Luminal Line Research Articles

Abstract P5-05-02: Regulation of glucose metabolism by the RUNX2 transcription factor has a negative impact on mitochondrial function in breast cancer cells

Abstract The RUNX2 transcription factor regulates breast cancer (BC) metastasis to bone and is itself a target of IGF-1 signaling pathways. We have shown that glucose can activate RUNX2 phopshorylation through IGF-1 receptor signaling and that RUNX2 was associated with inhibition of pyruvate dehydrogenase (PDHA1) activity and repression of mitochondrial respiration in BC cells. However, the mechanisms by which RUNX2 alters mitochondrial function and supports an oncogenic phenotype are not completely known. RUNX2 expression in a luminal BC cell line increased expression of glycolytic genes, and sensitivity to glucose starvation. However, RUNX2 knockdown in a triple-negative BC cell line inhibited expression of glycolytic genes. RUNX2 also repressed mitochondrial oxygen consumption rates (OCR), a measure of oxidative phosphorylation while overexpression of SIRT6, a NAD-dependent histone deacetylase, increased respiration in RUNX2-positive cells. RUNX2 repressed SIRT6 expression directly at the transcriptional and post-translational levels. High SIRT6 expression was observed in normal mammary tissue and cells that did not express RUNX2 but endogenous SIRT6 expression was low in malignant BC tissues or cell lines, which expressed high levels of RUNX2. These results suggest that SIRT6, a known tumor suppressor, was a critical mediator of these RUNX2-regulated metabolic changes. These results support a hypothesis whereby RUNX2 upregulation during BC progression leads to inactivation of the SIRT6 tumor suppressor to promote tumorigenesis. It will be important to investigate further changes in mitochondrial function to understand the precise mechanisms by which RUNX2 regulates BC metabolism. Citation Format: Kim MS, Choe M, Cho H, Polster BM, Girnun GD, Passaniti A. Regulation of glucose metabolism by the RUNX2 transcription factor has a negative impact on mitochondrial function in breast cancer cells. [abstract]. In: Proceedings of the Thirty-Eighth Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2015 Dec 8-12; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2016;76(4 Suppl):Abstract nr P5-05-02.

Aortic Curvature Instead of Angulation Allows Improved Estimation of the True Aorto-iliac Trajectory

Supra- and infrarenal aortic neck angulation have been associated with complications after endovascular aortic aneurysm repair. However, a uniform angulation measurement method is lacking and the concept of angulation suggests a triangular oversimplification of the aortic anatomy. (Semi-)automated calculation of curvature along the center luminal line describes the actual trajectory of the aorta. This study proposes a methodology for calculating aortic (neck) curvature and suggests an additional method based on available tools in current workstations: curvature by digital calipers (CDC). Proprietary custom software was developed for automatic calculation of the severity and location of the largest supra- and infrarenal curvature over the center luminal line. Twenty-four patients with severe supra- or infrarenal angulations (≥45°) and 11 patients with small to moderate angulations (<45°) were included. Both CDC and angulation were measured by two independent observers on the pre- and postoperative computed tomographic angiography scans. The relationships between actual curvature and CDC and angulation were visualized and tested with Pearson's correlation coefficient. The CDC was also fully automatically calculated with proprietary custom software. The difference between manual and automatic determination of CDC was tested with a paired Student t test. A p-value was considered significant when two-tailed α<.05. The correlation between actual curvature and manual CDC is strong (.586-.962) and even stronger for automatic CDC (.865-.961). The correlation between actual curvature and angulation is much lower (.410-.737). Flow direction angulation values overestimate CDC measurements by 60%, with larger variance. No significant difference was found in automatically calculated CDC values and manually measured CDC values. Curvature calculation of the aortic neck improves determination of the true aortic trajectory. Automatic calculation of the actual curvature is preferable, but measurement or calculation of the curvature by digital calipers is a valid alternative if actual curvature is not at hand.

Abstract LB-207: ΔNp63α regulates quiescence, stem or progenitor activity of normal and malignant breast cells in a cell type-specific manner

Abstract [Introduction] Despite apparent resection of tumors, breast cancer patients often suffer relapse years after due to remnant dormant tumor cells. Nevertheless, the molecular mechanism regulating dormancy of breast cancer cell is still unclear. On the other hand, relapsed tumor is generally believed to be derived from cancer stem cells retaining normal stem cell property, which can undergo quiescence. ΔNp63α, an N-terminally truncated isoform of p51/p63 protein was shown to play crucial roles in the maintenance of stem cells within mammary epithelium. Furthermore, both tumor suppressive and oncogenic roles of ΔNp63α have been reported in mammary carcinogenesis. Thus, we investigated the role of ΔNp63α in the regulation of normal and malignant breast cells. [Method] MCF10A, normal human mammary epithelial cells and breast cancer cell lines of different subtypes were introduced with doxycycline-inducible constructs of ΔNp63α. These cells were subjected to cell cycle analysis, western blot, BrdU-Ki-67 immunostaining. Stemness was determined by mammosphere formation assay combined with flow cytometry analysis of cell surface markers. Drug response was studied using MTT assay. Also luminal breast cancer cell line, MCF7, was subjected to combined mRNA-miRNA microarray analysis. [Result] The induction of ΔNp63α in MCF7 luminal breast cancer cell line led the cells to acquire phenotype typical to quiescent luminal progenitor-like cells. Furthermore, the BMP and Wnt signaling pathways emerged as regulators of ΔNp63α-induced quiescence by microarray analysis. Interestingly, these quiescent cells exhibited down-regulation of BRCA1-dependent DNA repair pathways making them more sensitive to DNA damaging drug, Doxorubicin, but more resistant to anti-mitotic drug, Paclitaxel. Conversely, induction of ΔNp63α in MCF10A normal basal cells resulted in increased cell proliferation and expansion of mammary stem-like cells. ΔNp63α expression also led to the increase of cells expressing breast cancer stem cell markers in both MCF7 and MCF10A cells. However, induction of ΔNp63α affected neither cell proliferation nor the increase of mammary stem- or luminal progenitor-like cells in more aggressive luminal (T47D) and basal (MDA-MB-231) breast cancer cells containing mutant p53. This result interestingly suggested a cell type-specific function of ΔNp63α. We also identified a microRNA network that might regulate quiescence in ΔNp63α-expressing breast cancer cells which will be presented at the meeting in detail. [Conclusion] Our results suggest that the role of ΔNp63α in regulating quiescence, stem or progenitor activity in normal and breast cancer cells is cell type-specific. Given the heterogeneity in the cellular origin of breast cancer subtypes, ΔNp63α-directed chemotherapeutic strategy might be instructive to target therapeutically resistant breast cancer cells. Citation Format: Md. Ruhul Amin, Yuiko Morita-Fujimura, Shuntaro Ikawa. ΔNp63α regulates quiescence, stem or progenitor activity of normal and malignant breast cells in a cell type-specific manner. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr LB-207. doi:10.1158/1538-7445.AM2015-LB-207

