bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 34: Inhibitory effects of betulinic acid isolated from Nelumbo nucifera (Lotus) on breast cancer-mediated bone destruction
Authors: Park Se-Young , Park Kwang-Kyun , Chung Won-Yoon
Published: August 2012

Most of cancer patients die, not because of the tumor in the primary site, but because of spreading to other site. In particular, bone metastasis is relatively common in patients with breast, prostate, and lung cancer. Many breast cancer patients suffer from bone metastasis with bone pain and pathological fractures. These metastases are predominantly osteolytic and develop when tumor cells interact with bone. Tumor and bone interact in a vicious cycle, where tumor-secreted factors stimulate bone cells, which in turn release growth factors and cytokines that act back on the tumor cells. Within the vicious cycle are many potential therapeutic targets for treatment of bone metastatic disease. Nelumbo nucifera (Lotus) has been known as a medicinal herb for treatment of cough, fever and hyperdipsia. Betulinic acid (BA) is a naturally occurring triterpenoid present in lotus. In this study, we found that BA reduced mRNA expressions of osteolytic factors in MDA-MB-231 human metastatic breast cancer cells. In particular, mRNA level of parathyroid hormone-related protein (PTHrP), the major component of vicious cycle within bone, was significantly inhibited by BA. Treatment with BA decreased receptor activator of nuclear factor kappa-B ligand (RANKL) /osteoprotegerin (OPG) ratio in PTHrP-treated human osteoblasts. In addition, BA dose-dependently inhibited the RANKL-induced osteoclast differentiation and bone resorption by reducing the secretion of matrix metalloproteinases (MMPs) and cathepsin K derived from osteoclasts, without any evidence of cytotoxicity. We further examined the effect of BA on ovariectomy-induced osteoporosis in mice. Serum levels of tartrate-resistant acid phosphatase isoform 5b, alkaline phosphate, calcium, osteocalcin and C-terminal telopeptide of type I collagen were significantly increased in ovariectomized (OVX) mice than in sham mice. BA inhibited increases in the serum levels of these osteoporotic markers in OVX mice. We analyzed bone volume, trabecular bone thickness, trabecular number and trabecular separation by micro-computer tomography (μ-CT) scanning. Taken together, BA may reduce the expression of PTHrP in MDA-MB-231 human metastatic breast cancer cells, RANKL/OPG ratio in PTHrP-stimulated human osteoblasts, and RANKL-induced osteoclastogenesis in bone marrow macrophages. BA may also prevent osteoporotic bone loss in OVX mice. Thus, BA can be beneficial agents for patients with secondary bone lesions associated with breast cancer.

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 34. doi:1538-7445.AM2012-34
Abstract 3400: A systems biology approach reveals common metastatic pathways in osteosarcoma
Authors: Flores Ricardo J. , Li Yiting , Vanucci Marina , Lau Ching C. , Man Tsz-Kwong , Lau Serrine S.
Published: August 2012

Background Osteosarcoma (OS) is the most common malignant bone tumor in children and adolescents. The survival rate of patients with metastatic disease remains very dismal. Nevertheless, metastasis is a complex process and a single-level analysis will not likely identify its key biological determinants. In this study, we used a systems biology approach to identify common metastatic pathways that are jointly supported by both mRNA and protein expression data in two distinct human metastatic OS models. Results mRNA expression microarray and N-linked glycoproteomic analyses were performed on two commonly used isogenic pairs of human metastatic OS cell lines, namely HOS/143B and SaOS-2/LM7. Pathway analysis of the up-regulated genes and glycoproteins separately revealed pathways associated to metastasis including RAS-related G-proteins signaling, cell cycle regulation, epithelial-to-mesenchymal-transition, cell adhesion, and WNT signaling. However, no common significant pathway was found between the two metastatic models, suggesting a very different biological nature of the cell lines. To address this issue, we used a topological significance analysis based on a “shortest path” algorithm to identify topological nodes that remained hidden from the independent transcriptomic and glycoproteomic analyses. Pathway analysis of the significant topological nodes revealed a striking concordance between the models and identified important common pathways, including “Cytoskeleton remodeling/TGF/WNT,” “Development/WNT signaling,” “Cytoskeleton remodeling/Cytoskeleton remodeling,” and “Cell adhesion/Chemokines and adhesion”. Of these, the “Cytoskeleton remodeling/TGF/WNT” was the top ranked common pathway from the topological analysis of the two cell line models. The up-regulation of proteins in the “Cytoskeleton remodeling/TGF/WNT” pathway in the SaOS-2/LM7 and HOS/143B models was further validated using a Reverse Phase Protein Array platform. Conclusions In this study, we used a systems biology approach by integrating genomic and proteomic data to identify key and common metastatic mechanisms in OS. The use of the topological analysis revealed hidden biological networks that are known to play critical roles in metastasis. Since WNT signaling has been previously implicated in OS and other tumors, further characterization of this common pathway and other topological pathways may lead to a novel therapeutic strategy for curing metastatic OS.

