bluuebunny/crossref_metadata_2025 huggingface dataset:
Is Accelerated Partial Breast Irradiation Appropriate Adjuvant Therapy for Patients with Ductal Carcinoma In-Situ?
Authors: Careaga E. , Hughes J. , Allen L. , Carella R. , Frazier T.
Published: December 2010

Introduction: Accelerated partial breast irradiation (APBI) using high-dose rate (HDR) balloon catheter breast brachytherapy following wide segmental resection of the breast has been shown to be equivalent to whole breast irradiation (WB-XRT) in the treatment of stage-I breast carcinoma. Because the majority of all breast cancers recur in the same quadrant as the original tumor, the advantages of this treatment compared with WB-XRT include concentration of radiation in the area where cancer is most likely to recur, shorter treatment duration, and greater patient acceptance. This study examined the rate of local/regional recurrence in patients diagnosed with stage-0 breast cancer, specifically ductal carcinoma in-situ (DCIS), following wide segmental resection of the breast and treatment with APBI using HDR balloon catheter breast brachytherapy.Methods: An IRB-approved, single-institution, retrospective chart review was conducted identifying 40 patients diagnosed with stage-0 breast cancer between March, 2004 and April, 2008 who were treated with wide segmental resection of the breast followed by APBI using HDR MammoSite™ balloon catheter breast brachytherapy. Outcome measures included rates of same-quadrant recurrence, ipsilateral second-primary occurrence, and regional recurrence.Results: Patient age ranged from 44 to 89 years (median = 69 years) with a median follow-up of 40 months (range: 16 to 60 months). All 40 patients had DCIS. One patient of 40 (2.5%) had DCIS with an additional focus of lobular carcinoma in-situ. 29 of 40 patients (72.5%) were estrogen receptor (ER) positive. Of these 29 patients, 17 were also progesterone receptor (PR) positive. One patient (2.5%) at 38 months experienced a local recurrence of DCIS with micro-invasion and a synchronous focus of invasive ductal carcinoma in a separate quadrant. This was subsequently treated with total mastectomy, sentinel lymph node mapping, and axillary lymph node dissection. No other patients experienced local-regional recurrence or ipsilateral primary breast cancer occurrence.Conclusion: Patients with DCIS can be effectively treated with APBI/HDR balloon catheter breast brachytherapy following wide segmental breast resection. The rate of local-regional recurrence was 2.5% at a median follow-up time of 40 months. This is consistent with that of patients with DCIS treated with wide segmental breast resection followed by WB-XRT. Long-term follow-up will be required to assure these rates remain favorable.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2116.
BUBR1 and MAD2: Novel Markers for Predicting Benefit from Adjuvant Anthracyclines?
Authors: Munro A. , Cameron D. , Thomas J. , Twelves C. , Bartlett J.
Published: December 2010

Background: We previously demonstrated the importance of duplication of CEP17 (C17) in predicting sensitivity to anthracyclines in two retrospective adjuvant trials (BR9601 and MA.5). Spindle checkpoint failure, and resultant chromosome instability (CIN), could be a mechanism for this observation. BubR1 and MAD2 are key components of the spindle assembly checkpoint which prevent progression to anaphase. Overexpression of BUBR1 or a reduction in MAD2 is associated with CIN and polysomy. Unlike MAD2, BubR1 competitively binds, phosphorylates and stabilises p53 during mitosis, at a site overlapping that of MDM2. Therefore, BubR1 is implicated in both spindle assembly and DNA damage checkpoints. We investigated BubR1 and MAD2 as predictive markers of anthracycline sensitivity in breast cancer.Methods: We evaluated cytoplasmic BubR1 and MAD2 protein expression in 321 tumours from patients in the BR9601 trial, which compared E-CMF with CMF as adjuvant therapy for breast cancer. Overall survival (OS) and relapse-free survival (RFS) were estimated using Kaplan-Meier curves.Results: BubR1 and MAD2 overexpression were detected in 49.2% and 25.1% of tumors, respectively. Both MAD2 and BubR1 overexpression were associated with ER negativity (p<0.0001, p=0.013) and high grade (p<0.0001, p=0.029). BubR1 overexpression was associated with C17 (p=0.0003) and high proliferation (Ki67, p<0.0001); similar trends were noted for MAD2. BubR1 overexpression was associated with reduced RFS (HR: 1.58, 95%C.I. 1.06-2.37, p=0.024) and OS (HR: 1.831 95%C.I. 1.161-2.890), whereas there was no clear association with outcome for MAD2.Interaction tests revealed no significant evidence that the benefit of the addition of epirubicin to CMF was confined to those tumours with either high levels of BubR1 or low levels of MAD2 (BubR1 HR: 1.70 95%C.I. 0.74-3.87 and MAD2 HR: 0.573 95%C.I. 0.143-2.296). However, combining overexpression of BubR1 with low expression of MAD2 selects for 33.0% (98/297) of patients and is associated overall with a poorer RFS (HR: 0.602 95%C.I. 0.402-0.902, p=0.014) but an increased RFS benefit from the addition of anthracycline to CMF (HR: 0.409 95%C.I. 0.208-0.803) than those with any other combination of BubR1 and MAD2 expression (HR: 0.718 95%C.I. 0.423-1.216). Similar results were noted with OS. However, neither treatment by marker interaction reached significance (RFS HR: 1.73 95%C.I. 0.74-1.08, p=0.208; OS HR 1.820 95%C.I. 0.707-4.686, p=0.215).Conclusion: BubR1 overexpression is associated with an aggressive phenotype and decreased RFS and OS in BR9601 patients. Tumours with BubR1 overexpression and/or low MAD2 expression showed a potential increased sensitivity to anthracycline therapy compared to those with low BubR1 levels and/or high MAD2 expression. Small trials (BR9601) are useful for hypothesis generating; further work in larger patient cohorts such as MA.5 or NEAT is warranted.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2124.
The Expression of Insulin-Like Growth Factor-1 Receptor (IGF-1R) in Early Breast Cancer.
Authors: Spears M. , Cunningham C. , Thomas J. , Kerr G. , Jack W. , Campbell F. , McKay L. , Kunkler I. , Cameron D. , Chetty U. , Bartlett J.
Published: December 2010