Abstract 5109: The CARMA3-Bcl10-MALT1 signalosome mediates NF-κB activation and cellular invasion in AGTR1-positive breast cancer

Abstract Angiotensin II (Ang II) is a potent vasoconstrictor and vascular pro-inflammatory mediator, known classically for its role in promoting arterial dysfunction. Many of these pathogenic effects result from activation of NF-κB in response to stimulation of the type-1 Ang II receptor (AGTR1), a G protein-coupled receptor. We previously discovered that a complex composed of three proteins, CARMA3, Bcl10 and MALT1 (CBM signalosome) mediates AGTR1-dependent activation of NF-κB in vascular cells. Despite its principal role in vascular pathobiology, we recently found that AGTR1 is aberrantly overexpressed in 10-20% of breast cancers (characterized as Luminal, ER+, PR+, Her2-). In this context, AGTR1 promotes cellular invasion and in vivo tumorigenesis, but the mechanisms driving this effect of AGTR1 have remained unclear. We hypothesized that stimulation of AGTR1 in this subset of breast tumors induces CBM-dependent NF-κB activation, thus contributing to tumorigenesis. To develop experimental models for studying the impact of AGTR1 in breast cancer (BC), we screened a panel of BC cell lines and found overexpression of endogenous AGTR1 in BT549 cells, among a few others, but no expression in most lines. To complement the BT549 line, we stably over-expressed exogenous AGTR1 in ZR75 cells (an AGTR1-, luminal line) to produce the ZR75-AGTR1 line. All BC lines tested, irrespective of AGTR1 status, expressed CARMA3, Bcl10 and MALT1. We found that Ang II treatment induces time-dependent phosphorylation of IκB, indicating NF-κB activation, in both BT549 and ZR75-AGTR1 cells, but not in control ZR75 cells. We then employed an NF-κB reporter assay to generate further evidence of NF-κB activation in ZR75-AGTR1 cells. We also demonstrated induction of several endogenous NF-κB target genes using real-time Q-PCR. Importantly, Ang II-dependent NF-κB activation is effectively blocked by pretreatment with losartan (AGTR1 antagonist) or with IKK-VI (IKKβ inhibitor), as well as by siRNA-mediated knockdown of Bcl10 or MALT1. These data indicate that Ang II dependent NF-κB activation in these cells is dependent on activation of AGTR1, proceeds through the canonical NF-κB machinery, and requires the CBM signalosome. Moreover, we showed that AGTR1 activation triggers a dramatic increase in migration and invasion of both BT549 and ZR75-AGTR1 cells, and that this response is completely blocked by siRNA-mediated Bcl10 knockdown. These results demonstrate for the first time that AGTR1 stimulation leads to NF-κB activation in a subset of breast cancer cells, and link this response to aggressive phenotypic behavior. These studies have significant clinical implications since AGTR1 antagonists, currently used for treatment of hypertension, might be effectively re-deployed for treatment of breast cancers that harbor AGTR1 overexpression. Inhibition of CBM-dependent signaling may also represent a novel treatment approach. Citation Format: Prasanna Ekambaram, Nathaniel Hubel, Jia-Ying Lee, Linda Klei, Vincent Concel, Philip Delekta, Scott Tomlins, Linda McAllister-Lucas, Peter Lucas. The CARMA3-Bcl10-MALT1 signalosome mediates NF-κB activation and cellular invasion in AGTR1-positive breast cancer. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5109. doi:10.1158/1538-7445.AM2015-5109

Abstract P2-04-05: Modeling luminal breast cancer heterogeneity: Combination therapies to suppress hormone receptor-negative subpopulations among receptor-positive ones

Abstract All Luminal breast cancers are heterogeneous, containing substantial numbers of estrogen (ER) and progesterone (PR) receptor-negative cells among the ER+PR+ ones. We have identified two such Luminal-derived ER–PR– cells: The first, we call "Luminobasal" (LB), are ER–PR– and cytokeratin 5 (CK5)-positive. The second, we call "Double Negative" (DN), are ER–PR– and CK5–. Currently, neither is targeted for treatment. Luminobasal cells: To address the relationships between true Luminal ER+PR+CK5– and Luminobasal ER–PR–CK5+ cells in Luminal cancers and tightly control their ratios we generated isogenic pure Luminal (pLUM) and pure Luminobasal (pLB) cells from solid tumor xenografts starting from the same parental T47Dco Luminal human breast cancer cell line. Cells were gene profiled and unique immunohistochemical (IHC) biomarkers for each were confirmed. Interaction dynamics studies show that in mixed-cell 3D colonies, pLUM or Luminal MCF-7 cells suppress growth of pLB cells. Similarly, in mixed-cell solid tumor xenografts, pLUM cells suppress pLB proliferation. Alarmingly, in mixed-cell models, monotherapy of a pLUM or MCF-7 subpopulation with the antiestrogen Fulvestrant inadvertently expands the number of pLB cells. This suggested that pLB cells also need to be treated. An 89 drug FDA-approved oncology library and high throughput screening methods were therefore used to show that pLB cells are specifically targeted by the EGFR inhibitors Gefitinib and Erlotinib. We then showed, in both mixed-cell 3D colonies and mixed-cell solid mouse tumors, that combination therapy using Fulvestrant plus Gefitinib constitutes a robust treatment strategy that targets both cell populations simultaneously. Double Negative cells: DN cells are EGFR– and therefore unaffected by EGFRi. However they express the Luminal marker Claudin-3 (CLD3). Among other conditions, DN cells arise as a discrete CLD3+ subpopulation within CLD3– pLB xenografts. This allowed us to use CLD3 for FACS isolation and purification of DN cells from xenografts. Purified cells were gene expression profiled, which showed that DN cells have a unique genomic signature that distinguishes them from LUM and LB cells. They express diagnostic markers suitable for IHC and FACS-sorting, some of which also present potential therapeutic targets. We propose that heterogeneous Luminal breast cancers may be best treated with combination therapies that include endocrine, EGFRi and/or DN inhibitors. Importantly, optimizing combination regimens that eradicate or suppress not only ER+PR+ cells but also diverse ER–PR– cells in Luminal disease requires that primary tumors be pre-screened for appropriate biomarkers, including but not limited to ER, PR, CK5 and EGFR. Citation Format: Allison L Scaling, Aaron J Knox, Maurcio P Pinto, Brian S Bliesner, James M Haughian, Hany A Abdel-Hafiz, Kathryn B Horwitz. Modeling luminal breast cancer heterogeneity: Combination therapies to suppress hormone receptor-negative subpopulations among receptor-positive ones [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-04-05.

