bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 5146: Reprogramming metastatic melanoma with the embryonic chick neural crest microenvironment
Authors: Kasemeier-Kulesa Jennifer C. , Romine Morgan , Seftor Elisabeth A. , Seftor Richard EB , Hendrix Mary JC , Kulesa Paul M.
Published: February 2011

Our previous work demonstrated that metastatic melanoma cells transplanted into the chick embryonic neural crest microenvironment invade into the host tissue, migrate along host neural crest cell migratory pathways without forming tumors, and assume a neural crest cell-like phenotype (Kulesa et al., 2006; Hendrix et al., 2007; Kasemeier-Kulesa et al., 2008). Here, we aim to elucidate the tissues and signals within the ancestral embryonic neural crest microenvironment capable of reprogramming metastatic melanoma cells. We find that in vitro co-culture of C8161 melanoma cells with various chick tissues, including head, branchial arches, and trunk, reveal that embryonic day 4 trunk tissue induces an overall 6% re-expression of MART-1, a marker of a melanocyte like phenotype. This MART-1 re-expression is highest in cells in close proximity to the co-cultured tissue. We also tested known differentiation factors present in embryonic day 4 trunk tissue, and found NGF to have a 5-fold increase of MART-1 re-expression on C8161 cells. Further work will help establish whether the neural crest embryonic microenvironment initiates a more stable, long-lived reprogramming of the aggressive, metastatic melanoma cells to a more benign neural crest cell phenotype.

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 5146.
Abstract 5148: A neural stem cells marker fatty acid binding protein 7 (FABP7) is involved in proliferation and invasion of glioblastoma cells
Authors: Rosa Antonella De , Rossi Marco , Pellegatta Serena , Tunici Patrizia , Malusa Federico , Magnoni Letizia , Raggiaschi Roberto , Bakker Annette
Published: February 2011

Malignant brain tumours are among the most deadly cancers. Current treatment strategies, such as surgery, chemotherapy and radiotherapy only modestly improve patient survival. The limited success of these strategies is largely due to a high incidence of tumor recurrence after the treatment. Recent evidences suggested that a rare population of stem-like cells (or tumour initiating cells) present in brain tumours might be responsible for the aggressiveness and recurrence of these tumours. These cells, named brain tumor stem-like cells (BTSC), share many properties of normal neural stem cells, including self renewal, extended proliferation, formation of neurospheres (when cultured in vitro in the presence of growth factors) and potential to differentiate into neurons and glial cells. Further investigations have suggested that studying this BTSC population in glioblastoma (GBM) is a more relevant system for exploring glioma biology than the adherently growing glioma cell lines.

In the current study we identified target genes and proteins that are differently expressed between adherent glioma cell lines and sphere growing BTSC, using microarray and differential proteomics, respectively.

Fatty acid-binding protein 7 (FABP7) was identified as overexpressed in BTSC compared to glioma adherent cell lines both at the gene as well as at the protein level.

FABP proteins are a family of small, highly conserved, cytoplasmic proteins that bind long-chain fatty acids and other hydrophobic ligands. FABPs are thought to play important roles in fatty acid uptake, transport, and metabolism. Functionally, FABPS were described to play a role in gene regulation, cell signaling, cell growth and differentiation. The functional role of FABP7 in glioblastoma was investigated by assessing the effect of silencing FABP7 on migration and cell proliferation of glioblastoma cells. We observed that down-regulating FABP7 expression significantly reduced in vitro cell proliferation and even more the migration of BTSC.

Since increased tumour infiltration was reported as a consequence of radiotherapy, the potential involvement of FABP7 in this process was evaluated. Irradiated BTSCs displayed significantly higher FABP7 protein levels and therefore we suggest that FABP7 may contribute to the radiotherapy-induced aggressive phenotype of BTSCs.

Moreover, further mechanistic investigations indicated that directly or indirectly reducing the expression of FABP7 significantly impacts on the behaviour of glioblastoma derived BTSC.

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 5148.
Abstract 5150: Differential epigenetic regulation of sonic hedgehog GLI1 on downstream target genes in medulloblastoma and astrocytomas
Authors: Shahi Mehdi H. , Fan Xing , Sinha Subrata , Afzal Mohammad , Eberhart Charles , Castresana Javier S.
Published: February 2011

Sonic hedgehog (Shh) signaling in astrocytic tumorigenesis has not been as thoroughly investigated as in medulloblastoma. We have attempted to understand the regulatory status of GLI1, the immediate activator of Shh signaling pathway on its downstream target genes PTCH1, Cyclin D2, Plakoglobin, NKX2.2 and PAX6. We attempted to correlate the expression pattern of GLI1 and its regulated genes in 14 cell lines (8 of astrocytoma and 6 of medulloblastoma) and 41 primary samples (27 astrocytomas and 14 medulloblastomas). We also determined the promoter methylation of Cyclin D2 and PTCH1 in the 14 cell lines and in a total of 58 primary samples (44 astrocytomas and 14 medulloblastomas).