Background: The insulin-like growth factor-1 receptor (IGF-1R) is expressed in normal breast epithelial cells and breast carcinomas. A number of studies have examined the over-expression of IGF-1R in breast cancer with the range of between 39% to 93%. However, many of these studies were carried out using ELISAs, radioimmunoassay with small patient cohorts. Phosphorylation of IGF-1R results in the activation of the MAPK and PI3K/Akt pathway. Activation of the PI3K/Akt pathway under the influence of IGF-1R plays an important role in maintaining the proliferation of breast cancer cells that are resistant to gefitnib, trastuzumab or chemoradiotherapy in vitro and in vivo. In the present study, we examined the expression of IGF-1R in early breast cancer and investigated the clinicopathological implications using immunohistochemistry and FISH.Methods: TMAs from a consecutive series (1981-98) of 1,812 women managed by wide local excision and postoperative radiotherapy (45Gy in 20-25 fractions) were collected following appropriate ethical review. Of 1686 cases stained, 198 received no adjuvant hormonal or chemotherapy, 1106 received tamoxifen only as adjuvant therapy and 144 received a combination of hormonal and chemotherapy. Median age at diagnosis was 56, 72% were post-menopausal, 23.9% node positive, median size was 1.5 cm. Samples were stained, using triplicate 0.6mm2 TMA cores for IGF-1R.Results: FISH for IGF1R gene amplification was successful in 343/408 cases (84%). Only 7 cases of IGF1R amplification were observed (2%). IGF1R expression was successfully evaluated in 1597 of 1686 (94.7%) cases. High levels of IGF-1R expression was weakly associated with tumour ER-a (correlation coefficient, 0.108; p=2.2 x 10-5) and PgR levels (correlation coefficient 0.162; p=1.86x10-10) and inversely correlated with EGFR expression (correlation coefficient -0.189; p=6.33x10-6). No association was noted between IGF1R expression and other molecular or clinical markers, such as tumour size. There was a trend for over-expression of IGF-1R to link with increased distant relapse free survival, particularly in ER positive breast cancers. This trend is time dependent, such that no difference in outcome is observed at 15 years.Conclusions: We have demonstrated in a large cohort of patients that IGF-1R gene amplification is a rare event, and over-expression is weakly associated with good prognostic features (ERa and PgR, lack of EGFR and better outcome).

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2128.
Stromal Caveolin-1 Levels Predict Early Ductal Carcinoma In Situ Progression to Invasive Breast Carcinoma.
Authors: Witkiewicz A. , Dasgupta A. , Schwartz G. , Nguyen K. , Rui H. , Lisanti M.
Published: December 2010

With the increase use of screening mammography the incidence of ductal carcinoma in situ (DCIS) increased and DCIS currently accounts for 20-30% of newly diagnosed breast cancers.The current classification for DCIS based on nuclear grade, architectural differentiation and presence of necrosis, does not adequately predict the likelihood of recurrence after breast conservingtherapy. Therefore, there is a critical need to identify novel predictors of DCIS progression and potential targets for therapy. Recently, we showed that an absence of stromal caveolin-1 (Cav-1)expression in invasive breast cancers is a powerful single independent predictor of early disease recurrence and metastasis. The aim of this study was to evaluate the stromal expression of Cav-1 in a cohort of DCIS patients treated with wide-excision and close follow-up, but without any form of chemo- or radiotherapy.Material and MethodsSeventy eighty DCIS cases were included in the study. Cav-1 expression in the tumor stroma was assessed by employing a standard immunoperoxidase method with monoclonal ani-Cav-1 antibody (BD Biosciences, San Jose, CA; 1:50 dilution).The staining was scored semi-quantitatively as negative (0; no staining), weak (1; either diffuse weak staining or strong staining in less than 30% of stromal cells) and strong (2; defined as strong staining of 30% of stromal cells).Statistical analysis was performed using the Fisher exact test or the Kruskal-Wallis test.ResultsThe overall recurrence rate was twenty-eight percent (50% recurred as DCIS, and the other 50% recurred with progression to invasive breast cancer). Nearly seventy percent of DCIS patients with any type of recurrence had reduced levels of stromal Cav-1 (score = 1 or 0). Eighty nine percent of DCIS patients that underwent recurrence with progression to invasive breast cancer had reduced or absent levels of stromal Cav-1. Conversely, ninety-seven percent of DCIS patients with high levels of stromal Cav-1 (score = 2) did not show any invasive recurrence over the duration of follow-up (4-208 months).Our results indicate that an absence of stromal Cav-1 is a novel biomarker that predicts early DCIS progression to invasive breast carcinoma.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2136.
Delineating the Secretome of Eleven Breast Cell Lines in the Quest for Novel Breast Cancer Biomarkers.
Authors: Pavlou M. , Pavlou M. , Diamandis E. , Diamandis E. , Diamandis E.
Published: December 2010