Comparison of Aortic Diameter and Area After Endovascular Treatment of Aortic Dissection

Different methods have been used to assess remodeling of the thoracic aorta after endovascular treatment of Stanford type B aortic dissections. Changes in morphology may be described using diameter, area, or volume. The aim of this study was to determine if aortic diameter measurements could be used to approximate aortic area in order to refine reporting standards. The study population encompassed 100 patients enrolled in the VIRTUE registry (designed to assess thoracic endografting with the Valiant Stent Graft System [Medtronic, Minneapolis, MN] for the treatment of type B aortic dissections). Diameter and area measurements of the true lumen, false lumen, and whole aorta were made using three-dimensional computed tomographic (3D CT) workstations, at different anatomic locations. Measurements included preoperative, postoperative, and follow-up scans. The Pearson test was used to determine general correlation between diameter and volume at each location. Scatter plots were drawn and linear regression models were used to draw a line of best fit. Comparison of these with nonlinear models was performed. Aortic true and false lumen diameter and area showed good correlation (p < 0.001) in the majority of anatomic locations. This relationship was present preoperatively and during follow-up (p < 0.001). The linear regression models fit well with high R(2) values. At very large aortic sizes nonlinear models were a slightly better fit, but this was not significant. Aortic diameter measurements correlate with luminal areas in patients with type B aortic dissection. This implies area increases proportionately with diameter over time. Therefore, diameter measurements using multiplanar reconstructions based on a central luminal line appear to be adequate when assessing aortic remodeling after endovascular treatment of aortic dissection.

Abstract 2719: Use of abiraterone against a luminal androgen receptor breast cancer cell line

Abstract Background: Data from this pre-clinical model suggests that the LAR TNBC subtype is unique and may respond to novel hormonal agents such as bicalutamide or abiraterone. The LAR subtype represents 11 % of all TNBC (which represent 20% of all breast cancers). Thus, positive studies of abiraterone could potentially extend the application of abiraterone to an additional 2% of all breast cancer patients, who could be treated in the metastatic, neoadjuvant, and adjuvant setting (for five years) with hormonal maintenance therapy. We, therefore, conducted studies of abiraterone on LAR TNBC cell line, MDA-MB-453. . Methods: i). Cell proliferation- Cell proliferation will be done by plating 500,000 cells in triplicate Petri dishes and measuring cell number over a six day period between parent cells and cellstreated with the following varying concentrations of abiraterone: 100 ng/ml, 250 ng/ml, and 400 ng/ml. ii).Cell cycle effects- Cells were incubated for 24 hours with abiraterone and then lifted from the plate with trypsin/EDTA (0.5%/0.1%) and fixed overnight in 80% ethanol. This was followed by staining in PBS with 10 ug/ml of propidium iodide for twenty minutes at room temperature. Flow cytometry was performed in our facility, and data was obtained on the percentage of the cell population in various points of the cell cycle. iii). Apoptosis and necrosis assay (Annexin-V-PI assay)- Cells were harvested at 72 hours post-seeding and incubation with abiraterone and washed with ice-cold PBS, followed by staining with 1ug/ml of PI and 5ul of Alexa Fluor® 488 annexin V in 100ul of annexin binding buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2). After the incubation at room temperature for 15 minutes, 400ul of ice-cold annexin binding buffer was added and stained cells were analyzed by Becton-Dickinson FACSCalibur Flow Cytometer. Results: Abiraterone caused a significant impact on cell proliferation and promoted apoptosis in the MDA-MB-453 population. Cell cycle effects were also noted with abiraterone treatment. Conclusion: Abiraterone has activity against MDA-MB-453, a breast cancer cell line known to express luminal androgen receptor. The results suggest that further studies may be warranted with this agent against this unique subtype of breast cancer. Citation Format: Krishna A. Rao, Vishnu Modur, Sumana Ghosh. Use of abiraterone against a luminal androgen receptor breast cancer cell line. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2719. doi:10.1158/1538-7445.AM2014-2719

Abstract 5135: A chromatin modifier from the 8p11 amplicon in luminal breast cancer