The dependence of the GLI1 modulated genes on expression of GLI1 was confirmed by knockdown experiments with the help of GLI1-siRNA. We also found that Shh-GLI1 signaling shows different patterns of regulation of downstream target genes in medulloblastomas and astrocytomas: Cyclin D2 and Plakoglobin were upregulated in medulloblastoma but downregulated in astrocytoma; however, PAX6 and NKX2.2 showed over all a low expression pattern of expression in both tumors. However, in several cell lines (A172, SW1783, T98G, CCF-STTG-1 and GOS-3), exposure to both 5-Aza-2′deoxycytidine and Trichostatin A increased the expression of the GLI1 downstream target gene Cyclin D2 (p=0.0014). These results were verified by promoter methylation studies in the 14 cell lines and the 58 primary samples. Our promoter methylation studies revealed a significant number of astrocytoma cell lines (63%) and primary astrocytomas (32%) showing that methylation of Cyclin D2 related to its own level of expression; something that did not occur in medulloblastoma. Compared to Cyclin D2, PTCH1 promoter methylation did not influence on PTCH1 expression in the cell lines and tumor samples.

Our results demonstrate that Shh-GLI1 signaling acts differentially for the regulation of target genes in medulloblastoma and astrocytoma. Secondly, PTCH1 and Cyclin D2 are under epigenetic regulation besides the transcriptional regulation exerted by GLI1. This finding may open new ways for treating patients by demethylating agents.

Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend.

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 5150.
Abstract 5155: BMP-2 inhibits the tumorigenicity of human osteosarcoma tumor-initiating cells
Authors: Wang Lin , Park Paul , Zhang Huina , Marca Frank La , Lin Chia-Ying
Published: February 2011

Bone morphogenetic proteins (BMPs), members of the transforming growth factor (TGF)-β superfamily, were originally identified as molecules that induce bone and cartilage formation and are now considered as multifunctional cytokines. More recently, there have been a number of studies showing the inhibitory effects of BMP-2 on tumorigenesis in many cancers. However, very little has been investigated to date about the role of BMP-2 in human osteosarcoma. In the present study we show that BMP-2 inhibits the tumorigenicity of an aggressive human osteosarcoma cell line, OS99-1. Based on the stem cell theory of cancer, a subpopulation of cells showing characteristics of stem cells was identified using high aldehyde dehydrogenase (ALDH) activity (ALDHbr cells) from xenografts. These stem-like cells were found to be highly tumorigenic and were classified as tumor initiating cells. In vitro, the growth of the sorted tumorigenic ALDHbr cells was significantly inhibited by the addition of 300 ng/ml of BMP-2 in the presence of 0.5% fetal bovine serum (FBS) for 48h, as evaluated by cell proliferation assay. In addition, using RT-PCR analysis, we found BMP-2 down-regulated the expressions of embryonic stem cell markers Oct3/4, Nanog, and Sox-2. Conversely there was an increase in the transcription of osteogenic markers Runx-2 and Collagen Type I in the sorted tumorigenic ALDHbr cells treated with the same BMP-2 solution for 48h compared with the same volume of vehicle. In vivo, tumor formation in animals was compared to a control receiving ALDHbr cells maintained under identical conditions without BMP-2. All animals receiving untreated ALDHbr cells developed large tumor masses that showed characteristic osteosarcoma features, including nuclear atypia, extensive neovascularization, and high mitotic activity. Conversely, ALDHbr cells treated with 30μg/animal of BMP-2 did not form significant tumors. Further immunostaining confirmed most Ki-67 positive cells were shown in the tumor sections from mice injected with ALDHbr cells without BMP-2, whereas few Ki-67 positive cells were present in the sections of tumor from ALDHbr cells treated with BMP-2. Based on these results, it appears that BMP-2 may suppress tumor growth by inducing the differentiation of tumor -initiating cells. BMP-2 or BMP-2-mimetic drugs, if properly delivered to tumor and combined with traditional therapies, may therefore provide a new therapeutic option.

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 5155.
Abstract 5156: Wnt5A enhances the malignant phenotype of lung cancer cells via non-canonical signaling
Authors: Ripley Robert T. , Rao Mahadev , Kemp Clinton D. , Mathur Aarti , Hussain Mustafa , Hong Julie A. , Zhang Mary , Schrump David S.
Published: February 2011

