MCM3 upregulation confers endocrine resistance in breast cancer and is a predictive marker of diminished tamoxifen benefit.

Resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer is a major clinical problem with poorly understood mechanisms. There is an unmet need for prognostic and predictive biomarkers to allow appropriate therapeutic targeting. We evaluated the mechanism by which minichromosome maintenance protein 3 (MCM3) influences endocrine resistance and its predictive/prognostic potential in ER+ breast cancer. We discovered that ER+ breast cancer cells survive tamoxifen and letrozole treatments through upregulation of minichromosome maintenance proteins (MCMs), including MCM3, which are key molecules in the cell cycle and DNA replication. Lowering MCM3 expression in endocrine-resistant cells restored drug sensitivity and altered phosphorylation of cell cycle regulators, including p53(Ser315,33), CHK1(Ser317), and cdc25b(Ser323), suggesting that the interaction of MCM3 with cell cycle proteins is an important mechanism of overcoming replicative stress and anti-proliferative effects of endocrine treatments. Interestingly, the MCM3 levels did not affect the efficacy of growth inhibitory by CDK4/6 inhibitors. Evaluation of MCM3 levels in primary tumors from four independent cohorts of breast cancer patients receiving adjuvant tamoxifen mono-therapy or no adjuvant treatment, including the Stockholm tamoxifen (STO-3) trial, showed MCM3 to be an independent prognostic marker adding information beyond Ki67. In addition, MCM3 was shown to be a predictive marker of response to endocrine treatment. Our study reveals a coordinated signaling network centered around MCM3 that limits response to endocrine therapy in ER+ breast cancer and identifies MCM3 as a clinically useful prognostic and predictive biomarker that allows personalized treatment of ER+ breast cancer patients.

CDX2 expression and perioperative patient serum affects the adhesion properties of cultured colon cancer cells.

BACKGROUND: Colon cancer is one of the most commonly diagnosed types of cancer with surgical resection of the tumor being the primary choice of treatment. However, the surgical stress response induced during treatment may be related to a higher risk of recurrence. The aim of this study was to examine the effect of surgery on adhesion of cultured colon cancer cells with or without expression of the tumour suppressor CDX2. METHOD: We enrolled 30 patients undergoing elective, curatively intended laparoscopic surgery for colon cancer in this study. Blood samples were drawn 1 day prior to surgery and 24 h after surgery. The samples of pre- and postoperative serum was applied to wild type colon cancer LS174T cells and CDX2 inducible LS174T cells and adhesion was measured with Real-Time Cell-Analysis iCELLigence using electrical impedance as a readout to monitor changes in the cellular adhesion. RESULTS: Adhesion abilities of wild type LS174T cells seeded in postoperative serum was significantly increased compared to cells seeded in preoperative serum. When seeding the CDX2 inducible LS174T cells without CDX2 expression in pre- and postoperative serum, no significant difference in adhesion was found. However, when inducing CDX2 expression in these cells, the adhesion abilities in pre- and postoperative serum resembled those of the LS174T wild type cell line. CONCLUSIONS: We found that the adhesion of colon cancer cells was significantly increased in postoperative versus preoperative serum, and that CDX2 expression affected the adhesive ability of cancer cells. The results of this study may help to elucidate the pro-metastatic mechanisms in the perioperative phase and the role of CDX2 in colon cancer metastasis.

Aurora kinase A as a possible marker for endocrine resistance in early estrogen receptor positive breast cancer.

