bluuebunny/crossref_metadata_2025 huggingface dataset:
Lapatinib can activate or supress estrogen receptor (ER) signaling in cell models of endocrine resistant breast cancer.
Authors: Leary AF , Martin L , Lykkesfeldt AE , Dowsett M , Johnston SR
Published: December 2010

Abstract #3031

Background  Growth factor (GF) signaling is frequently upregulated in hormone resistant breast cancer (BC) and provides the rationale for using the dual EGFR/HER2 inhibitor, lapatinib (LAP) in an effort to overcome endocrine resistance. Hormone resistant BC can also vary in its degree of continued ER dependence. We previously demonstrated potent synergy between LAP and TAM in cell models of acquired endocrine resistance to tamoxifen (TAMR cells), and to estrogen deprivation (LTED cells)1. This study aimed to characterize the phenotype of these two models with a focus on the ER pathway, and to determine the biological basis for the observed synergy between LAP and TAM.  Methods  LTED cells and TAMR cells were both derived from hormone-sensitive MCF7 parental cells as previously described1. The differential effects of LAP, TAM, estrogen deprivation (ED) or the combination were assessed by proliferation assays, ERE reporter assays, quantitative PCR and western blot analyses.  Results  In LTEDs: Increased ER expression and ERE transactivation were seen in LTEDs compared to MCF7 cells, as well as continued sensitivity to the ER downregulator, fulvestrant (FUL), indicating that in LTEDs, ER activity is not only enhanced but is also a significant driver of growth. In LTEDs, LAP alone suppressed ER activity as measured by both ERE transactivation and ER phosphorylation at Ser118; the most effective suppression of ER activity was achieved with a combination of LAP+TAM/ED where the effects were additive.  In TAMRs: In contrast, TAMRs demonstrated decreased ER activity (low basal ERE transactivation and loss of PgR) and ER independent proliferation (FUL had minimal growth inhibitory effects despite ER downregulation). In TAMRs, neither LAP nor TAM had significant antiproliferative effects on their own: LAP alone decreased pMAPK/pAkt but increased basal ERE transactivation and transcription of the ER-responsive gene pS2. TAM alone inhibited ERE activation but increased HER2 gene and protein expression. Inhibiting both HER2 and ER pathways with LAP+TAM resulted in synergistic growth inhibition.  Conclusions  In LTED cells with enhanced ER function attributable in part to GF receptor activation, the synergistic antiproliferative effects from LAP and TAM or ED may be largely due to enhanced inhibition of ER signaling. In contrast in TAMR cells with suppressed ER activity, LAP alone appears to enhance ER signaling, while TAM alone inhibits ER activity but increases HER2 expression. In TAMRs, the combination of TAM+LAP is required to interrupt both ER and GF signaling. These results suggest that the basis for synergy between LAP and endocrine therapy in hormone resistant BC may vary depending on the nature of the cross-talk between the ER and GF pathways.  1SABCS, 2006 Abs 303.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3031.
Association of the polymorphisms CYP19 (TTTA)n in treatment response to hormone therapy based in aromatase inhibitors in breast cancer patients.
Authors: Murillo-Ortiz B , Castillo-Valenzuela R , Martínez-Garza S , Moreno-Perez A , Silva A , Ruiz-García E , Astudillo-de la Vega H
Published: December 2010

Abstract #3033

Background   Case-control studies have reported inconsistent results concerning breast cancer risk and polymorphisms in genes that control endogenous estrogen biosynthesis. We report findings from the first study in Mexican women examining associations between female with breast cancer and polymorphisms CYP19 (TTTA repeated polymorphism).  Methods   We conducted a study among 180 healthy women and 70 women with breast cancer underwent hormone therapy with aromatase inhibitor. DNA and questionnaire data was obtained. Tandem repeated (TTTA)n polymorphism in CYP19 gene was determined by PCR followed by electrophoresis on denaturalizing acrilamide gel stained with silver nitrate. Differences were visualized with Gel-Doc BioRad. Estrone, estradiol and FSH levels were measured by RIA and IRMA. We used likelihood-based statistical methods to examine allelic associations.  Results:  250 women (age 55 ± 12 years) were included. BMI was 30 ± 7.1 Kg/m2. We found a distribution of different CYP19 allele frequencies. In healthy women the allele frequencies with 6 (32.7 %) and 7 (21.6 %) tandem repetitions were the most frequent, in women with breast cancer the alleles with 6(29%) and 10(26%) tandem repeated were the most frequent. A relationship between hormonal levels and number of (TTTA) repeated was not found. Anastrozol reduced significantly estrona and estradiol. Surpriseling we found in a patients with 10 or more (TTTA)n repeated an association with a mayor tumoral activity (p=0.04).  Conclusion   This study indicates that status of CYP19 >10 TTTA repeated might be related to increased breast cancer risk and with the clinical response (aromatase inhibitor). Because of this is the first study to report an association between CYP19 >10 TTTA repeated and treatment hormonal response in breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3033.
The developmental protein HOXc11 as a mediator of endocrine resistance in breast cancer.
Authors: McCartan DP , McIlroy M , Eary S , Hill AD , Young LS
Published: December 2010

