EGFR and HER2/ErbB2 pathways

O. Segatto, Regina Elena Cancer Institute

RTK activity regulates cellular programmes crucial to cell transformation and tumour progression

The hallmarks of cancer

RTKs have intrinsic tyrosinekinase activity which is activated

upon ligand binding

RTKs are bistable systems, i.e. they transit from an “off” to an

“on” state with no intermediate states

Kinase domain

Extracellular domain

COOH tail

Ligand binding induces kinase activation and receptor self-phosphorylation on specific Tyr residues

Specific p-Tyr sites generated by RTK autophosphorylationinitiate downstream signalling by acting as docking sites

for cellular proteins containing SH2 or PTB domains.

All SH2 and some PTB domains bind to p-Tyr. Specificity of molecular recognition is dictated by residues C-terminal

to p-Tyr for SH2 domains and N-terminal to p-Tyr for PTB domains.

The human genome encodes 139 SH2-containing and 49 PTB-containing proteins.

EGFR

PLC-

IP3 + DAG

Activation of downstream signalling: enzymes containing an SH2 domain bind to pY-EGFR and are relocated to the cell surface

Relocation onto the EGFR allows PLC- to be activated via Tyr phosphorilation and “induced proximity”

PIP2

RAS

EGFR

PI-3K-AKTRAF-ERKSOSGRB2

Activation of downstream signalling: SH2 adaptors bind to pY-EGFR and relocate enzymes to the cell surface

RAS

GTP (active)

GDP (inactive)

Direct plasmamembrane-to-nucleus signalling throughengagement of STAT proteins

SH2 and PTB domains are present in enzymes, adaptors and membrane-bound scaffolding proteins

Formazione del “signalosoma” su RTK: perché?

Concentrazione di effettori enzimatici in membrana

Attivazione rapida e reversibile di funzioni enzimatiche

Attivazione integrata di vie di segnalazione multiple

Amplificazione del segnale

Polarizzazione del segnale

RTK activation needs to be tightly controlled

Aberrant RTK activity is linked to cell transformation

Focus

Understanding the “design principles” of ErbBactivation, as deviation from these design principlesmay underline oncogenic conversion of ErbB RTKs

Intramolecular interactions lock receptors in an inactive conformation, which is released upon ligand binding

Restricted ligand availability limits receptor activation

Feedback inhibition restricts receptor activity

Key design principles

a) on-demand-only activationb) prevention of unwanted activationc) tight monitoring of receptor activity

Operational principles

Intramolecular interactions lock EGF receptors in a monomeric inactive conformation, which is released upon ligand binding

Part 1: structural transitions in the extracellular domain

Xuewu Zhang et al., Cell, Volume 125, Issue 6, 1137-1149, 13 June 2006

Intramolecular interactions lock receptors in an inactive conformation, which is released upon ligand binding

Part 2: structural transitions in the catalytic domain

Ligand-induced EGFR dimerizationreleases the intra-molecular inhibitionof EGFR kinase via inter-molecularallosteric activation

• Intramolecular interactions lock receptors in an inactive conformation, which is released upon ligand binding

• Restricted ligand availability limits receptor activation

• Feedback inhibition restricts receptor activity

Operational principles

Restricted ligand availability limits receptor activation

ErbB ligands are synthesised by stromal cells as membrane-bound precursors. Cleavage by proteases releases the soluble form

• Intramolecular interactions lock receptors in an inactive conformation, which is released upon ligand binding

• Restricted ligand availability limits receptor activation

• Feedback inhibition restricts receptor activity

Operational principles

PP P PPP P P

PTPX

EGFR inhibitors

Feedback inhibition restricts EGF receptor activity

Downregulation depletes receptors and ligands, leading to cellular refractoriness to further homologous stimulation

Inducible feedback regulators?

Feedback inhibition restricts receptor activity

MIG6 is an inducible feedback inhibitor that suppresses EGFR catalytic activation by binding to a dimer interface located in the COOH lobe of the EGFR kinase domain

Xuewu Zhang, Kerry A. Pickin, Ron Bose, Natalia Jura, Philip A. Cole & John KuriyanNature 450, 741-744(29 November 2007)

wt KO KO + Gefitinib

EGFR signalling activity is the result of a dynamicequilibrium between mechanisms of signal

generation and signal extinction

RTKs are bistable systems, i.e. they transit from an “off” to an

“on” state with no intermediate states

Intramolecular interactions lock receptors in an inactive conformation, which is released upon ligand binding

Restricted ligand availability limits receptor activation

Feedback inhibition restricts receptor activity

Oncogenic conversion of EGFR and ERBB2 releases the receptors from these constrains, thus allowing unabated signalling activity

ErbB receptors at work: the network context

Thomas Jefferson, United States Declaration of Independence, 1776

… whereas ErbB receptors, ligands and ligand-receptor combinations are not!