Background: Close to 200,000 new cases of breast cancer are estimated in United States for 2009, rendering breast cancer the most frequently diagnosed cancer in women. Patients diagnosed with early stage breast cancer have significantly improved survival rates compared to late stage patients, underlining the need for identification of biomarkers for early detection. In this study the secretome of 11 breast cell lines was analyzed by tandem mass spectrometry to identify novel breast cancer biomarkers.Materials and Methods: To reflect disease heterogeneity, three cell lines for each breast cancer type (estrogen/ progesterone receptor positive, triple negative, HER2/neu amplified) along with two near-normal cell lines were selected (HCC-1428, BT483, MCF-7, MDA-MB-231, HCC-1143, HCC-38, SK-BR-3, HCC-202, UACC-812). The conditioned media were lyophilized to dryness and the proteins were denaturated, reduced and trypsin digested. The peptides were separated by strong cation chromatography and the resulted fractions were analyzed in a linear ion-trap coupled to an orbitrap mass analyser. All samples were run in duplicate and spectra were searched with Mascot and X!Tandem engines. Scaffold software was used to cross-validate Mascot and X!Tandem results.Results and Discussion: Over 800 unique proteins were identified in the conditioned media of each cell line, resulting in more than 4,000 proteins from the eleven breast cell lines. More than 40% of the proteins were identified with at least 2 unique peptides and the reproducibility between replicates of the same cell line was approximately 70-80%. Using an in-house developed program, we compared the proteomes of all the cell lines to distinguish unique and common proteins and we determined the cellular localization and the biological function for each protein. Then, we focused on extracellular and plasma membrane proteins, which constitute approximately 30% of total proteins, since secreted or shed proteins are more likely to enter the circulation and serve as potential biomarkers. This list of candidates was compared to the existing nipple aspirate fluid proteome to select proteins that have been also detected in breast microenvironment. These proteins were further analyzed based on Unigene database and appropriate literature to select the final list of 10 candidates for validation by immunoassays.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2137.
Molecular Biomarkers of Columnar Cell Lesions Identified by Proteomic Approaches.
Authors: Pape-Zambito D. , Kistler J. , Chen X. , Li X. , Chen Y. , Cai K. , Wu H. , Huang M. , Yeung A. , Patchefsky A. , Daly M. , Godwin A.
Published: December 2010

The widespread increase in the use of screening mammography has led to a significant rise in the detection and diagnosis of benign breast disease (BBD), which encompasses a wide variety of non-cancerous changes including columnar cell lesions (CCL). Epidemiologic studies provide evidence for a relationship between BBD and subsequent risk of developing breast cancer. Observational studies have reported CCL are found adjacent to areas of tubular carcinoma and ductal and lobular carcinomas in situ and recently published molecular data state that genetic abnormalities frequently found in low grade ductal carcinoma in situ are also found in CCL. All these data support the hypothesis that CCL are the earliest morphologically distinguishable precursor lesions to low grade ductal carcinoma in situ and invasive carcinoma. However, BBD is heterogeneous and there is limited understanding of the biological importance associated with the spectrum of lesions. The lack of accurate molecular-based risk categories, therefore, hinders the ability of assigning prognostic significance to benign biopsy findings. The purpose of our study was to identify putative molecular markers of cancer progression from epithelial cells of BBD lesions. Regions of ductal hyperplasia (DH) and normal breast epithelium were isolated from mastectomy specimens using laser capture microdissection and extracted proteins were analyzed through proteomic approaches. Of the 1,500 total protein spots resolved by 2-dimensional electrophoresis, 98 were differentially expressed between DH and normal breast epithelial proteomes. Twenty-two proteins or related isoforms were identified using mass spectrometry. Two proteins of particular interest, NAD(P)H: quinone oxidoreductase 1 (NQO1) and galectin-3, were further investigated by immunohistochemistry (IHC) on cases diagnosed with BBD lesions. NQO1 was expressed sporadically in overtly normal epithelial cells lining breast ducts, but was overexpressed in BBD and tumor tissues. Notably, we found that a subpopulation of DH tissues with CCL stained positively for NQO1 and negatively for galectin-3. This finding has significance because CCL can only be diagnosed morphologically and the presence of CCL in biopsy specimens may indicate a precancerous lesion. However, sectioning artifacts associated with core biopsies can hinder morphological diagnosis of CCL, thus a means to additionally identify these lesions using IHC has tremendous value. Together, immunohistochemical staining of NQO1 and galectin-3 may prove to be clinically relevant in the identification of CCL in biopsy samples.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2142.
Albumin-Binding and Angiogenic Domains of SPARC Located at Its C-Terminus.
Authors: Knauer D. , Hwang L. , Lowe C. , Hwang J. , Norng M. , Trieu V. , Desai N.
Published: December 2010

