| الملخص: |
HER2-amplified tumours are associated with poor prognosis, higher metastatic rate and shortened overall survival. Despite improvement in clinical activity, many patients still develop resistance to anti-HER2 targeting agents. This highlights the need for better understanding of HER2-driven biology to aid patient stratification and new therapeutic strategies. This study identifies HER2-binding partners in an ER-positive background, as well as their role in mediating pathway activation and response to therapy. HER2 phosphorylation status was also assessed regarding activation of downstream signalling cascades and receptor interactions. To investigate the role of HER2 phosphorylation sites (Y1248 and Y1221/22) in ER-positive HER2-positive background, two HER2-overexpressing model systems were generated: doxycycline inducible expression and stable expression systems. The HER2-negative MCF7 cell line was transduced with lentiviral vectors containing HER2-wild type (HER2-WT), HER2 single phospho-site mutants (HER2-Y1248F or HER2-Y1221/22F) or a HER2-double mutant (HER2-DM). The HER2 phospho-site mutants did not have an effect on proliferation, suggesting a potential redundancy in this pathway. HER2-Y1221/22F overexpressing MCF7 cells displayed reduced migratory ability compared to HER2-WT cells, which was coupled with reduced signalling through the ERK/MAPK pathway. In addition, the three phospho-site mutant HER2-overexpressing MCF7 cells demonstrated reduced anchorage-independent colony formation ability. RET was identified as a binding partner for HER2 through mass spectrometry analysis. Inhibition of HER2 tyrosine kinase activity using lapatinib resulted in decreased levels of both phospho-HER2 and phospho-RET, indicating that HER2 is required for RET activation. To elucidate the role of RET in downstream pathway activation, RET knockdown showed reduced phospho-ERK levels in HER2-positive cells. Phospho-RET expression levels were decreased in HER2-Y1248F mutant cells, suggesting that HER2 auto-phosphorylation is required for efficient RET activation. This implicates RET as an important mediator of MAPK activation in ER+HER2+ setting and highlights its importance as a drug target. To investigate the role of HER2 phosphorylation sites (Y1248 and Y1221/22) in ER-positive HER2-positive background, two HER2-overexpressing model systems were generated: doxycycline inducible expression and stable expression systems. The HER2-negative MCF7 cell line was transduced with lentiviral vectors containing HER2-wild type (HER2-WT), HER2 single phospho-site mutants (HER2-Y1248F or HER2-Y1221/22F) or a HER2-double mutant (HER2-DM). The HER2 phospho-site mutants did not have an effect on proliferation, suggesting a potential redundancy in this pathway. HER2-Y1221/22F overexpressing MCF7 cells displayed reduced migratory ability compared to HER2-WT cells, which was coupled with reduced signalling through the ERK/MAPK pathway. In addition, the three phospho-site mutant HER2-overexpressing MCF7 cells demonstrated reduced anchorage-independent colony formation ability. RET was identified as a binding partner for HER2 through mass spectrometry analysis. Inhibition of HER2 tyrosine kinase activity using lapatinib resulted in decreased levels of both phospho-HER2 and phospho-RET, indicating that HER2 is required for RET activation. To elucidate the role of RET in downstream pathway activation, RET knockdown showed reduced phospho-ERK levels in HER2-positive cells. Phospho-RET expression levels were decreased in HER2-Y1248F mutant cells, suggesting that HER2 auto-phosphorylation is required for efficient RET activation. This implicates RET as an important mediator of MAPK activation in ER+HER2+ setting and highlights its importance as a drug target. Finally, a 63-gene expression signature which detects HER2 phosphorylation at Y1248 was assessed in two breast cancer clinical cohorts. The phospho-HER2 signature had better prognostic value for predicting recurrence-free and overall survival than standard clinical HER2 testing. Moreover, the phospho-HER2 signature identified a poor prognosis subgroup within HER2-positive breast cancer patients, treated with either trastuzumab or endocrine therapy. The signature was applied across a panel of breast cancer cell lines and demonstrated a correlation with phospho-HER2 expression levels determined by western blot. Moreover, the phospho-HER2 signature showed an inverse correlation with response to lapatinib. |