Abstract #3034

The nuclear receptor coactivator, SRC-1, has been implicated as an important mediator of resistance to endocrine therapy in breast cancer through work from our group and others. Using mass spectrometry we identified the homeobox protein HOXc11 as an interacting partner for the nuclear receptor coactivator SRC-1 in endocrine resistant breast cancer cells. Homeobox genes control cell differentiation. The processes of neoplasia and normal development are closely linked, therefore it is little surprise that increasing evidence implicates these genes in neoplasia development.  We propose that the interaction between SRC-1 and HOXc11 plays an important role in the development of endocrine resistance. Our aim was to identify potential HOXc11/SRC-1 target genes and assess the clinical significance of these genes.  Co-immunprecipitation assays confirmed the SRC-1 and HOXc11 interaction after treatment with tamoxifen for 2 hours in the endocrine resistant LY2 breast cancer cell line, an interaction that was not present in endocrine sensitive MCF-7 cells under the same treatment conditions. A target gene for HOXc11, S100β, has previously been identified in neuronal cells. Based on this we sought the presence of either protein at the promoter region for S100β. Chromatin immunoprecipitation (ChIP) assays in LY2 cells confirmed recruitment of both HOXc11 and SRC-1 to the promoter region of S100β after treatment with tamoxifen at both 45 minutes and 2 hours when compared to control.  To assess the clinical significance of S100β, we used immunohistochemistry on a paraffin embedded tissue microarray to examine itÂs expression in a large cohort of breast cancer patients (n=560). S100β was a strong predictor of reduced disease free survival in patients receiving adjuvant endocrine treatment (Hazard ratio 5.82; p<0.0001). This also showed that expression of S100β was associated with expression of HOXc11 and SRC-1. Given that S100β is a secreted protein, we hypothesized that elevated levels could be detected in patients with breast cancer at time of diagnosis. Using an ELISA to measure S100β levels, we found elevated levels in one quarter of breast cancer patients, pre treatment, when compared to age matched controls.  Investigation into non steroidal SRC-1 binding partners in endocrine resistant breast cancer identified the homeobox protein HOXc11. A target gene for the interaction between SRC-1 and HOXc11, S100β, is a secreted protein and raises the exciting possibility that it may offer potential as a biomarker for endocrine resistance in breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3034.
Novel non-steroidal p160 coactivator interactions in tamoxifen resistant breast cancer.
Authors: McIlroy M , McCartan D , Early S , Hill AD , Young LS
Published: December 2010

Abstract #3038

While most breast cancer patients initially respond to endocrine therapy, 30-40% of cases relapse within 5 years of treatment. Translational studies from our group and others have provided substantial evidence of a significant role for p160 family of Steroid Receptor Co-activator (SRC) proteins in the development of resistance to endocrine therapy. In this study we have investigated novel non-steroidal SRC-1 interactions in endocrine resistant breast cancer.  A mass-spectrometry-based screen was used to identify proteins which differentially interact with SRC-1 in endocrine resistant compared with endocrine sensitive breast cancer cells. The developmental protein, HOXC11, was confirmed as an SRC-1 interacting partner. HOXC11 was shown to be over-expressed in tamoxifen-resistant cells using immunocytochemistry and was observed to translocate to the nucleus and peri-nuclear region when cells were treated with tamoxifen. In a breast cancer TMA (n=560) HOXC11 was found to correlate with disease recurrence and metastasis (local and distant). Moreover, in addition to associating with SRC-1, HOXC11 was found to be a strong predictor of reduced disease-free survival on endocrine treatment (Hazard ratio: 5.79; p<0.0001).  Bioinformatic analysis identified S100β as a target gene of the HOXC11 transcription factor. Interestingly, the s100 family of calcium binding proteins has been associated with poor outcome and reduced disease-free survival in melanoma. Reporter assays were used to confirm HOXC11 as a mediator of S100β transcription. Regulation of the target gene in the presence of tamoxifen was shown to be modified by activation of protein kinase A (PKA) which has previously been implicated in the enhancement of tamoxifenÂs agonist profile. Here we have defined HOXC11 as a non-steroidal interacting partner of SRC-1; furthermore, this interaction appears to be regulated by the activation of PKA. HOXC11 expression in our breast cancer patient cohort associated with poor survival and the development of metastatic disease. This study provides evidence of a novel mechanism at play in endocrine resistant breast cancer cells.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3038.
Persistence of isolated tumor cells in the bone marrow of breast cancer patients predicts increased risk for relapse – a European pooled analysis.
Authors: Naume B , Fehm T , Wiedswang G , Jückstock J , Borgen E , Rack B , Synnestvedt M , Braun S , Sommer H , Solomayer E , Pantel K , Friese K , Janni W
Published: December 2010

Abstract #304

Purpose:  The prognostic significance of DTC in the BM of breast cancer pts at the time of primary diagnosis has already been confirmed by a large pooled analysis. In view of the lack of early indicators for secondary adjuvant treatment, we here evaluated whether the persistence of DTC after adjuvant therapy confers a risk for relapse and breast cancer-related death.  Patients and Methods:  We analyzed BM aspirates of 723 pts from academic breast cancer units in Oslo (n=356), Munich (n=228) and Tuebingen (n=139) during recurrence-free follow-up at a mean interval of 31.7 months (standard deviation [std] 19.4 months) after primary diagnosis of breast cancer pT1-4, pN0-3 pM0. Carcinoma cells were detected using a standardized immunoassay with the monoclonal antibodies A45-B/B3 (Munich, Tuebingen), or AE1 and AE3 (Oslo), directed against cytokeratin (CK). Pts were followed for a median of 54.5 months (SD 25.1 months) after primary diagnosis.  Results:  Persistent DTC in the BM was detected in 15.5% of the pts (n=112). The Kaplan-Meier estimate for mean distant relapse-free survival was 162.5 months (157.9 – 167.0 95%CI) in pts with negative and 102.4 months (94.2 – 110.6, 95% CI, p< .0001, log rank test) in pts with positive BM status. The mean overall survival was 170.1 months, (167.7 – 172.5 95% CI), compared to 113.4 months in patients with persistent DTC in BM (107.3 – 119.5, p < .0001). In multivariate Cox regression analysis, allowing for BM status, tumor size, nodal status and histopathological grading, presence of DTC was an independent prognostic factor for reduced breast cancer specific survival ((RR 3.42, 1.64 – 7.13 95% CI, p= .001).  Conclusion:  Persistent DTC significantly predicts an increased risk for subsequent relapse, and, therefore, might serve as a potent monitoring tool for the follow-up of breast cancer pts. Future therapeutic intervention trials are currently being initiated to assess the benefit of secondary adjuvant treatment based on DTC detection as stratification parameter.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 304.
Outcomes of triple negative breast cancer in African-Americans vs. Caucasians: a community-based study.
Authors: Shah MM , Patel JK , Fischbach NA
Published: December 2010