Background: SPARC (Secreted Protein Acidic and Rich in Cysteine) is an albumin-binding glycoprotein overexpressed in breast cancer and is associated with poor prognosis and shorter overall survival. We have previously shown that SPARC expression appeared to correlate with response to nanoparticle albumin-bound (nab) paclitaxel (Abraxane) in head-and-neck and pancreatic-cancer patients. To further define the role of SPARC, recombinant-human SPARC (rhSPARC) was characterized to identify its albumin-binding and angiogenic domain.Materials and methods: The albumin-binding site on SPARC was defined by testing the binding of Alexa 488-labeled BSA to immobilized rhSPARC in a solid-phase-binding assay and in a competitive-binding assay in presence of increasing concentration of various SPARC-derived peptides. The angiogenic activity of rhSPARC was evaluated using a HUVEC tube-formation assay.Results: The SPARC albumin-binding assay revealed a pattern of saturable and specific binding with an estimated Kd of 700 µM very near the known plasma concentration of albumin (600 µM). Competitive binding with Cathepsin K-digested SPARC and SPARC-derived peptides defined the albumin-binding domain to be amino acids 209-223 in the C-terminal region. In the HUVEC tube-formation assay, rhSPARC was pro-angiogenic at 10 µg/ml and anti-angiogenic at 100 µg/ml. The angiogenic domain of SPARC was located to the C-terminal of SPARC.Discussion: The albumin-binding domain of SPARC was located to residues aa209-223, supporting its role as a target for nab-technology-based drug delivery. We confirmed that SPARC can promote angiogenesis at physiological concentrations, contributing to the role of SPARC in a more aggressive tumor phenotype. The angiogenic domain of SPARC is located within a 54-aa sequence of its C-terminus. SPARC could be a potential therapeutic target and a biomarker for predicting response to nab-paclitaxel.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2144.
Anastrozole Reduces Progesterone and Proliferation Index in Short Term Hormone Therapy. A Prospective Placebo Double Blind Study.
Authors: Mattar A. , Logullo A. , Facina G. , Nonogaki S. , Soares F. , Gebrim L.
Published: December 2010

Background: Identification of new biomarkers with potential predictive and prognostic role has contributed unequivocally to breast cancer treatment. Although traditionally endocrine therapy is based on Hormonal receptors status (estrogen - ER and progesterone- PR), some patients become hormone resistant. In order to identify a possible profile associated to hormonal resistance, some biomarkers have been assessed after short period primary hormone therapy (HT).Objectives: To compare the expression of Ki-67, Bcl2, Bax, Bak, ER and e PR in postmenopausal women with ER positive invasive ductal carcinomas (IDC), prior and after tamoxifen and anastrozole in neoadjuvant treatment.Methods: Fifty-eight patients with palpable ER-positive IDC (stage II and III) were double-blind randomized in a prospective placebo controlled study with three neoadjuvant HT groups for twenty-six days prior to surgical treatment: Anastrozole 1mg/day (n= 18), Placebo (n=25) and Tamoxifen 20mg/day (n= 15). Pre and post HT samples were disposed in tissue micro array blocks and submitted to immunohistochemical essay. Biomarkers status (Ki-67, Bcl2, Bax, Bak, ER and e PR) were obtained comparing each immunohistochemical evaluation of pre and post-surgery samples using semi-quantitative Allred's method. Statistical analysis were performed using the GEE (General Estimation Equations) and Anova tests with significant p ≤ 0,05.Results: ER and PR status did not change significantly in placebo group (96% -96% and 76-72%) and KI-67 index variation was 40 to 52% (p>0,05), as expected. In Tamoxifen group, ER status was also stable during treatment (100-100%). However, in anastrozole group, ER status -originally 100% - lowered to 89%,(p>0,05) and also PR expression rates lowered significantly from 56% to 28% in post treatment (p=0,0007). Anastrozole group also showed a significant reduction in KI-67 status from 78% in the pre-treatment KI-67 to 39% in post treatment samples (p=0,0079). Tamoxifen group showed a slight increase of PR expression from 73% to 93% along a non-statistical significant variation of KI-67 status 67% pre-treatment to 47% post-treatment. The other biomarkers (RE, Bak, Bax, Bcl-2 )did not show any variation in the three groups evaluated.Conclusions: a significant reduction of PR and ki-67 status was associated to Anastrozol treatment (26 days, 1mg/day). Short primary HT was not associated to any variation in apoptosis-related proteins (Bak, Bax, Bcl-2) regardless the drug used.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2145.
Targeting DNA Replication before It Starts: Cdc7 as a Therapeutic Target in p53 Mutant Her2 and Triple Negative Breast Ca.
Authors: Sainsbury R. , Proctor I. , Rodriguez S. , Loddo M. , Tudzarova S. , Stoeber K. , Williams G.
Published: December 2010

Based on protein expression profiles of core regulatory proteins involved in the G1–S and G2–M phase transitions, we have identified three distinct cell cycle phenotypes in a series of 200 breast cancers: (I) a G0 “out-of-cycle” state (18% of cases); (II) a G1 arrested/delayed state (24% cases); and (III) accelerated S-G2-M phase progression (58% of cases). The accelerated cell cycle progression phenotype had a higher risk of relapse when compared with G0 and G1-delayed/arrested phenotypes (HR=3.90 (1.81-8.4, p<0.001) and was associated with Her2 and triple negative subtypes (p<0.001). High-grade tumours with the G1 delayed/arrested phenotype showed an identical low risk of relapse compared with well-differentiated G0 tumours. In addition to its prognostic significance, the cell cycle phenotype also impacts on individualised therapeutic decisions. It is only patients showing the actively cycling, aggressive cell cycle phenotype that are likely to benefit from conventional chemotherapeutic S- or M-phase-directed agents or from the new generation of targeted cell cycle inhibitors that are now entering clinical trials.The DNA replication initiation factor Cdc7 is an emerging anti-cancer target. Cdc7 inhibition results in an abortive S phase and potent cancer cell killing. Specificity is based on normal cells undergoing a reversible G1 arrest following Cdc7 inhibition due to activation of a novel cell cycle checkpoint that is lost or impaired in cancer cells. Our analysis of the molecular circuitry underlying this replication origin activation checkpoint reveals that G1 arrest is dependent on three non-redundant checkpoint axes coordinated through the Forkhead transcription factor FoxO3a and p53. We show that only breast cancers displaying the accelerated cell cycle phenotype express elevated Cdc7 levels and are therefore highly represented in p53 mutant Her2-subtype and triple negative tumours. Breast cancers of the luminal subtype expressing low levels of Cdc7 undergo a cytostatic G1 arrest after Cdc7 inhibition due to their p53 wild type status, a checkpoint response mimicking untransformed cells. In contrast Her2 and triple negative tumours show a marked response to Cdc7 inhibitors with potent cancer-cell-specific killing as a result of (i) overexpression of the target protein and (ii) impairment of the origin activation checkpoint due to p53 lesions. Thus integrating cancer cell cycle phenotypic data with a molecular analysis of the origin activation checkpoint serves as a predictor of response to pharmacological Cdc7 inhibitors.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2148.
Cyclin D1 Is a Direct Target of JAG-Mediated Notch Signaling in Breast Cancer.
Authors: Reedijk M. , Reedijk M. , Reedijk M. , Cohen B. , Shimizu M. , Ng N. , Bukhman Y. , Pan J. , Dering J.
Published: December 2010

