bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 3764: Loss of β2SP (ELF) enhances the stem-like traits and invasiveness of HCC cells.
Authors: Zhi Xiuling , Lin Ling , Shivapurkar Narayan , Jogunoori Wilma , Mishra Lopa , He Aiwu R
Published: August 2014

β2SP(ELF) is one of adapter proteins for Smad3/Smad4 complex formation during TGF-β signal transduction. 40% of β2SP+/- mice spontaneously develop hepatocellular carcinoma (HCC) and most cases of human HCC, gastric cancer, and lung cancer demonstrate significant reductions in β2SP expression. In our study, we investigated the possible mechanism for loss of β2SP induced HCC tumorigenesis. Recent studies have highlighted the involvement of cancer stem cells during the formation and progression of various types of cancers, epithelial cells that have undergone EMT (epithelial mesenchymal transition) acquire stem-cell properties during cancer development. Giving the role of TGF-beta signaling in stem cell differentiation and EMT during embryonic development, we hypothesize loss of β2SP results in alteration of liver stem cell that may contribute to the development of HCC in β2SP+/− mice. It has been shown that EpCAM-positive adult liver cells has stem cell property and can differentiate into functional hepatocytes in xenograft model. We found that EpCAM-positive hepatocyte number doubled in β2SP+/− mouse compared to wild type by fluorescence-activated cell sorter (FACS). Furthermore EpCAM level in β2SP+/− mouse liver was three times higher than that in the liver of wild type mouse. To study the mechanism by which Epcam is regulated by β2SP, HCC cells were transfected by β2SP siRNA. Inhibition of β2SP increased the expression of Epcam, and EMT markers vimentin was increased and E-cadherin expression was decreased in PLC/PRF/5 and SNU449 cells, while TGF-β1 and Smad3 rescued the expression of Epcam and E-cadherin, but not vimentin. We also found that suppression of β2SP expression promotes adhesion, migration and cloning formation of PLC/PRF/5 and SNU449 cells. Our data suggests loss of β2SP enhances the stem-like traits and invasiveness of HCC cells.

Citation Format: Xiuling Zhi, Ling Lin, Narayan Shivapurkar, Wilma Jogunoori, Lopa Mishra, Aiwu R He. Loss of β2SP (ELF) enhances the stem-like traits and invasiveness of HCC cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3764. doi:10.1158/1538-7445.AM2013-3764
Abstract 3768: Autophagy: a mechanism of resistance to therapy in breast cancer.
Authors: Dave Bhuvanesh , Mitra Sucharita
Published: August 2014

Autophagy is an evolutionarily conserved lysosomal pathway for degradation of cytoplasmic proteins, macromolecules, and organelles. It is defined as a cell survival pathway which allows cells to survive nutrient and environmental stress. Tumor initiating cells (TICs) survive radiation, endocrine therapy, and chemotherapy, making them the primary cause of relapse in breast cancer patients. Our previous studies have identified autophagy as the mechanism of survival in these treatment-resistant populations in letrozole- as well as chemotherapy-treated patient samples. In this study we confirmed our previous finding by using a real time PCR low density array of 84 genes related to autophagy in 9 paired pre- vs. post-letrozole treated clinical samples, where 60 out of 84 genes demonstrated a statically significant change (p<0.05). Autophagy inhibitors like N-acetyl cysteine and catalase demonstrate a significant reduction in TICs using mammosphere forming efficiency (MSFE) (p<0.05) and flow cytometry of CD44+/CD24- (p<0.05) cells in three triple negative breast cancer cell lines (MDA-MB231, SUM159 and BT549). Recent evidence from our group had determined a role for hypoxia and reactive nitrogen species in TIC inhibition. Here we demonstrate that both catalase and N-acetyl cysteine inhibit TICs by a mechanism that significantly reduces inducible nitric oxide synthase and HIF-1α, implicating both hypoxia and reactive nitrogen species in the process. We will be confirming our finding in-vivo using patient-derived and cell line human xenografts and further examine the cross talk between hypoxia and reactive nitrogen species as it relates to autophagy. The understanding of this process may allow us to target TICs in a novel mechanistic way.

Citation Format: Bhuvanesh Dave, Sucharita Mitra. Autophagy: a mechanism of resistance to therapy in breast cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3768. doi:10.1158/1538-7445.AM2013-3768
Abstract 3771: Establishment of primary cultures of cancer stem cells from patients with Li-Fraumeni Syndrome, carrier of TP53 p.R337H Brazilian founder mutation.
Authors: Mambelli Lisley I. , Kerkis Irina , Hainaut Pierre , Achatz Maria Isabel W.
Published: August 2014

