bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract P4-05-01: Molecular Interactions between Human Adipocytes and Breast Cancer Cells May Underline Increased Breast Cancer Incidence in Obese Postmenopausal Women
Authors: Siriwardhana N , Moustaid-Mossa N , Wimalasena J. 
Published: April 2011

Abstract
There is an increasing body of evidence that obesity, metabolic syndrome, insulin resistance, and diabetes may substantially increase the risk and modify the outcome of a variety of cancers including postmenopausal breast cancer (BC). In has been proposed that stromal adipose tissue is activated in these metabolic diseases and that the activated stroma regulates function of cancer cells. Much of the stroma in the breast is made of adipocytes (AD) and AD and resident immune cells, such as macrophages, secrete cytokines/chemokines which have direct regulatory actions on BC cells. The latter may, in turn, regulate the behavior of AD. We have initiated studies where cultured AD from obese individuals is co-cultured with BC cells (such as MCF-7 and MDA MB 231 cells) in a 3 dimensional (3D) Matrigel co-culture system or non tumorigenic MCF10A cells.We quantitatively measured the changes in 26pro-inflammatory/anti-inflammatory cytokines(using Millipore multiplex ELISA) secreted into culture media. Of the 12 differentially regulated cytokines the expression levels of 6 cytokines (Eotaxin, G-CSF, and IL1α. IL-6, IL-8 and MCP-1) were significantly different between treatments. Secretion of Eotaxin, G-CSF, IL-6 and MCP-1) cytokines from obese adipocytes were significantly higher than the lean adipocyte and the opposite was recorded for IL-8. Further, the secretion of G-CSF, IL-6 and MCP-1 was significantly different in the order of MCF10A &lt;MCF7 &lt; MDA MB 231. In the co-cultures with adipocytes IL-6, IL-8 and MCP-1 showed different secretion levels when lean or obese adipocytes were cocultured with BC cells(e.g. the IL-6 secretion was increased in the order of MCF10A &lt;MCF7 &lt; MDA MB 231 in lean cells but in obese cells the order was changed to MDA MB &lt;MCF10A &lt; MCF7). Further, we measured the differences in 3D acini sizes in the 3D co-cultures, MCF7 cell formed irregular and growth unrestricted structures and when compared to the lean adipocytes diameter measured was 17% higher in the presence of obese adipocytes. The same pattern was observed when monolayer culture media from obese adipocytes were transferred into growing MCF7 cells and growth measured by MTT assay. These data suggest that adipocytes from lean and obese women regulate BC functions differently and such differences may underlie the promotional effect of obesity in BC. Further studies are underway to dissect the molecular mechanisms by which differentially regulated cytokines influence the behavior of BC cells.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-05-01.
Abstract P4-05-03: Quantification of an Individual's Response to Drug Therapy Utilising a Novel Three-Dimensional Assay with Primary Breast Cancer Explants
Authors: Leeper AD , Thomas JSJ , Dixon JM , Harrison DJ , Katz E. 
Published: April 2011

Abstract
Background: Breast cancer is not a single disease but one that displays both inter-and intra-tumoral heterogeneity. The principal models for breast cancer research are currently immortalised breast cancer cell lines as they are easily propagated, tractable to genetic modification and yield reproducible results However, it is not possible to use a cell line to predict the disease course of an individual patient. Moreover, epithelial cells grown on plastic in two dimensional cultures fail to recapitulate their three dimensional interaction with the tumour microenvironment, which is believed to be integral to tumour progression.
Purpose: Using a collagen based three dimensional assay system and fresh breast cancer biopsies obtained at surgical resection, we have developed a novel assay which demonstrates ongoing proliferation and invasion of an individual's breast cancer. The proliferation and invasion of the breast cancer can be modified using currently available anti-cancer drugs. The effect of these drugs on an individual patient's cancer may then be quantified.
Results: The model is amenable to investigation both in real-time and following formalin fixation. Using live microscopy and time delay image capture techniques we are able to study proliferation and invasion over a timecourse. Subsequent formalin fixation and sectioning enables two dimensional immunofluorescence quantification of drug effect at a cellular level. Three dimensional scanning and quantification, utilising Optical Projected Tomography and Velocity software enables one to demonstrate the effect of a drug on invasion at a macroscopic level. Conclusions: This assay has provided a method of sustaining an individual's primary breast tissue in long-term culture. Moreover, it provides a method of observing different patterns of invasion. Through the addition of currently available and novel therapeutics we are able to quantify how residual disease may respond to a battery of different treatments. In turn this may improve the ability of the clinician to decide on optimal adjuvant therapy, resulting in individually tailored therapy.
Figure available in online version.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-05-03.
Abstract P4-06-04: Rho-Family GTPase Activity in Invading Mammary Tumor Cells Visualized by Two-Photon Excitation Laser Scanning Microscopy
Authors: Kamioka Y , Matsuda M 
Published: April 2011