Abstract #3043

Background: Breast cancers with a triple negative tumor (TNT) subtype are associated with inferior outcomes and more recently racial disparities in patients with TNT have been demonstrated with African American women experiencing worse outcomes compared to Caucasians. This study examines association of race (AA vs. Caucasians) and breast cancer outcomes in a large community hospital TNT population.  Methods: Breast cancers of the TNT subtype were reviewed from a hospital-based tumor registry. Cases diagnosed from 1999 to 2006 were included in the study. Multivariate regression analyses were conducted including socio-demographic, and treatment factors. Endpoints were local/systemic recurrence and death.  Results: Of 1827 newly diagnosed cases of breast cancer from 1999- 2006, 93 were of the TNT subtype. A higher number of AA (17.58%) had TNT compared to Caucasians (3.7%). In patients with TNT, AA presented at a younger age (mean age 51.78 years vs. 58.52 years, (<50 years) (p value=0.07), had more advanced disease at diagnosis (stage 3) (5.38% vs. 1.08%; p value 0.001) and had a poor insurance status (uninsured and Medicaid) (9.68% vs. 4.30%; p value 0.004). There was no difference in the histologic grade of the tumors. AA had a higher rate of local recurrence (4.30% vs. 2.15%; p value 0.029), systemic recurrence (19% vs. 10%; p value 0.02) [Figure 2] and death (17.20% vs. 13.98%; p value 0.005) [Figure 1] compared to Caucasians.        Even after adjusting for insurance status and treatment modalities, AA had a higher mortality rate compared to Caucasians (p value 0.014).  Conclusions: The high prevalence of TNTs among AA women and poorer survival in spite of adjusting for insurance and treatment modalities, suggests an underlying biologic variation with respect to race/ ethnicity.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3043.
Deletion of STAT5 prevents HER2/Neu/ErbB2-induced mammary tumor development.
Authors: Yamaji D , Robinson GW , Muller WJ , Hennighausen L
Published: December 2010

Abstract #3045

HER2 (ErbB2/Neu in rodents) is overexpressed in a number of breast cancers and it activates several intracellular signaling pathways by recruiting adaptor proteins. Among them, Shc or Gab2 signaling mediates ErbB2/Neu-induced mammary tumor progression. HER2 activates the transcription factors STAT5A/B but their contribution to the etiology of breast cancer remains to be elucidated. STAT5 mediates signals from a wide variety of cytokine receptors, including the EGFR and ErbB4. Further, constitutive activation of STAT5 has been observed in human breast cancer, leukemia and many solid tumors. To address whether STAT5 mediates HER2/ErbB2/Neu-induced breast cancer progression, both Stat5 genes were deleted specifically in ErbB2/Neu oncogene (known as HER2 in human)-expressing mammary epithelial cells in mice. Briefly, transgenic mice carrying floxed Stat5 alleles (Stat5fl/fl mice) were mated with transgenic MMTV/NIC mice expressing both activated Neu/ErbB2 and Cre recombinase from the same bicistronic transcript due to the presence of an internal ribosome entry site (IRES) between the two cDNA sequences (NIC) under the control of mouse mammary tumor virus (MMTV) long terminal repeat. Whole mount staining of mammary tissue of MMTV/NIC mice demonstrated that mammary glands of control virgin NIC mice (MMTV/NIC;Stat5+/+) exhibited hyperplastic lesions at 5 months of age, while no lesions were detected in NIC mice that lacked STAT5 (MMTV/NIC;Stat5fl/fl). Histological analysis revealed the absence of hyperplastic or neoplastic lesions in MMTV/NIC;Stat5fl/fl mammary glands. These results indicate that inactivation of Stat5 results in the absence of hyperplasias in spite of overexpression of ErbB2/Neu in mammary epithelium and suggest that activation of STAT5 contributes to the development of HER2-induced breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3045.
A multi-marker QPCR panel for the detection of circulating tumor cells predicts survival in breast cancer patients.
Authors: Molloy TJ , Bosma AJ , Baumbusch LO , Borgen E , van't Veer LJ , Naume B
Published: December 2010