Li-Fraumeni syndrome (LFS), an inherited cancer predisposition syndrome, is associated with germline mutations in TP53. It is characterized by high risk of multiple, early cancers. In Brazil, a variant form of LFS is exceedingly frequent due to a widespread founder TP53 mutation, p.R337H, detected in about 0.3% of the general population in Southern Brazil. This mutation occurs in p53 oligomerization domain and its effect on p53 oligomerization is supposed to be dependent upon pH conditions. Recent studies indicate that p53 plays a critical role in regulating differentiation and asymmetric divisions of stem cells. The main objective of this study is to isolate and to characterize Cancer Stem Cells (CSC) in patients with germline mutations in TP53 gene with Li-Fraumeni syndrome and Li-Fraumeni-like Syndrome. We have isolated and characterized CSC from tumors of p.R337H mutation carriers. After informed consent, surgical resection fragments were dissociated and brought in culture. Adherent cells and spheroids were derived from different tumor types. Spheroids and stromal cells derived from a breast cancer (BC) were further analyzed by immunofluorescence and flow cytometry to demonstrate positive immunolabeling for CD44+, CD24−, Oct4, Ki67 and Sox2 antibodies. Time-lapse videomicroscopy showed rapid growth, frequent asymmetric division and absence of senescent phenotypes for least 17 passages. Moreover, we showed by Colony Forming Units assay (CFU) that stromal cells are very clonogenic, once 103 cells initially plated were able to form 442 new colonies after 15 days in culture. It was also verified the differentiation potential of stromal cells for the following lineages: adipogenic, osteogenic and muscle-like cells. The adherent stromal cells as well as oncoesferas are able to self-renew and to proliferate in vitro for long periods without losing its main features. These properties are similar to stem cell possessing the p53 inactive. However, we observed that treatment of adherent cells with doxorubicin (DNA damaging agent) causes accumulation of p53 and causing induction of cell cycle arrest with low doses of this drug, and cell death / apoptosis in higher doses. Our findings using CSC-p.R337H can provide a very useful model for better understanding the role of specific mutation, as well as the TP53 tumor biology.

Citation Format: Lisley I. Mambelli, Irina Kerkis, Pierre Hainaut, Maria Isabel W. Achatz. Establishment of primary cultures of cancer stem cells from patients with Li-Fraumeni Syndrome, carrier of TP53 p.R337H Brazilian founder mutation. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3771. doi:10.1158/1538-7445.AM2013-3771
Abstract 3780: Tumorigenicity and partial clear cell conversion of the epithelioid, but not fibroblastoid, subset of a sarcomatoid renal carcinoma cell line.
Authors: Hsieh Chin-Hsuan , Chen Hung-Chang , Chuang Cheng-Keng , Pang See-Tong , Chang Ying-Hsu , Liao Shuen-Kuei
Published: August 2014

The biology of sarcomatoid renal cell carcinoma (RCC) and its conversion from and to the clear cell RCC cells are not fully understood. In this study, we analyzed the sarcomatoid RCC cell line, RCC52, derived from a clear cell RCC lesion with extensive sarcomatoid differentiation. When grown as monolayers, RCC52 cells consisted of mostly epithelioid cells and partly fibroblastoid cells, and were all PAX-2 negative, suggestive of in vitro growth of the sarcomatoid, but not clear cell, component. Immunohistology of xenografts resulted from subcutaneous injection of tumor cells revealed that only the parental line and all epithelioid sublines tested could develop solid tumors consisting of mostly sarcomatoid cells and PAX-2+ clear cells in areas. Our findings revealed (i) the coexistence of the two phenotypically distinct epithelioid and fibroblastoid subsets in this sarcomatoid RCC52 cell line, (ii) the dissociation of the abilities to display tumorigenicity and migratory/invasive potential by the epithelioid and fibroblastoid subsets, respectively, (iii) the capability of only epithelioid subset to undergo partial conversion to a clear cell type, and (iv) the identification of epithelioid subset as the niche for cancer stem cells within the RCC52 cell line. Importantly, using CD44 and CD24 markers in cytofluorometric analysis, we identified the phenotypes of the epithelioid and fibroblastoid subsets to be CD44+/CD24− and CD44+/CD24+, respectively. Collectively, our findings highlight the phenotypical and functional properties of the two distinct subsets in a sarcomatoid RCC. Thus, improved understanding of tumor heterogeneity, metastatic niche and stromal progression has profound consequence of metastatic disease, and promises to open up new strategies for therapy of cancer.

Citation Format: Chin-Hsuan Hsieh, Hung-Chang Chen, Cheng-Keng Chuang, See-Tong Pang, Ying-Hsu Chang, Shuen-Kuei Liao. Tumorigenicity and partial clear cell conversion of the epithelioid, but not fibroblastoid, subset of a sarcomatoid renal carcinoma cell line. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3780. doi:10.1158/1538-7445.AM2013-3780
Abstract 3783: Talin-1 phosphorylation promotes invasion and migration of prostate tumor cells through activation of β1 integrins.
Authors: Jin Jung-Kang , Lee Yu-Chen , Lin Sue-Hwa , Gallick Gary E.
Published: August 2014

