Biological significance of human epidermal growth factor receptor 2 (HER2) in breast cancer: effect of oestrogen receptor

Background: HER2 gene amplification and protein overexpression defined as HER2 positivity (HER2+) breast cancer (BC) is encountered in 15-25% of cases and is characterised by an aggressive behaviour and poor outcome. Despite the high clinical efficacy of anti-HER2 targeted therapy, the response and clinical behaviour of HER2+ tumours is variable. There is evidence indicating that the response of HER2+ BC to anti-HER2 targeted therapy and chemotherapy is related to Oestrogen Receptor (ER) expression. In addition global gene expression profiling studies have demonstrated that ER and HER2 are the main determinant of BC molecular profiles and that HER2+/ER+ (luminal B) are molecularly distinct from HER2+/ER- (HER2 positive) tumours. It is hypothesised that ER+/HER2+ BC is also a distinct molecular class when compared to tumours with single positive or double negative expression. Therefore, this study aimed to investigate the biological impact of ER expression in HER2+ BC with consideration of the molecular classification of BC and key pathways related to expression and behaviour of both proteins in an attempt to understand their variable biological significance and relationship to treatment response and potentially to identify new therapeutic targets. Methods: Methods included assessment of proteins with known associations with HER2 and ER status and correlating their expression with clinicopathological variables, molecular classes, different key BC proteins and outcome. For this purpose, Immunohistochemistry (IHC) was used to stain a number of key targets, including Mitogen Activated Protein Kinases (MAPKs), Phosphatidylinositol 3 kinase (PI3K)/Akt/mammalian target of Rapamycin (mTOR) pathway members and other proteins related to HER2 and ER and proliferation in a large well-characterised uniformly treated and annotated cohort of 1835 patients with primary BC. In addition, a cohort of 197 primary BC patients treated with Trastuzumab between 2003 and 2012, were also included. Reverse Phase Protein Array (RPPA) was used to quantify protein expression in six BC cell lines. To assess the effect of HER2 on cell lines with and without ER expression, two HER2 negative cell lines (MCF-7 and MDA-MB-231) were transfected with HER2. Results: The majority of MAPKs pathway members (pan Extracellular Signal- Regulated Kinase (ERK1/2), nuclear phosphorylated (p)-ERK1/2, p-c-jun-N terminal Kinase (JNK1/2), pan p38, p-p38 and p-ATF2 and p-C-JUN) showed positive associations with good prognostic variables and longer survival in the whole (unselected) cohort and in ER+ tumours but many of these associations were lost with HER2 co-expression. Such associations were infrequently observed within ER-HER2+ cases. HER2 overexpression was associated with downregulation of phosphorylated MAPKs within the whole cohort and within ER+ BC (significant for nuclear p-ERK1/2, p-ATF2 and p-p38), but ERK1/2 and p-p38 were associated with HER2 positivity within ER- tumours implying their context specific function. In addition, pan ERK1/2, p-p38 and p-ATF2 were independent predictors of better survival in BC and in ER+ BC. RPPA confirmed the IHC findings and showed similar association where the expression of MAPKs was different in ER+HER2+ cell lines compared to ER-HER2+ and ER+HER2- ones. Regarding the PI3K/Akt/mTOR pathway, p-mTORC1 and Phosphatase and Tensin homolog (PTEN) were negatively associated with HER2 overexpression in ER+ tumours but were (in addition to Akt and PI3K) positively associated with HER2 in ER- tumours. Meanwhile, mTOR exhibited positive associations with favourable prognostic factors within ER+ BC which were decreased with HER2 co-expression and with ER loss. Additionally, p-mTORC1 was associated with prolonged breast cancer specific survival (BCSS) within Akt+ tumours but not within the whole cohort or other subgroups. In this study, using RPPA, mTOR and PTEN were positively associated with ER and negatively with HER2 in ER+ cell lines and p-mTORC1 was positively associated with HER2 in ER- cell lines in addition to other members. Importantly, PI3K, Akt, p-mTORC1 and its downstream p-S6K showed increased expression within ER+HER2+ cell lines compared to ER-HER2+ cell lines but PTEN expression was increased in ER-HER2+ vs ER+HER2+ cell lines. When the biological significance of HER2 and KI67-LI was investigated in ER+ tumours, both HER2 and KI67-LI were associated with poor prognostic variables and adverse outcome in the ER+ tumours. Although KI67-LI rather than HER2 was associated with downregulation of luminal associated biomarkers, HER2 positivity was associated with worse outcome in ER+ tumours, indicating that HER2+ BC are distinct aggressive tumours regardless of their proliferative activity. Investigation of other proteins related to HER2 and ER pathways revealed that nuclear form of both the Carboxyl-terminus of Hsp-70-Interacting Protein (CHIP) and the stem cell protein, Sry-Related HMG Box 9 (SOX9), were negatively associated with HER2. CHIP was positively associated with ER, ER-associated proteins and prolonged outcome in the unselected BC and in ER+ BC but not in ER+/HER2+ and ER-/HER2+ tumours. The phosphorylated form of ER at Serine (SER) 118 was positively associated with good prognostic variables and negatively with HER2 in the unselected series and in the ER+ BC group with an observed decrease in these associations within ER+/HER2+ tumours. Increased loss of association was encountered and even some unfavourable associations were observed within ER-HER2+. Furthermore, it was associated with prolonged survival in ER+ tumours and was a predictor of prolonged survival in patients receiving tamoxifen therapy. Clustering analysis to predict class memberships based on HER2 and ER expressions using a large panel of biomarkers related to ER, HER2 and key pathways’ proteins, generated a decision tree that could be a future model for patients’ stratification which indicated the overwhelming driving effect of HER2 expression. Conclusions: ER+/HER2+ BC is a distinct biological group, having some luminal features but is associated with worst outcome owing to the co-expression of HER2 independently from ER influence. The investigation of MAPKs, PI3K/Akt/mTOR pathways and other proteins highlighted their differential expressions and associations (with key proteins related to ER and HER2) within different BC subgroups based on ER and HER2 expressions indicating that ER+HER2+ stands as a group with unique features from those with single positive or double negative expression. Finally, development of a decision tree is a potentially promising tool for patients’ stratification. Breast cancer cell line studies using the high throughput technique, RPPA, showed good concordance with IHC results, implying that further in vitro studies using relevant cell line models could be possible