Abstract The chromosome 8p11-p12 amplicon is present in 12-15% of breast cancers, resulting in an increase in both copy number and expression of several chromatin modifiers in these tumors, including KAT6A, WHSC1L1, and ASH2L. Previous analyses in SUM52 breast cancer cells showed amplification and over-expression of KAT6A, and subsequent RNAi screening identified KAT6A as a potential driving oncogene. Also known as MYST3, KAT6A is a histone acetyltransferase (HAT) with intrinsic HAT activity towards itself and lysine residues on histone 2B, histone 3, and histone 4. Previously identified as a fusion partner with CREB binding protein (CREBBP) in acute myeloid leukemia, KAT6A is amplified in 8.7% of invasive breast cancers according to the Cancer Genome Atlas (TCGA). Knockdown of KAT6A with several shRNAs in SUM52 cells, a luminal breast cancer cell line harboring the 8p11 amplicon, resulted in reduced growth rate compared to non-silencing controls and profound loss of clonogenic capacity both in mono-layer and in soft agar. The normal cell line MCF10A, however, did not exhibit slower growth with knockdown of KAT6A, nor did other breast cancer cell lines without KAT6A amplfied. Interestingly, SUM52 cells with KAT6A knockdown formed fewer mammospheres in culture compared to non-silencing controls, suggesting a possible role for KAT6A in breast cancer stem cell activity and self-renewal. Further analysis of cell surface markers CD24 and CD44 in KAT6A knockdown cells revealed an increase in cells double positive for CD24 and CD44 expression, a characteristic of increased cell differentiation. Previous work from our laboratory identified FGFR2 as a driving oncogene in SUM52 cells. While FGFR inhibition can completely arrest the growth of these cells, it is completely reversible. Thus, exposure of SUM52 cells to FGFR inhibitors does not significantly affect the colony forming ability of the cells. By contrast, the colony forming efficiency of SUM52 KAT6A knockdown cells in the presence of the FGFR inhibitor PD170374 was significantly reduced compared to cells with KAT6A knockdown only. These data suggest that KAT6A may be a potential novel oncogene in breast cancers bearing the 8p11 amplicon and that it interacts with FGFR2 to regulate expression of transformed phenotypes. While there are other putative oncogenes in the amplicon, the identification of KAT6A as a driving oncogene suggests that chromatin-modifying enzymes are a key class of oncogenes in these cancers, and play an important role in the selection of this amplicon in luminal B breast cancers. Citation Format: Brittany Turner Ivey, Steve Guest, Stephen P. Ethier. A chromatin modifier from the 8p11 amplicon in luminal breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 5135. doi:10.1158/1538-7445.AM2014-5135

Abstract 3015: Annexin A3 is selectively expressed in MET-like as compared to EMT-like breast cancer stem cells

Abstract Increasing evidence has demonstrated that breast cancers are driven by a cellular subset that displays stem cell properties. These cells mediate tumor metastasis and contribute to therapeutic resistance. We recently reported that breast cancer stem cells (BCSCs) are capable of inter-converting between two cellular states in a process regulated by the tumor microenvironment. CD44+/CD24- CSCs are characterized by expression of epithelial-mesenchymal transition (EMT) genes, invasive and largely quiescent, whereas mesenchymal-epithelial-like (MET) stem cells are characterized by expression of Aldehyde dehydrogenase (ALDH), epithelial morphology and self-renewal. In order to further characterize these two alternative populations of BCSCs, we carried out gene expression analysis of sorted ALDH+ and CD44+/24- populations from a series of primary human breast cancers and established cell lines. We used the CSC markers to fractionate MCF7 cells and performed proteomic analysis utilizing liquid chromatography-tandem mass spectrometry. Differentially expressed proteins were identified using t-test and ANOVA based on spectra counts. Both microarray and proteomic analysis identified Annexin A3 (ANXA3) to be consistently upregulated in ALDH/MET stem cell populations compared to matched CD44+/24-/EMT-like CSCs. These results were validated through qPCR and western blotting in utilizing the luminal cell line MCF7 as well as the claudinlow cell line SUM 159. In the latter, ANXA3 was upregulated 2-5 fold at both the mRNA and protein levels in ALDH+ compared to ALDH- populations. Further, immunofluorescence studies using confocal microscopy demonstrated co-expression of ANXA3 and ALDH in MCF7 and SUM 159 cells. To determine the clinical significance of ANXA3 in breast cancer, we performed immunohistochemical analyses on tissue microarrays of breast cancer patients. ANXA3 expression was found to be significantly higher in breast cancers compared to normal breast tissue with over 75% of primary human tumors demonstrating ANXA3 staining. ANXA3 was expressed at significantly higher levels in luminal compared to basal and claudinlow tumors. Furthermore, 42% of HER2+ tumors also showed moderate to high expression of ANXA3. These results suggest that ANXA3 is predominantly expressed in luminal breast cancers, where it marks the ALDH+ epithelial-like CSC populations. The functional role of ANXA3 in breast CSCs is currently under investigation. Citation Format: Deol S. Yadwinder, Sean McDermott, David M. Lubman, Jenny C. Chang, Song Nie, Yang Cong, Alice Turdo, Ebrahim Azizi, Tahra K. Luther, Shawn G. Clouthier, Max Wicha. Annexin A3 is selectively expressed in MET-like as compared to EMT-like breast cancer stem cells. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 3015. doi:10.1158/1538-7445.AM2014-3015

Abstract 2470: Cd177, a novel metastasis suppressor of breast cancer

Abstract Mammary glands contain luminal and basal epithelial cells. We recently found that both luminal and basal epithelial cells can initiate malignant mammary tumors with forced ErbB2/Her2/c-Neu expression (referred to as luminal and basal tumor-initiating cells, respectively). Basal tumor-initiating cell (TIC)-derived tumors had a greater incidence and larger volume than luminal TIC-derived tumors. Transcriptomic analysis was performed to evaluate the underlying transcriptional difference between ErbB2-driven luminal and basal TIC-derived tumors. CD177 was found to be downregulated within basal TIC-derived tumors. CD177 is an important membrane glycoprotein on neutrophils and serves as a marker for myeloproliferative diseases. The role of CD177 in solid tumors is unknown. Our initial analysis revealed that loss of CD177 expression is linked to higher metastasis incidence and shorter survival in breast cancer patients, but not other diagnostic markers like estrogen-, progesterone-, or HER2-receptors. Silencing CD177 in 4T1 cells, a mouse mammary tumor cell line, led to a significant increase for colonic growth in soft agar and pulmonary metastasis in mice. Overexpressing CD177 in MCF7 cells, a human luminal mammary tumor cell line, resulted in a significant decrease for colonic growth in soft agar. We also found that genetic deletion of CD177 in female mice results in significantly increased Lineage-CD24+CD49fhi basal epithelial cells, the major tumor-initiating cells for ErbB2-induced breast cancer. Collectively, we identified a novel potential metastasis-suppressor for human breast cancer with important prognostic value. Future studies will involve in the mechanistic understanding of how CD177 regulates metastasis of breast cancer. Citation Format: Qing Xie, Nicholas Borcherding, Ryan Kolb, Weizhou Zhang. Cd177, a novel metastasis suppressor of breast cancer. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 2470. doi:10.1158/1538-7445.AM2014-2470

Abstract 604: Styrene compounds induce apoptosis in triple negative breast cancer cell lines.