Integrins have been shown to play a critical role in cell survival. We recently demonstrated that integrins are constitutively activated in highly metastatic prostate cancer cell PC3-mm2. Activation of integrins increases their binding affinity with extracellular matrix, resulting in increased migration and cancer metastasis. Talin is a 270 kDa cytoskeletal protein that has been shown to regulate integrin activity. Previous reports showed that the S425 residue in the N-terminal domain of talin was phosphorylated by Cdk5 to increase talin stability. In this study, we determined whether talin S425 phosphorylation regulates β1 integrin activation. We found that β1 integrin activation in prostate tumor cells correlates with talin S425 phosphorylation but not total talin levels. To examine the role of talin-pS425 in β1 integrin activation, endogenous talin-1 in PC3-mm2 cells was knocked down by shRNA, followed by re-expression of a phosphorylation-deficient S425A or phosphorylation-mimicking S425D talin-1 mutant. Re-expressing phosphorylation-deficient S425A-talin-1 in talin-1-knockdown PC3-mm2 cells showed reduced activation of β1 integrins, and decreased migration and invasion, compared to cells re-expressing wild type talin-1. In contrast, re-expressing the phosphorylation-mimicking S425D mutant in talin-1-knockdown PC3-mm2 cells restored β1 integrin activation and increased migratory and invasive properties of these cells. Inhibition of Cdk5 by roscovitine, a Cdk5 inhibitor, decreases talin-1 S425 phosphorylation and also inhibits downstream integrin signaling, including FAK and Src phosphorylation. Treatment of PC3-mm2 cells with roscovitine decreases β1 integrin activation and migration of PC3-mm2 cells. These results suggest that phosphorylation of talin-1 at S425 plays a role in promoting β1 integrin activation, leading to increased metastatic potential of prostate tumor cells.

Citation Format: Jung-Kang Jin, Yu-Chen Lee, Sue-Hwa Lin, Gary E. Gallick. Talin-1 phosphorylation promotes invasion and migration of prostate tumor cells through activation of β1 integrins. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3783. doi:10.1158/1538-7445.AM2013-3783
Abstract 3789: The alternatively spliced PRMT1 isoform PRMT1v2 promotes breast cancer cell survival and invasiveness.
Authors: Baldwin Robert Mitchell , Morettin Alan , Paris Genevieve , Goulet Isabelle , Cote Jocelyn
Published: August 2014

Breast cancer is the leading cause of cancer-related deaths in women. Despite intensive treatment to overcome this disease, recurrence is common and often incurable due to the highly aggressive nature of the recurrent tumour. Protein arginine methylation is catalyzed by protein arginine methyltransferases (PRMTs) and plays an important role in many cellular processes. Aberrant PRMT expression has been observed in several common cancer types, however their precise contribution to the cell transformation process and cancer progression is not well understood. Previously, we reported that the PRMT1 gene generates several alternatively spliced isoforms and our initial biochemical characterization of these isoforms revealed that they exhibit distinct substrate specificity and sub-cellular localization. Here we have focused on the PRMT1v2 isoform, which is the only predominantly cytoplasmic isoform and has increased relative expression in breast cancer cell lines and tumours. Specific depletion of PRMT1v2 using RNA interference caused a significant decrease in breast cancer cell survival due to an induction of apoptosis. Furthermore, depletion of PRMT1v2 in MDA-MB-231, an aggressive breast cancer cell line, resulted in significantly decreased cell invasion. Alternatively, PRMT1v2 overexpression in a non-aggressive breast cancer cell line, MCF7, was sufficient to enhance their invasiveness. This novel activity is specific to PRMT1v2, as overexpression of other isoforms did not enhance invasion. It also requires both proper subcellular localization and methylase activity. Consistent with an invasive phenotype, PRMT1v2 overexpression caused altered cell morphology and reduced cell-cell adhesion. Finally, we observed a significant reduction in β-catenin protein levels, a critical cell-cell adhesion protein, and show that this reduction is pivotal to PRMT1v2-enhanced cell invasion. Our study underscores the importance of characterizing the distinct functional differences between PRMT1 isoforms. Overall, we demonstrate a specific role for PRMT1v2 in promoting breast cancer cell survival and invasion, and propose that it may represent a promising therapeutic target.

*RMB is a Postdoctoral Fellow of the Canadian Institute of Health Research

Citation Format: Robert Mitchell Baldwin, Alan Morettin, Genevieve Paris, Isabelle Goulet, Jocelyn Cote. The alternatively spliced PRMT1 isoform PRMT1v2 promotes breast cancer cell survival and invasiveness. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3789. doi:10.1158/1538-7445.AM2013-3789
Abstract 3793: S100A8/A9 promotes invasion and migration through p38 MAPK-dependent NF-κB activation in gastric cancer cells.
Authors: Kwon Chae Hwa , Moon Hyun Jung , Park Hye Ji , Choi Jin Hwa , Park Do Youn
Published: August 2014