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 3400. doi:1538-7445.AM2012-3400
Abstract 3408: Gene expression profiling of MCF10A series of Ha-Ras transformed human breast epithelial cells: Gene expression involved in EMT pathway
Authors: Yang Sihyung , Cho April E. , Lee Eunhye , Lim Soojeong , Novak Raymond F. , Kim So Hee
Published: August 2012

MCF10A series (10A, 10AT, 10ATG3B, 10CA1h and 10CA1a) of breast epithelial cells were examined for gene expression involved in epithelial-mesenchymal transition (EMT) process. This model represents a unique system for examining early and progressive alterations that occur in cells. Microarray analysis showed more than 720 genes are highly expressed (>2 fold, P<0.05) but over 870 genes significantly decreased (<0.5 fold, P<0.05) compared to control 10A. Ontology analysis of 759 highly expressed genes in malignant 10CA1a showed genes involved in metabolism were the greatest. 72 genes progressively increased and 392 genes gradually decreased with the tumorigenicity. 12 genes in EMT were significantly reduced (<0.5 fold, P<0.05) and 24 genes significantly increased (>2 fold, P<0.05) from microarray and 12 genes significantly decreased (P<0.01) and 18 genes were highly expressed (P<0.01) from PCR array. Based on two analyses, BMP7, COL5A2, DSC2, GSK3B, RGS2, TSPAN13 and VIM were highly expressed but MAP1B, TIMP1 and WNT5A significantly decreased. Collectively, altered gene levels in EMT process may correlate with increasing risk of tumorigenicity and these genes may serve as an early prognostic indicator of tumorigenic potential. Supported by grant from NRF (R-2009-006-6765).

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 3408. doi:1538-7445.AM2012-3408
Abstract 3425: Prognostic significance of BRMS1 promoter methylation in operable breast cancer
Authors: Chimonidou Maria , Stathopoulos Efstathios , Georgoulias Vasilis , Lianidou Evi S.
Published: August 2012

Backround: Breast Cancer Metastasis Suppressor gene-1 (BRMS1) suppresses breast cancer metastasis in multiple experimental models by reducing solitary cell survival and inhibiting growth initiation. Loss of BRMS1 expression has been shown to predict reduced disease-free survival in subsets of breast cancer patients. The BRMS1 promoter region contains a promoter-associated CpG island (CGI) that encompasses the transcriptional start site.The aim of our study was to investigate the methylation status of BRMS1 in breast tumors (FFPES) of operable breast cancer patients and evaluate its prognostic significance. Patients and methods: Using methylation-specific PCR (MSP), we examined BRMS1 promoter methylation in 116 fixed paraffin-embedded tissues (FFPEs): 5 pairs of breast tumors and their surrounding non-cancerous tissues, 4 non-cancerous tissues, 10 benign breast tumors (fibroadenomas), 10 normal breast tissues and 82 breast tumors. All breast tumors were isolated from patients with operable breast cancer. Results: Methylation of BRMS1 promoter was observed in 0/19 (0%) noncancerous breast tissues, in 0/10 (0%) benign breast tumors (fibroadenomas) and in 31/82 (37.8 %) breast cancer tumors. During the follow-up period 22/82 (26.8%) patients relapsed and 20 (24.4%) patients died. BRMS1 methylation was detected in 13/22 (59.1%) patients that relapsed and in 12/20 (60.0%) of patients who died. Disease-free interval (DFI) and overall survival (OS) were significantly associated with BRMS1 promoter methylation (P=0.007). Conclusion: We show for the first time that BRMS1 promoter methylation status in FFPEs from operable breast cancer patients provides important prognostic information and merits to be further evaluated and validated in a larger cohort of patients. This finding is consistent with other studies that showed significant down-regulation of BRMS1 in some breast tumors, especially in metastatic disease, because of epigenetic silencing.

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 3425. doi:1538-7445.AM2012-3425
Abstract 343: Inhibitory effect of oncolytic adenovirus on transforming growth factor- β-induced epithelial-mesenchymal transition in human cancer cells
Authors: Hashimoto Yuuri , Tazawa Hiroshi , Urata Yasuo , Kagawa Shunsuke , Fujiwara Toshiyoshi
Published: August 2012

Background: Epithelial-mesenchymal transition (EMT) is a tumor progression-related process by which epithelial cells lose their epithelial characteristics and acquire mesenchymal properties. EMT promotes the malignant phenotypes including invasion, metastasis and drug resistance, resulting in tumor recurrence and poor prognosis. Therefore, to prevent tumor progression, EMT targeted therapy is required. Oncolytic virotherapy is a promising strategy for anticancer therapy. Although a variety of oncolytic adenoviruses have been developed to induce tumor-selective cell death, the effect of oncolytic adenovirus on the EMT-induced tumor progression remains unclear. In this study, we investigated the biological effect of oncolytic adenovirus on EMT in human cancer cells. Methods: We previously generated a telomerase-specific replication-selective oncolytic adenovirus (OBP-301; Telomelysin), in which the human telomerase reverse transcriptase promoter drives the expression of E1A and E1B for virus replication. Transforming growth factor-β (TGF-β) was used to induce EMT in human lung (A549) and pancreatic (Panc-1) cancer cells. To investigate whether OBP-301 infection affects TGF-β-induced EMT in these cells, western blot and real-time PCR analysis for EMT-related markers were performed. We also examined cell mobility using transwell migration and invasion assays. In vitro antitumor effect of OBP-301 and chemotherapeutic agents in TGF-β-treated cancer cells were assessed by trypan blue exclusion assay. Results: Administration of TGF-β induced mesenchymal characteristics, including spindle-like cell morphology, down-regulation of E-cadherin, up-regulation of vimentin and high motility, in A549 and Panc-1 cells. When TGF-β-treated cancer cells were infected with OBP-301, suppression of E-cadherin expression was attenuated. Conversely, the expression levels of mesenchymal markers, vimentin and N-cadherin, and EMT-inducing transcription factors, Snail, Slug and ZEB1, were reduced. OBP-301 infection also inhibited TGF-β-induced enhancement of migration and invasion in cancer cells. Moreover, OBP-301 efficiently killed TGF-β-treated cancer cells, whereas TGF-β-treated cancer cells were resistant to cisplatin and docetaxel. Conclusion: Our results suggest that telomerase-specific oncolytic adenovirus OBP-301 inhibits TGF-β-induced EMT in human cancer cells. OBP-301 has therapeutic potential against cancer cells that have undergone EMT. Therefore, OBP-301 might be a promising anticancer agent to suppress EMT-mediated cancer progression and metastasis.