Abstract #305

The detection of circulating tumor cells (CTCs) in the blood of cancer patients is a promising tool for risk stratification, treatment tailoring, and monitoring of the disease state. We previously developed a QPCR-based detection platform for the semi-quantitation of circulating tumor cells in the peripheral blood of breast cancer patients. This CTC assay combines gene expression data from 4 tumor marker genes into a single score using a quadratic discriminant analysis, and is optimized such that positivity indicates tumor cell presence, and negativity indicates tumor cell absence. An initial cohort of metastatic patients demonstrated that score positivity was correlated to a significantly shorter time to disease progression.  The current study aimed to validate these results by using the CTC assay in an independent, retrospective patient series consisting of 111 non-metastatic stage I-III breast cancer patients (median followup 7.3 years), 16 metastatic breast cancer patients, and 28 healthy controls. Assay score positivity was observed in 14 out of 16 (88%) metastatic patients, 16 out of 111 (14%) non-metastatic stage I-III patients, and 0 out of 28 (0%) healthy female controls.  In the early stage patient group, a positive CTC score was correlated to significantly poorer relapse-free survival (HR = 3.6, 95% CI = 1.7-7.9, p < 0.01) and overall survival (HR = 3.2, 95% CI = 1.6-6.1, p < 0.01). The assay was particularly powerful in patients with no lymph node metastases at time of diagnosis (relapse-free survival HR = 13.0, 95%CI = 3.0-55.7, p < 0.01, overall survival HR = 6.3, 95% CI = 1.9-21.1, p < 0.01). This platform compared very favorably to the traditional immunohistochemistry-based assay for disseminated tumor cell detection in bone marrow utilizing pan-anticytokeratin staining (AE1/AE3), which was not a significant predictor of survival in this patient group (HR = 1.3, 95% CI = 0.6-2.4, p = 0.49). In multivariate analyses, our CTC assay was a significant, independent predictor of overall survival.  Work is currently ongoing to expand the study to include an additional 500-600 patients which should serve as a robust validation of the assay. This CTC detection platform represents an objective assay with both high specificity and prognostic power, and could be an effective clinical tool for outcome prediction in breast cancer patients.  This work was supported by the Sixth Framework Program of the European Commission as part of the international DISMAL collaboration for research into disseminated epithelial malignancies.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 305.
The antitumorgenic actions of estrogen receptor beta in breast cancer cells.
Authors: Wimalasena J , Baek S , Sukhthankar M , Strom A
Published: December 2010

Abstract #3056

Recent studies have suggested that ERβ may oppose actions of ERα in breast cancer cells and clinical studies indicate that loss of ERβ expression in ERα positive tumors is associated with a poor prognosis and perhaps resistance to tamoxifen therapy. Clinical evidence indicates that in post menopausal women hormone replacement therapy significantly decreased incidence of colon cancer, suggesting that estrogens have a protective role . We have recently found that in SW 480 colon cancer cells, (ER negative) stable expression of ERβ decreased SKP2, c-myc but not p27kip1 mRNA measured by real time PCR. These results were confirmed for c-myc and SKP2 at the protein level. However, ERβ expression increased only p27 protein levels. Addition of E2 did not regulate effects of ERβ expression.  Similar results have been observed with T47D (ER positive breast cancer cells) stably over expressing ERβ. In the latter cells and MCF7 (ER alpha positive) cells over expressing ERβ, E2 induced β catenin protein expression was blocked by ERβ. The latter also blocked AKT and ERK activation by E2 and induced apoptosis, in an E2 independent fashion. ERβ expression also blocked E2 induced growth in MCF 7 cells. Antibody array analysis showed that ERβ regulated >100 proteins at or above the 2 fold level in T47D breast cancer cells,where as 2D PAGE/MS showed that over 60 proteins changed significantly, both types of analysis and Western blots indicate that ERβ regulates Sp1,HSP70,β catenin, c-Jun, JNK,c-Jun ,BAD among other proteins. Antibody arrays and MS have therefore revealed unsuspected targets of ERβ action. Thus, ERβ appears to oppose several actions of ERα in breast cancer cells and block colon cancer cell growth, independently of E2. Design of selective ERβ activating agents or delivery of ERβ using gene therapy may be of substantial utility in the treatment of breast and colon cancer. Category27.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3056.
No clinical relevance of standardized immunocytological and molecular methods for detection of disseminated tumor cells in bone marrow of patients with primary breast cancer.
Authors: Kaul S , Sonnauer M , Bartik B , Deckert A , Zeifang F , Bastert G , Fersis N
Published: December 2010

Abstract #306

Background: Bone marrow samples from 2093 patients operated for primary breast cancer (stage T1-4, N0/N+, M0/M1) at the University Hospital Heidelberg between 1999 und 2007 were analysed for disseminated tumor cells (DTC) by standard cytology on cytospin slides. In parallel a subgroup of 250 samples was analysed by immunobead-RT-PCR methods for CK19 expression.  Material and Methods: Mononuclear cells were enriched by Ficoll density-gradient centrifugation. Cytospin-slides (mean 6) with 1x106 BM-cells were fixed in 3.5% buffered formalin at RT and endogenous AP and Ig were blocked with 10% acetic acid and 2.28% NaJO4. DTC were identified by CK antibodies 5D3 and A45-B/B3 (automated immunostainers, AP-techniques). Slides were analysed by automated picture analysis with the ACIS I and II systems (ChromaVision). Criteria for classification of disseminated tumor cells were in compliance with the publication of Fehm et al. (Cancer, Vol.107, 2006). 1x107 BM cells were used for immunomagnetic enrichment of epithelial cell types using antibodies targeting the cell membrane glycoproteins MUC1 and EpCAM. The markers cytokeratin 19 (CK19) and MG1 were analysed by real-time qRT-PCR using primers and FAM-labeled TaqMan probes of the UniversalProbeLibrary system (Roche AG, Basel, CH). The newly developed embedded tumor cell calibrator (ETC) technique was introduced for cell quantification.  Results: Small cells characterized by an excentrically located small and clear nucleus were detected in 56% of the bone marrow samples. These cytokeratin-positive cells were identified as bone marrow stroma stem cells. Identical cell types were present in 27 samples from normal BM-donors. In contrast, tumor cells were characterized by strong and irregular cytoplasmic cytokeratin staining, a granular nucleus and a clearly enlarged nuclear size. These DTC had a mean value for the parameter area of 309 (n=13) according to the ACIS picture analysis. Using published tumor classification critera 40 (1.9%) from 2093 patients with primary breast carcinoma were scored DTC positive. At the basis of 2x106 BM-cells the molecular detection methods of a) endpoint RT-PCR for CK19 and b) real-time RT-PCR for CK19 and MG1 had positivity rates of 3.5% and 3.8%, respectively.  Discussion: We have shown that bone marrow stroma cells are positive for various epithelial antigens including CK. Elimination of these cells either by size exclusion in picture analysis or by immunomagnetic separation results in tumor cell detection rates of less than 4% in 2x106 bone marrow cells. We conclude that cytological DTC analysis and the technique of immunomagnetic enrichment followed by molecular CK19 detection have no clinical relevance in patients with primary breast cancer.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 306.
Breast tumor cells with PI3K mutation or HER2 amplification are selectively addicted to Akt signaling.
Authors: She Q , Chandarlapaty S , Ye Q , Lobo J , Haskell KM , Leander KR , DeFeo-Jones D , Huber HE , Rosen N
Published: December 2010

