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

Clinicopathological and prognostic significance of mitogen-activated protein kinases (MAPK) in breast cancers

Background Mitogen-activated protein kinases (MAPKs) are signalling transduction molecules that have different functions and diverse behaviour in cancer. In breast cancer, MAPK is related to oestrogen receptor (ER) and HER2. Methods Protein expression of a large panel of MAPKs (JNK1/2, ERK, p38, C-JUN and ATF2 including phosphorylated forms) were assessed immunohistochemically in a large (n = 1400) and well-characterised breast cancer series prepared as tissue microarray. Moreover, reverse phase protein array was applied to quantify protein expression of MAPKs in six breast cancer cell lines with different phenotypes including HER2-transfected cells. Results MAPKs expression was associated with clinicopathological variables characteristic of good prognosis. These associations were most significant in the whole series and in the ER? subgroup compared to other BC classes. Most of MAPKs showed a positive association with ER, BCL2 and better outcome and were negatively associated with the proliferation marker Ki67 and p53. Association of MAPK with HER2 was mainly seen in the ER- subgroup. Reverse phase protein array confirmed immunohistochemistry results and revealed differential expression of MAPK proteins in ER? and ER- cell lines. Conclusions MAPKs are associated with good prognosis and their expression is mainly related to ER. Studying a large panel rather than individual biomarkers may provide improved understanding of the pathwa

Expression of CDK7, cyclin H and MAT1 is elevated in breast cancer and is prognostic in estrogen receptor- positive breast cancer

Purpose: CDK-activation kinase (CAK) is required for the regulation of the cell-cycle and is a trimeric complex consisting of Cyclin Dependent Kinase 7 (CDK7), Cyclin H and the accessory protein, MAT1. CDK7 also plays a critical role in regulating transcription, primarily by phosphorylating RNA polymerase II, as well as transcription factors such as estrogen receptor-α (ER). Deregulation of cell cycle and transcriptional control are general features of tumor cells, highlighting the potential for the use of CDK7 inhibitors as novel cancer therapeutics. Experimental Design: mRNA and protein expression of CDK7 and its essential co-factors cyclinH and MAT1, were evaluated in breast cancer samples to determine if their levels are altered in cancer. Immunohistochemical staining of >900 breast cancers was used to determine the association with clinicopathological features and patient outcome. Results: We show that expression of CDK7, cyclinH and MAT1 are all closely linked at the mRNA and protein level and their expression is elevated in breast cancer compared with the normal breast tissue. Intriguingly, CDK7 expression was inversely proportional to tumour grade and size and outcome analysis showed an association between CAK levels and better outcome. Moreover, CDK7 expression was positively associated with ER expression and in particular with phosphorylation of ER at serine 118, a site important for ER transcriptional activity. Conclusions: Expression of components of the CAK complex, CDK7, MAT1 and Cyclin H are elevated in breast cancer and correlates with ER. Like ERα , CDK7 expression is inversely proportional to poor prognostic factors and survival

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

Prognostic and biological significance of proliferation and HER2 expression in the luminal class of breast cancer

The definition of Luminal-B subclass of breast cancer (BC) varies in literature. In this study, we have compared the proliferation status; assessed using KI67 labeling index (KI67-LI), and HER2-expression in estrogen receptor positive (ER+) BC to assess their impact on the biological and clinical characteristics of luminal-BC. 1547 (73.8 %) well-characterized clinically annotated stage I–III ER + BC were assessed for expression of KI67, HER2 (ASCO guidelines), and a large panel of relevant biomarkers (no = 37). 46.3 % of the cases show high KI67-LI (>13 %) and 8.4 % show HER2+ and both markers are positively associated with younger age, higher tumor grade and poorer outcome. High KI67-LI and HER2+ are associated with upregulation of ER-coactivators and proliferation-related markers and with downregulation of good prognostic markers. High KI67-LI is associated with larger size, advanced stage, and lymphovascular invasion (LVI) and with downregulation of luminal-enriched and DNA-damage repair markers. In contrast, HER2+ is associated with upregulation of ER-regulated proteins and E-cadherin. When analysis is restricted to high KI67-LI subgroup, HER2+ shows an association with upregulation of differentiation-associated proteins and E-cadherin. Conversely, within HER2+ class, high KI67-LI maintains its association with downregulation of differentiation-associated/luminal-enriched proteins. Outcome analyses indicate that both markers are independently associated with shorter survival but HER2+ is associated with a worse outcome. Although both are associated with high proliferation and poor prognosis within ER + BC, HER2+ is less frequent than high KI67-LI. Unlike KI67, HER2 seems to independently drive the aggressive behavior of ER+ tumors without downregulation of luminal proteins

