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 ERα ERα ERα ERα Negative Positive Her-2 Normal Basal-like Luminal A Luminal B Positive Breast-like � Histological grade: degree of differentiation � Histological type: growth pattern

Receptor dimerization and horizontal crosstalk

� 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

End Point Breast Adenocarcinoma Cell Viability: MCF-7 (ERα positive ) SK-Br-3 (Her-2-positive) MTT mAb Cell Cycle Efficacy Efficacy Kinetics Kinetics Receptor Density: Cell Cycle: Affibody Molecule PI Staining Trastuzumab: Her-2Targeting mAb Epidermal Growth Factor Late Early (EGF): Apoptosis Apoptosis Her-1 ligand Homogenous Apoptosis-Necrosis: Heregulin-1β (HRG-1β): Caspase Assay: Her-3 and Her-4 Ligand AnnexinV - PI DEVD Substrate

MCF-7 SK-Br-3 Trastuzumab (100µg/ml) 96.39 % (±2.69 ) 74.17 % (±1.60) ( EGF + T: P< 0.001 ) EGF 101.70 % (±1.84) 81.43 % (±2.46) ( EGF + T: P< 0.001 ) EGF + Trastuzumab EGF + Trastuzumab 81.97 % (±1.99) 81.97 % (±1.99) 86.02 % (±1.60) 86.02 % (±1.60) (Trastuzumab: P < o.o5 ) � 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

MCF-7 SK-Br-3 Trastuzumab (100µg/ml) 96.39 % (±2.69 ) 74.17 % (±1.60) ( HRG: P< 0.001 ) HRG 107.01 % (±2.42) 115.40 % (±2.08) ( Trastuzumab: P< 0.001) HRG + Trastuzumab HRG + Trastuzumab 89.90 % (±1.95) 89.90 % (±1.95) 118.10 % (±2.71) 118.10 % (±2.71) ( HRG: P< 0.001 ) ( Trastuzumab P< 0.001 ) � 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

Untreated Control G1 Phase Control S Phase Control G2 Phase Control ��� ��� ��� ��� ��� ��� ��� ��� ������������������� G1 �� �� �� �� S G2 �� �� �� �� �� �� �� �� �� �� �� �� � � � � ���� ��������� ������ �� ��������

��!�" ������#���$ ��� *** ��� ��� ������� ����������������� G1 �� S G2 �� Sub�G1 �� �� � �������� ��� �� "� Time (Hours) Cell cycle analysis in MCF-7 cells G1, S, G2 and sub-G1 phases expressed as a percentage after deconvolution

� 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

Unstained Control Untreated Control (SK-Br-3) Positive Control ��� ��� ��� ��� ��� ��� (����%�������&�������� (����%�������&�������� (����%�������&�������� ��� ��� ��� ��������!'�� ��������!'�� ��������!'�� �� �� �� �� �� �� �� �� �� �� �� �� � � � %���� ��& )��&����*� � %�������&

+,�-��. ���/���� *** ��� ** *** ��� ������ *** *** (����%�������&������� ��� ��� ** ��������!'�� * �� �� �� �� � %�������& � �)! �)!���� )�'� Her-2 receptor density analysis in SK-Br-3 cells X-mean of fluorescent intensity (FL1) expressed as a percentage of untreated control

� 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

� 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

� Acknowledgements � Funding: CANSA and RDP � Roche Pharmaceuticals: Trastuzumab � Personnel and Colleagues: Department of Pharmacology � Personnel and Colleagues: Department of Pharmacology