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 343. doi:1538-7445.AM2012-343
Abstract 3434: EphB3 mediates kinase-dependent suppression of non-small cell lung cancer metastasis
Authors: Li Guo , Ji Xiao-dan , Xie Dong
Published: August 2012

Eph family is one of the largest subfamily of receptor tyrosine kinases. Their important but controversial roles in cancer development have drawn growing attentions in recent years. We have recently reported that EphB3 acts as a tumor promoter in human non-small-cell lung cancer (NSCLC) independent of its kinase activity (Cancer Research. 71(3):1156, 2011). In the present study, we find that despite overexpression of EphB3 in NSCLC, the expression of its cognate ligands, ephrin-B1 and ephrin-B2, is significantly down-regulated, leading to reduced tyrosine phosphorylation of EphB3. Activation of EphB3 kinase with either ligand stimulation or genomic engineering (Y608/614E) in EphB3-overexpressing NSCLC cells inhibits cell migratory capability in vitro as well as in vivo metastatic seeding. Furthermore, we have identified a novel EphB3-binding protein, the receptor for activated C-kinase 1 (RACK1), which mediates the assembly of a ternary signal complex comprising protein phosphatase 2A (PP2A), Akt and itself in response to EphB3 activation, leading to reduced Akt phosphorylation and subsequent inhibition of cell migration. Our study reveals a novel tumor-suppressing signaling pathway associated with kinase-activated EphB3 in NSCLC, and provides a potential therapeutic strategy by activating EphB3 signaling, thus inhibiting tumor metastasis.

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 3434. doi:1538-7445.AM2012-3434
Abstract 3440: Effect of neoadjuvant therapy for the number of lymph node micrometastasis in esophageal squamous cell carcinoma
Authors: Lee Tomohiro , Tanaka Hiroaki , Yoshii Mami , Sakurai Katsunobu , Nagahara Hisashi , Kimura Kenjirou , Amano Ryousuke , Noda Eiji , Kubo Naoshi , Muguruma Kazuya , Yamada Nobuya , Yashiro Masakazu , Maeda Kiyoshi , Onoda Naoyoshi , Sawada Tetsuji , Nakata Bunzo , Ohira Masaichi , Ishikawa Tetsurou , Hirakawa Kosei
Published: August 2012

[Introduction] Esophageal squamous cell carcinoma (ESCC) often develops lymph node metastasis. Several study demonstrated that micrometastasis (MM) in lymph nodes was an independent prognostic factor for ESCC. Recently effect of neoadjuvant therapy including chemotherapy and chemoradiontherapy has been reported. The aim of this study was to investigate the affect of on MM in lymph nodes for ESCC. [Materials and methods] We retrospectively reviewed clinical data of consecutive162 patients with ESCC who underwent esophagectomy accompanied with systemic lymphadectomy in our institution from 2000 to 2010. Firstly, we analyzed the relapse free survival of 90 patients who underwent surgical resection without neoadjuvant therapy and assessed the impact of MM in lymph nodes on prognosis of ESCC by immunohistochemistry using anti-cytokeratin antibody. Next we examined MM in lymph nodes of 72 patients who underwent surgical resection with neoadjuvant therapy and evaluate the effect of neoadjuvant therapy on MM in lymph nodes. [Results] Of 5414 lymph nodes from 90 patients with cStage II/III (surgery group), we detected 311(5.1%) of overt metastasis detected by H&E staining and 124(2.3%) of MM in lymph nodes. The clinicopathological factors related to the postoperative relapse included pTNM Stage and number of lymph node metastasis with MM. Otherwise, in neoadjuvant Group, Of 3466 lymph nodes from 72 patients, we detected 154(4.4%) of overt metastasis and 19(0.4%) of MM. In neoadjuvant group, the rate of MM was decreased from 2.3% to 0.4% as compared to that of surgery group. Importantly, the rate of MM in upper mediastinal region was decreased by neoadjuvant therapy. [Conclusion] We demonstrated the significance of micrometastasis in lymph nodes for prognosis of esophageal cancer. Our findings suggested that neoadjuvant therapy may reduce the number of micrometastasis. Neoadjuvant therapy should be estimated as the standard therapy against esophageal squamous cell carcinoma.