Abstract #3061

Background: Activation of the PI3K/Akt pathway is a common event in human cancers and is responsible for key aspects of the transformed phenotype. This pathway is activated in most breast cancers, but by different mechanisms including HER2 amplification, PI3K mutation and PTEN inactivation. Inhibition of this pathway is thus thought to be clinically useful, but the functional output of PI3K/Akt signaling and the corresponding clinical effects of its inhibition may be due to the mechanisms by which it is activated. The objective of this study was to determine the role of Akt activation in breast cancer as a function of mechanism of activation and whether inhibition of Akt signaling is a feasible approach to therapy.  Methodology/Principal Findings: Using a panel of human breast cancer cell lines that harbor HER2 amplification, PI3K or PTEN mutations, or wild-type of PI3K and PTEN, and a selective, PH-domain dependent, allosteric inhibitor of Akt1 and Akt2 (AKTi-1/2), and a variety of biochemical, molecular, and cellular techniques as well as in vivo xenograft models, we show that AKTi-1/2 resulted in the selective inhibition of breast tumor cell lines with either mutant PI3K or amplified HER2, but not those in which PI3K/Akt signaling is not mutationally activated. Cellular sensitivity was due to induction of G1 arrest and of apoptosis and associated with inhibition of Akt signaling, loss of D-cyclin expression, and dephosphorylation of Rb. Most importantly, AKTi-1/2 effectively inhibited Akt kinase and its downstream effectors in vivo and caused complete suppression of the growth of breast cancer xenografts with PI3K mutation or HER2 amplification, including models of the latter selected for resistance to Herceptin. Furthermore, chronic administration of the drug was well-tolerated, causing only transient hyperglycemia without gross toxicity to the host despite the pleiotropic normal functions of Akt.  Conclusions/Significance: Our data demonstrate the exquisite dependence of PI3K-mutated or HER2-amplified breast cancer cells on Akt signaling, and that effective inhibition of Akt in tumors is feasible and effective in vivo. These findings suggest that direct inhibition of Akt may represent a therapeutic strategy for breast cancers that are addicted to the pathway and in patients with clinical resistance to Herceptin.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3061.
Orally active gnRH-antagonist AEZS-115 inhibits growth of human ovarian and breast cancers in vitro.
Authors: Engel JB , Ivanisevic M , Guenther E , Dietl J , Kaemmerer U , Hoenig A
Published: December 2010

Abstract #3065

Introduction: AEZS-115 is an orally active peptidomimetic antagonist of GnRH, which has been shown to block the pituitary GnRH receptor in nanomolar range. Recently an increasing number of human cancers have been demonstrated to express receptors for GnRH, which mediate growth promoting signals. Accordingly, we tested the antitumor activity of AEZS-115 in in vitro models of human breast and ovarian cancer.  Material and Methods: Human ovarian SKOV3, Ovcar 3 as well breast cancer cell line MDA-MB 468 were analyzed for GnRH receptor expression by immunocytochemistry. To screen for a putative anti-tumor effect, these cell lines were incubated with increasing concentrations of AEZS-115, peptidic GnRH-antagonist Cetrorelix and GnRH-agonist Triptorelin (1, 10, 100 µm) for 48 hours and the number of viable cells was determined by crystal violet staining as well as by ATP-dependent luminometric assay.  Results: Cell lines SKOV3, OVCAR3 and MDA-MB 468 expressed GnRH I receptors as demonstrated by immunehistochemistry. Both GnRH-antagonists dose dependently inhibited growth of all three cell lines, while GnRH-agonist Triptorelin showed marginal growth inhibition. At 10 µM AEZS-115 inhibited cell growth by 40-60%, at 100 µM growth inhibition was 60-80%. Inhibition with Cetrorelix at 100 µM ranged from 20-40%, while only minor effects on cell growth were seen at 10 µM. These results obtained by crystal violet staining were confirmed by additional luminometric evaluation of the ATP content after treatment with the respective substances.  Conclusions: To our knowledge here we report for the first time in vitro anti-cancer activity for a peptidomimetic GnRH antagonist.  AEZS-115 showed substantial anti-tumor activity in human ovarian and breast cancer cell lines, while GnRH-agonist Triptorelin showed only minor growth inhibition. Surprisingly AEZS-115 was somewhat more potent than peptidic GnRH-antagonist Cetrorelix. Due to the good antitumor-activity and the possible oral administration AEZS-115 should be a promising candidate for in vivo studies.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3065.
Molecular mechanisms for leptin regulation of VEGF in breast cancer cells.
Authors: Xu Y , Watters A , Leibovich SJ , Mann D , Rueda BR , Gonzalez RR
Published: December 2010

