S. Tovey

AKT activation predicts outcome in breast cancer patients treated with tamoxifen

Oestrogen receptor (ERα) expression is a strong predictor of response to endocrine therapy. The PI3K/AKT/mTOR signal transduction pathway has been implicated in endocrine resistance in vitro. The present study was carried out to test the hypothesis that AKT activation mediates tamoxifen resistance in clinical breast cancer. Immunohistochemistry (IHC) using AKT1‐3, pan‐AKT, pAKT (Thr‐308), pAKT (Ser‐473), pER (Ser‐167), and pHER2 antibodies was performed on 402 ERα‐positive breast carcinomas from patients treated with tamoxifen. High pAKT (Ser‐473) activity (p = 0.0406) and low AKT2 expression (p = 0.0115) alone, or in combination [high pAKT (Ser‐473)/low AKT2; ‘high‐risk’ patient group] (p = 0.0014), predicted decreased overall survival in tamoxifen‐treated patients with ERα‐positive breast cancers. There was no significant association between tumour levels of AKT expression or activity and disease‐free survival (DFS); however, the ‘high‐risk’ patient group was significantly more likely to relapse (p = 0.0491). During tamoxifen treatment, neither AKT2 nor pAKT predicted DFS. Finally, activation of AKT, via phosphorylation, was linked to activation of both HER2 and ERα in this patient cohort. The data presented here show that the PI3K/AKT/mTOR pathway is associated with relapse and death in ERα‐positive breast cancer patients treated with tamoxifen, supporting in vitro evidence that AKT mediates tamoxifen resistance. Patients with a ‘high‐risk’ expression profile were at increased risk of death (hazard ratio 3.22, p = 0.002) relative to ‘low‐risk’ patients, highlighting the potential that tumour profiling, with multiple IHC markers predictive of therapeutic response, may improve patient selection for endocrine therapies, eg tamoxifen or aromatase inhibitor‐based treatments. Copyright

Observer variation in immunohistochemical analysis of protein expression, time for a change?

Aim : Immunohistochemical analysis of protein expression is central to most clinical translational studies and defines patient treatment or selection criteria for novel drugs. Interobserver variation is rarely analysed despite recognition that this is a key area of potential inaccuracy. Therefore our aim was to examine observer variation and suggest the revision of current standards.

The ERBB4/HER4 Intracellular Domain 4ICD Is a BH3-Only Protein Promoting Apoptosis of Breast Cancer Cells

ERBB4/HER4 (referred to here as ERBB4) is a unique member of the epidermal growth factor receptor (EGFR) family of receptor tyrosine kinases. In contrast to the other three members of the EGFR family (i.e., EGFR, ERBB2/HER2/NEU, and ERBB3), which are associated with aggressive forms of human cancers, ERBB4 expression seems to be selectively lost in tumors with aggressive phenotypes. Consistent with this observation, we show that ERBB4 induces apoptosis when reintroduced into breast cancer cell lines or when endogenous ERBB4 is activated by a ligand. We further show that ligand activation and subsequent proteolytic processing of endogenous ERBB4 results in mitochondrial accumulation of the ERBB4 intracellular domain (4ICD) and cytochrome c efflux, the essential and committed step of mitochondrial regulated apoptosis. Our results indicate that 4ICD is functionally similar to BH3-only proteins, proapoptotic members of the BCL-2 family required for initiation of mitochondrial dysfunction through activation of the proapoptotic multi-BH domain proteins BAX/BAK. Similar to other BH3-only proteins, 4ICD cell-killing activity requires an intact BH3 domain and 4ICD interaction with the antiapoptotic protein BCL-2, suppressed 4ICD-induced apoptosis. Unique among BH3-only proteins, however, is the essential requirement of BAK but not BAX to transmit the 4ICD apoptotic signal. Clinically, cytosolic but not membrane ERBB4/4ICD expression in primary human breast tumors was associated with tumor apoptosis, providing a mechanistic explanation for the loss of ERBB4 expression during tumor progression. Thus, we propose that ligand-induced mitochondrial accumulation of 4ICD represents a unique mechanism of action for transmembrane receptors, directly coupling a cell surface signal to the tumor cell mitochondrial apoptotic pathway.