We have previously demonstrated that expression of the Notch ligand, JAG1 is associated with the basal breast cancer phenotype and disease recurrence. Herein we report on a genomics approach to elucidate mechanisms downstream of JAG1 that promote breast cancer growth. In a survey of 46 breast cancer cell lines we found that triple negative (TN; basal and mesenchymal ER-, PR- and Her2-negative) lines express JAG1 at significantly higher levels than do HER2+ or luminal (ER+) Her2- cell lines. The TN breast cancer cell lines HCC1143 and MDA MB231, which express JAG1 and demonstrate growth inhibition with RNA interference-induced down-regulation of JAG1, were selected for further study. We used microarray profiling of tumor cells transfected with JAG1 siRNA to identify JAG1-regulated genes. Differentially expressed genes (p ≤ 0.005; fold change ≥ 1.5) were identified for further study. Among the JAG1-regulated genes identified, cyclin D1 was found to be a direct target of NOTCH1 and NOTCH3. We show that JAG1 down-regulation results in reduced cyclin D1 expression, reduced direct binding of Notch to the cyclin D1 promoter, and inhibition of cell cycle progression through the cyclin D1-dependant G1/S check-point. Furthermore, we show that cyclin D1 and JAG1 expression correlate in basal breast cancer expression data sets. These data suggest a model whereby JAG1 promotes cyclin D1-mediated proliferation of TN breast cancer cells.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2150.
Function of HES6 an Inhibitor of HES1, in the Breast Cancer Cell Lines T47D and MCF-7 Is to Up-Regulate E2F-1 and Increased Proliferation.
Authors: Strom A. , Hartman J. , Lam E. , Lin C. , Gustafsson J. , Gustafsson J.
Published: December 2010

Hormone resistance remains as a major obstacle in breast cancer treatment. The majority of breast tumors are dependent on estrogens to grow, therefore anti-estrogens such as Tamoxifen is an effective treatment option. We have found that a transcription factor Hes6, earlier described in the nervous tissue, is up-regulated in hormone-independent breast cancer cells compared to normal estrogen sensitive breast cancer cells. By using a lenti-virus system, we have over-expressed Hes6 in T47D cells which are estrogen sensitive. This resulted in increased proliferation. We also found that estrogen treatment of MCF-7 breast cancer cells induced expression of endogenous Hes6 in the G1-phase. We could not find any binding of estrogen receptor α to the HES6 gene but instead found a binding site up-steam of the promoter in the ASCL1 gene. ASCL1 is a known inducer of HES6 and we are showing that ASCL1 is up-regulated by 17β-estradiol in MCF-7 cells. To find the mechanism behind the tumorogenic effects of Hes6, we analysed important factors of the cell-cycle. We found that the G1-phase factor E2F-1 was up-regulated in response to increased Hes6 expression. Since HES6 have been shown to be an inhibitor of HES1, this is in agreement with a previous study where we found that HES1 inhibited proliferation by binding to the promoter of and down-regulating E2F-1 expression. E2F-1 is an important limiting factor in late G1-phase of the cell cycle and can drive cell proliferation.We believe that the HES1-HES6 interplay is important in antiestrogen-resistant breast cancer. Studies are now ongoing to investigate if Hes6 is up-regulated in estrogen-independent human breast cancer samples.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2153.
nWASP, the Neural Wiskott-Aldrich Syndrome Protein, Plays a Pivotal Role in the Control of Endothelial Migration and Angiogenesis.
Authors: Jiang W. , Martin T. , Mansel R.
Published: December 2010