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 3440. doi:1538-7445.AM2012-3440
Abstract 3455: p53 status determines the radio-sensitizing effect of an Akt kinase inhibitor in malignant gliomas
Authors: Patel Disha R. , Gordon Nicolaus , Chakravarti Arnab , Palanichamy Kamalakannan
Published: August 2012

Purpose: It has been reported that PI3K-Akt pathway, which plays a role in cell survival and apoptosis, is deregulated in many cancer types. In gliomas, amplification and constitutive activation of Akt, which can result from amplifications in TKIs including EGFR, IGFR1α, PDGFRα, etc. and mutations in PI3K, PTEN, etc. are thought to play a role in radiation resistance. Some have tried to modulate the PI3K/Akt pathway by targeting molecules upstream of Akt such as Ras and PI3K, while others have used inhibitors targeting molecules downstream of Akt, such as mTOR. These inhibitors have been shown to reduce cancer cell growth in vitro and in vivo, but have had limited success in clinical settings as single agents. In our study, we have investigated the use of a phosphatidylinositol ether lipid analogue (PIA) that directly inhibits Akt activation as a radiosensitizer for GBM. Materials and Methods: We utilized multiple established (U87, LN18, LN229) and primary (VC3, MGH8, OSU61) cell lines with varying molecular and genetic signatures to determine radiosensitizing effects of PIA. We performed Annexin V, clonogenic survival, and COMET assays on glioma cells treated with PIA, radiation (RT), and a combination of PIA and RT. Western blot was performed using these cell lines to examine the changes in expression of PI3K-Akt pathway, apoptotic, and pro-survival proteins following treatment. Furthermore, we sequenced tumor suppressor genes p53 and PTEN in all cell lines used in this study. Gene silencing of p53 was done in U87 cells via a lentiviral transfection of shRNA. Results and Conclusions: PIA decreases the activation of Akt to a considerable extent (∼80%), induces apoptosis, and reduces clonogenicity of all cell lines tested. A subset of cell lines did show enhanced radiosensitization when PIA is combined with RT. Further detailed investigation of differential radiosensitizing effects led us to discover the link between p53 status, Akt kinase inhibition, and radiosensitization. GBMs with intact p53 had no radiosensitizing effect after Akt kinase inhibition, whereas radiosensitization was observed if either p53 is null or mutated. This observation was further validated using isogenic U87 p53 knockdown cells. Surprisingly, MGMT status, a predictive marker of outcome in GBM, did not determine radiosensitizing effects of PIA in the GBM cells tested. Through this preclinical study, we have identified which subset of GBM patients will derive benefit from Akt kinase inhibition and the investigations conducted justify future clinical trials and lays ground work for phase I study.

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 3455. doi:1538-7445.AM2012-3455
Abstract 347: The role of the epithelial-mesenchymal transition in the maintenance of stemness in neural and glioma stem cells
Authors: Fernandez Nestor , Wu Megan , Das Sunit
Published: August 2012

Despite advances in treatment, Glioblastoma multiforme (GBM) continues to carry a horrible prognosis with an average overall survival of only 14 months [Stupp et al, 2005]. GBMs are heterogeneous tumors that contain a subpopulation of stem-cell like cells called glioblastoma stem cells (GSCs). GSCs appear to be chemotherapy- and radiation-resistant and are thought to be responsible for GBM progression [Kang et al, 2007; Bao et al, 2006]. More effective GBM therapies must target GSCs, underscoring the importance of understanding GSC biology. Our study examines the epithelial-mesenchymal transition (EMT) and its converse, the mesenchymal-epithelial transition (MET), in the context of GSCs and neural stem cells (NSCs). We hypothesize that the zinc-finger enhancer binding transcription factor (ZEB)/miR-200 feedback loop is relevant in the context of GSCs and is modulated by TGF-β and BMP. We have found that ZEB1 and miR-200 define divergent glioma cell populations: qRT-PCR shows differential expression of ZEB1 and miR-200 in mature human astrocytes, human fetal NSC, and human GSCs (GliNS1). In GSCs, BMP antagonism (noggin) or TGF-β stimulation results in increased ZEB1 expression. Conversely, BMP4 stimulation increases miR-200b/c, while noggin or TGF-β treatment decrease miR-200b/c expression. BrdU proliferation studies show increased proliferation with TGF-β stimulation and decreased proliferation with BMP treatment in three GSC lines. TGF-β signaling also confers an invasive phenotype in GSCs while BMP4 treatment decreases invasion, suggesting a role for TGF-β and BMP parallel to that seen in EMT and MET. Western blot analysis reveals that treatment of GSCs with TGF-β induces phosphorylation of Stat3, which has been shown to be a driver of EMT [Carro MS, 2010]. Interestingly, BMP and TGF-β treatment does not change Sox2 protein expression, suggesting that the GliNS1 GSCs retain their stem cell identity. However, Bmi1 increases with BMP stimulation and decreases with TGF-β treatment, while ID1 increases with BMP treatment, suggesting that the stem-ness in GSCs is modulated by these cytokines. In conclusion, BMP and TGF-β control the ZEB1/miR-200 feedback loop in GSCs and affect cell proliferation and invasive capability. We are continuing studies to determine the relevance of TGF-β and BMP signaling to patients with GBM.