Can Molecular Markers Predict When to Implement Treatment with Aromatase Inhibitors in Invasive Breast Cancer

Purpose: Resistance to tamoxifen is linked to overexpression of HER2, and aromatase inhibitors show particular benefit in progesterone receptor (PR)–negative patients. We previously reported reduced survival in patients overexpressing HER1, HER2, and HER3. We now show that both HER1-3 and PR status predicts for early relapse in estrogen receptor (ER)–positive tamoxifen-treated breast cancer patients. Experimental Design: Tissue microarray technology was used to analyze 402 ER-positive tamoxifen-treated patients. Immunohistochemistry using epidermal growth factor receptor, HER2, HER3, HER4, and PR antibodies was done. Kaplan-Meier life table and Cox Regression analysis (log-rank testing of differences in breast cancer–related relapse on tamoxifen) was done. Results: HER1-3 (but not HER4) overexpression predicted for early relapse on tamoxifen (P = 0.0060). PR-negative cases were also significantly more likely to relapse while on tamoxifen (P= 0.017). HER1-3-positive and/or PR-negative patients combined as a “high-risk” group were significantly more likely to relapse on tamoxifen in univariate (P < 0.0001) and Coxs multivariate analysis (P = 0.0069). However, this applied to early relapse on tamoxifen only, as any disease relapse after 3 years of tamoxifen was unrelated to PR/HER status. Conclusions: We show that HER1-3 and PR status can identify time-dependent de novo tamoxifen resistance with risk declining markedly after 3 years of tamoxifen treatment. These results parallel data from the ATAC and Intergroup Exemastane Study trials which suggest that whereas PR-negative patients derive greater benefit from initial aromatase inhibitor treatment, PR status has no effect on response when given as delayed treatment to those disease free on tamoxifen after 3 years.

Outcome and Human Epithelial Growth Factor Receptor (HER) 1-4 status in invasive breast carcinomas with proliferation indices evaluated using bromodeoxyuridine (BrdU) labelling

BackgroundWe have shown previously that whilst overexpression of HER1, 2 and 3 is associated with poor prognosis in breast cancer, HER4 is associated with a good prognosis. Cell proliferation is a key component of aggressive cancers and is driven by growth factors. In this study bromodeoxyuridine-derived proliferation indices are correlated with clinical outcome and HER1-4 status to further clarify the differing roles for the HER family at a biological level.Patients and Methods78 invasive breast cancers had BrdU in vivo labelling to determine the labelling index (BLI) and the potential tumour doubling time (Tpot). Long term clinical follow up was available for these patients. Using immunohistochemistry we established the HER1-4 status in 55 patients from the BrdU cohort.ResultsWe demonstrate a significant correlation between high BLI values and breast cancer specific death (p = 0.0174). Low Tpottimes were also significantly correlated with breast cancer specific death (p = 0.0258). However BLI did not independently predict survival in Coxs multiple regression analysis when combined with other prognostic factors such as size, grade and nodal status.Tumours found to be positive for HER 1, 2 or 3 had significantly (p = 0.041) higher labelling indices, with HER1 also showing significantly higher indices when considered independently (p = 0.024). Conversely HER4 positivity significantly correlated (p = 0.013) with low BLI values in line with previous data associating this receptor with good prognosis tumours.ConclusionsThese results support the hypothesis that HER1-3 are associated with driving tumour proliferation whilst HER4 is involved in a non-proliferative or even protective role.