Abstract
Background: Rho-family GTPases play critical roles in the regulation of tumor cell motility and invasion. Despite the vast accumulated knowledge of Rho-family GTPase regulation in two-dimensional culture models, their three-dimensional spatio-temporal regulation in vivo remains poorly understood because of technical difficulties. The ability of tumor cells to migrate in vivo depends on both physical and biochemical characteristics of the tumor microenvironment, i.e., extracellular matrix, stromal cells and myeloid cells as well as tumor cell properties. Therefore, development of methods for directly measuring the activity of Rho-family GTPases in vivo is required for further understanding the mechanism of tumor invasion. Material and Methods: To visualize the activity of Rho-family GTPases in vivo, we developed fluorescence resonance energy transfer (FRET)- based probes comprising of fluorescent proteins. The probes were stably expressed in 4T1 mouse mammary tumor cells by means of retrovirus-mediated gene transfer. 4T1 mouse mammary tumor cells highly expressing FRET probes were selected by fluorescence activated cell sorting (FACS) and implanted subcutaneously in nude mice. The 4T1 cells formed tumors within 5 to 10 days. The activity of Rho-family GTPases was examined by two-photon excitation laser scanning microscopy (TPLSM). The collagen matrix surrounding tumors was visualized by second harmonic signal. TexasRed-dextran was injected into nude mice intravenously before observation for the visualization of blood vessels. Results: We found that 4T1 mouse mammary tumor cells assumed an elongated, mesenchymal phenotype in vivo and in three-dimensional culture models. Cell motility in vivo was significantly slower than in 2D cell culture models. In the in vivo models, the elongated tumor cells demonstrated cell movement along aligned collagen fibers away from the primary tumor mass. Furthermore, we observed that Rac1 activity was higher at the tumor-stroma borders than at the tumor cores, using FRET imaging.
Discussion: In this study, we successfully monitored the activity of Rho-family GTPases in vivo by FRET imaging. We also confirmed that 4T1 mouse mammary tumor cells mainly exhibited a mesenchymal phenotype when these tumor cells invaded into the surrounding stromal tissue. Recent studies have shown that some tumor cells exhibit two different modes of individual cell movement in 3D cell culture models: mesenchymal movement is characterized by an elongated cell shape and a dependence upon ECM proteolysis, whereas in amoeboid movement, cells have a rounded morphology, and are less dependent on proteases. We hereby demonstrate the utility of a three-dimensional, in vivo imaging technique toward understanding the mechanisms underlying tumor invasion.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-04.
Abstract P4-06-05: Silencing of Cyclin D1 Increases Breast Cancer Cell Migration through Increased Id1 Expression and Induction of Epithelial-Mesenchymal Transition
Authors: Tobin NP , Lundgren K , Sims A , Landberg G. 
Published: April 2011