Abstract Triple negative breast cancer (TNBC) is a rare basal-like subtype of breast cancer that is highly aggressive due to its invasiveness and metastatic properties. TNBC is characterized by the loss or low expression of estrogen receptor, progesterone receptor, and HER2 proteins which have been targeted and established as somewhat effective therapy regiments in other breast cancer subtypes including luminal breast cancer. Since triple negative tumors are missing these established drug targets, there has been little advancement in the establishment of drug therapies to specifically target this breast cancer subtype and therefore the need for novel therapeutics in treating TNBC still remains. Previous reports have suggested that PARP inhibitors show promise in potentiating the effectiveness of other known chemotherapeutics. Styrene compounds are volatile organic compounds that have a similar structural composition to some known PARP inhibitors. Our group has developed styrene compounds composed of a 3-nitro group on the A ring and varying aryl groups in the B ring of the styrene, and then tested their biological effects on luminal and TNBC cell lines. In this study, four different styrene compounds were used to treat the luminal breast cancer cell line HCC70 and the triple negative breast cancer cell line HCC1806. The breast cancer cells were exposed to a 10, 100, and 1000 μM titration over a 24hr time period. The apoptotic effect on these cell lines were analyzed and measured via qualitative and quantitative methods. We show here that light microscopy revealed a classic apoptotic dose-response to all of the styrene compounds in the cell lines. We also used the cell viability and MTT assays to reveal a dose-response decrease in cell viability and increase in cell death to the titration of the styrene compounds. This preliminary data show that these compounds have the ability to induce apoptosis in luminal and triple negative breast cancer cell lines. Our preliminary results lead us to believe that these styrenes could represent a new chemotherapeutic treatment for Triple Negative Breast Cancer and potentially other breast cancer subtypes. Citation Format: Checo J. Rorie, Shaina L. Richardson, Agape C. Lucas, Phillip A. Thomas, Kashenya M. Gurley, Marion A. Franks. Styrene compounds induce apoptosis in triple negative breast cancer cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 604. doi:10.1158/1538-7445.AM2013-604

Migration of fenestrated aortic stent grafts

This article reports the incidence, timing, and related sequelae for proximal and distal migration of the Zenith Fenestrated AAA Endovascular Graft (Cook Medical, Bloomington, Ind) used to treat abdominal aortic aneurysms. A prospectively maintained database at a tertiary referral hospital was used to identify 83 patients who underwent endovascular repair using the Zenith fenestrated stent graft. Inclusion criteria included a postoperative computed tomography (CT) scan within 6 weeks of implantation and at least one additional follow-up CT scan (>5 months) available electronically at our institution. Eligible patients underwent assessment of stent graft migration using a CT-based central luminal line (CLL) technique. The proximal and distal margins of the stent graft were measured using CLLs relative to vascular landmarks on all available follow-up CT scans. Migration was defined as stent graft movement ≥4 mm. Fifty-five patients were included in this study, mean age was 74± 7 years, and 89% were men. Mean preoperative aneurysm diameter was 67± 9 mm. In these 55 patients, fenestrations were applied to 162 target vessels with the commonest design accommodating two renal arteries (RAs) and the superior mesenteric artery (SMA). Median follow-up was 24 (range, 5-97) months; 80% of patients (n= 44) had both the proximal and two distal attachment sites assessed for evidence of migration. Twelve iliac limbs in 11 patients were excluded from analysis due to occlusion of one internal iliac artery precluding CLL assessment (n= 7), or image quality issues (n= 5). Using CLLs and based on those patients who exhibited migration, the median proximal and distal migration distances were+5.0 (range, +4.0 to+8.1) mm and -5.0 (range, -4.3 to -21.3) mm, respectively. Kaplan-Meier analysis for proximal migration revealed migration rates of 14% and 22% at 12 and 36 months, respectively. Distal migration rates were lower at 3% and 8%, respectively. There have been no incidences of late rupture or open conversion. Of the patients with proximal migration, two patients lost a single target vessel (two RAs) and three patients were reported to have target vessel stenosis (two SMAs, one RA). These cases did not require reintervention. Both suprarenal fabric extension and visceral artery stenting are known to provide additional fixation for fenestrated aortic stent grafts. Despite this, minor proximal migration still occurs in up to one quarter of fenestrated endovascular repair patients by 4 years. We believe this is mainly due to the engagement of the barbs of the anchoring stent. Distal migrations occur with lower frequency.

Abstract P6-02-06: A 3D tri-culture model of normal mammary gland. A tool for breast cancer initiation studies