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 347. doi:1538-7445.AM2012-347
Abstract 3473: Beta catenin inhibitor ICG-001 attenuates proliferation of hepatic tumor initiating cells in Pten deficient models
Authors: Medina Vivian Galicia , Kassa Anketse D. , Stiles Bangyan L.
Published: August 2012

Progenitor or tumor initiating cells (TICs) are “altered” stem cells with the capacity to form solid tumors. Tumor suppressor PTEN (phosphatase and tensin homologue deleted on chromosome ten) is aberrantly expressed in liver cancers. Liver specific Pten (Pm) mice develop liver cancer following an extensive phase of chronic lipid accumulation and demonstrate escalating levels of hepatic injury markers from 6-12M, prior to hepatic progenitor cell proliferation. In addition, TUNEL analysis revealed that hepatocytes from Pm mice undergo extensive apoptosis relative to control mice. We hypothesize that hepatocyte cell death induced by hepatic injury presents an opportunity for TICs to proliferate and consequently form mixed lineage tumors. Attenuation of hepatic injury by Akt2 deletion reduces progenitor cell proliferation and delays tumor development. Our analyses also revealed that the Wnt/β-Catenin signaling pathway is the likely molecular mediator of TIC proliferation in our Pm model. Wnt ligands including 7a and 10a as well as Wnt signaling receptor Fzd 2 are induced. In this study, we investigate the role of the Wnt/β-Catenin pathway in the activation of hepatic progenitor cells in the Pten deletion liver cancer model using novel Wnt/β-catenin inhibitor ICG-001. ICG-001 is a small molecule which specifically inhibits β-Catenin/CBP interaction. Coactivator CBP/β-Catenin/T cell factor (TCF) mediated transcription has been reported as critical for stem cell/progenitor cell proliferation. Here we demonstrate that ICG-001 significantly attenuates proliferation of hepatic cancer stem cell line P0 in vitro and also inhibits hepatotoxin induced proliferation of hepatic TICs in vivo in liver Pten null mice. Our preliminary results indicate that targeting the cancer stem cell niche may prove to be a viable approach to inhibit liver tumorigenesis.

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 3473. doi:1538-7445.AM2012-3473
Abstract 3478: CD44+CD24low+ progenitors in ER negative breast cancer have higher Notch1 activation, self-renewal, and chemo resistance and generate CD44+CD24neg cells and tumors that metastasize
Authors: Azzam Diana J. , Elger Katherine Drews , Zhao Dekuang , Ranganathan Prathibha , Capobianco Tony , Minn Andy J. , Creighton Chad , Picon Manuel , Sun Jun , Pan Chendong , Wander Seth A. , Ashry Dorraya El , Slingerland Joyce M.
Published: August 2012

CD44 surface expression is associated with self renewal in several cancers. While CD44+/CD24neg/low/ESA+ breast cancer subpopulations are enriched for cancer stem cells (CSC), the relative contributions of CD24 negative versus low subpopulations to stemness and metastasis is poorly defined. Here we show that CD44+/CD24low+ CSC give rise to CD44+/CD24neg progeny with reduced tumorigenicity and altered drug sensitivities. CD44+/CD24low+ subpopulations in MDA-MB-231 and in primary dissociated ER negative breast cancers show greater sphere and soft agar colony formation, Notch1 activation, Sox2 and Nanog expression and chemo resistance compared to CD44+/CD24neg. CD44+/CD24low+ can self-renew and give rise to CD44+/CD24neg cells, while CD44+/CD24neg progeny are exclusively CD44+/CD24neg. Tumorigenicity was increased and metastasis arose exclusively from orthotopic CD44+CD24low+ xenografts. CD44+/CD24low+ had greater expression of metastasis- and embryonic stem cell- associated and Notch pathway activated genes than CD44+/CD24neg cells. Moreover, MDA-MB-231 cells overexpressing Notch1 intracellular domain (N1-ICD) had higher Sox2 expression, and higher indices of CSC self-renewal (higher % CD44+/CD24low+, ALDH1 activity, sphere and soft agar colony formation), all of which were abrogated by Sox2 knockdown. Gamma secretase inhibition reduced ES-TFs and self-renewal in CD44+CD24low+ progenitors, but had no effect on proliferation or survival of CD44+CD24neg cells supporting further clinical development of Notch targeting drugs for cancer treatment in humans. Thus, CD44+CD24low+ and CD44+CD24neg CSC in ER negative breast cancer have distinct properties. CD44+CD24low+ can self-renew and generate CD44+CD24neg progeny. Orthotopic xenograft CD44+CD24low+ tumors arose with reduced latency with higher frequent, were larger, and the sole source of metastasis. Notch1 activation of Sox2 drives CD44+CD24low+ self-renewal and is blocked by GSI providing a strong rationale for use of GSI as CSC targeting agents in ER negative breast cancer.