Abstract
Backround: The oncogene cyclin D1 is overexpressed in many breast cancers and despite proliferation activating properties, has been linked to a less malignant phenotype. We have previously shown that down-regulation of cyclin D1 in actively cycling breast cancer cells significantly increased their migratory capacity whilst also decreasing proliferation. Gene expression analysis and animal modeling have highlighted an increase in the inhibitor of differentiation-1 (Id1) gene following cyclin D1 gene (CCND1) silencing. Notably, increased Id1 is associated not only with enhanced migratory and invasive features of breast cancer cells but also with prevention of cell differentiation and induction of an epithelial-mesenchymal-like (EMT) phenotype. Here we sought to determine if the increase in cell motility following cyclin D1 silencing was through increased Id1 expression and induction of an EMT phenotype, and confirm our findings in a large patient cohort.
Materials and Methods: Expression of cyclin D1, Id1 and SNAI2 was determined in MDA-MB-231 and ZR75 cells by qPCR and western blot, whilst cell migration was monitored by transwell assay. Gene expression of CCND1, ID1 and EMT markers was analysed from a pooled cohort of batch-mean centered publically available data sets, with data analysis performed in SPSS.
Results: Silencing of cyclin D1 increased cell migration 1.38 and 1.72 fold in MDA-MB-231 (P&lt;0.001), and ZR75 (P&lt;0.001) cells respectively, an effect abolished by silencing of Id1. qPCR analysis confirmed an increase in Id1 (1.73) and SNAI2 (1.50) expression following siRNA treatment of cyclin D1, an effect reversed following siRNA treatment of Id1. CCND1 gene exression was negatively, while Id1 was positively, associated with the EMT markers Vimentin (P&lt;0.001), SNAI2 (P&lt;0.001) and TWIST1 (P&lt;0.001) in 1,100 breast tumors. In ER+ patients CCND1 was negatively correlated to Id1 expression (0.050) and in CCND1 low patients increasing Id1 expression was predictive of worse cumulative survival (0.027). Conclusion: Decreased CCND1 expression enhances MDA-MB-231 and ZR75 cell migration through increased Id1 expression and induction of EMT. Moreover, both genes are correlated to EMT markers in breast cancer patients and in a subset of the cohort, confer a worse survival. These results may explain the conflicting data regarding cyclin D1 expression in breast carcinomas and solidifies the rationale of the protein as a clinical target.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-05.
Abstract P4-06-07: PI3K Mutations Are More Common in Low Grade DCIS
Authors: Sakr R , Chandarlapaty S , Andrade V , Giri D , Muhsen S , Wooyul P , Morrow M , Rosen N , King T. 
Published: April 2011

Abstract
Background: DCIS is a non-obligate precursor to invasive breast cancer yet it remains difficult to predict which lesions will progress or over what time interval. Factors associated with a higher risk of progression include high histologic grade and HER2 amplification. HER2 signaling via the PI3K pathway is associated with accelerated invasiveness in laboratory models and mutations in the helical domain of PI3K have been associated with a worse prognosis in invasive cancer. We hypothesized that PI3K pathway mutations are associated with higher risk of progression in DCIS and therefore would be more common in high grade DCIS.
Methods: 195 cases of pure DCIS were identified from the MSKCC breast service database (1999-2003). All cases were reviewed by a single pathologist to assign histologic grade. Sections were obtained from archived formalin-fixed paraffin embedded (FFPE) blocks for manual microdissection to isolate pure DCIS lesions for DNA extraction. Multiplex array (Sequenom®) genotyping for PIK3CA was performed on prePCR amplified DNA. Comparisons were made between high and low grade DCIS using Fisher's exact test.
Results: Among 195 pure DCIS cases, 89 were classified as high grade and 106 as low grade. Sequenom® analysis was informative in all cases. PIK3CA mutations were identified in 4/89 (4%) cases of high grade DCIS and in 24/106 (23%) cases of low grade DCIS (p=0.000). All 4 mutations in high grade DCIS were located in the kinase domain, whereas in low grade DCIS, 9/24 (37%) mutations were in the kinase domain (H1047R) and 15/24 (67%) mutations were in the helical domain (E542K, E545K, N345K).
Conclusion: PI3K mutations were relatively uncommon in pure high grade DCIS as compared to low grade DCIS. PI3K mutations in low grade DCIS were observed in both the kinase and helical domain. These findings support the hypothesis that breast tumorigenesis differs by grade and PI3K mutations may be more prominent in low grade carcinogenesis. The significance of helical domain mutations in low grade lesions requires further investigation.

Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-07.
Abstract P4-06-09: Genetic Signature Discriminating Metastatic from Non-Metastatic Small Tumors
Authors: Ellsworth RE , Valente AL , Field LA , Kane JL , Love B , Tafra L , Shriver CD. 
Published: April 2011