Objective: Previously, we reported that cigarette smoke condensate dramatically increases tumorigenicity of lung cancer cells via epigenetic silencing of Dickkopf-1(Dkk-1), which encodes an inhibitor of Wnt stem cell signaling. Furthermore, we demonstrated that knock-down of Dkk-1 up-regulates Wnt5A in lung cancer cells. Expression of this non-canonical Wnt ligand correlates with adverse outcome in lung cancer patients. The present study was undertaken to ascertain if Wnt5A directly modulates the malignant phenotype of lung cancer cells. Methods: Calu-6, H358, A549, and H841 lung cancer lines were transduced with lentiviral vectors expressing Wnt5A or control sequences. Cell count, scratch, and matrigel assays were used to evaluate in-vitro proliferation, migration, and invasion of transduced cells. Similar experiments were performed with exogenous recombinant Wnt5A (rWnt5A). Nude mouse xenograft experiments were used to assess tumorigenicity of lung cancer cells transduced with Wnt5A or vector controls. Canonical and non-canonical Wnt signal pathways were evaluated using western blot, promoter-reporter, and focused quantitative RT-PCR array techniques. Results: Wnt5A transduced lung cancer cells exhibited significantly increased proliferation, migration, and invasion relative to vector controls (p<0.05; 2-tailed t-test); this phenomenon was pronounced under low serum conditions, and was observed following exposure of parental cells to rWnt5A. Relative to vector controls, Calu-6 and H358 cells constitutively expressing Wnt5A exhibited increased tumor take (p<0.05) and size of subcutaneous xenografts (p<0.05). Whereas microarray and Wnt Super Array experiments demonstrated upregulation of genes implicated in canonical as well as non-canonical Wnt signaling, western blot and promoter-reporter assays revealed that Wnt5A predominantly mediated activation of non-canonical signaling. Calu-6 and H358 cells constitutively expressing Wnt5A exhibited upregulation of the nuclear orphan receptor, ROR2, as well as activation of c-jun terminal N-kinase (Jnk) and c-jun kinase, which mediate non-canonical Wnt signaling. Consistent with these findings, rWnt5A abrogated rWnt3A-mediated activation of β-catenin signaling in lung cancer cells. Conclusion: Wnt5A enhances proliferation, migration, invasion, and tumorigenicity of cultured lung cancer cells via non-canonical pathways. Collectively, these findings warrant evaluation of inhibitors of Wnt5A-mediated signaling for lung cancer therapy.

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 5156.
Abstract 5157: Functional and regulatory role of src kinase in podoplanin expressing glioma cancer stem cells
Authors: Ezhilarasan Ravesanker , Goodman Lindsey D. , Love Patrice N. , Aldape Kenneth D. , Sulman Erik P.
Published: February 2011

The majority of patients with malignant gliomas, the most common and most lethal brain tumor, develop disease recurrence despite aggressive, multi-modality therapies. This treatment resistance has been attributed to a small population of tumor-initiating glioma cancer stem cells (GSCs). We recently described a new marker of GSCs, podoplanin (pdpn), based on analyses of gene expression profiling data and have found it to more accurately identify the stem cell population than other described markers, such as CD133, and to play a functional role stem cell phenotype. Pdpn expressing cells formed neurospheres at high rates in vitro, were more resistant to ionizing radiation, and initiated tumors in vivo. Pdpn belongs to a mesenchymal gene signature that is characteritic of poor patients survival from malignant gliomas. To identify the mechasism of pdpn within GSCs, we evaluated putuative binding partners to this transmembrane glycoprotein. One such partner, target of myb-like-1 (tom1l1) has been shown to regulate the Src oncogene pathway. The cytoplasmic tyrosine kinases of the Src family (SFK) play important roles in signal transduction induced by growth factors leading to DNA synthesis, cytoskeletal rearrangement, and receptor endocytosis. We therefore hypothesized that pdpn's role GSCs is due to its interaction with the Src pathway. Our aim for this study was to analyze downstream signaling events of Src in association with pdpn and its functional significance in glioma stem cell maintanence, invasion, and chemoradiation resistance. Using primary GSC and established glioma cell lines engineered to either overexpress or silence PDPN, we screened for SFK protein activation (phosphorylation) by immunblot. Changes in Src activation were correlated to in vitro invasion assays, in vivo tumor initiation, and radiation resistance. Preliminary data confirms binding of tom1l1 to pdpn and regulation of SFK activation. Engineered clones with shRNA targeting pdpn and/or tom1l1 were examined by expression profiling for downstream transcriptional changes. In summary, our data suggest a role of SFKs in the regulation of stemness by pdpn. These data integrate well with ongoing trials using Src-kinase inhibitors which may directly target the pdpn-expressing, GSC population in tumors and perhaps overcome the treatment resistance of these critical cells.

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 5157.
Abstract 5159: Kruppel-like factor 9 modulates the growth and differentiation of glioblastoma-derived neoplastic stem cells
Authors: Ying Mingyao , Xia Shuli , Laterra John
Published: February 2011

Glioblastoma (GBM) is the most prevalent brain tumor with high frequency of recurrence. During the past decade, the identification of neoplastic stem-like cells in diverse human cancers including GBM represents an important conceptual advance in cancer biology and suggests new therapeutic strategies. We found that retinoic acid (RA) induces growth arrest and cell differentiation of GBM-derived stem-like cells (GBM-SCs). RA treatment also inhibited the growth of GBM-SC xenografts in mice. We studied gene expression changes during RA-induced GBM-SC differentiation and found that kruppel-like factor 9 (KLF9) expression increased 8-fold after RA treatment. KLF proteins are evolutionarily conserved zinc finger-containing transcription factors that bind to GC-rich DNA regions. KLF9 is known to function as a progesterone receptor cofactor to regulate progesterone-responsive genes but its function in cancer and stem cell biology is essentially unknown. We used lentivirus-based methods to modulate KLF9 expression in GBM-SCs. KLF9 gain-of-function induced GBM-SC differentiation and inhibited GBM-derived neurosphere formation in clonogenic and neurosphere cell growth assays. KLF9 gain-of-function also prevented the growth of GBM-SC subcutaneous xenografts in mice. Knockdown of KLF9 suppressed RA-induced differentiation of GBM-SCs. These data indicate that KLF9 and possibly other KLF family members modulate transcriptional mechanisms that regulate GBM-SC growth, differentiation, and possibly tumor initiation. Currently, we are utilizing gene expression microarray and chromatin immunoprecipitation assays to study the KLF9 target genes. Our findings provide new insights pertaining to the molecular regulation of the GBM-SC phenotype and could lead to innovative therapeutic strategies for targeting the stem-like cell pool in malignant brain tumors.