BACKGROUND: Cell culture studies have disclosed that the mitotic Aurora kinase A is causally involved in both tamoxifen and aromatase inhibitor resistant cell growth and thus may be a potential new marker for endocrine resistance in the clinical setting. MATERIAL AND METHODS: Archival tumor tissue was available from 1323 Danish patients with estrogen receptor (ER) positive primary breast cancer, who participated in the Breast International Group (BIG) 1-98 trial, comparing treatment with tamoxifen and letrozole and both in a sequence. The expression of Aurora A was determined by immunohistochemistry in 980 tumors and semi quantitively scored into three groups; negative/weak, moderate and high. The Aurora A expression levels were compared to other clinico-pathological parameters and outcome, defined as disease-free survival (DFS) and overall survival (OS). RESULTS: High expression of Aurora A was found in 26.9% of patients and moderate in 57.0%. High expression was significantly associated with high malignancy grade and HER2 amplification. High Aurora A expression was significantly more frequent in ductal compared to lobular carcinomas. We found no significant association between Aurora A expression and DFS or OS and no evidence of interaction between Aurora A expression and benefits from tamoxifen versus letrozole. CONCLUSIONS: Aurora A expression in breast tumors was associated with high malignancy grade III and with HER2 amplification. A trend as a prognostic factor for OS was found in patients with high Aurora A expression. No predictive property was observed in this study with early breast cancer.

Williams syndrome transcription factor (WSTF) acts as an activator of estrogen receptor signaling in breast cancer cells and the effect can be abrogated by 1α,25-dihydroxyvitamin D3.

A majority of estrogen receptor positive (ER+) breast cancers are growth stimulated by estrogens. The ability to inhibit the ER signaling pathway is therefore of critical importance in the current treatment of ER+ breast cancers. It has been reported that 1α,25-dihydroxyvitamin D3 down-regulates the expression of the CYP19A1 gene, encoding the aromatase enzyme that catalyzes the synthesis of estradiol. Furthermore, 1α,25-dihydroxyvitamin D3 has also been reported to down-regulate the expression of estrogen receptor α (ERα), the main mediator of ER signaling. This study reports a novel transcription factor critical to 1α,25-dihydroxyvitamin D3-mediated regulation of estrogenic signaling in MCF-7 breast cancer cells. We have investigated the molecular mechanisms for the 1α,25-dihydroxyvitamin D3-mediated down-regulation of CYP19A1 and ERα gene expression in human MCF-7 breast cancer cells and found that Williams syndrome transcription factor (WSTF) plays a key role by binding to the promoters of CYP19A1 and ERα. Although sometimes reported as an inhibitor of gene expression, we found that WSTF acts as an activator of the promoter activity of both CYP19A1 and ERα. Silencing of WSTF by siRNA transfection resulted in decreased aromatase-dependent cell growth as well as decreased ER signaling in the cells. When cells were treated with 1α,25-dihydroxyvitamin D3, WSTF was dissociated from the promoters and the promoter activities of CYP19A1 and ERα were decreased. We have measured the expression of WSTF in ER-positive tumor-samples from breast cancer patients and found that WSTF is expressed in the majority of the investigated samples and that the expression is higher in cancer tissue than in normal tissue. However, we were not able to show any significant association between the WSTF expression in the tumor and the disease free and overall survival in this patient group who have received adjuvant tamoxifen treatment, nor between the WSTF expression and the expression of ERα, progesterone receptor or HER2. The major conclusions of this study are that WSTF acts as an activator of ER signaling in MCF-7 breast cancer cells, that this action can be inhibited by 1α,25-dihydroxyvitamin D3, and that the expression of WSTF is higher in breast cancer tissue than in normal tissue. WSTF may by a new target for treatment of estrogen-dependent breast cancer cell growth.

Assessment of perioperative stress in colorectal cancer by use of in vitro cell models: a systematic review.

BACKGROUND: The perioperative period is important for patient outcome. Colorectal cancer surgery can lead to metastatic disease due to release of disseminated tumor cells and the induction of surgical stress response. To explore the overall effects on surgically-induced changes in serum composition, in vitro model systems are useful. METHODS: A systematic search in PubMed and EMBASE was performed to identify studies describing in vitro models used to investigate cancer cell growth/proliferation, cell migration, cell invasion and cell death of serum taken pre- and postoperatively from patients undergoing colorectal tumor resection. RESULTS: Two authors (MG and TK) independently reviewed 984 studies and identified five studies, which fulfilled the inclusion criteria. Disagreements were solved by discussion. All studies investigated cell proliferation and cell invasion, whereas three studies investigated cell migration, and only one study investigated cell death/apoptosis. One study investigated postoperative peritoneal infection due to anastomotic leak, one study investigated mode of anesthesia (general anesthesia with volatile or intravenous anesthetics), and one study investigated preoperative intervention with granulocyte macrophage colony stimulating factor (GMCSF). In all studies an increased proliferation, cell migration and invasion was demonstrated after surgery. Anesthetics with propofol and intervention with GMCSF significantly reduced postoperative cell proliferation, whereas peritoneal infection enhanced the invasive capability of tumor cells. CONCLUSION: This study suggests that in vitro cell models are useful and reliable tools to explore the effect of surgery on colorectal cancer cell proliferation and metastatic ability. The models should therefore be considered as additional tests to investigate the effects of perioperative interventions.