S100A8 and S100A9 (S100A8/A9) are low-molecular weight members of the S100 family of calcium-binding proteins. Recent studies have reported S100A8/A9 promote tumorigenesis. We have previously reported that S100A8/A9 is mostly expressed in stromal cells and inflammatory cells between gastric tumor cells. However, the role of environmental S100A8/A9 in gastric cancer has not been defined. We observed in the present study the effect of S100A8/A9 on migration and invasion of gastric cancer cells. S100A8/A9 treatment increased migration and invasion at lower concentrations that did not affect cell proliferation and cell viability. S100A8/A9 caused activation of p38 mitogen-activated protein kinase (MAPK) and nuclear factor-κB (NF-κB). The phosphorylation of p38 MAPK was not affected by the NF-κB inhibitor Bay whereas activation of NF-κB was blocked by p38 MAPK inhibitor SB203580, indicating that S100A8/A9-induced NF-κB activation is mediated by phosphorylation of p38 MAPK. S100A8/A9-induced cell migration and invasion was inhibited by SB203580 and Bay, suggesting that activation of p38 MAPK and NF-κB is involved in the S100A8/A9 induced cell migration and invasion. S100A8/A9 caused an increase in matrix metalloproteinase 2 (MMP2) and MMP12 expression, which were inhibited by SB203580 and Bay. S100A8/A9-induced cell migration and invasion was inhibited by MMP2 siRNA and MMP12 siRNA, indicating that MMP2 and MMP12 is related to the S100A8/A9 induced cell migration and invasion. Taken together, these results suggest that S100A8/A9 promotes cell migration and invasion through p38 MAPK-dependent NF-κB activation leading to an increase of MMP2 and MMP12 in gastric cancer.

Citation Format: Chae Hwa Kwon, Hyun Jung Moon, Hye Ji Park, Jin Hwa Choi, Do Youn Park. S100A8/A9 promotes invasion and migration through p38 MAPK-dependent NF-κB activation in gastric cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3793. doi:10.1158/1538-7445.AM2013-3793
Abstract 3795: Vangl2 and Prickle1 expression, interaction and regulation of Wnt/PCP signaling in medulloblastoma and neuroblastoma.
Authors: Dyberg Cecilia H. , Papachristou Panagiotis , Lagercrantz Hugo , Johnsen John I , Wickström Malin
Published: August 2014

Background /Aims: Wnt are a family of intercellular signaling factors that regulates a wide range of functions during embryonic development including cell proliferation, cell fate determination, differentiation, cell polarity and cell migration. They bind to Frizzled receptors and thereafter proceed via Dishevelled to several intracellular pathways known as the canonical and non-canonical signaling pathways. Activation of the canonical pathway results in an accumulation of cytoplasmic beta-catenin levels. Increased Wnt/beta-catenin signaling is associated with many forms of cancer, including the embryonic neural childhood tumors medulloblastoma and neuroblastoma. The non-canonical Wnt signaling pathways can inhibit the canonical signaling pathway, although the mechanism is still unclear. In this study, we investigate the roles of Prickle1 and Van Gogh-Like 2 (Vangl2), two planar cell polarity (PCP) genes in regulation of the cytoskeleton, Wnt/beta-catenin activity and tumorigenicity in neuroblastoma and medulloblastoma.

Methods: mRNA and protein expression of human Prickle1, Vangl2 and beta-catenin were examined in neuroblastoma and medulloblastoma cell lines. siRNA and cDNA transfection were performed together with co-immunoprecipitation and immunocytochemistry.

Results and conclusions: We show that Prickle1 and Vangl2 are expressed in neuroblastoma and medulloblastoma cell lines at different levels. Decreased Prickle1 expression correlates with increased levels of Wnt/beta-catenin in both medullo- and neuroblastoma cell lines, demonstrating a downregulation of PCP components and a possible activation of the canonical Wnt signaling pathway. Immunoprecipitation demonstrated that Prickle1 is present in complex with Vangl2. However, the exact mode of interaction between Prickle1 and Vangl2 remains to be eluciated. Microarray analysis revealed that low expressions of Prickle1 or Vangl2 are both associated significantly worse clinical outcome in neuroblastoma. Additionally, siRNA experiments revealed that knockdown of Prickle1 and Vangl2 increased cell proliferation of neuroblastoma cells. Taken together this indicate that the non-canonical Wnt pathway may be an interesting therapeutic target in embryanol neural childhood tumors.

Citation Format: Cecilia H. Dyberg, Panagiotis Papachristou, Hugo Lagercrantz, John I Johnsen, Malin Wickström. Vangl2 and Prickle1 expression, interaction and regulation of Wnt/PCP signaling in medulloblastoma and neuroblastoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3795. doi:10.1158/1538-7445.AM2013-3795
Abstract 3796: Endophilin II promotes breast tumor progression and metastasis.
Authors: Baldassarre Tomas , Meens Jalna , Craig Andrew W.
Published: August 2014

Invadopodia are filamentous actin-based membrane protrusions with extracellular matrix (ECM) degrading activity that are implicated in tumor progression and metastasis. A key step in invadopodia formation involves the recruitment of the membrane type 1 matrix metalloprotease (MT1-MMP) to invadopodia precursors. Surface expression of MT1-MMP promotes ECM degradation and cell invasiveness, and this is dependent on Src kinase-induced phosphorylation and inhibition of endocytic proteins. In fibroblasts, MT1-MMP internalization was regulated by Endophilin II (Endo II), a widely expressed protein implicated in synaptic vesicle uncoating and recycling in neurons. Here, we tested the role of Endo II in invadopodia formation in basal-like breast cancer cells, and in tumor progression and metastasis in mice. High levels of Endo II expression were observed in basal breast cancer cell lines with high Src kinase and ECM-degrading activity. Immunohistochemistry staining also identified Endo II expression in primary breast tumors and lymph node metastases. To define the functions of Endo II, we achieved stable silencing of Endo II in two basal breast cancer cell lines (MDA-MB-231, HCC1806). Although Endo II was not required for uptake of receptors via clathrin-mediated endocytosis (EGFR, TfR), we observed a significant reduction in numbers of invadopodia and ECM degradation upon Endo II silencing. Consistent with invadopodia defects in vitro, Endo II silencing led to a significant reduction in tumor growth and metastases in mouse orthotopic tumor xenografts assays performed with both basal breast cancer cell models. This study provides novel insights into roles of Endo II as a positive regulator of invadopodia and basal breast cancer metastasis.