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 5159.
Abstract 5160: Tumor cells potentiate mesenchymal stem cells-pericytes transition and requirement through PDGF-BB-NRP-1 axis
Authors: Dhar Kakali , Dhar Gopal , Majumder Monami , Haque Inamul , Banerjee Sushanta K. , Mehta Smita , Vanveldhuizen Peter J. , Banerjee Snigdha
Published: February 2011

New blood vessel formation or angiogenic switch is an essential event in the development of solid tumors and their metastatic growth. Tumor blood vessel formation and remodeling is a complex and multi-step processes. The differentiation and recruitment of mural cells including vascular smooth muscle cells and pericytes are essential steps in tumor angiogenesis. However, the role of tumor cells in differentiation and recruitment of mural cells is unknown. In this study we focus on the role of tumor cells in governing the differentiation of mesenchymal stem cells (MSCs) to pericytes and their recruitment in tumor angiogenesis process. We show that C3H/10T1/2 mouse embryonic mesenchymal stem cells, under the influence of different tumor cells-derived conditioned media, differentiate into mature pericytes. These differentiated pericytes, in turn, are recruited to bind with capillary-like networks formed by endothelial cells on the matrigel under in vitro condition. The degree of recruitment of pericytes into in vitro neo-angiogenesis is tumor cell phenotype specific. Interestingly, the invasive cells recruit less pericytes as compared to non-invasive cells. We identify tumor cell-secreted platelet derived growth factor (PDGF-BB) as a crucial factor controlling differentiation and recruitment processes through a cross-talk with NRP-1. These new insights into the roles of tumor cell-secreted PDGF in MSCs-fate determination may help in developing new antiangiogenic strategies to prevent the tumor growth and metastasis and also to treat cancers more effectively.

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 5160.
Abstract 5174: Characterization of glycolipid ligands for E-selectin
Authors: Marshall Jocelyn , Abram Courtney , Wood S. Matthew , Nimrichter Leonardo , Burdick Monica M.
Published: February 2011

The prevention, treatment, and curing of cancer are three of the most researched and highly anticipated areas of modern medicine. One of the greatest barriers to making advances within these fields is the lack of understanding of the molecular mechanisms responsible for cancer metastasis. Head and neck cancer (HNC) is the fifth most diagnosed cancer in the United States, according to the American Cancer Society, and claims the lives of many of its victims because it is often not diagnosed until metastasis has already occurred. After the onset of metastasis, the five-year survival rate drops to below thirty-five percent. Therefore, the study of how metastasis occurs is essential to finding a treatment for HNC.

It is believed that metastasis occurs when cancer cells break free from the primary tumor and travel through the blood stream, attaching themselves onto endothelium of blood vessel walls and in turn, forming secondary tumors in new organs. The cell adhesion mediated by glycolipids on cancer cells to E-selectin on vascular endothlelium may be a significant contributor to cancer metastasis. E-selectin plays a significant role in the first two steps of the adhesion cascade, tethering and rolling, which initiate the recruitment process. In our preliminary studies, it has been shown that HNC cells express glycolipids that can bind to E-selectin. Flow cytometry shows that HNC cells express sialofucosylated molecules that could act as E-selectin ligands, and flow chamber adhesion assays confirm that HNC cells bind to cytokine-stimulated endothelial cells in a wholly E-selectin-dependent manner. Enzyme treatment further reveals that sialofucosylated glycolipids are major adhesive ligand(s) for E-selectin. Through the use of specialized flow chambers mimicking physiological conditions, optical tweezers, gas chromatography-mass spectrometry, the mediators of these adhesive interactions can be studied in depth, leading to the discovery of characteristics of cancer glycolipids that promote adhesion.

The purpose of this investigation is to gain further insight as to which specific glycolipids located on HNCcells are potent ligands for E-selectin binding. With increased understanding of selectin-ligand adhesion interactions, the identity and properties of molecules responsible for metastasis can be used to determine possible therapeutic methods.