A high level of estrogen-stimulated proteins selects breast cancer patients treated with adjuvant endocrine therapy with good prognosis.

BACKGROUND: Adjuvant endocrine therapy has significantly improved survival of estrogen receptor α (ER)-positive breast cancer patients, but around 20% relapse within 10 years. High expression of ER-stimulated proteins like progesterone receptor (PR), Bcl-2 and insulin-like growth factor receptor I (IGF-IR) is a marker for estrogen-driven cell growth. Therefore, patients with high tumor levels of these proteins may have particularly good prognosis following adjuvant endocrine therapy. PATIENTS AND METHODS: Archival tumor tissue was available from 1323 of 1396 Danish breast cancer patients enrolled in BIG 1-98, a randomized phase-III clinical trial comparing adjuvant letrozole, tamoxifen or a sequence of the two drugs. Immunohistochemical staining for ER, HER-2, PR, Bcl-2 and IGF-IR was performed and determined by Allred scoring (ER, PR and Bcl-2) or HercepTest (HER-2 and IGF-IR). RESULTS: Data on all five markers were available from 969 patients with ER-positive, HER-2-negative tumors. These patients were classified in ER activity groups based on the level of PR, Bcl-2 and IGF-IR. High ER activity profile was found in 102 patients (10.5%) and compared with the remaining patients, univariate and multivariate analysis revealed HR (95% CI) and p values for disease-free survival (DFS) of 2.00 (1.20-3.22), 0.008 and 1.70 (1.01-2.84), 0.04 and for the overall survival (OS) of 2.33 (1.19-4.57), 0.01 and 1.90 (0.97-3.79), 0.06, respectively. The high ER activity profile did not disclose difference in DFS or OS according to treatment with tamoxifen or letrozole (p = .06 and .09, respectively). CONCLUSIONS: Stratifying endocrine-treated patients in ER activity profile groups disclosed that patient with high ER activity profile (10.5%) had significantly longer DFS and OS, and the profile was an independent marker for DFS. High ER activity is a marker for estrogen-driven tumor growth. We suggest further analyses to disclose whether the ER activity profile or other markers associated with estrogen-driven growth may be used to identify ER-positive high-risk breast cancer patients who can be spared adjuvant chemotherapy.

High CDK6 Protects Cells from Fulvestrant-Mediated Apoptosis and is a Predictor of Resistance to Fulvestrant in Estrogen Receptor-Positive Metastatic Breast Cancer.

PURPOSE: Resistance to endocrine therapy in estrogen receptor-positive (ER+) breast cancer remains a major clinical problem. Recently, the CDK4/6 inhibitor palbociclib combined with letrozole or fulvestrant was approved for treatment of ER+ advanced breast cancer. However, the role of CDK4/6 in endocrine resistance and their potential as predictive biomarkers of endocrine treatment response remains undefined. EXPERIMENTAL DESIGN: We investigated the specific role of increased CDK6 expression in fulvestrant-resistant cells by gene knockdown and treatment with palbociclib, and evaluated the effect in cell proliferation, apoptosis, and kinase activity. Furthermore, we evaluated CDK6 expression in metastatic samples from breast cancer patients treated or not with fulvestrant. RESULTS: We found increased expression of CDK6 in two fulvestrant-resistant cell models versus sensitive cells. Reduction of CDK6 expression impaired fulvestrant-resistant cell growth and induced apoptosis. Treatment with palbociclib resensitized fulvestrant-resistant cells to fulvestrant through alteration of retinoblastoma protein phosphorylation. High CDK6 levels in metastatic samples from two independent cohorts of breast cancer patients treated with fulvestrant (N = 45 and 46) correlated significantly with shorter progression-free survival (PFS) on fulvestrant treatment (P = 0.0006 and 0.018), whereas no association was observed in patients receiving other first- or second-/third-line endocrine treatments (N = 68, P = 0.135 and 0.511, respectively). CONCLUSIONS: Our results indicate that upregulation of CDK6 may be an important mechanism in overcoming fulvestrant-mediated growth inhibition in breast cancer cells. Patients with advanced ER+ breast cancer exhibiting high CDK6 expression in the metastatic lesions show shorter PFS upon fulvestrant treatment and thus may benefit from the addition of CDK4/6 inhibitors in their therapeutic regimens. Clin Cancer Res; 22(22); 5514-26. ©2016 AACR.