Abstract
Background: The widespread use of mammographic screening resulted in increased diagnosis of small (&lt;2 cm; T1) tumors. Small tumors are associated with better prognosis, including a lower likelihood of developing metastasis, than larger tumors. Although this lower propensity to metastasize suggests that less aggressive treatments may be warranted in patients with T1 tumors, a subset of patients with small tumors (10-20%) will be diagnosed with lymph node metastasis.
Methods: Frozen breast specimens were collected from women with T1 tumors and either negative (n=29) or positive (n=15) lymph node status. RNA was isolated from pure tumor cell populations after laser microdissection. Gene expression data was generated using HG U133A 2.0 arrays (Affymetrix). Differential expression was determined using Mann-Whitney testing using a P-value &lt; 0.001 to define significance. Results for ESR1 were validated by immunohistochemistry.
Results: Tumor characteristics did not differ significantly between groups in terms of age at diagnosis, grade, HER2 or PR status; however, tumors from patients with positive lymph nodes (47%) were significantly (P&lt;0.05) more frequently ER negative compared to node negative (14%) patients. Gene expression analysis revealed 17 genes that were differentially expressed between node negative and node positive tumors: 6 with higher expression in node positive, including AURKA, and 11 with higher expression in node negative patients, including ESR1 and EPHX2. Of note, ESR1 was expressed at &gt;4X higher levels in tumors without metastasis, in agreement with IHC findings.
Conclusions: Small metastatic tumors differ in gene expression from those without metastasis. EPHX2 has been implicated as a metastasis suppressor while AURKA has been implicated as a metastasis promoter. These results suggest that small tumors have different propensities to metastasize and the genetic signature may serve as a new molecular tool to discriminate metastatic and non-metastatic small tumors, allowing appropriate treatment and risk assessment to be performed.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-09.
Abstract P4-06-10: Altered Activity of RhoA and RhoC GTPases Phosphorylate C-Jun N-Terminal Kinase and Induce Cellular Dormancy in Breast Cancer Cells
Authors: Chatterjee M , van Golen KL 
Published: April 2011

Abstract
Tumor dormancy has been a very real problem in breast cancer survivors where recurrences are common and usually fatal. Molecular mechanisms involved in this process are often poorly understood and are a “hot-spot” in breast cancer research today.
Ongoing efforts in our laboratory has shown that Rho GTPases, specifically RhoA and RhoC, play a significant role in dormancy of breast cancer. We have currently established two in vitro models of breast tumor dormancy — one involving growth of the breast cancer cell line MCF7 on fibronectin and subsequent treatment with basic fibroblast growth factor and the other involving treatment of MCF7 cells with the farnesyl transferase inhibitor FTI-L-744,832. In both these models, we find high levels of active RhoC and almost undetectable levels of active RhoA GTPases in quiescent breast tumor cells. Investigating further downstream we found c-jun N-terminal kinase (JNK) is phosphorylated in these dormant cells but not p38mitogen activated protein kinase(MAPK) or extracellular signalregulated kinase(ERK1/2). On inhibiting JNK with a pharmacological inhibitor specific for JNK, SP600125, we found dormancy could be interrupted and cell proliferation was induced. JNK has been implicated in inducing autophagy in cells by activating autophagy gene Atg5 and Beclin1 (BCN1) [Byun et al., Carcinogenesis, 2009]. Since autophagy can be a mode of survival for the dormant tumor cell [Matthew et al., Cancer Cell, 2006] we explored the possibility of autophagy being induced in these dormant tumor cells via JNK by employing the autophagy marker LC3-II. We found upregulation of the autophagy marker, thus revealing autophagy as a strategy for survival for these tumor cells in unfavorable tumor microenvironment. We are currently investigating the molecular mechanisms underlying the induction of dormancy in breast tumor cells by Rho GTPases through JNK and the dormant tumor survival strategy through autophagy.
We believe that targeting these implicated molecules like RhoA and RhoC GTPases, JNK or some of the autophagy genes might yield therapeutic benefits for breast cancer patients in remission and wipe out fears of deadly metastatic recurrences thus prolonging survival.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-10.
Abstract P4-06-15: BP1 Upregulates Twist and Induces EMT in Breast Cancer Cells
Authors: Hwang B , Rheey J , Fu SW , Berg PE. 
Published: April 2011