Amplified in Breast Cancer 1 in Human Epidermal Growth Factor Receptor–Positive Tumors of Tamoxifen-Treated Breast Cancer Patients

Purpose: Amplified in breast cancer 1 (AIB1) is a member of the p160/steroid receptor coactivators family and is involved in estrogen-dependent gene transcription by reducing the antagonistic activity of tamoxifen-bound estrogen receptor-α (ER-α). The present study was carried out to test the hypothesis that AIB1 protein expression and/or gene amplification mediates tamoxifen resistance in breast cancer. Experimental Design: Immunohistochemistry using AIB1 antibody and fluorescence in situ hybridization using probes specific for AIB1 and chromosome 20 was done on 402 ER-α–positive tamoxifen-treated breast cancers. Results: AIB1 overexpression was not associated with relapse during treatment with tamoxifen. In contrast, high AIB1 expression in patients with human epidermal growth factor receptor (HER) 2– and HER3-overexpressing tumors or tumors expressing one or more of HER1, HER2, or HER3 (HER1-3 positive) was associated with an increased risk of relapse on tamoxifen [hazard ratio, 2.20; 95% confidence interval, 1.07-3.52 (P = 0.0416); hazard ratio, 2.42; 95% confidence interval, 1.32-4.43 (P = 0.0030), respectively]. AIB1 gene amplification was observed in 18 of 362 (5%) patients. High AIB1 gene copy number had no effect on overall or disease-free survival. Conclusions: Data presented here support a role for AIB1 expression on relapse during tamoxifen treatment in hormone-responsive HER-expressing clinical breast cancers and support clinical evidence, suggesting a cross-talk between ER-α and growth factor receptor pathways through changes in expression of specific coactivator proteins, such as AIB1. This study highlights the potential that tumor profiling, using multiple markers of treatment response, may improve patient selection for endocrine treatment, such as tamoxifen or aromatase inhibitors.

Sphingosine kinase 1 induces tolerance to human epidermal growth factor receptor-2 and prevents formation of a migratory phenotype in response to sphingosine 1-phosphate in estrogen receptor-positive breast cancer cells

ABSTRACT We demonstrate here a new concept termed “oncogene tolerance” whereby human EGF receptor 2 (HER2) increases sphingosine kinase 1 (SK1) expression in estrogen receptor-positive (ER+) MCF-7 HER2 cells and SK1, in turn, limits HER2 expression in a negative-feedback manner. The HER2-dependent increase in SK1 expression also limits p21-activated protein kinase 1 (p65 PAK1) and extracellular signal regulated kinase 1/2 (ERK-1/2) signaling. Sphingosine 1-phosphate signaling via S1P3 is also altered in MCF-7 HER2 cells. In this regard, S1P binding to S1P3 induces a migratory phenotype via an SK1-dependent mechanism in ER+ MCF-7 Neo cells, which lack HER2. This involves the S1P stimulated accumulation of phosphorylated ERK-1/2 and actin into membrane ruffles/lamellipodia and migration. In contrast, S1P failed to promote redistribution of phosphorylated ERK-1/2 and actin into membrane ruffles/lamellipodia or migration of MCF-7 HER2 cells. However, a migratory phenotype in these cells could be induced in response to S1P when SK1 expression had been knocked down with a specific siRNA or when recombinant PAK1 was ectopically overexpressed. Thus, the HER2-dependent increase in SK1 expression functions to desensitize the S1P-induced formation of a migratory phenotype. This is correlated with improved prognosis in patients who have a low HER1-3/SK1 expression ratio in their ER+ breast cancer tumors compared to patients that have a high HER1-3/SK1 expression ratio.

Ras/Raf-1/MAPK Pathway Mediates Response to Tamoxifen but not Chemotherapy in Breast Cancer Patients

Purpose: The expression and activation of the Ras/Raf-1/mitogen-activated protein kinase (MAPK) pathway plays an important role in the development and progression of cancer, and may influence response to treatments such as tamoxifen and chemotherapy. In this study we investigated whether the expression and activation of the key components of this pathway influenced clinical outcome, to test the hypothesis that activation of the MAPK pathway drives resistance to tamoxifen and chemotherapy in women with breast cancer. Experimental Design: Breast tumors from patients at the Glasgow Royal Infirmary and others treated within the BR9601 trial were analyzed for expression of the three Ras isoforms, total Raf-1, active and inactive forms of Raf-1 [pRaf(ser338) and pRaf(ser259), respectively], MAPK, and phospho-MAPK using an immunohistochemical approach. Analyses were done with respect to disease free-survival and overall survival. Results: Expression and activation of the Ras pathway was associated with loss of benefit from treatment with tamoxifen but not chemotherapy. Overexpression of pRaf(ser338) was associated with shortened disease-free and overall survival time in univariate analyses. Multivariate analysis suggested pRaf(ser338) was independent of known prognostic markers in predicting outcome following tamoxifen treatment (P = 0.03). Conclusion: This study suggests that activation of the Ras pathway predicts for poor outcome on tamoxifen but not chemotherapy, and identifies pRaf(ser338) as a potential marker of resistance to estrogen receptor–targeted therapy. In addition, it suggests that expression of pRaf(ser338) could identify patients for whom tamoxifen alone is insufficient adjuvant systemic therapy, but for whom the addition of chemotherapy may be of benefit.