Some ligands do not lead to EGFR degradation: TGF and Epiregulin drive complete and fast EGFR recycling, Amphiregulin drives both fast and slow EGFR recycling.

These ligands may be continuously re-used by the cell and also allow the EGFR to escape down-regulation.

High gain of signalling potency

ErbB2 and ErbB3 are non-authonomous receptors: ErbB2does not bind to any known ligand, whereas ErbB3 has nokinase activity.

Under physiological conditions ErbB2 and ErbB3 signal only in thecontext of ligand-induced heterodimers

ErbB2/HER2: an atypical RTK

Sequence divergence in the extracellular region generatesa) inability to bind ligandb) extended conformation of the dimerization arm

ErbB2 is the hierarchically dominant ErbB receptor in dimer assembly

ErbB2 is refractory to endocytosis/downregulation

EGFR:ErbB2 heterodimers gain signalling potency due to decelerated ligand off-rates and refractoriness to endocytosis/downregulation

ErbB2 is a powerful signal amplifier

Strength and duration of signals generated by ErbB RTKs depend on the nature of the ligand:dimer combination

Citri et al. Nature Reviews Molecular Cell Biology 7, 505–516 (July 2006)

Towards the system level…

Oncogenic conversion of EGFR and ERBB2 in human tumours: mechanisms and therapeutic opportunities

Oncogenic conversion is caused by genetic lesions, which drive the constitutive signalling activity of EGFR and ERBB2.

Oncogenic signalling by EGFR and ERBB2 differs in quantitative and qualitative terms from physiological signalling.

This may create a state of “oncogene addiction” and cause tumour cells to become exquisitively sensitive to drugs that target EGFR and/or ERBB2.

Inactive wtEGFR L858R EGFR mutant

EGFR mutations in lung cancer generate constitutively active kinases

Yun, C-H et al., 2007, Cancer Cell, 11:217-227

Mutations cause constitutive activation of the EGFR kinase

Mutations are associated to EGFR copy gain

Mutations render EGFR refractory to down-regulation

Mutations sensitize tumour cells to EGFR kinase inhibitors

Compound effects of mutational activation of EGFR in NSCLC

ERBB2 is activated by gene amplification and attendant over-expression – mutational activation is very rare

Case study: breast cancer, ERBB2 subtype

ERBB2 overexpression drives constitutive homo-dimerization

ERBB2 over-expression is associated to increased ERBB3 expression, with ERBB3 being a necessary signalling subunitof ERBB2

RTKs are bistable systems, i.e. they transit from an “off” to an

“on” state with no intermediate states

Intramolecular interactions lock receptors in an inactive conformation, which is released upon ligand binding MUTATIONS, OVEREXPRESSION

Restricted ligand availability limits receptor activation AUTOCRINE PRODUCTION of LIGANDS

Feedback inhibition restricts receptor activityREFRACTORINESS to DOWN-REGULATION (severalmechanisms)

Oncogenic conversion of EGFR and ERBB2 releases the receptors from these constrains, thus allowing unabated signalling activity

Quantity (strength and duration)…

… but also quality

Signal promiscuity imposed by ERBB2

average: 7.2 average: 17

Jones, RB et al., 2006 Nature, 439:168-174

Over-expression modifies the quality of signals generated by EGFR and ERBB2

Jones, RB et al., 2006 Nature, 439:168-174

Genetic lesions of EGFR and ERBB2 in humantumours lead to constitutive signalling activity which differs from physiological signalling in quantitative and qualitative terms

Cetuximab inhibits ligand-dependent activation of EGFR

Targeting ERBB2 oncogenic signalling with therapeutic antibodies

Kinase inhibitors target the ATP-binding pocket of EGFR and ErbB2

Yun, C-H et al., 2007, Cancer Cell, 11:217-227

Mutations in the EGFR kinase may increase its affinity for competitive ATP inhibitors such as Gefitinib

Different mutations may display different sensitivity to competitive ATP inhibitors: shall drugs get personal?

Targeting the ErbB network, rather than any individual ErbB RTK may result in a therapeutic advantage

Citri et al. Nature Reviews Molecular Cell Biology 7, 505–516 (July 2006)

Conclusions

Oncogenic signalling by EGFR and ErbB2 originates from thesubversion of key regulatory principles of receptor activation

Oncogenic conversion grants EGFR and ErbB2 full operationalautonomy as well as evasion from negative regulation

Targeting EGFR and ErbB2 in tumours must take into accountthe “design principles” of oncogenic signalling by EGFR and ErbB2, including aberrant network activation

In memoria di Matthias Kraus, 1956-2009

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Citri et al. Nature Reviews Molecular Cell Biology 7, 505–516 (July 2006)

Inactive wtEGFR