Abstract
Background: We have cloned a gene, BP1, which is a member of the homeobox gene family of transcription factors. Our recent studies have shown that BP1 may play a role in breast cancer cell survival, aggressiveness and metastasis. BP1 protein (pBP1) is expressed in 80% of invasive ductal breast tumors, and is associated with estrogen receptor negativity and tumors of African American women, both associated with aggressive tumors. BP1 is also expressed in metastatic tumors, shown by immunostaining of 46 samples of inflammatory breast cancer; all cases were BP1 positive. Nine cases had metastasized, and all nine metastatic lymph nodes were BP1 positive. BP1 positive cells were observed in lymph channels and blood vessels. Here, we demonstrated BP1 induces the epithelial to mesenchymal transition (EMT), resulting in increased migratory ability. EMT, a process by which cancer cells lose their epithelial features and gain mesenchymal markers, enables tumor cells become more invasive, migratory and can lead to metastasis. Twist, a basic helix-loop-helix (bHLH) transcription factor which triggers EMT, is activated by BP1.
Materials and Methods: RNA levels of markers of EMT were verified by real-time PCR and their protein levels by Western blotting and, in some cases, by confocal microscopy. Chromatin immunoprecipitation (ChIP) was performed to verify the BP1 binding site on the Twist promoter. A scratch test was used to measure migratory ability. BP1 knockdown by siRNA transfection was also performed. Recombinant BP1 (rpBP1) was produced in E. coli.
Results: Increased expression levels of Twist were observed on microarrays after probing with RNA from MCF-7 cell lines overexpressing BP1. We demonstrated that BP1 can upregulate Twist expression in two different breast cancer cell lines, MCF-7 and Hs578T cells, by binding to the Twist promoter. BP1 upregulates mesenchymal marker expression and down-regulates epithelial marker expression, consistent with EMT. BP1 also promotes breast cancer cell migratory ability, shown by the scratch test. In addition, cells grown in medium supplemented with rpBP1 showed increased Twist expression and migration. Experiments evaluating the effects of siBP1 on EMT are underway.
Conclusions: BP1 stimulates Twist expression in MCF-7 and HS578T breast cancer cells, resulting in a more mesenchymal cell phenotype. We therefore hypothesize that BP1 induces EMT by upregulating Twist expression, and may result in metastasis. If BP1 is involved in EMT, it may be a good target for therapy.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-15.
Abstract P4-06-16: TWEAK/Fn14 Signaling Axis as a Driver of Breast Cancer Cell Invasion and Metastasis
Authors: Stone AE , Willis AL , Niska JR , Black MA , Ramos P , Mancini CM , Cunliffe HE. 
Published: April 2011

Abstract
The molecular events associated with the multi-step process of cell invasion and metastasis are poorly understood. Tumor necrosis factor receptor family member fibroblast growth factor-inducible 14 (Fn14) and its ligand TWEAK (tumor necrosis factor-like weak inducer of apoptosis) have recently emerged as a pro-invasive signaling mechanism and indicator of poor prognosis in breast cancer. Our laboratory has recently reported enhanced Fn14 expression in a significant proportion of breast tumor samples, but not in normal breast epithelium. Analysis of publicly available breast cancer data indicates that high Fn14 expression positively correlates with HER2/neu positivity, estrogen receptor negativity, high tumor grade, positive lymph nodes, and metastasis. Modulation of Fn14 expression in breast cancer cell lines either by exogenous overexpression or siRNA-mediated blockade is sufficient to regulate breast cancer cell invasive behavior in vitro. However, the mechanisms leading to Fn14 overexpression, and how activation of the TWEAK/Fn14 signaling axis contributes to breast tumor malignant progression remain unclear. We hypothesized that genomic alterations and inappropriate autocrine or paracrine growth factor signaling may play a role in aberrant Fn14 regulation. Using array genomic hybridization and fluorescence in situ hybridization, we found increased Fn14 gene copy number relative to centromere 16 in two out of four Fn14-expressing, invasive breast cancer cell lines and in 28% of primary breast tumors. We also investigated the role of exogenous growth factors known to play important roles in the breast tumor microenvironment in stimulating Fn14 expression in vitro. TWEAK, EGF, IGF1, HRG and HGF/SF were sufficient to stimulate Fn14 expression de novo in a variety of biologic contexts. Many of these growth factors converge on the MAPK signaling axis. We have confirmed a functional role for the MAPK pathway in facilitating Fn14-mediated invasion. Consistent with this observation, Fn14 expression in primary breast tumors is high in the context of K-RAS mutation. We are currently evaluating the presence of important key regulatory regions within the Fn14 promoter that may be contributing Fn14 over expression in breast cancer cells. To define mechanisms by which Fn14 contributes to a malignant cell phenotype we have identified an Fn14-specific invasion signature using gene expression microarray profiling. Analysis of this data points to key malignant mechanisms likely to be impacted by Fn14 activation within different biologic contexts that warrants further investigation.
Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P4-06-16.