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 5174.
Abstract 5178: Homing of circulating prostate cancer cells to bone is regulated by alpha 1,3 fucosyltransferase 7
Authors: Barthel Steven R. , Wiese Georg K. , Cho Jaehyung , Opperman Matthew J. , Hays Danielle L. , Siddiqui Javed , Pienta Kenneth J. , Furie Bruce , Dimitroff Charles J.
Published: February 2011

Circulating cancer cells are known to bind vascular surfaces and then to extravasate into target tissues. How such binding occurs is an essential, yet ill-defined step in metastasis. We and others have proposed that cancer cells exploit identical adhesive determinants as hematopoietic cells in order to bind microvascular endothelial cells. In particular, sialyl Lewis X (sLe(X)) on prostate cancer (PCa) cells is thought to promote metastasis by mediating PCa cell binding to microvascular endothelial (E)-selectin. Nonetheless, regulation of sLe(X) and related E-selectin ligand expression in PCa cells is a poorly understood factor in PCa metastasis. Here, we describe an important glycobiological mechanism regulating E-selectin-mediated adhesion and metastatic potential of PCa cells to the bone marrow. We demonstrate that alpha1, 3 fucosyltransferases (FT) 3, 6, and 7 are markedly elevated in bone- and liver-metastatic PCa tissue and dictate synthesis of sLe(X) and E-selectin ligands on metastatic PCa cells. Upregulated FT3, FT6, or FT7 expression induced robust PCa PC-3 cell adhesion to bone marrow endothelium and to inflamed postcapillary venules in an E-selectin-dependent manner. Membrane proteins, CD44, carcinoembryonic antigen (CEA), podocalyxin-like protein (PCLP), and melanoma cell adhesion molecule (MCAM) were major scaffolds presenting E-selectin-binding determinants on FT-upregulated PC-3 cells. Strikingly, elevated FT7 expression promoted PC-3 cell trafficking to and retention in bone marrow through an E-selectin dependent event. These results highlight alpha1, 3 FTs as potent enhancers of PCa cell metastatic efficiency to the bone marrow via an E-selectin-dependent trafficking mechanism.

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 5178.
Abstract 518: Plasminogen activator inhibitor-1 (PAI-1) protects tumor cells from extrinsic apoptosis
Authors: Fang Hua , DeClerck Yves A.
Published: February 2011

Plasminogen-activator inhibitor-1 (PAI-1) is a member of the serine protease (serpin) family of inhibitors that control the proteolytic activity of urokinase and tissue-type PA. The expression of PAI-1 in most cancer types has been shown to be a marker of poor rather than favorable outcome, suggesting that PAI-1 has a pro-tumorigenic activity. The observation that PAI-1 positively contributes to angiogenesis has in part provided a mechanism in support of the pro-tumorigenic effect of PAI-1. Consistent with such a role, we have previously reported that PAI-1 protects endothelial cells (EC) from FasL-mediated apoptosis by controlling FasL cleavage by plasmin (Bajou et al, Cancer Cell, 2008). Here we hypothesized that PAI-1 may play a similar protective role in cancer cells by preventing them from undergoing apoptosis. We observed that downregulation of PAI-1 by small interfering RNA (siRNA) induces apoptosis in breast cancer MDA-MB-231 cells (14.1 ±1.1% apoptotic cells in PAI-1 siRNA-treated cells vs. 1.5 ± 0.5% apoptotic cells in scramble control; p < 0.001) and increases caspase-3 activity by 5 fold. Consistent with an involvement of the extrinsic apoptotic pathway, we observed that downregulation of PAI-1 in MDA-MB-231 cells did not alter mitochondrial membrane potential but resulted in an increase in caspase-8 activity by 3 fold. PAI-1 silencing also enhanced the production of urokinase (uPA) and the cell-associated activity of plasmin, and inhibited migration and invasion in vitro. The data thus suggest that like in EC, PAI-1 protects tumor cells from extrinsic apoptosis and provides an additional mechanism explaining the pro-tumorigenic activity of PAI-1.

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 518.
Abstract 5180: MUC4 mucin upregulates N-cadherin: A novel mechanism for human ovarian tumor metastasis
Authors: Ponnusamy Moorthy Palanimuthu , Lakshmanan Imayavaramban , Jain Maneesh , Batra Surinder K.
Published: February 2011

The human MUC4 protein is a high molecular weight transmembrane glycoprotein which is over-expressed in ovarian tumors and plays a role in increasing the cellular motility of the ovarian cancer cells. Previous reports have indicated that gain of expression of N-cadherin in tumor cells is associated with increased motility and metastasis. We hypothesize that MUC4 upregulates N-cadherin and plays a major role in metastasis of ovarian cancer. The aim of the present study was to analyze the mechanism of MUC4 induced cellular motility, invasion and metastasis in ovarian cancer cells. For this we have used a MUC4 gene construct (entire coding sequence along with 10% of the central tandem repeat region) for ectopic expression to determine the metastatic function of MUC4 in human ovarian cancer cells (SKOV3). Interestingly, we observed that N-cadherin was up-regulated in MUC4 overexpressing ovarian cancer cells (SKOV3-MUC4) compared to control (vector transfected) cells. We further discovered that N-cadherin upstream pathways such as FAK, MKK7, JNK and c-Jun are activated in the MUC4 expressing SKOV3 cells. Additionally, inhibition of pFAK led to the downregulation of N-cadherin by inhibiting the activation of MKK7, JNK and c-Jun in MUC4 overexpressing SKOV3 cells. On other hand, knockdown of N-cadherin decreased the activation of ERK, AKT and MMP9 in the MUC4 expressing SKOV3 cells. Inhibition of pFAK, pJNK and knockdown of N-cadherin in the MUC4 overexpressing cells further led to a significant decrease in cellular motility. Upon tumorigenisis and metastasis analysis with NOD-SCID mice, the SKOV3-MUC4 cells produced significantly larger tumors and demonstrated a higher incidence of metastasis to distant organs (peritoneal wall, colon, intestine, stomach, lymph nodes, liver and diaphragm) than the SKOV3-vector cells. Taken together, our result suggests that MUC4 mediated upregulation of N-cadherin plays a major role in promoting the metastatic property of ovarian cancer cells.