Abstract #3071

Background: We have previously reported that in vitro and in vivo leptin signaling mediates proliferation of mouse 4T1 mammary tumor (MT) cells and levels of VEGF and VEGFR2. Therefore, we hypothesize that leptin can contribute to MT growth by augmenting angiogenesis through the regulation of VEGF expression. To test this we assessed whether leptin regulates VEGF promoter activity in mouse MT cells and if leptin-induced factors (HIF-1α, NFκB, AP1, SP1) regulate the VEGF promoter. Materials and Methods: Mouse MT cells (4T1, EM6 and MMT) were transiently transfected with molecular engineered luciferase-reporters (pGL3) for mouse VEGF promoter (full-length) and transcription factor-binding deletions for hypoxia responsive element (HRE), AP1, SP1 and NFκB. Leptin dose-response effects on reporter activity and VEGF levels (protein and mRNA) were investigated using ELISA, Western blot and real-time PCR, respectively. To further assess leptin regulation of VEGF expression in MT cells the effects of hypoxia (CoCl2, hypoxia mimetic agent) alone and combined with leptin on VEGF promoter activity were investigated. To assess which leptin-induced signaling pathways/transcription factors are linked to VEGF regulation in MT cells inhibitors for: (1) JAK2/STAT3, MEK-1/MAPK and PI-3K/Akt-1 pathways and; (2) target molecules NFκB; SP1; AP1 and HIF-1α were used. The effects of leptin/CoCl2 and inhibitors on luciferase activity of transfected cells were compared using pRL-TK renilla luciferase as control and the Dual-Luciferase Reporter Assay System. Results: Our data suggest that leptin signaling can regulate the transcriptional activity of VEGF gene in breast cancer cells by activating gene transcription at several sites of the VEGF promoter. Leptin-induced HIF-1α has a relevant role in the regulation of VEGF expression in MT cells. Leptin can also increase VEGF-levels (protein and mRNA) through the specific activation of AP1, SP1, and NFκB. PI-3K activity was mainly linked to leptin regulatory effects on VEGF. However, diverse mechanisms for leptin regulation of VEGF expression were found in the different MT cells. Discussion: Our results provide novel information on the molecular mechanisms for leptin-induced angiogenic effects in breast cancer. Our data further suggest that disruption of leptin signaling could impact angiogenesis and MT growth by inhibiting VEGF expression. Thus targeted disruption of leptin-signaling could serve as method of prevention/treatment of breast cancer. This work was supported in part by the Susan G. Komen Foundation for the Cure (to R.R.G.; BC 504370); UAB Breast SPORE Award (to R.R.G) and Contraceptive Research and Development Program (CONRAD; CIG-07-114 to R.R.G).

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3071.
Rapamycin-regulated gene expression signature determines prognosis in breast cancer.
Authors: Akcakanat A , Zhang L , Tsavachidis S , Meric-Bernstam F
Published: December 2010

Abstract #3072

Background: Mammalian target of rapamycin (mTOR) is a serine/threonine kinase involved in multiple intracellular signaling pathways promoting tumor growth. mTOR is aberrantly activated in a significant portion of breast cancers and is a promising target for treatment. Rapamycin and its analogues are in clinical trials for breast cancer treatment. Patterns of gene expression (metagenes) may also be used to simulate a biologic process or effects of a drug treatment. In this study, we tested the hypothesis that the gene-expression signature regulated by rapamycin could predict disease outcome for patients with breast cancer.  Methods: MDA-MB-468 breast cancer cell line is triple negative and PTEN null. Sensitivity of MDA-MB-468 breast cancer cell line to rapamycin was determined by in vitro growth assays and in vivo tumor studies. Total RNA was extracted from cell lines treated with rapamycin or DMSO for 24 hours and xenografts treated with DMSO or rapamycin for 1 or 22 days. Total RNA was used for expression profiling and hybridized to Affymetrix HG-U133 Plus 2.0 arrays. We used two well-described primary breast cancer datasets of 251 (Miller et al, Proc. Natl. Acad. Sci. USA 2005;102:13550) and 286 (Wang et al, Lancet. 2005;365:671) patients from the public domain and assessed the prognostic capability of rapamycin-regulated gene expression signatures.  Results: Colony formation and sulforhodazine B (IC50 < 1 nM) assays, and xenograft animals showed that MDA-MB-468 cells were sensitive to treatment with rapamycin. The comparison of in vitro and in vivo gene expression data identified a 31-gene expression signature that was up- regulated by rapamycin treatment in vitro as well as in vivo (FDR of 0.1). From the 31 genes on HG-U133 Plus 2.0 array, 20 that were included on HG-U133A array were used for analysis. In the Miller dataset, rapamycin metagene index (RMI) did not correlate with tumor size or lymph node status. High (0.75 percentile) RMI was significantly associated with longer survival (P=0.015). On multivariate analysis, RMI (P=0.029), tumor size (P=0.015) and lymph node status (P=0.001) were prognostic. In the Wang dataset, RMI predicted time to disease relapse (P=0.009).  Conclusion: Rapamycin-regulated gene expression signature predicts clinical outcome in breast cancer. This supports the central role of mTOR signaling in breast cancer biology and provides further impetus to pursue mTOR-targeted therapies for breast cancer treatment.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3072.
Cyr61 (CCN1) is a novel target for Zometa (zoledronic acid) in breast cancer bone metastasis.
Authors: Espinoza I , Liu H , Dimri M , Lupu R
Published: December 2010