Abstract The mechanisms involved in breast cancer initiation are not well understood. This may in part be due to a lack of an in vitro model that faithfully recapitulates the morphology, phenotype and in vivo architecture of the human mammary gland. Most in vitro models of normal breast have relied on the use of reconstituted basement membrane gels to induce luminal epithelial cell polarity and have neglected the role of myoepithelial cells and fibroblasts in this process. We aimed to develop a 3D in vitro culture system of normal breast which includes three of the major functional cell types embedded in a more physiologically relevant collagen 1 matrix. We used this system to investigate the mechanisms behind breast cancer initiation via genetic manipulation of some well known oncogenes and tumour suppressors involved in breast cancer progression. Myoepithelial cells (Myo1089) and fibroblasts (generated in house) were isolated and immortalised from breast reduction mammoplasty samples collected with ethical approval. Following characterisation, these were virally transfected with Enhanced Green Fluorescent Protein (EGFP) and dsRed protein respectively to enable tracking. 3D cultures were established in collagen 1 and included the non-tumorigenic luminal epithelial cell line HB2, EGFP Myo1089 cells and dsRed fibroblasts. Cells were cultured for 3 weeks in Transwell™ cell culture inserts. Following fixation these were analysed by H&amp;E, confocal microscopy and immunohistochemistry. Morphology and immunostaining profiles were compared to sections of normal breast tissue. Immunohistochemical characterisation using the following antibodies: E-cadherin, epithelial membrane antigen, vimentin, laminin 5, collagen 4 plus luminal and basal cytokeratins, demonstrated polarised epithelial structures with lumen formation and basement membrane production with a similar immunostaining profile to normal breast tissue. The importance of including myoepithelial cells and fibroblasts in maintaining these structures was demonstrated. We established this model is amenable to genetic manipulation by overexpressing Her2 in HB2 cells, and knocking out ERβ1 in EGFP Myo1089 cells and Rac1 and Dock4 in dsRed fibroblast cell lines using shRNA techniques. These were included in separate models with morphological and phenotypic effects determined by confocal microscopy. In summary we have developed an in vitro model of normal breast tissue that includes three of the major functional breast cell types cultured in a physiologically relevant 3D matrix. We have validated the morphology and protein expression profile against human breast tissue specimens and confirm that this is a suitable model of normal breast. The model is suitable for experimental manipulation and cell behaviour can be easily visualised using standard laboratory techniques. We conclude this is a robust in vitro model of normal breast tissue offering an alternative cost-effective method of studying genes involved in breast cancer initiation. Citation Information: Cancer Res 2012;72(24 Suppl):Abstract nr P6-02-06.

Abstract 3362: Tumor initiating cells are sensitive to paclitaxel encapsulated in polymeric nanoparticles

Abstract Tumor initiating cells (TICs) represent a small fraction of tumor cells, and are considered to be key drivers of tumor growth, drug resistance and recurrence. TICs are highly resistant to conventional chemotherapy, possibly due to overexpression of drug efflux transporters and other protective mechanisms. Because drug resistance and tumor relapse are significant problems, approaches to eliminate TICs are urgently needed. Recent studies show that tumor populations that are depleted of TICs demonstrate a return of the TIC sub-population 1 suggesting the possibility of de-differentiation of non-TICs to TICs. We hypothesized that sustained delivery of a cytotoxic agent to the tumor population could potentially reduce TIC fraction and consequently increase the therapeutic effectiveness of chemotherapy. To test this hypothesis, we encapsulated paclitaxel, a microtubule stabilizing agent, in PLGA nanoparticles formulated using an FDA-approved polymer, poly (lactide-co-glycolide). Our previous studies have shown nanoparticles can sustain the intracellular delivery of encapsulated drugs like paclitaxel 2. The effectiveness of nanoparticle-encapsulated paclitaxel in reducing TIC fraction was investigated by standard cancer stem cell assays- mammosphere formation and anchorage-independent growth on soft agar in MCF-7, an ER+ luminal breast cancer cell line. Results obtained in these studies show that drug encapsulated PLGA formulations effectively decreased mammosphere formation and soft-agar colony formation when compared to free drug treatment. In mammosphere assays, paclitaxel solution treatment did not significantly affect the mammosphere formation relative to untreated controls, whereas paclitaxel loaded PLGA nanoparticles treatment decreased the number of mammospheres to 50 ± 33% of untreated controls. In soft-agar colony formation assay, paclitaxel solution treatment increased the number of colonies to 122 ± 10% of untreated controls. In contrast to this, paclitaxel loaded PLGA nanoparticles treatment decreased the number of colonies to 66 ± 8% of untreated controls. Future studies will investigate the effectiveness of these formulations on TICs in mouse models of breast cancer. References 1 Gupta, P. B. et al., Cell 146 (4), 633; Chaffer, C. L. et al., Proc Natl Acad Sci U S A 108 (19), 7950. 2 Patil, Y. B. et al., Biomaterials 31 (2), 358. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3362. doi:1538-7445.AM2012-3362

The accuracy of computed tomography central luminal line measurements in quantifying stent graft migration

This study evaluated the accuracy of central luminal line (CLL) measurements in quantifying stent graft migration. The bias of the CLL technique together with observer variability were assessed. Stent grafts were deployed in plastic aortic phantoms at fixed locations from two side branches. Each phantom was filled with iodinated contrast, and a 2-mm multislice computed tomography (CT) scan was performed. The stent graft was then displaced caudally, its new location determined, and again, a CT scan performed. This created a series of 15 cases with known stent graft migration. CLLs were used to measure stent graft position on the CT scans and calculate migration (3 observers). In vivo stent graft migration was then evaluated in a similar manner using a series of follow-up CT scans from nine patients (2 observers). All CLL measurements were performed independently and were repeated on a separate occasion. The mean difference in CLL migration between the actual and observed measurements (bias) in the aortic phantoms was <1 mm. The 95% confidence intervals for the bias were within the interval (-1 and 1 mm), and the 95% limits of agreement were within -3 mm and +3 mm. The 95% limits of agreement for measurements within and between observers were -4 to 2 mm and -2 to 2 mm, respectively. The phantom study generated a coefficient of repeatability (RC) of 1 mm for within-observer measurements. Clinically, CLLs generated 95% limits of agreement within and between observers of -3 to 4 mm (RC, 2 mm) and -3 to +3 mm, respectively. Bias from CLL-determined migration is small and insignificant from a practical point of view. A small amount of measurement variability within and between observers does exist; it should be feasible to detect changes in stent graft position that are ≥4 mm.

PD03-03: Identification of Transcription Factors Critical for the Growth of Basal Breast Cancer.