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 3478. doi:1538-7445.AM2012-3478
Abstract 3487: Distant delivery of glioma-restricted oncolytic adenovirus loaded into a neural stem cell-based carrier prolongs the survival of animals with human glioblastoma
Authors: Ahmed Atique U. , Thaci Bart , Tobias Alex , Alexiades Nikita G. , Han Yu , Ulasov Ilya I. , Aboody Karen , Lesniak Maciej S.
Published: August 2012

Despite recent advancements in the treatment of brain tumors, the prognosis for patients with malignant glioblastoma (GBM) still remains poor due to the invasive nature of these tumors. In this context, our lab and others have investigated the unique inherent tumor tropic properties of neural stem cells (NSCs) as a novel platform for the targeted delivery of oncolytic adenovirus for anti-glioma therapy. NSCs hold great promise for glioma therapy because of their inherent ability to target satellite tumor foci throughout the brain. In this study, we report that the infection/loading of NSCs with oncolytic adenovirus upregulate various chemoattractant receptors such as CXCR4 (10-fold, P=0.0054), VEGFR2 (5-fold, P=0.0143) and significantly enhances glioma specific migration of NSCs both in vitro and in vivo. In a human glioma xenograft animal model, virus loaded NSCs retain a majority of their tumor homing capacity with minimal effect on their viability, and are capable of delivering a therapeutic payload to tumor cells located far from the original implanted site. Moreover, NSC-based cell carriers significantly reduce the non-specific distribution of therapeutic virus in the animal brain. Finally, distance delivery of NSCs-loaded with CRAd-S-pk7, a glioma restricted oncolytic adenovirus, in multiple orthotropic xenograft models of human glioma have shown the ability to inhibit tumor growth and increase median survival by about 38 days (54%) as compared to the animal group treated with the CRAd-S-pk7 alone (P=0.0416). Our data suggest that NSC-based cell carriers have the potential to improve the therapeutic efficacy of anti-glioma virotherapy by not only protecting the therapeutic virus from the host immune system, but also by enhancing targeted delivery of therapeutic virus at disseminated tumor sites.

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 3487. doi:1538-7445.AM2012-3487
Abstract 351: 2-Methoxyestradiol targets RON kinase and FLIP to inhibit epithelial-mesenchymal transition in prostate cancer
Authors: Batth Izhar Singh , Gong Jingjing , Bedolla Roble G. , Weintraub Susan T. , Kumar Addanki Pratap
Published: August 2012

Prostate Cancer (PCA) is the second leading cause of cancer-related deaths among men in the United States. Despite significant progress in our understanding of prostate biology, early detection and treatment, the five-year survival for hormone refractory metastatic prostate cancer (HRPCA) patients is less than 30%. Current therapies targeting PCA are not effective against the metastatic stage. 2-Methoxyestradiol (2-ME2) is a metabolic product of 17-β-estradiol and is found in trace amounts in the body; it has shown anti-tumorigenic properties in multiple cancer models. Previous studies from our lab have shown that 2-ME2 can induce apoptosis in PCA cells by blocking cell cycle progression, and also inhibits PCA growth and progression in TRAMP mice. However, the precise mechanism of action of 2-ME2 has not yet been elucidated. In order to classify the mechanism behind PCA inhibition by 2-ME2, we tested its effectiveness on an array containing genes associated with epithelial-mesenchymal transition (EMT). We identified Recepteur d'Origine Nantais (RON) as one of the most significantly downregulated genes in response to 2-ME2 treatment. RON has been shown to promote cell invasiveness, migration, survivability, and proliferation in many cancers such as breast, colon, lung, and pancreatic. However, very little is known about RON's signaling mechanism in PCA. We've found RON to be highly increased in advanced stage PCA cell lines. RON knockdown shows a concomitant decrease in the expression of FLICE-like inhibitory protein (FLIP). FLIP is an anti-apoptotic protein which prevents death receptor-induced apoptotic signaling. FLIP has been found to promote EMT as well as androgen-independent PCA growth. Stimulation with the RON ligand HGF yielded elevated expression of RON and FLIP in PCA cell lines while treatment with 2-ME2 caused downregulation. Immunoprecipitation experiments showed RON to be associated with the androgen-induced proliferation inhibitor (APRIN) in PCA cells. Our data suggest that RON/FLIP crosstalk plays a role in prostate cancer. Therefore, targeting RON/FLIP signaling could have potential therapeutic benefit for metastatic PCA. Supported by NIH CA 135451 (APK).

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 351. doi:1538-7445.AM2012-351
Abstract 3512: Expanded human regulatory iNKT cells exhibit direct cytotoxicity against hematolymphoid tumor targets
Authors: Luszczek Wioleta , Morales-Tirado Vanessa , van der Merwe Marie , Kudo Ko , Campana Dario , Pillai Asha
Published: August 2012