Expression of transmembrane protein 26 (TMEM26) in breast cancer and its association with drug response.

We have previously shown that stromal cells desensitize breast cancer cells to the anti-estrogen fulvestrant and, along with it, downregulate the expression of TMEM26 (transmembrane protein 26). In an effort to study the function and regulation of TMEM26 in breast cancer cells, we found that breast cancer cells express non-glycosylated and N-glycosylated isoforms of the TMEM26 protein and demonstrate that N-glycosylation is important for its retention at the plasma membrane. Fulvestrant induced significant changes in expression and in the N-glycosylation status of TMEM26. In primary breast cancer, TMEM26 protein expression was higher in ERα (estrogen receptor α)/PR (progesterone receptor)-positive cancers. These data suggest that ERα is a major regulator of TMEM26. Significant changes in TMEM26 expression and N-glycosylation were also found, when MCF-7 and T47D cells acquired fulvestrant resistance. Furthermore, patients who received aromatase inhibitor treatment tend to have a higher risk of recurrence when tumoral TMEM26 protein expression is low. In addition, TMEM26 negatively regulates the expression of integrin ß1, an important factor involved in endocrine resistance. Data obtained by spheroid formation assays confirmed that TMEM26 and integrin ß1 can have opposite effects in breast cancer cells. These data are consistent with the hypothesis that, in ERα-positive breast cancer, TMEM26 may function as a tumor suppressor by impeding the acquisition of endocrine resistance. In contrast, in ERα-negative breast cancer, particularly triple-negative cancer, high TMEM26 expression was found to be associated with a higher risk of recurrence. This implies that TMEM26 has different functions in ERα-positive and -negative breast cancer.

Aurora kinase B is important for antiestrogen resistant cell growth and a potential biomarker for tamoxifen resistant breast cancer.

BACKGROUND: Resistance to antiestrogen therapy is a major clinical challenge in the treatment of estrogen receptor α (ER)-positive breast cancer. The aim of the study was to explore the growth promoting pathways of antiestrogen resistant breast cancer cells to identify biomarkers and novel treatment targets. METHODS: Antiestrogen sensitive and resistant T47D breast cancer cell lines were used as model systems. Parental and fulvestrant resistant cell lines were subjected to a kinase inhibitor library. Kinase inhibitors preferentially targeting growth of fulvestrant resistant cells were identified and the growth inhibitory effect verified by dose-response cell growth experiments. Protein expression and phosphorylation were investigated by western blot analysis. Cell cycle phase distribution and cell death were analyzed by flow cytometry. To evaluate Aurora kinase B as a biomarker for endocrine resistance, immunohistochemistry was performed on archival primary tumor tissue from breast cancer patients who have received adjuvant endocrine treatment with tamoxifen. RESULTS: The selective Aurora kinase B inhibitor barasertib was identified to preferentially inhibit growth of fulvestrant resistant T47D breast cancer cell lines. Compared with parental cells, phosphorylation of Aurora kinase B was higher in the fulvestrant resistant T47D cells. Barasertib induced degradation of Aurora kinase B, caused mitotic errors, and induced apoptotic cell death as measured by accumulation of SubG1 cells and PARP cleavage in the fulvestrant resistant cells. Barasertib also exerted preferential growth inhibition of tamoxifen resistant T47D cell lines. Finally, high percentage of Aurora kinase B positive tumor cells was significantly associated with reduced disease-free and overall survival in 261 ER-positive breast cancer patients, who have received tamoxifen as first-line adjuvant endocrine treatment. CONCLUSIONS: Our results indicate that Aurora kinase B is a driving factor for growth of antiestrogen resistant T47D breast cancer cell lines, and a biomarker for reduced benefit of tamoxifen treatment. Thus, inhibition of Aurora kinase B, e.g. with the highly selective kinase inhibitor barasertib, could be a candidate new treatment for breast cancer patients with acquired resistance to antiestrogens.