Abstract Background: Basal breast cancers are aggressive, poor prognosis tumors that occur commonly in young women and in African American women. Profiling of breast tumor mRNA has demonstrated that there are differences in gene expression between the basal and luminal subtypes of breast cancer. In this study, we identified response elements in the genes that define basal breast cancers, and identified transcription factors that are critical for the growth of basal breast cancer cells. Materials and Methods: We performed promoter analysis using 4 published microarray studies (PMID: 12829800; PMID: 19435916; PMID: 17157792; PMID: 11562467) to select genes that are highly expressed in basal tumors compared to luminal tumors. For this analysis we selected 61 genes highly expressed in a set of basal tumors in any of the 4 microarray studies. We next used the online tool, CORE_TF, along with the MATCH algorithm minimizing for the sum of false positives and false negatives to identify binding motifs within the promoter (defined from −1 kb to the first exon) of each gene. The frequency of binding motif occurrence for these 61 basal genes was compared to the frequency within the promoters of 3000 randomly selected genes. Significance was tested using an exact binomial test with a cutoff of p&amp;lt;0.05. RNA expression of motif-identified transcription factors was then analyzed in-silico using all 10 datasets in Oncomine™ that contained annotation for triple-negative status. Expression in triple negative samples was compared to expression in non-triple-negative samples with a cutoff of p &amp;lt;0.05. We next performed siRNA knockdown studies to determine whether the identified TFs regulate basal breast cancer growth. Basal and luminal cells transfected with control and specific siRNAs were grown in triplicate and mean cell counts at day 6 were compared. Results: Promoter analysis identified 24 binding motifs that were over-represented in basal breast tumor genes compared to a random set of 3000 genes. TransFac analysis indicated that 47 transcription factors bind the 24 identified motifs. Oncomine analysis showed that 8 of the 47 transcription factors were significantly more highly expressed in basal as compared to non-basal tumors. Identified transcription factors include FOXC1, FOXM1, CDC5L, E2F3, CEBP and NF-Y. siRNA to FOXM1 in 2 basal breast cell lines reduced growth by &amp;gt;70% after 6 days, whereas, in the luminal cell line MCF7, growth was reduced by 15%. Discussion: This study identified transcription factors that are highly expressed in basal breast tumors (as compared to non-basal breast tumors). siRNA knockdown studies showed that FOXM1 is critical for basal breast cancer cell growth. These results suggest that transcription factors highly expressed in basal breast cancers may be novel targets for the treatment of this disease. These studies were supported by a Promise grant from the Susan G. Komen for the Cure (PB, SGH), and by the Norman E. Brinker Award for Research Excellence (PB). Citation Information: Cancer Res 2011;71(24 Suppl):Abstract nr PD03-03.

Abstract 4519: In vitro cell and in vivo animal studies of the demethylating agent in treating HER2-amplified breast cancer

Abstract PURPOSE: The recent evidence shows that HER2-amplified breast cancers exhibit more frequent CpG island methylation than other types of breast cancers, suggesting epigenetic alterations are involved in HER2-mediated tumorigenesis. In this study, we addressed the possibility of the use of the demethylating agent (5-aza-2’-deoxycytidine, decitabine) for treatment of HER2-overexpressing breast cancer. EXPERIMENTAL DESIGN: Two cell model systems were used in this study: murine primary Her2-overexpressing tumor cell line (A6O5, derived from the tumor naturally developed in HER2/neu transgenic mice) and human HER2-overexpressing luminal breast cancer cell line MCF7-HER2-18. Cells were cultured in regular cell culture dishes with the serum-containing medium (adhesive culture) or in ultra-low attachment dishes with the serum-free sphere culture medium (tumorsphere culture) for drug testing. Tumors developed from A6O5 cells injected into mammary glands of HER2/neu transgenic mice were used as the xenograft model. The early spontaneous mammary tumors naturally developed in HER2/neu transgenic mice (size &amp;lt; 5 mm) were used for drug treatment. RESULTS: The in vitro growth of A6O5 was more significantly inhibited by decitabine than that of non-tumorigenic murine mammary epithelial cells. Consistently, MCF7-HER2-18 cells were more sensitive to decitabine-induced growth suppression than its control vector-transfected cell line MCF7-Neo. These results suggest that HER2 overexpression renders cancer cells more sensitive to the effect of decitabine. Surprisingly, the tumorsphere formation of A6O5 and MCF7-HER2-18 was dramatically abrogated by decitabine, suggesting that self-renewal of cancer stem cells in these two cell lines was annihilated by decitabine. Importantly, decitabine inhibited the in vivo growth of A6O5 xenograft tumors. For proof-in-principle of this study, we treated the HER2/neu transgenic mice bearing early developing mammary tumors with decitabine. Indeed, decitabine effectively suppressed the in vivo tumor growth of the decitabine-treated group compared to that of the vehicle-treated group. CONCLUSIONS: This study for the first time explores the potential of the demethylating agent in the treatment of HER2-amplified breast cancers using in vitro cell and in vivo animal models. These findings shed light on the translational application of epigenetic modifiers with the HER2-signaling blockers to treatment of HER2-amplified breast cancer patients. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 4519. doi:10.1158/1538-7445.AM2011-4519

Abstract P6-08-01: Endothelial-Like Phenotype of Triple-Negative Breast Carcinoma Cells and Implications for New Molecular Targets