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 5180.
Abstract 5182: Sialofucosylated glycosphingolipids expressed on head and neck cancer cells mediate adhesion to endothelial cells
Authors: Wood Steven M. , Marshall Jocelyn , Abram Courtney , Burdick Monica M.
Published: February 2011

The development of innovative strategies can be used to detect and prevent cancer metastasis once the molecular mediators are known. Cancer metastasis is the spread of cancer from a primary site, from which cancer cells break away and travel to a new organ via the bloodstream or the lymphatic system. However, the molecular signals responsible for the cancer cells to travel from the primary site to a new location are largely unknown. It has been hypothesized that cancer invasion is similar to leukocyte migration during response of vascular tissue to harmful stimuli. In this process, leukocytes travel through the bloodstream, attach to the vessel endothelium, and migrate through the endothelium to the site of injury. Using leukocyte migration as a model for cancer metastasis it is hypothesized that E-selectin is a major molecular mediator for head and neck squamous cell carcinoma (HNSCC) metastasis. E-selectin is expressed on the surface of vascular endothelial cells and initiates cell-cell interactions by binding to proteins and lipids on the surface of cancer cells that have sialofucosylated carbohydrate epitopes. We have previously shown that HNSCCs bind to E-selectin expressed on stimulated human umbilical vein endothelial cells (HUVECs) under physiological shear stress. Also, it has been shown that glycosphingolipids isolated from HNSCCs support adhesive interactions with E-selectin-transfected Chinese hamster ovary cells. To further reveal the role of sialofucosylated glycosphingolipids in cell-cell interactions, polystyrene microsphere beads were utilized, in an in vitro model of cell adhesion under fluid flow conditions. Polystyrene beads were coated with sialyated non-fucosylated gangliosides (GM1) or sialofucosylated glycosphingolipids from HNSCCs. The expression of glycosphingolipids on the newly coated microspheres was checked by flow cytometry to ensure that the lipids adsorbed to the beads. When coated microspheres were perfused over cytokine-stimulated HUVECs, the microspheres with sialofucosylated glycosphingolipids tethered and rolled on the endothelium similar to HNSCCs, while the microspheres coated with GM1 did not bind to the endothelium. Treatment of the sialofucosylated glycosphingolipid coated microspheres with sialidase or fucosidase also eliminated adhesion of the beads to the endothelium. Our study signifies the importance of the role that glycosphingolipids play in adhesion to endothelial E-selectin under physiological shear stress. Further studies are needed to determine if sialofucosylated glycosphingolipids should be targeted for patients with HNSCCs.

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 5182.
Abstract 5184: Breast cancer cells express glycoprotein and glycolipid ligands for E-selectin
Authors: Shirure Venktesh S. , Burdick Monica M.
Published: February 2011

E-selectin, an adhesion molecule expressed by endothelial cells, has been implicated in various cancer metastatic processes. The hypothesis of the present study is that endothelial E-selectin is involved in the adhesion of breast cancer cells under physiological flow conditions. The characterization of E-selectin ligands expressed by BT-20 and MDA-MB-468 human breast cancer cells was performed. E-selectin ligands were broadly classified into glycoprotein and glycolipid ligands. Purified fractions of glycolipids containing sialic acid residues, known as gangliosides, were found to be E-selectin ligands at wall shear stress of 0.80 dynes/cm2. Immunostaining of breast cancer cells for various proteins known for E-selectin activity on non breast cancer cells revealed that P-selectin glycoprotein ligand-1 (PSGL-1), CD-43, podocalyxin like protein (PCLP), carcinoembryonic antigen (CEA) were absent, but CD44 was expressed by both breast cancer cell lines. Dynamic antigen capture assay at wall shear stress of 0.80 dynes/cm2 indicated that CD44 expressed by breast cancer cells is an E-selectin ligand; however, E-selectin ligand activity of CD44 was weak compared to LS-174T colon carcinoma cells. Efforts were then made to characterize novel glycoprotein ligands using immunoprecipitation and western blotting techniques. BT-20 cells showed presence of protein E-selectin ligands. The characterization of glycans decorating protein E-selectin ligands was performed by immunoprecipitation and western blot staining with various carbohydrate antibodies. In summary breast cancer cells express CD44 and novel glycoproteins as well as glycolipids as E-selectin ligands. Further characterization and identification of novel E-selectin ligands can lead to new diagnostic and prognostic approaches.