SRC drives growth of antiestrogen resistant breast cancer cell lines and is a marker for reduced benefit of tamoxifen treatment.

The underlying mechanisms leading to antiestrogen resistance in estrogen-receptor α (ER)-positive breast cancer is still poorly understood. The aim of this study was therefore to identify biomarkers and novel treatments for antiestrogen resistant breast cancer. We performed a kinase inhibitor screen on antiestrogen responsive T47D breast cancer cells and T47D-derived tamoxifen and fulvestrant resistant cell lines. We found that dasatinib, a broad-spectrum kinase inhibitor, inhibited growth of the antiestrogen resistant cells compared to parental T47D cells. Furthermore western blot analysis showed increased expression and phosphorylation of Src in the resistant cells and that dasatinib inhibited phosphorylation of Src and also signaling via Akt and Erk in all cell lines. Immunoprecipitation revealed Src: ER complexes only in the parental T47D cells. In fulvestrant resistant cells, Src formed complexes with the Human Epidermal growth factor Receptor (HER)1 and HER2. Neither HER receptors nor ER were co-precipitated with Src in the tamoxifen resistant cell lines. Compared to treatment with dasatinib alone, combined treatment with dasatinib and fulvestrant had a stronger inhibitory effect on tamoxifen resistant cell growth, whereas dasatinib in combination with tamoxifen had no additive inhibitory effect on fulvestrant resistant growth. When performing immunohistochemical staining on 268 primary tumors from breast cancer patients who had received tamoxifen as first line endocrine treatment, we found that membrane expression of Src in the tumor cells was significant associated with reduced disease-free and overall survival. In conclusion, Src was identified as target for treatment of antiestrogen resistant T47D breast cancer cells. For tamoxifen resistant T47D cells, combined treatment with dasatinib and fulvestrant was superior to treatment with dasatinib alone. Src located at the membrane has potential as a new biomarker for reduced benefit of tamoxifen.

The broad-spectrum metalloproteinase inhibitor BB-94 inhibits growth, HER3 and Erk activation in fulvestrant-resistant breast cancer cell lines.

Breast cancer cells can switch from estrogen receptor α (ER)- to human epidermal growth factor receptor (HER)-driven cell growth upon acquiring antiestrogen resistance. HER ligands are cleaved by metalloproteinases leading to release of active HER ligands, activation of HER receptors and consequently increased cell growth. In this study, we investigated the importance of HER receptors, in particular HER3, and HER ligand shedding for growth and signaling in human MCF-7 breast cancer cells and MCF-7-derived sublines resistant to the antiestrogen fulvestrant. The HER3/HER4 ligand heregulin 1ß induced phosphorylation of HER3, Akt and Erk, and partly rescued fulvestrant-inhibited growth of MCF-7 cells. HER3 ligands were found to be produced and shed from the fulvestrant-resistant cells as conditioned medium from fulvestrant-resistant MCF-7 cells induced phosphorylation of HER3 and Akt in MCF-7 cells. This was prevented by treatment of resistant cells with the metalloproteinase inhibitor TAPI-2. Only the broad-spectrum metalloproteinase inhibitor BB-94, and not the more selective inhibitors GM6001 or TAPI-2, which inhibited shedding of the HER ligands produced by the fulvestrant-resistant cells, was able to inhibit growth and activation of HER3 and Erk in resistant cells. Compared to MCF-7, fulvestrant-resistant cells have increased HER3 phosphorylation, but knockdown of HER3 had no inhibitory effect on resistant cell growth. The EGFR inhibitor gefitinib exhibited only a minor growth inhibition, whereas the pan-HER inhibitor CI-1033 exerted growth arrest. Thus, neither HER3 nor EGFR alone are the main driver of fulvestrant-resistant cell growth and treatment should target both receptors. Ligand shedding is not a treatment target, as receptor activation occurred, independent of release of ligands. Only the broad-spectrum metalloproteinase inhibitor BB-94 could abrogate HER3 and Erk activation in the resistant cells, which stresses the complexity of the resistance mechanisms and the requirement of targeting signaling from HER receptors by multiple strategies.