Abstract #3074

Background: Cyr61, currently known as CCN1, is a pro-angiogenic factor highly expressed in triple negative breast carcinomas. CCN1 binds, among others, to αvβ3 integrin receptor, which is concomitantly expressed with CCN1 in triple negative carcinomas. It has clinically established that a greater part of the tumor initially diagnose as triple negative eventually metastasize to the bone. Women with advanced breast cancer often develop bone metastases. Currently, many patients receive bisphosphonate treatment as a support treatment to maintain bone density. Zometa (Zoledronic acid=ZOL), a third generation amino-bisphosphonate, inhibits bone resorption and it might prevent development of new osteolytic lesions; lesions induced by tumor metastases. Provided that: 1) ZOL has a direct anti-tumor activity on breast cancer cells in vitro, 2) CCN1:αvβ3 is an autocrine loop shown to be involved in breast cancer cells which metastasize to the bone, 3) ZOL inhibits αvβ3 expression in the cell surface of endothelial cells, and CCN1 induces the expression of αvβ3 expression.  Material and Methods: Using MTT assay we analyzed the effect of ZOL in the viability of cells overexpressing CCN1:αvβ3. The study whether the effect of ZOL was at the transcriptional level of CCN1 a Luciferase reporter assay was performed. To study whether FOXO3a binding site on CCN1 promoter was an important site for ZOL effect on CCN1 we used a CCN1-promoter containing a deletion in the FOXO3a binding site and we performed the Luciferase reporter assay. We also analyze the effect of ZOL in the anchorage-independent growth by soft agar assay and in the branching and morphogenesis in 3D Matrigel culture.  Results: We found that: a) ZOL regulates the expression of CCN1 expression in triple negative breast cancer cell lines; b) the IC50 for Zometa for CCN1:αvβ3 expressing cells is lower (about 12μM) than the IC50 for cells that express low to undetectable levels of CCN1:αvβ3 cells; c) ZOL specifically inhibited CCN1 at a protein and transcription expression levels in a concentration-dependent manner; d) FOXO3a is the site by which ZOL induces transcriptional activation of CCN1; e) ZOL induced specific inhibition of anchorage independent growth of CCN1:αvβ3 cells in a dose dependent manner, and f) ZOL induces Mesenchimal-Epithelial Transition (MET) of CCN1:αvβ3 expressing cells and inhibited the generation of branching and morphogenesis in 3D in Matrigel culture of CCN1:αvβ3 expressing cells.  Discusion: Collectively, these data demonstrated for first time a specific effect of ZOL on a pro-angiogenic factor involved in triple negative carcinomas; providing a new model for the development of a therapeutic strategy for ZOL.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3074.
Epstein-Barr virus (EBV) in fine needle aspirates containing inflammatory breast cancer cells.
Authors: Li C , Pusztai L , Cohen EN , Symmans F , Wang B , Lee B , Hortobagyi GN , Cristofanilli M , Reuben JM
Published: December 2010

Abstract #3084

BACKGROUND: Inflammatory breast cancer (IBC) is the most aggressive manifestation of primary breast cancer and its clinical presentation and the peculiar geographic distribution have suggested a possible viral etiology. Mouse Mammary Tumor, Human Herpes Virus 8 or Epstein-Barr virus (EBV) may play significant roles in the pathogenesis of the disease and account for the peculiar lymphangiogenic mechanism of growth and disease progression. In particular, EBV late membrane protein (LMP)-1 plays a critical role in tumor invasion lymphangiogenesis. In this study, EBV DNA was measured in each FNA sample from patients with IBC and locally advanced breast cancer (LABC) using real time PCR directed against 5 different targets within the EBV genome.  PATIENTS AND METHODS: Sixty-eight fine needle aspirates (FNA) of breast cancer from patients with newly diagnosed IBC (n=18) and LABC (n=50) were examined. The FNA specimens were collected for the pharmacogenomic research program before any systemic therapy started. Median age of subjects was 53 yrs (range: 26-83 yrs). Total DNA was extracted from the FNA using the QIAmp Kit. EBV DNA were detected by quantitative polymerase chain reaction (QPCR) directed at five different targets within the EBV genome (BamH1W, LMP1, EBNA1, LMP2, and BZLF1) that encode products that are highly conserved across all EBV strains and important for viral pathogenesis. DNA extracted from Namalwa cells were used to construct the standard (R2 = 0.993); HL60 cells were used as EBV-negative controls. Data were collected and analyzed by Sequence Detection System software (Applied Biosystems Incorporated, ABI, Foster City, CA).  RESULTS: EBV DNA was detected in freshly obtained breast cancer FNA samples using Q-PCR. The Q-PCR assays were sensitive to as low as 1 copy of EBV DNA per reaction. Of 68 breast tumor FNA specimens, EBV genes were detected in FNA from 4/18 (22%) IBC patients (range, 1-4 genes) and 5/50 (10%) LABC patients (range, 1-3 genes). Among the 9 positive specimens, BamH1W was detected in 6; EBNA1 in 5; LMP1 in 4; BZLF1 in 2; and LMP2 in 1, respectively.  DISCUSSION AND CONCLUSIONS: Q-PCR is an effective method for detecting EBV DNA in FNA of breast cancer patients. The two most frequently detected EBV DNA sequences were BamH1W (6 of 9) and EBNA1 (5 of 9). BamH1W is a sequence that is frequently replicated throughout the EBV genome and EBNA1 is a reliable latent gene responsible for maintaining infection for life of the host. LMP-1 is a potential viral oncogene and was detected in 4/9 patients. The detection of these EBV sequences suggest latent EBV infections in IBC and LABC patients. The clinical significance of these findings warrants further investigation.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3084.
Cytochrome P450 2D6 variants and their association with breast cancer survival.
Authors: Abraham JE , Maranian M , Driver K , Greenberg D , Shah MN , Earl HM , Dunning AM , Pharoah PP , Caldas C
Published: December 2010