Abstract Background. Triple Negative Breast Cancers (TNBCs) still represent a question mark in breast cancer biology and a primary issue in clinics. A promising approach for an efficient targeted treatment of TNBCs seems to be represented by antiangiogenic therapies. Methods. Effects of different compounds on proliferation of TNBC was evaluated in vivo and in vitro respectively by xenograft tumor volume analysis and SRB assay. Vasculogenic Mimicry (VM) properties were evaluated in vitro and in vivo respectively by tube formation assay and Transmission Electron Microscopy (TEM). Results. Treatment of triple negative (TN) MDA-MB-231 xenografts with Sunitinib induced tumor regression, versus only a slight growth inhibition in tumors derived from MCF7 luminal cell line, whereas Bevacizumab determined only a modest decrease in tumor growth in both models. Accordingly, the efficacy of Sunitinib in blocking in vitro growth of breast cancer cell lines was higher in MDA-MB-231 cells in comparison with MCF7 cells (IC50 at 72h in MDA-MB-231 was 5 uM vs 25 uM in MCF7, as evaluated by SRB), and sensitivity to Bevacizumab was comparable between the two different cell lines. Investigating the undifferentiated nature of TNBCs, we observed that these tumors present an endothelial like phenotype and behavior, as supported respectively by the expression of endothelial markers, and the formation of vascular-like channels in vitro. In fact, all six TN cell lines evaluated (MDA-MB-231, MDA-MB-157, MDA-MB-468, BT-549, BT-20 and HCC1937) were able to form vascular channels when seeded on the murine tumor-derived basement membrane (Matrigel), whereas luminal (MCF7, T47D and ZR-75-1) and HER2- positive (MDA-MB-361, BT474 and SKBr3) breast carcinoma cell lines did not exhibit vascular structures. Then, we evaluated the VM in xenograft tumors derived from MDA-MB-231, MCF7 and MDA-MB-361 using transmission electron microscopy (TEM). MDA-MB-231 xenografts displayed channel-like structures formed by tumor cells encompassing erytrocytes, whereas in MCF7 and MDA-MB-361 xenografts the endothelial lining delimiting blood vessels was clearly visible. Notably, blood vessels surrounded by tumor cells were also identified in human TN specimens processed for TEM, and these structures were significantly more frequent in TN compared to non-TN tumors. 60% reduction in TN vascular channel formation in vitro by an anti-bFGF monoclonal antibody along with no effect using anti-VEGF antibody indicated that TN breast carcinoma cells can generate vascular channels through bFGF-mediated pathway. Silencing of different receptors involved in bFGF signal (i.e. FGFR2 and PDGFR β) abrogated VM in TN cells. Finally, TNBC cells able to perform vascular-like channels were found to express significantly higher (p= 0.003) levels of FGFR related genes, described associated with basal-like BC aggressiveness, compared to all the other tested cell lines. Conclusions. Our findings point to the possibility that TNBC cells are maintained by proangiogenic signals and that increased sensitivity to Sunitinib probably relies on the specific impairment of PDGFRβ and FGFR2-mediated pathways, which might represent a possible specific therapeutic target. Partially supported by AIRC and Italian Bureau of Health. Citation Information: Cancer Res 2010;70(24 Suppl):Abstract nr P6-08-01.

Variability of vascular CT measurement techniques used in the assessment abdominal aortic aneurysms

Purpose The aim of this project is to assess the variability of six CT measurement techniques for sizing abdominal aortic aneurysms (AAAs). Method 37 CT scans with known AAAs were loaded on to a departmental picture archiving and communication system (PACS). A team of three observers, with experience in aortic CT measurements and the PACS performed a series of 2D and 3D measurements on the abdominal aorta. Each observer was asked to measure 3 quantities; anterior–posterior AAA diameter, maximum oblique AAA diameter, maximum aneurysm area using both 2D and 3D techniques. In order to test intra-observer variability each observer was asked to repeat their measurements. All measurements were taken using electronic callipers, under standardised viewing conditions using previously calibrated equipment. 3D measurements were conducted using a computer generated central luminal line (CLL). All measurements for this group were taken perpendicular to the CLL. Results A total of 972 independent measurements were recorded by three observers. Mean intra-observer variability was lower for 2D diameter measurements (AP 1.3 ± 1.6 mm; 2D Oblique 1.2 ± 1.3 mm) and 2D areas (0.7 ± 1.3 cm 2) when compared to inter-observer variability (AP 1.7 ± 1.9 mm; Oblique 1.6 ± 1.7 mm; area 1.1 ± 1.5 cm 2). When comparing 2D with 3D measurements, differences were comparable except for 3D AP diameter and area which had lower inter-observer variability than their 2D counterparts (AP 2D 1.7 ± 1.9 mm, 3D 1.3 ± 1.3 mm; area 2D 1.1 ± 1.5 cm 2, 3D 0.7 ± 0.7 cm 2). 3D area measurement was the only technique which had equal variability for intra- and inter-observer measurements. Overall observer variability for the study was good with 94–100% of all paired measurements within 5.00 mm/cm 2 or less. Using Pitman's test it can be confirmed that area measurements in the 3D plane have the least variability ( r = 0.031) and 3D oblique measurements have the highest variability ( r = 0.255). Conclusion 3D cross-sectional area measurement techniques have the lowest variability and should be preferred for repeatable measurements of AAAs where possible. Results confirm that both inter- and intra-observer variability exists for all measurement techniques.

Abstract 4226: A luminal epithelial cell line isolated from PTEN−/−;P53−/− mouse protospheres forms adenocarcinoma in vivo

Abstract Pten deletion in the mouse prostate epithelium results in the expansion of a prostatic stem/progenitor cell subpopulation, which co-fractionates with basal epithelial cells. Progenitor cells in the luminal layer of Pten null prostates also have been suggested as tumor initiating cells. Here we describe the isolation of a protosphere-derived, androgen independent cell line with a luminal epithelial phenotype that displays properties of prostate progenitor cells and forms adenocarcinoma in-vivo. The cell line was generated from Rosa26-CreERPtenfl/flP53fl/fl prostate cells. Cells were plated to form protospheres, which originate from stem/progenitor cells, and the deletion of Pten and p53 was induced by tamoxifen. After spheres were serially passaged for 3 generations, a monolayer cell line capable of growth in minimal serum-free media was established. A clonal line also was generated from the parental cell line. In order to determine the differentiated cell phenotype of the parental and the clonal cell lines, cells were stained over multiple generations for markers of epithelial (CK5, CK8, CK14) and neuroendocrine (beta 3 tubulin) cell lineages. The cell line is entirely CK8 positive with a minor component of CK8/CK14 double positive cells. Cells are capable of forming protospheres both in Matrigel and in suspension, a property of stem/progenitor cells. In addition, based on lentiviral reporter assays, a minor population of cells in the parental and the clonal cell line expressed Nanog, Oct4 and Notch dependent activity. Interestingly, Nanog and Oct4 positive cells showed higher sphere forming ability as compared to negative cells. In order to investigate their tumorigenic potential, the parental and the cloned lines were injected subcutaneously into athymic nude mice. Visible tumor formation was observed after 2 weeks, and histological analysis revealed adenocarcinoma and poorly differentiated carcinoma. In addition to CK8+ cells within the tumors, there were rare CK5+ and neuroendocrine cells, suggesting plasticity in the differentiation potential. In conclusion, we have isolated a prostate luminal epithelial cell line and derivative clones with a defined background (Pten and p53 null) that have stem-like and tumor initiating properties in vitro and in vivo. Future experiments are aimed at deciphering the molecular mechanisms regulating growth and differentiation downstream of Pten and p53 loss. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 4226.