Introduction. nWASP, the neural Wiskott-Aldrich Syndrome Protein, is a member of the Wiskott-Aldrich syndrome (WAS) protein family which are known regulators of actin polymerization. Via interactions with actin, ERM (ezrin/moesin/radixin) family proteins, and other cytoskeletal associated proteins, nWASP is a key cell migration mediator. It has been shown that nWASP is aberrantly expressed in human breast cancer and that a reduction of nWASP is linked to a poor clinical outcome. Over-expression of nWASP in breast cancer cells also reduces their aggressiveness in vitro. In the present study, we investigated the potential influence of nWASP on the biological functions of endothelial cells and the angiogenic aspect of endothelial cells.Materials and methods. A panel of human cell lines of variety origins (n=43) were screened for the presence/absence of nWASP expression. A human vascular endothelial cell line, HECV, was found to be negative for nWASP expression and was chosen as a cell model in the present study. An expression construct for human nWASP was prepared using a mammalian expression vector, pcDNA3/V5/GFP/TOPO vector. HECV cells were transfected with the expression construct, or control vectors. The impact of nWASP over-expression in the endothelial cell line on the adhesiveness, migration and in vitro angiogenesis was assessed using a panel of in vitro models.Results. HECV cells, following transfection with the nWASP construct, successfully expressed nWASP, a subline subsequently established from which was so named as HECVnWASPexp7. Using an electric cell impedance sensing (ECIS) method, it was shown that HECVnWASPexp7 cells showed a significantly reduced adhesiveness to matrix surface (333.4±1.4 ohm), p<0.01 when compared with the wild type cells (HECVwt 419.7±1.6 ohm) and transfection control (HECVpControl 387.9±1.46 ohm). In a similar fashion, HECVnWASPexp7 cells also displayed a marked reduction in cell migration when compared with control cells. Using an in vitro microtubule formation assay, it was shown that both HECVwt and HECVpControl cells responded well in their microtubule formation to an angiogenic factor, hepatocyte growth factor (HGF). However, HECVnWASPexp7cells were less responsive to HGF when assessed for microtubules. We have further shown that in response to an nWASP inhibitor, Wiskostatin, HECVnWASPexp7 cells markedly increased their tubule formation, a clear contrast to the wild type and control cells.Conclusion. It is concluded that nWASP is a key regulator of the migratory property of vascular endothelial cells and that this effect on cell migration is reflected by changes in the angiogenic activities of the cells. Collectively, nWASP is a potential intrinsic angiogenic regulator in endothelial cells.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2156.
Impact of IL17B on Endothelial Cells and Angiogenesis.
Authors: Sanders A. , Guo X. , Mason M. , Jiang W.
Published: December 2010

Introduction:Tumour angiogenesis plays a vital role in cancer development and spread. In the current study, IL17B was found to be unregulated in endothelial cells in response to Hepatocyte Growth Factor (HGF). IL17B shares structural similarities to IL17A, which has been previously proposed to promote angiogenesis and tumourgenicity, though currently the role of this cytokine in cancer or angiogenesis is largely unknown.Methods:HECV gene expression in response to HGF treatment was examined over a wide range of genes using micro array analysis to detect differential gene expression over the U133+2 chip. Recombinant human IL17B (rhIL17B) was subsequently used to treat human HECV endothelial cells over a range of concentrations. The impact of rhIL17B on HECV cell motility and angiogenic potential was assessed using Matrigel migration/wounding and tubule formation.Results:Micro array analysis detected a significant increase in IL17B expression in response to 4 hour treatment of HECV human endothelial cells with 40ng/ml HGF (p < 0.05 vs HGF untreated HECV cells). rhIL17B negatively influenced HECV tubule formation at the higher concentrations with substantial reduction in tubule formation being seen following treatment with 250ng/ml rhIL17B. Higher concentrations of rhIL17B were also seen to reduce the capacity of HECV cells to migrate in a scratch wounding assay and significant differences in migrated distance were observed following 75 minute incubation (P < 0.05 vs control untreatment group).Conclusions:Micro array analysis suggests that IL17B gene expression in the HECV human endothelial cell line can be upregulated in response to treatment with HGF. Subsequently high levels of rhIL17B were seen to negatively impact on the tubule formation and migratory capacity of this cell line. Together this data suggests that IL17B is unlikely to play a key role in the pro-angiogenic response initiated through HGF signalling but at higher concentrations may itself be able to negatively impact the angiogenic potential of the HECV endothelial cell line.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2157.
Mast Cells Positive to Tryptase Correlate with Angiogenesis in Early Breast Cancer.
Authors: Troilo S. , Patruno R. , Zizzo N. , Troilo P. , Catino A. , Gadaleta C. , Ranieri G.
Published: December 2010

Background: Literature data indicate that mast cells (MCs) are involved in tumor angiogenesis due to the release of several pro-angiogenetic factor among which tryptase, a serine protease stored in MCs granules, is one of the most active. In benign breast lesions and in primary breast cancer, tryptase-positive MCs are more numerous as compared to chymase-positive MCs. Recently, it has been demonstrated that tryptase-positive MCs are involved in angiogenesis in sentinel lymph nodes with micrometastases from patients with breast cancer. However no data has been published regarding the correlations between MCs positive to tryptase and angiogenesis in primary breast cancer tissue. In the present study, we have evaluated the correlations between the number of MCs positive to tryptase (MCDPT), the area occupied by MCs positive to tryptase (MCAPT), microvascular density (MVD) and endothelial area (EA) in a series of T1-3, N0-2, MO female breast cancer, by means of immunohistochemistry and image analysis methods.Material and Methods: Bioptic specimens were collected from 88 female breast cancer patients who had undergone breast cancer surgery. For the evaluation of MCDPT, MCAPT, MVD and EA, a three layer biotin-avidin-peroxidase system was utilized. Briefly, six-micrometers thick serial sections of formalin-fixed and paraffin-embedded bioptic tumor samples were obtained. Then sections were microwaved at 500 W for 10 min. and treated with a 3% hydrogen peroxide solution. Sections were incubated with human-specific monoclonal antibodies anti-CD34 (QB-END 10; Bio-Optica Milan, Italy) diluted 1:50 for 1h at room temperature and anti-tryptase (clone AA1; Dako, Glostrup, Denmark) diluted 1:100 for 1h at room temperature. The bound antibody was visualised using biotinylated secondary antibody, avidin-biotin peroxidase complex, and and 3-amino-9-ethylcarbazole. The five most vascularized areas ("hot spot") were selected at low magnification and both individual vessels were counted at x 400 magnification. In serial sections each single tryptase-positive MC was counted. Single brown stained endothelial cells and red MCs positive to tryptase were also evaluated in terms of immunostained area at x 400 magnification.Results: Our data demonstrated a significantly (r= ranging from 0.78 to 0.89; p: ranging from 0.001 to 0.002 by Pearson's analysis respectively) correlation between MCDPT, MCAPT, MVD, EA each to other, whereas no correlation concerning MCDPT, MCAPT, MVD, EA and the main clinical pathological features was found.Discussion: These preliminary results suggest that tryptase-positive MC play a key role in breast cancer angiogenesis and that inhibition of tryptase may be a new antiangiogenic strategy for the treatment of breast cancer.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2159.
Blockade of Notch Signaling by GSI Induces Apoptosis in Human Breast Cancer Cells through Various Molecular Mechanisms Involving Survivin and BH3-Only Members of the Bcl-2 Family.
Authors: Séveno C. , Campone M. , Campone M. , Juin P. , Barillé-Nion S.
Published: December 2010