CD1d-restricted invariant natural killer T (iNKT) cells are rare but potent innate regulatory cells capable of immune modulation via robust production of Th1/Th2 cytokines, as well as tumor immunosurveillance via direct cytotoxicity. Protocols to expand iNKT cells and augment their cytotoxicity would broaden their application in allogeneic transplantation and adjuvant anti-tumor immunotherapy. We have optimized a protocol for ex vivo expansion of highly purified human CD3+CD4negVα24+ iNKT cells from human peripheral blood mononuclear cells (PBMC). PBMCs were stimulated with an iNKT-specific glycolipid (α-GalCer), recombinant IL-2 and IL-7 and sorted to > 98% purity at day 7. Sorted iNKT cells were further expanded in the presence of irradiated allogeneic PBMCs, Vα24-specific TCR stimulation, and low-dose IL-2 and IL-7 for 21 days. This results in roughly 10^3-fold expansion, with peak yields of at day 21-28 of expansion (range 1.5 x 10^5 - 10.6 x 10^6 iNKT cells from 1 x 10^8 - 3.5 x 10^8 starting PBMC). At 28 days, these iNKT cells secrete high levels of IL-13, TNF-α, GM-CSF, and IL-4, moderate IL-2 and IFN-γ in anti-CD2/CD3/CD28 bead-stimulated Luminex supernatant assay. Day 28 expanded iNKT cells are dose-dependent suppressors of sorted autologous CD3+CD8+ effector T cells in 72-hr mixed leukocyte reaction (MLR). Anti-CD2/CD3/CD28 activation of these iNKT cells induced expression of high levels of key cytolytic effector molecules, including granzyme B. We therefore measured cytotoxicity of activated day 28 iNKT cells against various hematolymphoid tumor targets following co-incubation of iNKT cells versus control effector populations with firefly luciferase-transduced RS4:11 and Nalm6 (B-ALL), U937 (monocytic), and K562 (CML) targets. iNKT cell effectors (E) demonstrated dose-dependent cytotoxicity against B-lymphoid tumor targets (T) (e.g.: Nalm6: 37.7% at E:T 2:1, 17.7% at E:T 1:1, 7.7% at E:T 0.5:1), with no significant cytotoxicity against myeloid targets (e.g.: K562: 11.8% at E:T 2:1, 6.7 at E:T 1:1, 1.8 at E:T 0.5:1). Our results indicate that human iNKT cells expressing high levels of Th2 and regulatory cytokines can be potently expanded ex vivo and manifest cytotoxicity against B-ALL targets, supporting their potential application in tailored immunotherapy in the pre- and post-transplant setting.

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 3512. doi:1538-7445.AM2012-3512
Abstract 3534: Human Th17 cells have stem cell-like features and promote long-term tumor immunity
Authors: Kryczek Ilona , Zhao Ende , Liu Yan , Wang Yin , Vatan Linda , Szeliga Wojciech , Moyer Jeffrey , Klimczak Aleksandra , Lange Andrzej , Zou Weiping
Published: August 2012

Th17 cells play a role in multiple pathological scenarios. However, their functional phenotype, survival pattern and the underlying controlling mechanisms are poorly investigated in humans. This significantly hampers clinical application of targeting Th17 cells. Now we have studied Th17 cells in the pathological microenvironments of graft-versus-host disease, ulcerative colitis and cancer in humans. Th17 cells are increased in the chronic phase of these diseases. Th17 cells phenotypically resemble to terminally differentiated memory T cells, but are different from central memory, exhausted and senescent T cells. Despite their phenotypic markers of terminal differentiation, in vivo adoptive transfusing experiments demonstrate that Th17 cells mediate and promote long-term anti-tumor immunity. Furthermore, Th17 cells have important functional features including high capacity of proliferative self-renewal, potent persistence and apoptotic resistance in vivo, and the plasticity of converting into other types of T helper cells. These features are accompanied with relatively specific gene signature in Th17 cells including abundant anti-apoptotic genes. Moreover, we investigated the molecular mechanisms controlling their functional characters. We demonstrate that hypoxia inducible factor (HIF)1α and Notch collaboratively control key anti-apoptosis Bcl-2 family gene expression and function in Th17 cells. Altogether, the data indicate that human Th17 cells may be a long-lived proliferating effector memory T cell population with unique genetic and functional characters. These characters may be important determinants in Th17 cell biology. Targeting Th17-associated signaling pathway would be therapeutically meaningful for treating patients with autoimmune disease and advanced tumor.

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 3534. doi:1538-7445.AM2012-3534
Abstract 3538: The HIF-1α hypoxia response in mouse tumor-infiltrating T lymphocytes induces functional CD137 (4-1BB) for immunotherapy
Authors: Palazon Asis , Martinez-forero Ivan , Teijeira Alvaro , Morales-Kastresana Aizea , Alfaro Carlos , Sanmamed Miguel Fernandez de , Hervas-Stubbs Sandra , Jure-Kunkel Maria , Aragones Julian , Melero Ignacio
Published: August 2012

The response to hypoxia modulates the expression of multiple genes. The tumor microenvironment of transplanted and spontaneous mouse tumors is profoundly deprived of oxygenation as confirmed by PET imaging. CD8 and CD4 tumor-infiltrating T lymphocytes (TILs) of transplanted colon carcinomas, melanomas and spontaneous breast adenocarcinomas are CD137 (4-1BB) positive, as opposed to their counterparts in tumor draining lymph nodes and spleen. Expression of CD137 on activated T lymphocytes is markedly enhanced by hypoxia and the prolyl-hydroxilase inhibitor DMOG. Importantly, hypoxia does not up-regulate CD137 in inducible HIF-1α knock-out T cells, and such HIF-1α-deficient T cells remain CD137-negative even when becoming TILs, in clear contrast to co-infiltrating HIF-1α-sufficient T cells. The fact that CD137 is selectively expressed on TILs was exploited to confine the effects of immunotherapy with agonist anti-CD137 monoclonal antibodies (mAb) to the tumor tissue, thereby avoiding liver inflammation, while still permitting synergistic therapeutic effects with PD-L1/B7-H1 blockade.