T47D breast cancer cells switch from ER/HER to HER/c-Src signaling upon acquiring resistance to the antiestrogen fulvestrant.

In this study, T47D cell lines resistant to the antiestrogen fulvestrant were established and analyzed to explore, whether a switch to HER signaling, as seen in fulvestrant resistant MCF-7 cell lines, is a general resistance mechanism. We find that parental T47D cells depend on ER and HER signaling for growth. Fulvestrant resistant T47D cells have lost ER expression and, although HER2 was over expressed, growth was only partially driven by HER receptors. Instead c-Src was important for resistant growth. Thus, the T47D and MCF-7 model system unravel different resistance mechanisms which may be important for fulvestrant resistant breast cancer patients.

Molecular alterations in AKT1, AKT2 and AKT3 detected in breast and prostatic cancer by FISH.

AIMS: The AKT family is implicated in cancer progression. There are three mammalian AKT isoforms located on chromosomes 14, 19 and 1, respectively. The aim of the study was to investigate genetic alterations of AKT in breast and prostatic cancers using fluorescence in situ hybridization (FISH). METHODS AND RESULTS: In oestrogen receptor(ER)-positive breast carcinomas, AKT1 was deleted in five (4.8%) and amplified in one (1%) carcinoma. Deletions of AKT2 were seen in 19 (21.1%) cases. No AKT2 amplifications were identified. Ten (9.9%) AKT3 amplifications but no deletions were seen. In prostatic cancer, AKT1 was amplified in one carcinoma (2.6%). No genetic changes were observed for AKT2 and AKT3. High frequencies of aneusomy for all chromosomes were observed in breast and prostatic carcinomas. CONCLUSIONS: In breast cancer AKT3 amplifications and AKT1 and AKT2 deletions were seen, which, to our knowledge, have not been shown by FISH before. Although these two cohorts cannot be directly compared, only one AKT1 amplification was identified in prostatic carcinomas. This indicates differences in the genetic changes underlying development of breast and prostatic cancers. To evaluate further the role of genetic changes of AKT in breast cancer progression, a cohort of both ER+ and ER- patients should be evaluated.

Breast cancer cells can switch between estrogen receptor alpha and ErbB signaling and combined treatment against both signaling pathways postpones development of resistance.

The majority of breast cancers are estrogen responsive, but upon progression of disease other growth promoting pathways are activated, e.g., the ErbB receptor system. The present study focuses on resistance to the pure estrogen antagonist fulvestrant and strategies to treat resistant cells or even circumvent development of resistance. Limited effects were observed when targeting EGFR and ErbB2 with the monoclonal antibodies cetuximab, trastuzumab, and pertuzumab, whereas the pan-ErbB inhibitor CI-1033 selectively inhibited growth of fulvestrant resistant cell lines. CI-1033 inhibited Erk but not Akt signaling, which as well as Erk is important for antiestrogen resistant cell growth. Accordingly, combination therapy with CI-1033 and the Akt inhibitor SH-6 or the Protein Kinase C inhibitor RO-32-0432 was applied and found superior to single agent treatment. Further, the resistant cell lines were more sensitive to CI-1033 treatment when grown in the presence of fulvestrant, as withdrawal of fulvestrant restored signaling through the estrogen receptor alpha (ERalpha), partly overcoming the growth inhibitory effects of CI-1033. Thus, the resistant cells could switch between ERalpha and ErbB signaling for growth promotion. Although parental MCF-7 cell growth primarily depends on ERalpha signaling, a heregulin-1beta induced switch to ErbB signaling rescued MCF-7 cells from the growth inhibition exerted by fulvestrant-mediated blockade of ERalpha signaling. This interplay between ERalpha and ErbB signaling could be abrogated by combined therapy targeting both receptor systems. Thus, the present study indicates that upon development of antiestrogen resistance, antiestrogen treatment should be continued in combination with signal transduction inhibitors. Further, upfront combination of endocrine therapy with pan-ErbB inhibition may postpone or even prevent development of treatment resistance.

