Investigating the impact of HER2 phosphorylation on breast tumorigenesis and treatment response

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. <br><br>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.<br><br>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. <br><br>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.<br><br>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. <br><br><br>