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 3538. doi:1538-7445.AM2012-3538
Abstract 3540: Prostate cancer-infiltrating mast cells suppress anti-tumor immunity through a TGFβ and IL-13 dependent mechanism
Authors: Watkins Stephanie K. , Hurwitz Arthur A.
Published: August 2012

Immune suppression is a major obstacle in the design of successful immune-based therapies for cancer. Using an experimental model of prostate cancer, we previously demonstrated the rapid induction of tolerance and suppressive activity in adoptively transferred tumor antigen-specific cytotoxic T cells. The mechanism of this tolerance induction involved T cell interactions with tumor-associated dendritic cells (TADCs) which express elevated levels of the forkhead box protein, FOXO3. We now propose a mechanism by which tumor-infiltrating mast cells exert immune suppressive activity on both T cells and DCs. Mast cells were identified as approximately 30% of the total CD45+ tumor-infiltrating leukocytes and were capable of degranulation and promoting immune suppression. Purified tumor-associated mast cells (TA-MC) spontaneously secreted IL-13 and TGF-β which were further increased by FcαR cross-linking or LPS stimulation. The suppression of Ag-specific T cells and the development of tolerogenic DC were found to be dependent on TA-MC secretion of TGF-β and IL-13 during in vitro co-culture. Further, blocking IL-13 and TGF-β in vivo prevented the induction of DC tolerogenicity and the induction of T cell tolerance, which, in turn, resulted in reduced tumor burden. Our findings reveal novel mast cell-mediated mechanisms that favor immune tolerance over anti-tumor immunity and therefore provide promising targets for the enhancement of cancer immunotherapy.

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 3540. doi:1538-7445.AM2012-3540
Abstract 3544: Role of tumor-infiltrating gammadelta T lymphocytes in breast cancer
Authors: Ma Chunling , Zhang Qunyuan , Ye Jian , Zhang Yanping , Wevers Eric , Schwartz Theresa , Hunborg Pamela , Vavares Mark A. , Hoft Daniel F. , Hsueh Eddy C. , Peng Guangyong
Published: August 2012

Breast cancer is the second leading cause of cancer-related deaths in women worldwide. Increasing evidence suggests that immunotherapy is a promising strategy for treating breast cancer, and understanding of the roles of different subtypes of tumor-infiltrating lymphocytes (TILs) in the tumor immunosuppressive microenvironment may be essential to cancer treatment and elimination. We recently discovered that enriched gammadelta1 T cell populations in the breast cancer TILs can suppress naïve and effector T cell responses and block the maturation and activities of dendritic cells (DC). In order to investigate the functional role of tumor-infiltrating gammadelta T cells in the immune pathogenesis of breast cancer, we further performed immunohistochemical staining to analyze gammadelta T cells, as well as CD4+, CD8+, IL-17-producing and FoxP3+ T cells in 50 freshly frozen tumor tissues from different stages of identified primary breast cancer and paired normal breast tissues. We found that the numbers of gammadelta T cells among tumor tissues were significantly increased compared with those in normal breast tissues in breast cancer patients. High numbers of gammadelta T cells were present in the patients with high tumor grades and lymph node metastases. Importantly, the patients with a high proportion of gammadelta T cells showed poorer survival rates in comparison to those with a low proportion of gammadelta T cells in breast cancer. These results indicate that gammadelta T cells constitute a dominant population existing in the breast tumor suppressive microenvironment that is negatively associated with clinical outcome, which may be an important cancer immunotherapeutic target for breast cancer.

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 3544. doi:1538-7445.AM2012-3544
Abstract 3573: Urine and serum biomarkers as early detection tools for ovarian cancer
Authors: Nolen Brian M. , Lomakin Aleksey , Bast Robert , Lu Karen , Marrangoni Adele , Lokshin Anna E.
Published: August 2012

Background: The measurement of biomarkers present in the bodily fluids of ovarian cancer patients represents an emerging strategy for early detection. To date, a great deal of related work has been devoted to blood, given its systemic exposure and extensive availability through tissue banks. However, blood is a dynamic biofluid with a proteome under continuous metabolic and homeostatic regulation. Alternatively, urine represents a biofluid that is inherently quiescent in that all molecular and proteolytic activity is largely complete upon sampling. Additional characteristics of urine, including its high level of temperature stability and low-complexity matrix resulting from renal filtration, suggest that this biofluid may serve as an effective alternative or companion to blood in biomarker analyses. Methods: A set of urines obtained from over 700 women, including patients diagnosed with ovarian cancer, patients diagnosed with benign pelvic lesions, and healthy donors, was tested for 39 biomarkers which demonstrated previous associations with ovarian cancer. In an effort to identify additional useful biomarkers, 122 candidate biomarkers were evaluated in a separate set of 150 sera obtained from ovarian cancer patients along with benign and healthy controls. Significantly altered biomarkers were identified in each analysis and the full biomarker dataset was examined by multivariate analysis to identify multianalyte panels capable of discriminating ovarian cancer patients from controls with high accuracy. Results: Numerous significantly altered biomarkers were identified in both urine and serum. The classification performance of several biomarkers was found to differ in urine vs. serum with an overall trend in favor of urine biomarkers. Separate urine and serum biomarker panels consisting of 3-4 analytes were identified which could discriminate ovarian cancer from healthy controls at a sensitivity of 96-97% at 95% specificity. Improvement upon this classification efficiency was observed when the best urine and serum biomarkers were combined into a single panel. The combination of urine HE4 and serum CA 125 was most effective in the discrimination of ovarian cancer from benign controls. Conclusions: Urine and serum biomarkers offer complementary information regarding ovarian cancer status. Their combined use may lead to the development of effective early detection tools.

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 3573. doi:1538-7445.AM2012-3573