Citation Format: Tomas Baldassarre, Jalna Meens, Andrew W. Craig. Endophilin II promotes breast tumor progression and metastasis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3796. doi:10.1158/1538-7445.AM2013-3796
Abstract 3798: Heat shock cognate protein HSPA8 (Hsc70) regulates migration and xenograft growth of breast tumor cell lines.
Authors: Pino Maria F. , Zhang Ge , Rosser Charles J. , Goodison Steve , Urquidi Virginia
Published: August 2014

In order to identify genes involved in the metastatic phenotype we had previously developed isogenic cell lines from the human breast carcinoma cell line MDA-MB-435 that have different metastatic phenotypes in immunocompromised mice. Proteomic profiling of the extracellular component of this model identified the heat shock cognate protein HSPA8 (Hsc70) as over-expressed in the metastatic cell line (M4A4) relative to its non-metastatic counterpart (NM2C5). Heat shock proteins are primarily implicated in a cytoprotective role when cells are exposed to stressful stimuli. They are intracellular molecular chaperones but are also found in the surface of embryonic stem cells and a variety of tumor cells. With the purpose of investigating the role of HSPA8 in tumor growth and metastasis, we first verified that this protein was expressed on the cell surface and in the conditioned medium of M4A4 cells, in addition to its intracellular location. Then we generated stable HSPA8 knockdown clones of the M4A4 cells and cell behavior was tested in a panel of analyses in vitro and xenograft growth was tested in immunocompromised mice. Knockdown of HSPA8 expression in M4A4 cells resulted in reduction of cell doubling time and of their migration potential in vitro. When individual M4A4-HSPA8 knocked down cell clones were injected into the mammary fat pad of immunocompromised mice significant reduction of the tumor size was observed relative to non-silencing control cells. The invasive and metastatic potential of the M4A4-HSPA8 knockdown clones as well as their transcriptional profile are currently under investigation. Our findings suggest that HSPA8 plays a role in cell growth in vitro and in vivo and may be involved in metastasis of breast cancer cells. Extracellular heat shock proteins have recently been shown to mediate a range of powerful effects in inflammatory, neuronal and immune cells through a number of cell surface receptors. The mechanisms involved in tumor dissemination and metastasis are potential targets for therapeutic intervention

Citation Format: Maria F. Pino, Ge Zhang, Charles J. Rosser, Steve Goodison, Virginia Urquidi. Heat shock cognate protein HSPA8 (Hsc70) regulates migration and xenograft growth of breast tumor cell lines. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3798. doi:10.1158/1538-7445.AM2013-3798
Abstract 3799: HTS performance and high-content analysis of antimigratory compound phenotypes using the Oris Pro 384 cell migration assay.
Authors: Fronczak Jennifer , Vollmer Laura , Krishnan Gopal , Vogt Andreas
Published: August 2014

The discovery of anti-metastatic agents that inhibit cancer cell motility has been hindered by a dearth of high-throughput screening (HTS) -compatible cell motility assays. The Oris™ Pro 384 well cell migration assay, developed by Platypus Technologies, is an innovative cell motility assay designed to enable HTS of potential anti-cancer compounds and wound healing agents on adherent tumor and endothelial cell lines. The assay utilizes a centrally located, non-toxic, biocompatible gel (BCG) to form a uniformly sized, cell-free detection zone into which cells migrate. The assay is logistically simple and does not require any mechanical processing steps, such as cell wounding or removal of physical barriers. The assay is fully compatible with laboratory automation, including robotic liquid handlers, plate washers, and high-content screening (HCS) readers.

A formal investigation of the accuracy, robustness, and HTS performance of the assay was conducted following guidelines in place at the University of Pittsburgh Drug Discovery Institute (UPDDI). Using MDA-MB-231 human breast cancer cells, the assay was optimized for migration kinetics, cell seeding density, and DMSO tolerance. In multi-day variability studies, the assay delivered signal-to-background ratios > 10 and Z-factors above 0.5 on three consecutive days, was insensitive to process errors or edge-effects, and passed UPDDI Assay Protocol Approval Committee (APAC) criteria. The assay delivered equal HTS performance on two different HCS platforms – the Thermo Scientific Cellomics ArrayScan II and the Molecular Devices ImageXpress®Ultra point scanning confocal reader. Screening of a test cassette of 1280 compounds with known biological activities identified several agents with mechanisms associated with cell motility. The multiparametric nature of the HCS assay permitted correlation of antimigratory activity with cellular toxicity. Our results validate the Oris™ Pro 384 cell migration assay as a logistically simple and information-rich cell motility analysis platform that meets the demands of large scale compound screening.

