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Tracey Hurrell Supervisor: Dr K. Outhoff Department of - - PowerPoint PPT Presentation
Tracey Hurrell Supervisor: Dr K. Outhoff Department of - - PowerPoint PPT Presentation
Tracey Hurrell Supervisor: Dr K. Outhoff Department of Pharmacology Faculty of Health Sciences University of Pretoria Molecular Subtypes ER ER ER ER Negative Positive Her-2 Normal Basal-like Luminal A Luminal B Positive
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Receptor dimerization and horizontal crosstalk
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Trastuzumab: anti-Her-2 mAb
Sub-domain IV of receptors extracellular domain Altered prognosis Her-2 metastatic disease
Her-2 amplification
Discordances up to 30% - primary and metastatic sites
Concept of dynamic receptor expression
Possibility of manipulating targets Provide explanation for emerging resistance
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Cell Viability: MTT Breast Adenocarcinoma
MCF-7 (ERα positive ) SK-Br-3 (Her-2-positive) Cell Cycle Kinetics mAb Efficacy End Point
Cell Cycle: PI Staining Homogenous Caspase Assay: DEVD Substrate Apoptosis-Necrosis: AnnexinV - PI Receptor Density: Affibody Molecule
Trastuzumab:
Her-2Targeting mAb
Epidermal Growth Factor (EGF):
Her-1 ligand
Heregulin-1β (HRG-1β):
Her-3 and Her-4 Ligand
Late Apoptosis Early Apoptosis Kinetics Efficacy
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MCF-7 SK-Br-3
Trastuzumab (100µg/ml) 96.39 % (±2.69 )
( EGF + T: P< 0.001 )
74.17 % (±1.60) EGF 101.70 % (±1.84)
( EGF + T: P< 0.001 )
81.43 % (±2.46) EGF + Trastuzumab 81.97 % (±1.99) 86.02 % (±1.60)
Excessive co-expression of EGFR and Her-2
Ligand-dependent cell death
▪ Presence of proliferative ligands
Trastuzumab - alter the extent of co-expression
EGF has less of an anti-proliferative effect
EGF + Trastuzumab 81.97 % (±1.99) 86.02 % (±1.60)
(Trastuzumab: P < o.o5 )
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MCF-7 SK-Br-3
Trastuzumab (100µg/ml) 96.39 % (±2.69 )
( HRG: P< 0.001 )
74.17 % (±1.60) HRG 107.01 % (±2.42) 115.40 % (±2.08)
( Trastuzumab: P< 0.001)
HRG + Trastuzumab 89.90 % (±1.95) 118.10 % (±2.71)
SK-Br-3: high levels of Her-2 and Her-3 receptors Her-2:Her-3: most potent mitogenic heterodimer pair
Co-operated signalling
▪ Synergistic action in cellular transformation
Potency of this dimer - trastuzumab negligible effects
HRG + Trastuzumab 89.90 % (±1.95)
( HRG: P< 0.001 )
118.10 % (±2.71)
( Trastuzumab P< 0.001 )
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Untreated Control G1 Phase Control S Phase Control G2 Phase Control
- G1
S G2
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- Cell cycle analysis in MCF-7 cells
G1, S, G2 and sub-G1 phases expressed as a percentage after deconvolution
- "
- G1
S G2 SubG1
Time (Hours)
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Trastuzumab: G1 cell cycle arrest
Her-2: regulate G1/S phase transitions Activity of cyclin-CDK complexes
EGF: mimic untreated control
Combination: G1 accumulation
HRG: accelerated S-phase entry (SK-Br-3)
Combination: mimic heregulin
▪ Potent stimulator - compensated for inhibition
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Unstained Control Untreated Control (SK-Br-3) Positive Control
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- Her-2 receptor density analysis in SK-Br-3 cells
X-mean of fluorescent intensity (FL1) expressed as a percentage of untreated control
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Trastuzumab: ability to influence receptor density
Conflicting and inconclusive Significant decrease in surface Her-2 protein
Her-family ligand binding
Internalisation of Her-2 containing heterodimers
▪ Further decreased by trastuzumab ▪ Insufficient cancer targeting
Unclear whether alterations maintained
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Surface Her-2 receptors are required - clinical benefit Endogenous Her-receptor ligands
Reduce surface Her-2 receptors Differentially altered parameters – trastuzumab
Supports the concept :
Receptor density and growth ligands
▪ Mutual importance in proliferation
Emergence of resistance to targeted therapy
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