Abstract #3089

Background: Cytochrome P450 2D6 (CYP2D6), a phase I drug metabolising enzyme, is involved in the metabolism of up to 25% of all drugs. The CYP2D6 gene is highly polymorphic with over 60 reported variant forms. Many of these have been classified by their effect on drug metaboliser status (poor, intermediate, extensive and ultra). Many variants have low minor allele frequencies (MAF) and metaboliser classification is not always certain. There is conflicting evidence for the potential predictive value of CYP2D6 genotype in patients treated with tamoxifen. In this study we investigate the association of the CYP2D6 gene variants with breast cancer survival in patients treated with tamoxifen.  Methods: Invasive breast cancer cases (n=6510), came from SEARCH (East Anglian, UK population-based study), 3155 of these cases had received Tamoxifen therapy. CYP2D6 single nucleotide polymorphisms (SNPs) for genotyping were selected using 2 approaches: (i) Known CYP2D6 functional variants (n=13; MAF>0.01), (ii) tag SNPs , that efficiently tagged all known CYP2D6 common variants on the HapMap database (n=5; MAF>0.05), such that known common SNPs had an rp2 of >0.8 with at least one tagSNP. (Pairwise correlation coefficient (rp2) is a measure of how well one SNP tags another.) The combination of these two approaches aims to capture all known functional variants as well as any, as yet undiscovered variants that may be tagged by the common tagSNPs. Genotyping was carried out using Taqman Assays by Design.  Results: Preliminary results, from this ongoing study, indicate that in patients treated with tamoxifen, one rare functional variant (carrier frequency = 0.02), CYP2D6*6, is associated with decreased breast cancer survival (P=0.007; HR=2.03; 95%CI=1.21-3.40), in comparison to patients who have not received hormone therapy (P=0.359; HR=0.52; 95%CI=0.13-2.10). None of the common tagSNPs (MAF>0.05) were associated with differences in survival (at P</=0.05 level).  Conclusion: We conclude that at least one variant of the CYP2D6 gene may affect breast cancer survival in patients treated with tamoxifen. CYP2D6*6 is associated with poor metaboliser status, resulting in impaired tamoxifen metabolism. Consequently genotyping for this CYP2D6 variant may help to determine which patients are unlikely to benefit from standard tamoxifen treatment.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3089.
Gene associations and risk of breast cancer.
Authors: Andavolu RG , Cone LA , Shu S , Izsac C , Andavolu MV , Luthra M
Published: December 2010

Abstract #3094

Background: Human BCCIPalpha (Tok-1alpha) was identified as a BRCA2 and CDKN1A (p21, or p21(Waf1/Cip1)) Interacting Protein. BCCIP binds to a highly conserved domain proximate to the C-terminus of BRCA2 protein and the C-terminal domain of the CDK-inhibitor p21. Previous reports showed that BCCIP alpha enhances the inhibitory activity of p21 toward CDK2 and that BCCIP alpha inhibits the growth of certain tumor cells. In cancers harboring wild type p53, the p53 transactivation activity may be compromised by other mechanisms. It is clear from previous reports that BCCIP functions through p53 to regulate the expression of p21Waf1/Cip1 and BCCIPalpha is an important cofactor for BRCA2 in tumor suppression. In this study, specific SNPs in BCCIP, CDKN2A, BRCA2 and KLF6 (tumor suppressor gene) were screened to evaluate the potential influence of these genes in the predisposition to breast cancer.  Materials & Methods: Blood was drawn from 204 primary breast cancer patients and 197 normal healthy subjects. An informed consent was obtained from all the study subjects. Genotyping was done using polymerase chain reaction followed by restriction enzyme digestion. SPSS version10.1 for windows software was used for data analysis.  Results: A relatively high frequency of the mutant alleles showing significant association with breast cancer was observed when compared with age, gender and ethnic matched control subjects (KLF6- p<0.0001; BRCA2- p<0.028; CDKN2A- p<0.018). Association among these gene variables were determined using spearman correlation coefficients. Significant correlations were observed among BCCIP-BRCA2 (p< 0.003); BCCIP-CDKN2A (p< 0.004), BRCA2-CDKN2A (p<0.037) genes.  Conclusions: Our results confirm the strong correlation of BCCIP gene with BRCA2 and CDKN2A gene. A strong association of KLF6 (tumor suppressor gene) gene with breast cancer was observed but no such correlation was identified with BCCIP, CDKN2A and/or BRCA2 suggesting that KLF6 gene may be involved in tumorigenesis using different regulating pathway. To better understand the role of these genes in the progression of breast carcinoma, gene expression studies will be performed using quantitative PCR.

Citation Information: Cancer Res 2009;69(2 Suppl):Abstract nr 3094.