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 5184.
Abstract 5188: Phosphoinositide 3-kinase γ: Implications in breast cancer metastasis
Authors: Xie Yan , Kirui Joseph K. , Jiang Haihong , Deng Caishu , Tu Yaping
Published: February 2011

Metastasis is the major cause of breast cancer mortality. Type I phosphatidylinositol 3-kinases (PI3Ks), including α, β, γ and δ, play a pivotal role in diverse cell processes such as proliferation and motility. Of these PI3Ks, PI3Kγ is especially intriguing because it is: 1) normally expressed primarily in white blood cells which have a physiological need to migrate and 2) is specifically activated by G-protein coupled receptors (GPCRs). We recently reported that aberrant Gi-coupled receptor signaling promotes breast cancer metastasis (Xie et al., Cancer Res. 69: 5743-5751). The goal of the present study was to determine roles of PI3Kγ in breast cancer metastasis. We first examined the levels of PI3Kγ in different breast cancer cell lines by RT-PCR and Western blot analysis. We then immunohistochemically analyzed PI3Kγ protein expression in histologically benign and neoplastic cells of archival human breast tissues. We also investigated effects of PI3Ks inhibitors on migration and invasion abilities of breast cancer cells in Transwell chamber assays. Finally, we determined effects of PI3Ks inhibitors on lamellipodia formation, a key step involved in cancer metastasis. Our data indicated that PI3Kγ mRNA and protein were not detected in a normal human breast epithelial cell line (MCF-10A) or non-metastatic breast cancer cells (MCF-7 and T47D), but was found in metastatic breast cancer MDA-MB-231 cells and MDA-MB-436 cells. In contrast, PI3Kα, β and δ are ubiquitously expressed in these cell lines. In addition, immunohistochemical staining using a PI3Kγ-specific antibody showed that the expression of PI3Kγ protein was significantly increased in invasive human breast carcinoma and in breast cancer metastases to a regional lymph node as compared to the adjacent benign breast tissues or the noninvasive ductal carcinoma in situ. Thus, PI3Kγ expression level correlates with the metastatic potential of breast cancer cells. Interestingly, migration and invasion of metastatic breast cancer cells were inhibited by a selective inhibitor of PI3Kγ but not α or β isoforms. Furthermore, blocking PI3Kγ but not α and β disrupted the Rac-dependent lamellipodia formation induced by the activation of Gi-coupled receptor CXC chemokine receptor 4, but not epidermal growth factor tyrosine kinase receptor. Taken together, these results indicate that up-regulated PI3Kγ conveys the metastatic signal initiated from GPCRs in breast cancer cells and suggest that PI3Kγ may be a novel therapeutic target for the treatment of breast cancers. Supported by Nebraska State LB692 (YX).

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 5188.
Abstract 520: Inhibition of uPAR and uPA reduces invasion and degradative potential in papillary thyroid carcinoma cells
Authors: Nowicki Theodore S. , Kummer Nicolas T. , Iacob Codrin , Suslina Nina , Moscatello Augustine , Schantz Stimson , Schaefer Steven , Shin Edward , Tiwari Raj K. , Geliebter Jan
Published: February 2011

Papillary thyroid cancer (PTC) is the most prevalent endocrine and thyroid malignancy. The ability of PTC to invade and migrate great distances contributes to distant metastases, which represent the most common cause of PTC-related death. The urokinase plasminogen activator (uPA) and the urokinase plasminogen receptor (uPAR) are key mediators of tumor invasion. Upon binding to uPAR, pro-uPA is converted to its active form, which is then capable of cleaving plasminogen to plasmin. Plasmin can then degrade components of the basement membrane and extracellular matrix, a prerequisite for tumor cell invasion and metastasis. The binding of uPA by uPAR also seems to mediate several signaling events that contribute to a migratory phenotype, as well as various growth signals. In this study, we analyzed uPA and uPAR expression in PTC and normal thyroid tissue, and examined in vitro how uPA and uPAR contribute to an invasive/metastatic phenotype, as well as the functional consequences of inhibiting this system. uPA and uPAR RNA were also significantly higher in patients with metastatic disease. Casein-plasminogen zymography and western blotting demonstrated increased active uPA secreted by BCPAP cells compared to NTHY-Ori-3-1. Fluorimetric assays revealed that BCPAP CM was able to activate plasminogen, resulting in measureable casein hydrolysis. This casein hydrolysis was prevented by the addition of several specific uPA inhibitors. The in vitro invasion phenotype of BCPAP cells was augmented by the addition of plasminogen, and this augmentation was reversed by inhibitory anti-uPA and anti-uPAR antibodies. Cells transfected with siRNA against uPAR demonstrated decreased migratory and invasive potentials as measured by Matrigel invasion assays. Finally, uPAR-knockdown cells exhibited decreased proliferation when compared to non-targeting siRNA transfectant cells. These data provide new functional evidence of the uPA/uPAR system's role in PTC invasion/metastasis and demonstrate the attractiveness of uPA and uPAR as molecular biomarkers and therapeutic targets.