ERK1/2 is related to oestrogen receptor and predicts outcome in hormone-treated breast cancer

The extracellular-regulated kinase (ERK) 1/2 is one of the members of the mitogen-activated protein kinases (MAPKs). MAPKs are transduction proteins that play a role in controlling diverse cellular functions including proliferation and survival. In breast cancer (BC), MAPKs are involved in oestrogen receptor (ER) and HER2 pathways. This study aims to assess the biological and clinical significance of ERK1/2 protein expression in BC. Immunohistochemistry was used to assess the expression of both total (ERK1/2) and phospholyated (p ERK1/2) ERK1/2 proteins in a large and well-characterised series of early stage BC (n = 1300) using tissue microarray technology. ERK1/2 expression was cytoplasmic, while p-ERK1/2 was observed in the nucleus (N-p-ERK1/2) and/or cytoplasm (C-p-ERK1/2). Both ERK1/2 and p-ERK1/2 were positiviely associated with markers of good prognosis including smaller size, lower grade, expression of hormone receptor and ER-related proteins and negatively associated with HER2, HER4, KI67 and p53. Outcome analysis showed an association between N-p-ERK1/2 and better outcome. In tamoxifen-treated cases, ERK1/2 expression was an independent prognostic marker of longer survival. ERK1/2 and p-ERK1/2 were associated with good prognosis. Importantly, positivity of ERK1/2 is independently associated with better outcome in tamoxifen-treated cases

Clinical and biological significance of glucocorticoid receptor (GR) expression in breast cancer

The glucocorticoid receptor (GR) is a member of the nuclear receptor superfamily of transcription factors, which exerts anti-proliferative and anti-apoptotic activities. The GR is expressed in a large proportion of breast cancer (BC) although levels generally decrease during cancer progression. This study aimed to determine the clinical and biological significance of GR expression using a large series of early-stage BC with long-term follow-up and BC cell lines. Immunohistochemistry was used to assess the expression of GR in 999 cases of primary invasive BC prepared as tissue microarrays. Reverse phase protein microarray was used to assess the expression of GR in MCF7 and MDA-MB-231 cell lines. Nuclear expression of GR was observed in 61.6 % of breast tumours and was associated with features of good prognosis including smaller tumour size and lower grade with less pleomorphism and low mitotic count. GR expression was positively correlated with expression of oestrogen (ER) and progesterone receptors. In ER-positive tumours, GR was associated with other features of favourable outcome including FOXA1, GATA3 and BEX1 expression, while low GR expression was associated with high Ki67, p53 and CD71 expression. GR expression is associated with features of good outcome but does not provide prognostic information independent of size, stage and grade. Understanding the receptor and its effects on BC behaviour is essential for avoiding any unwanted effects from the use of glucocorticoids in routine oncology practice

The mammalian target of rapamycin complex 1 (mTORC1) in breast cancer: the impact of oestrogen receptor and HER2 pathways

The mammalian target of rapamycin complex 1 (mTORC1) is a downstream of the PI3K/Akt pathway which affects cancer development. mTORC1 has many downstream signalling effectors that can enhance different cellular responses. This study aims to investigate the expression of mTORC1 in breast cancer (BC) and correlate it with key clinicopathological and molecular features of BC especially to proteins related to oestrogen receptor (ER) and HER2 pathways in different BC classes. Moreover, mTORC1 expression was assessed in 6 BC cell lines including ER+ and ER− cell lines with and without HER2 transfection. Immunohistochemistry was used to assess the expression of phospho (p) mTORC1 in a large (n = 1300) annotated BC series prepared as tissue microarray. Reverse phase protein array (RPPA) was used to assess its expression in the different BC cell lines. The expression of p-mTORC1 was cytoplasmic with moderate/high expression noted in 44 % of BC. p-mTORC1 expression was associated with clinicopathological variables characteristic of good prognosis. Positive correlation with ER, ER-related proteins AKT, PI3K and luminal differentiation markers were observed in the whole series and in the ER+HER2− subgroup. Association with HER2 was mainly observed in the ER-negative class. RPPA indicated that p-mTORC1 expression was mainly related to ER expression and with better outcome in the Akt positive tumours. p-mTORC1 is associated with good prognostic features. Its expression is related to ER and ER related proteins in addition to AKT and PI3K. Its relation with HER2 expression is mainly seen in the absence of ER expression