Is expression or activation of Src kinase associated with cancer-specific survival in ER-, PR- and HER2-negative breast cancer patients?

The aim of the current study was to assess the expression levels of c-Src and phosphorylated Src kinase in human breast cancers and to establish if these are linked to oestrogen receptor, progesterone receptor, and human epidermal growth factor receptor 2 status or patient survival. Tissue microarray technology was used to analyze 314 breast cancer specimens. Immunohistochemistry was performed using antibodies to c-Src, Y419Src, and Y215Src, and expression was assessed using the weighted histoscore method. High cytoplasmic c-Src kinase and high membrane phosphorylated activated Y419Src kinase was associated with decreased disease-specific survival. In contrast, phosphorylated activated nuclear and cytoplasmic Y215Src kinase expression levels were significantly associated with improved disease-specific survival. When the cohort was subdivided according to ER/PR/HER2 status, the ER-negative subgroup (105 patients) was associated with improved disease-specific survival and was found to be independent by multivariate analysis with a hazard ratio of 0.4 (interquartile range 0.2-0.8). High cytoplasmic c-Src expression was associated with decreased survival; high expression of activated c-Src (Y215) was associated with improved survival. This was potentiated in the ER/HER2-negative subgroup. Hence, administration of Src kinase inhibitors aiming to decrease phosphorylation should be approached with caution, especially in ER-negative patients. It is therefore essential to appropriately identify with the correct biomarkers which patients are most likely to respond to Src inhibitors.

HER4 in breast cancer: comparison of antibodies against intra- and extra-cellular domains of HER4

IntroductionWe have previously linked HER4 expression with increased survival in breast cancer. However, other reports have associated HER4 with adverse prognostic significance. One possible explanation for the conflicting reports may be that these results are antibody dependent. The HER4 protein is enzymatically cleaved, which may alter the function of its intracellular domain (ICD). We have therefore compared the staining patterns of antibodies against its intracellular and extracellular domains using tissue microarray technology.MethodsImmunohistochemistry was performed and evaluated on tumours from 402 tamoxifen treated oestrogen receptor positive patients. The HFR1 antibody recognises the ICD of HER4 and thus recognises both the intact receptor and the cleaved ICD. The H4.77.16 clone recognises an extracellular domain of HER4 and thus detects the full length receptor only.ResultsBoth antibodies demonstrated nuclear, cytoplasmic and membranous staining. Concordance between the membrane staining patterns was high (88.44%, kappa 0.426). The HFR1 antibody, however, demonstrated generally higher levels of cytoplasmic staining (concordance 74.77%, kappa 0.351). The antibodies demonstrated very different patterns of nuclear staining. Over 60% of patients stained with the H4.77.16 had no nuclear staining whereas the vast majority showed staining with the HFR1 antibody (concordance 40.12%, kappa 0.051). Neither antibody demonstrated relationships between membranous or cytoplasmic HER4 staining and survival, although associations were seen with known poor prognostic markers. Cases with H4.77.16-determined nuclear staining had significantly poorer survival outcomes.ConclusionThe difference in antigen site may explain the different staining patterns we have seen with respect to location; with each antibody appearing to select for distinct compartments. Thus, HFR1 may select for cytoplasmic and nuclear HER4 ICD, whilst H4.77.16 selects for membranous HER4 and/or HER4 being recycled in cytoplasm or nucleus. This ability to distinguish between site and function of HER4 and its fragments is particularly important, with recent evidence highlighting the different functions of nuclear and mitochondrial HER4.