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 520.
Abstract 5201: Erk activation of p21 activated kinase-1 is critical for medulloblastoma migration
Authors: Yuan Liangping , MacDonald Tobey J.
Published: February 2011

We previously identified that overexpression of the platelet-derived growth factor receptor (PDGFR) is associated with metastatic medulloblastoma (MB) and showed that PDGF treatment increases ERK activity and promotes MB cell migration. In this study, we investigated whether ERK regulates Rac1/Pak1 signaling and is critically linked to MB cell migration. Herein we demonstrate that PDGF-BB treatment of MB cells induces concomitant activation of PDGFRβ, MEK1/ ERK, Rac1 and Pak1, but suppresses Rho activity, which together significantly promotes cell migration. Conversely, cells transfected with either PDGFRβ or Pak1 siRNA or treated with an inhibitor of Rac1 (NSC23766) or N-myristoyltransferase-1 (Tris-dipalladium) are unable to activate Rac1 or Pak1 in response to PDGF, and consequently, are unable to undergo PDGF-mediated cell migration. Furthermore, we also demonstrate that either chemical inhibition of MEK/ERK (U0126) or stable downregulation of PDGFRβ by shRNA similarly results in the loss of PDGF-induced ERK phosphorylation and abolishes Rac1/Pak1 activation and cell migration in response to PDGF. However, specific depletion of Pak1 by siRNA has no effect on PDGF-induced ERK phosphorylation, indicating that in MB cells ERK signaling is Pak1-independent, but PDGF-induced migration is dependent on ERK-mediated activation of Pak1. Finally, using tissue microarrays, we detect phosphorylated Pak1 in 53% of medulloblastomas and show that immunopositivity is associated with unfavorable outcome. We conclude that Rac1/Pak1 signaling is critical to MB cell migration and is functionally dependent on PDGFRβ/ERK activity.

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 5201.
Abstract 5203: Domains that distinguish Akt1 from Akt2 in cell migration
Authors: Natarajan Sharmila B. , Zhou Guo-Lei , Field Jeffrey M.
Published: February 2011

Domains that distinguish Akt1 from Akt2 in cell migration

Sharmila B. Natarajan, Guo-Lei Zhou and Jeffrey Field

Department of Pharmacology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104

The Akt family of kinases, Akt1, Akt2 and Akt3 are activated in many cancers where they play critical role in cell survival, metabolism and migration. There are distinctive roles for Akt1 and Akt2 in cell migration where, depending on the cell type, loss of Akt1 can either stimulate or inhibit cell migration. Previous work from our lab demonstrated that loss of Akt2 stimulated cell motility and that, based on chimeras, the kinase domains of Akt1 and Akt2 are interchangeable. Specificity is conferred by the PH domain and a linker domain that separates the PH domain from the kinase domain. To address regions that are required to rescue Akt1 knockouts, which have reduced cell motility, we performed rescue experiments with mouse embryo fibroblasts from Akt1 knockout mice. Our results are in concordance with a study by E. K. Kim et al., (Cell Signaling, 2008) where they found that the linker region is again likely to confer specificity. Importantly, we found that a new construct, in which only the linker domain of Akt1 was swapped into Akt2, stimulated cell migration of Akt1 knockouts. Additionally, we find that both Akt1 and Akt2 associate with the Pak protein kinase but through different mechanisms. These studies suggest that differences in interaction with Pak may underlie the opposing effects of Akt1 and Akt2 on cell migration.

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 5203.
Abstract 5213: KiSS1R signaling in breast cancer
Authors: Zajac Matthew M. , Babwah Andy , Bhattacharya Moshmi
Published: February 2011

KiSS1R, a G-protein coupled receptor (GPCR), and its ligands, collectively known as the kisspeptins, were originally discovered to have anti-metastatic effects. In melanoma, the expression of KiSS1R and the kisspeptins correlates with positive prognosis. However, in breast tumors, higher mRNA expression of KiSS1R and kisspeptins has been shown to correlate with progressing tumour grade. In large, the mechanism(s) by which kisspeptin-signaling via KiSS1R regulates breast cancer cell migration and invasion, required for metastasis, is unknown, and is the focus of this study. Commonly targeted in breast cancer therapy, aberrant signaling of the epidermal growth factor receptor (EGFR) is able to lead to increased migration and invasion, culminating in a more aggressive metastatic phenotype. EGFR's ability to interact with other GPCRs, whether directly by transactivation or indirectly by down-stream crosstalk, is well known. We have found that Kp-10, the most potent kisspeptin, stimulates the migration and invasion of highly invasive human MDA-MB-231 cells stably expressing FLAG-KiSS1R. In addition, we have observed co-localization of FLAG-KiSS1R and EGFR-GFP in human embryonic kidney (HEK 293) cells in response to Kp-10 or EGF stimulation. Stimulation of MDA-MB-231 cells with Kp-10 resulted in the activation of EGFR. Taken together, our findings suggest a potential mechanism by which kisspeptins and KiSS1R may stimulate breast cancer cell migration and invasion.

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 5213.