The Notch pathway is multifunctional and controls key nodes in cell signaling. In most cases, its deregulation has oncogenic effects, and aberrant activation of Notch signaling has been evidenced in breast cancer (Stylianou S, CR 2006). In particular, a functional Notch pathway appears to be of critical importance in the maintenance of basal breast cancer (Lee CW, CR 2008, BCR 2008). Moreover, because Notch signaling is repressed by both ER (Rizzo P, CR 2008) and HER2 (Osipo C, Oncogene 2008) oncogenic pathways, its reactivation during tamoxifen or trastuzumab treatment may restrain the efficiency of these inhibitors. Thus, Notch signaling inhibitors, such as competitive inhibitors of the g-secretase, GSI, that prevent proteolytic processing of the Notch receptor and the activation of its intracellular domain, are being actively investigated for the treatment of breast cancer, even though the mechanisms that might underlie their effects are still poorly understood.We first observed that GSI XII (z-Ile-Leu-CHO) was more effective in inducing apoptosis, as a single agent, on ER-/PR-/Her2- breast cancer cell lines than GSI X (z-Leu-Leu-Nle-CHO). This effect was associated with decreased expression of the anti-apoptotic proteins Survivin and XIAP and increased expression of, depending on cell lines, either NOXA or PUMA, two pro-apoptotic BH3-only members of the Bcl2 family.In the opposite, ER+ breast cancer cell lines were more sensitive to induction of apoptosis by GSI X, but this was manifest only in combination with Tamoxifen. Interestingly, Tamoxifen treatment of these cells induced both GSI X-sensitive Notch transcriptional activity and Survivin expression. Moreover, Survivin knock-down by RNA interference increased Tamoxifen sensitivity. Thus, Survivin might be an important actor of survival signal triggered by Notch (re)activation during ER pathway inhibition. Our results corroborate previous data showing the interest to use GSI in breast cancer but underline also the need to unravel the sub-type and context dependent molecular events involved in their biological activity.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2160.
mTORC1 Activity Contributes to the Mcl-1 Dependence of HER2 Amplified Breast Cancer Cells.
Authors: Campone M. , Campone M. , Noel B. , Gouraud W. , Gouraud W. , Jézéquel P. , Jézéquel P. , Barillé-Nion S. , Juin P.
Published: December 2010

Whether or not certain subtypes of mammary tumors specifically rely on a limited number of anti-apoptotic mechanisms for their maintenance, and how therapy might affect these mechanisms, is currently ill characterized.We have analyzed published gene-expression profiles of breast cancer patients for which HER2 status, evaluated by immunohistochemistry, was also available. A statistically significant enrichment in the expression of Mcl-1 in HER2 amplified breast tumors, compared to other breast tumors, was found.In vitro, knock down of Mcl-1 expression by RNA interference proved sufficient to induce apoptosis in the HER2 amplified cell line BT474. Combined RNA interference experiments showed that apoptosis induced by Mcl-1 knock down in these cells (ie, their “Mcl-1 dependence”) relied on the expression of the pro-apoptotic BH3-only protein Bim. This indicates that the Bim/Mcl-1 balance (possibly regulated by direct physical interactions between these two proteins) constitutes an integrating point towards which the numerous survival pathways that are known to lie downstream of HER2 should converge, to the very least in these cells.While seeking for signaling pathways that might significantly affect Mcl-1 dependence downstream of HER2, using diverse small molecule inhibitors, we found that mTORC1 inhibition in BT474 cells not only leaves Mcl-1 expression levels unaltered, but also prevents induction of apoptosis by Mcl-1 depletion. mTORC1 inhibition enhances the activity of the survival kinases Akt and ERK (p44 and p42). HER2 signaling is involved in this activation, since it is prevented by trastuzumab treatment.These data suggest that, due to the structure of the signaling network mTORC1 is embedded in (which includes the HER2 driven negative feedback loop described here), its activity locks HER2 amplified cells in a Mcl-1 dependent state. They also imply that combining inhibitors of mTORC1 with inhibitors of the HER2 pathway (such as trastuzumab) might restrain the effects on cell survival of the former, and enhance their clinical efficiency in HER2 amplified breast cancers.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2161.
The Cell of Breast Cancer Origin Affects Tumor Estrogen Receptor Status.
Authors: Toneff M. , Du Z. , Li Y.
Published: December 2010