Citation Format: Jennifer Fronczak, Laura Vollmer, Gopal Krishnan, Andreas Vogt. HTS performance and high-content analysis of antimigratory compound phenotypes using the Oris Pro 384 cell migration assay. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3799. doi:10.1158/1538-7445.AM2013-3799
Abstract 380: Serine-Arginine protein kinase 1 (SRPK1) - A potential target of angiogenesis in prostate cancer.
Authors: Mavrou Athina , Oltean Sebastian , Bates Dave
Published: August 2014

Angiogenesis is required for all tumors to grow and metastasize and is mainly induced by the vascular endothelial growth factor (VEGF). Alternative spliced isoforms associated with cancer progression have been described in all areas of hallmarks of cancer, including angiogenesis. VEGF mRNA is alternatively spliced at the terminal exon, to produce two families of isoforms. The pro- angiogenic family, VEGFxxx, was shown to be up-regulated in all cancers studied so far, where as the anti- angiogenic family is preferentially expressed in normal non-angiogenic tissues, but down-regulated in most cancers. One of the factors involved in the regulation of VEGF alternative splicing is SRPK1. This is a protein kinase that phosphorylates the splicing factor SRSF1 and favours the production of the pro-angiogenic VEGF isoform. We therefore tested the hypothesis that down - regulation of SRPK1 in prostate cancer cells would increase the production of the anti-angiogenic isoform, preventing angiogenesis and tumor growth.

PC-3, DU145 and LnCap prostate cancer cells showed an increased expression of SRPK1 when compared to Primary Prostate epithelial cells. Stable SRPK1 knock-down (KD) in PC-3 and LnCap prostate cancer cells was obtained using a lentiviral system (Dharmacon). This resulted in a reduction in splice factor expression, mainly SRSF1, and induced a switch in the expression of VEGF towards the anti-angiogenic isoform, VEGF165b, when compared to control cells (transfected with non-targeting shRNA). PC-3 SRPK1 KD and control cell lines were injected subcutaneously in male nude mice. Knock down of SRPK1 resulted in an inhibition of tumour growth, as shown by the significant difference in tumor size between the SRPK1 KD (250.0 ± 82.28 n=9) and control cell line (994.7 ± 243.5 n=9)(Day 29 post-injection). In addition to the reduction in tumor volume, SRPK1 inhibition resulted in decrease in vascular density of the tumors, as shown by the significant reduction in blood vessel number in PC-3 SRPK1 KD (2.714 ± 0.3686 n=7) mice compared with control (4.292 ± 0.5426 n=8). These in vitro and in vivo data suggest that the regulation of VEGF splicing by SRPK1 alters prostate cancer tumour growth and angiogenesis.

Citation Format: Athina Mavrou, Sebastian Oltean, Dave Bates. Serine-Arginine protein kinase 1 (SRPK1) - A potential target of angiogenesis in prostate cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 380. doi:10.1158/1538-7445.AM2013-380
Abstract 3800: Osteonectin suppresses proliferation and migration of human prostate cancer cells.
Authors: Shin Minkyoung
Published: August 2014

Osteonectin/SPARC(Secreted Protein Acidic and Rich in Cysteine)/BM-40 is an extracellular protein that has been studied in ECM(Extra Cellular Matrix) organization, immune modulation, proliferation, adhesion, invasion and migration in various cancers. Advanced prostate cancer (PCa) is significantly affected by cross-talk among cancer cells, stromal cells and ECM. Such communication is particularly could contribute to migration and invasion of prostate cancer cells. However, the precise mechanism in migration and invasion of PCa cells are poorly understood. In this report, we investigated the effect of osteonectin on proliferation, migration and invasion of PCa cells. Indeed, Prostate-derived stromal cells (PrSC) has been shown to express high mRNA levels of osteonectin and secret relatively high levels of osteonectin. We revealed that osteonectin from PrSC and refined osteonectin protein also inhibited activation of AKT-p21 signaling, which plays an important role in prostate cancer cell growth, and affected down-regulation of migration and invasion of LNCaP cells. Together, these studies support a conclusion that interaction of stromal cells and PCa cells as osteonectin play an important role in progression of prostate cancer.

Citation Format: Minkyoung Shin. Osteonectin suppresses proliferation and migration of human prostate cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3800. doi:10.1158/1538-7445.AM2013-3800
Abstract 3818: TEL-AML1-induced STAT3 activation is essential for t(12;21)-associated childhood leukemia.
Authors: Mangolini Maurizio , de Boer Jasper , Walf-Vorderwülbecke Vanessa , Pieters Rob , den Boer Monique , Williams Owen
Published: August 2014

The t(12;21)(p13;q22) translocation is the most common chromosomal abnormality in pediatric leukemia, present in up to 25% of children with pre-B cell Acute Lymphoblastic Leukemia (ALL). This translocation involves two transcription factors, TEL(ETV6) and AML(RUNX1), both of which have crucial roles in regulating hematopoiesis.