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.

Development of new predictive markers for endocrine therapy and resistance in breast cancer.

Today, the decision to treat breast cancer patients with endocrine therapy relies solely on tumor expression of two predictive factors, the estrogen receptor and the progesterone receptor. Expression of these hormone receptors are, however, not a guarantee for a response to treatment and patients who experience response at first may become resistant after prolonged treatment. This paper describes the use of preclinical models to identify mechanisms and new markers for endocrine sensitivity and resistance and the translation of these data to clinical utility.

Expression of tumor necrosis factor alpha converting enzyme in endocrine cancers.

Tumor necrosis factor alpha converting enzyme (TACE) mediates shedding of human epidermal growth factor receptor-4 (HER4). Recent data suggest that released HER4 intracellular domain (4ICD) induces apoptosis in breast cancer. TACE expression, as measured by immunohistochemical analysis, was observed in 183 of 383 breast carcinomas, 39 of 217 ovarian carcinomas, and 16 of 24 and 17 of 24 hormonesensitive and hormone-insensitive prostate carcinomas, respectively. HER4 expression was detected in breast carcinomas by using 2 antibodies recognizing an extracellular or intracellular epitope. TACE expression was predominantly seen in tumors with high levels of 4ICD and membranous HER4. Apoptotic activity was measured by the terminal deoxynucleotidyl transferase-mediated nick-end labeling (TUNEL) assay and cleaved caspase-3 staining in breast carcinomas. There was no significant association between cleaved caspase-3 or TUNEL positivity and 4ICD, whereas TUNEL positivity was seen predominantly in tumors with high levels of internalized HER4. The data presented herein show TACE expression in endocrine cancers and further support a role for TACE in breast cancer apoptosis.

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-alpha (ER-alpha). 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-alpha-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-alpha 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.

Bad expression predicts outcome in patients treated with tamoxifen.

AIMS: Activation of the PI3K/Akt signal transduction pathway has been linked to endocrine resistance in tamoxifen treated breast cancer patients. Activation of the PI3K/Akt pathway causes phosphorylation of Bad leading to modulation of cellular apoptosis. The present study was carried out to test the hypothesis that disruption of apoptosis in breast cancer, via Akt activation, is linked with hormone resistance. METHODS: Immunohistochemistry (IHC) was performed on 402 oestrogen receptor (ER) positive breast cancers using antibodies against Bad, pBad (ser 112), Bcl-2, Bcl-xl and Bax. RESULTS: Bad, pBad (ser 112), Bcl-2 and Bax expression was observed in the cellular cytoplasmic compartment only. Patients, whose tumours had high levels of Bad expression, had a significantly improved disease-free survival when compared to patients whose tumours had low levels of Bad expression (P = 0.049). Activation of the PI3K/Akt pathway by either heregulin or oestrogen had no effect on expression of Bad, Bcl-2, Bax or Bcl-xl. However, heregulin increased pBad (ser 112) expression. DISCUSSION: Data presented here shows that Bad expression is associated with relapse in tamoxifen-treated breast cancer patients, supporting our hypothesis that the apoptosis pathway is involved in tamoxifen resistance.

AKT activation predicts outcome in breast cancer patients treated with tamoxifen.

Oestrogen receptor (ERalpha) 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 ERalpha-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 ERalpha-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 ERalpha in this patient cohort. The data presented here show that the PI3K/AKT/mTOR pathway is associated with relapse and death in ERalpha-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.