It has been known for some time that breast cancer phenotype is dependent upon the initiating oncogene. However, it has only recently been demonstrated that breast cancer phenotype, including tumor histopathology, gene expression profile, and metastatic potential, may also depend upon the cellular subtype from which a tumor arises. Breast tumor estrogen receptor (ER) status is a common prognostic factor, and approximately 70% of all breast tumors are ER+; that is, they expresse ER in ≥5% of tumor cells. However, it remains unknown which factors determine breast cancer ER status. It is possible that the cell of tumor origin plays a role in determining the ER status of the resulting tumor. Our lab has developed a method to somatically introduce oncogenes into a developmentally normal mouse mammary gland with temporal control using the modified avian retrovirus RCAS. We have generated transgenic mice that express the RCAS receptor TVA under mammary cell selective markers and demonstrated our ability to infect individual cells and induce mammary tumors. In this study we used an RCAS vector encoding polyoma virus middle T antigen (PyMT) to infect transgenic adult MMTV-tva or WAP-tva mice. The MMTV LTR is though to be active in several different mammary cell types including luminal epithelial, progenitor, and stem cells. WAP is primarily active in more highly differentiated mammary epithelial cells. Tumors induced by RCAS-PyMT differ in histopathology between the two lines. Those arising in MMTV-tva mice have a predominately solid morphology, while those arising in WAP-tva mice display a predominately papillary morphology. Interestingly, we found that the majority of tumors arising in MMTV-tva mice are ER+ (61%), while tumors arising in WAP-tva mice are predominately ER- (95%). Our data suggest that tumors induced by the same oncogene from distinct cellular subtypes in the mouse mammary epithelium can significantly vary in histopathology, and that tumor ER status can be dependent upon the cell of tumor origin.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 2163.
A Prospective Study of Breast Cancer Incidence and Stage Distribution in Women with a BRCA1 or BRCA2 Mutation under Surveillance with and without Magnetic Resonance Imaging.
Authors: Warner E. , Hill K. , Causer P. , Plewes D. , Jong R. , Yaffe M. , Wong J. , Wright F. , Sun P. , Narod S.
Published: December 2010

Introduction: The sensitivity of MRI for the detection of pre-clinical breast cancer exceeds that of mammography and of other screening tests. If MRI screening leads to reduced mortality in women with a BRCA1 or BRCA2 mutation, then it is expected that the incidence of advanced stage breast cancers should be reduced in a cohort of women undergoing regular MRI screening, compared to conventional screening.Methods: We followed 1275 women with a BRCA1 or BRCA2 mutation for a mean of 3.2 years for incident breast cancers. 445 women were enrolled in an MRI screening trial in Toronto and 830 controls, from elsewhere in Canada and the United States, underwent conventional screening. The cumulative incidences of DCIS, of early-stage and of late-stage breast cancer were estimated at six years in the two cohorts.Results: There were 41 cases of breast cancer diagnosed in the MRI-screened cohort and 76 cases of breast cancer diagnosed in the control cohort. The cumulative incidence of DCIS or stage I breast cancer at six years was 12.7% in the MRI-screened cohort and was 9.5% in the control group (p = 0.02; log rank test). The cumulative incidence of stage II – IV breast cancers at six years was 2.0% in the MRI-screened cohort and was 7.1% in the control group (p = 0.02; log rank test). The adjusted hazard ratio for the development of stage II – IV breast cancer associated with membership in the MRI-screened cohort was 0.30 (95% CI: 0.12 to 0.72; p = 0.008).Conclusion: Annual surveillance with MRI is associated with a significant reduction in the incidence of advanced stage breast cancer in BRCA1 and BRCA2 carriers.Funding for this study is generously provided by the Canadian Breast Cancer Research Alliance.

Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 26.
A QPCR-Based Circulating Tumor Cell Detection Platform Predicts Survival in 776 Early Stage Breast Cancer Patients.
Authors: Molloy T. , Bosma A. , Baumbusch L. , Borgen E. , van't Veer L. , Naume B.
Published: December 2010

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 platform for the detection of CTCs in peripheral blood, which proved to be prognostic in a cohort of metastatic breast cancer patients.In the current study, we aimed to build on this work by determining the prognostic value of our CTC assay in an independent, prospectively collected, retrospective patient series of 776 non-metastatic, stage I-III breast cancer patients (median follow-up 7.3 years). Assay positivity was observed in 83 patients (10.7%), while maintaining 96% specificity in a group of 25 healthy female controls (however a second blood sample from the healthy control that gave a positive result was found to be negative). CTC positivity as determined by our assay corresponded to a significantly poorer relapse-free survival (RFS) (HR = 1.9, 95% CI = 1.3-2.9, p = 0.02) and overall survival (HR = 1.8, 95% CI = 1.1-2.8, p = 0.02) in this group. This platform demonstrated similar prognostic power to the pan-anticytokeratin (AE1/AE3) immunohistochemistry-based detection of disseminated tumor cells (DTCs) in the bone marrow of these patients (RFS HR = 1.9, 95% CI = 1.3-2.7, p = 0.02). Importantly, multivariate analyses including tumor size, histological grade, lymph node status, and hormone receptor status, both CTC and DTC status remained significant, independent predictors of RFS (CTC status HR = 1.6, 95% CI = 1.0-2.6, p = 0.037; DTC status HR = 1.6; 95% CI = 1.1-2.5, p = 0.023). However, CTC status was prognostic in both lymph node negative patients (HR = 2.0, p = 0.04) and lymph node positive patients (HR = 1.7, p = 0.043), whereas DTC status was only prognostic in lymph node positive patients (HR = 2.0, p = 0.002).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(24 Suppl):Abstract nr 3002.