Our lab has previously established a list of TEL-AML1 transcriptional target genes using Affymetrix GeneChip global gene expression analysis of mouse fetal liver hematopoietic progenitor cells (HPC) over-expressing the fusion cDNA. This analysis demonstrated deregulated expression of genes associated with STAT3 signaling, known to be one of the most important pathways required for proliferation and maintenance of multipotency in cancer stem cells.

In this study, we analyze the importance of STAT3 activity in a mouse model of TEL-AML1 overexpression, in human TEL-AML1 positive leukemia cells and primary human leukemic samples. Our results show that activation STAT3, via RAC1 induction, is necessary for the survival, proliferation and self-renewal of TEL-AML1 positive leukemia through transcriptional regulation of MYC expression.

In conclusion we show a novel signaling pathway important for the maintenance of TEL-AML1 leukemia that constitutes a promising novel therapeutic strategy for the treatment of TEL-AML1 leukemia.

Citation Format: Maurizio Mangolini, Jasper de Boer, Vanessa Walf-Vorderwülbecke, Rob Pieters, Monique den Boer, Owen Williams. TEL-AML1-induced STAT3 activation is essential for t(12;21)-associated childhood leukemia. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3818. doi:10.1158/1538-7445.AM2013-3818
Abstract 3819: DKK2 mediates osteolysis, invasiveness and metastatic spread in Ewing sarcoma.
Authors: Richter Günther H. , Burdach Stefan , Hauer Kristina
Published: August 2014

Ewing sarcomas (ES) are malignant osteolytic bone and soft tissue tumors mostly affecting children and young adults, and are characterized by early lung and distant bone metastases. Here we identified the dickkopf 2 homolog (DKK2) as a highly over-expressed gene in ES, compared to normal tissues. DKK family members are increasingly recognized to be deregulated in bone metastasis. Using RNA interference, we show that DKK2 promotes anchorage-independent colony formation and proliferation of ES cells in vitro and tumorigenicity in vivo. Analysis of the invasion potential in vitro and in an orthotopic xenotransplantation model revealed a strong correlation of DKK2 expression and ES invasiveness that is possibly mediated by its downstream effector matrix metalloproteinase 1 (MMP1). Gene expression analyses proved DKK2 to differentially regulate genes such as CXCR4, PTHrP, RUNX2 and TGFβ1, that are associated with homing, invasion and growth of cancer cells in bone tissue as well as genes important for osteolysis, like HIF1α, JAG1, IL6 and VEGF. Moreover, we observed that DKK2 promotes bone infiltration and osteolysis in vivo, identifying DKK2 as a key factor for osteotropic malignancy. Interestingly, suppression of DKK2 expression increases neuronal differentiation, but at the same time decreases chondrogenic and osteogenic differentiation potential of ES cells. These data provide evidence that DKK2 is a key player in ES invasiveness and osteolysis, validating a critical role of DKK2 for malignancy and the differential phenotype of ES. Findings further suggest a broadly important role for a member of a family of antagonists of the Wnt pathway in driving bone invasion and osteolysis with potential implications for bone invasion in cancer generally.

Citation Format: Günther H. Richter, Stefan Burdach, Kristina Hauer. DKK2 mediates osteolysis, invasiveness and metastatic spread in Ewing sarcoma. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3819. doi:10.1158/1538-7445.AM2013-3819
Abstract 3833: A novel 3D remodelable hydrogel for bioengineering tumor .
Authors: Han Bo , Fang Josephine , Yang Zhi , Nimni Marcel , Tayag Charisse
Published: August 2014

The need for new tools and technologies to decrease the cost of drug discovery and reduce animal testing has long been recognized. Three-dimensional (3-D) cell cultures have great potential for reducing costly late-stage drug failures by providing an in vivo-like early stage evaluation of efficacy and toxicity. We have developed a novel 3D culture system, which will serve as a simple and rapid method to examine cell proliferation, migration, apoptosis, and tissue formation in a physiologically relevant 3-D environment, similar to the in vivo setting, than standard 2-D arrays. This transglutaminase crosslinked gelatin (T-gel) system provides a native ECM scaffold for cell-cell and cell-matrix interaction; its transparency property enables routine observation with ease; the proteolysis sensitivity scaffold supports cell migration and invasion; the controllable rigidity mimics physiological and pathological environments. Importantly, 3D gel models have great potential as drug evaluation tools as they exhibit in vivo-like cell-cell and cell-ECM interaction, possess metabolic, proliferative and diffusion gradients. It is simple to use on a large scale. Compare to Matrigel, Tgel is with well defined ingredients, wide range of handling properties, easy to prepare at room temperature, and little batch to batch variation. It supports both anchorage dependent and independent cell growth due to hydrogel and natural ECM network properties. We make 3D cultures more relevant by including two or more cell types in different configurations co-cultures. Cells can be tracked on their formation of aggregates, migration (individual or collective), invasion direction (random or directional), speed, and responses to hypoxic and nutritional gradients. At the end of test, viable cells can be easily removed from the 3D T-gel for further experimentation. T-gel can also be used as a carrier for xenograft tumor formation in nude mice models. Other applications include cell growth and differentiation studies, metabolism/toxicology Studies, in vitro/vivo angiogenesis assay, hESC and iPSC culture and Differentiation.

Citation Format: Bo Han, Josephine Fang, Zhi Yang, Marcel Nimni, Charisse Tayag. A novel 3D remodelable hydrogel for bioengineering tumor . [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3833. doi:10.1158/1538-7445.AM2013-3833
Abstract 3840: Development of an innovative 3D cell culture system to study tumor-stroma interactions in cancer cells.
Authors: Amann Arno , Zwierzina Marit , Gamerith Gabriele , Bitsche Mario , Huber Julia M. , Vogel Georg , Blumer Michael , Koeck Stefan , Kelm Jens , Hilbe Wolfgang , Zwierzina Heinz
Published: August 2014

Introduction: We describe a novel 3D co-culture method using non-small cell lung cancer (NSCLC) cell lines in combination with a lung fibroblast cell line in the hanging drop technology. This method allows the investigation of tumour-stroma interactions and addresses the importance of having a more in vivo like cell culture model. Compared to other 3D models, it better reflects tumour growth in its microenvironment since no addition of growth factors or artificial extracellular matrix (ECM) substitutes are required.

Methods: 96-well hanging drop microtiter plates (InSphero AG, Zürich, Switzerland) were used for the production of 3D mono- and co-cultures. In these hanging drops the two NSCLC cell lines A549 and Colo699 were cultivated either alone or co-cultured with lung fibroblasts. The viability of tumour spheroids was confirmed after five and ten days by using Annexin V/Propidium Iodide staining for flow-cytometry. Tumour fibroblast spheroid aggregation was displayed either by scanning electron microscope, semi-thin sections, immunofluorescence or by immunohistochemistry (IHC). In addition to conventional histology (H&E), protein expression of e-cadherin, vimentin, Ki67 and α-smooth muscle actin (ASMA) was investigated by IHC.

Results: Lower viability was seen in A549 monocultures compared to co-cultures, whereas Colo699 monocultures showed better viability compared to co-cultures. Ki67 expression varied significantly between mono- and co-cultures in both tumour cell lines. The co-cultivation with fibroblasts led to an increased expression of this protein indicating increased proliferation. Furthermore, a vimentin upregulation and an e-cadherin downregulation could be detected during the course of the cultivation indicating a transition to a more mesenchymal phenotype.

Conclusion: We demonstrate that the hanging drops technology is a promising technique for the generation of tumour spheroid co-cultures. Furthermore, these spheroids allow the investigation of tumour-stroma interactions and a better reflection of in vivo conditions of cancer cells in their microenvironment. Our method holds potential to contribute to the development of anti-cancer agents and support the search for predictive biomarkers.

Citation Format: Arno Amann, Marit Zwierzina, Gabriele Gamerith, Mario Bitsche, Julia M. Huber, Georg Vogel, Michael Blumer, Stefan Koeck, Jens Kelm, Wolfgang Hilbe, Heinz Zwierzina. Development of an innovative 3D cell culture system to study tumor-stroma interactions in cancer cells. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3840. doi:10.1158/1538-7445.AM2013-3840
Abstract 3841: 3D spheroid culture model for studying cancer progression: fine tuning the extracellular environment.
Authors: Benton Gabriel J. , Arnaoutova Irina , George Jay
Published: August 2014

There is a need for more comprehensive and physiologically predictive cancer progression models. Tumor cell aggregates or spheroids are more demonstrative of tumors in vivo as they display several physiological traits including comparable morphology, reduced proliferation rates, elevated cell survival, cell-cell bond formation, tumor dormancy, and a hypoxic core. Embedding these spheroids in a physiological hydrogel composed of basement membrane proteins provides networks on which cells can travel out of the spheroid, mimicking early events during metastasis. Recent studies implicate the tumor microenvironment as a key component of the cancer progression process, and changes in tissue rigidity and collagen I content have been associated with this pathological process. The purpose of this study is to model the effects of differing extracellular matrix composition on morphology and invasiveness in different metastatic cell types during early metastatic events. Here, human cell lines representing different cancer types were grown in spheroid culture and subjected to embedding in hydrogels of differing extracellular matrix protein composition. The cell panel includes MDA-MB-231 for breast cancer, PC-3 for prostate cancer, U-87 MG for glioblastoma and HT-1080 for fibrosarcoma. Spheroids were photographed every 24 hours, and photographs were subjected to image analysis. The composition of the extracellular matrix hydrogel had a significant effect on the morphology of the invading cells as well as the rate of invasion. Increasing the collagen I content to model a more fibrotic environment resulted in fewer but longer process formation, representing a more motile cancer model; however, collagen I, alone, was insufficient to support robust invasion. In conclusion, the 3D spheroid invasion model suggests that the composition of the extracellular matrix proteins is important for cancer cell behavior during metastasis.

Citation Format: Gabriel J. Benton, Irina Arnaoutova, Jay George. 3D spheroid culture model for studying cancer progression: fine tuning the extracellular environment. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 3841. doi:10.1158/1538-7445.AM2013-3841