Non-invasive Cell Lines Research Articles

Astemizole inhibits cell proliferation in human prostate tumorigenic cells expressing ether í -go-go-1 potassium channels

Prostate cancer (PC) is the main cause of cancer mortality in men worldwide. Therefore, novel treatments for PC are needed. Ether í -go-go-1 (Eag1) potassium channels display oncogenic properties, and have been suggested as early tumor markers and therapeutic targets for different cancers. These channels are overexpressed in many human tumors including PC. Astemizole targets several molecules involved in cancer including Eag1 channels, histamine receptors and ABC transporters. Here we studied Eag1 mRNA expression and protein levels in the non-tumorigenic and non-invasive human prostate RWPE-1 cell line, and in the tumorigenic and highly invasive human prostate WPE1-NB26 cell lines. The effect of astemizole on cell proliferation and apoptosis was also studied. The human prostate cell lines RWPE-1 and WPE1-NB26 were cultured following the provider´s instructions. Eag1 mRNA expression and protein levels were studied by real time RT-PCR and immunocytochemistry, respectively. Cell proliferation and apoptosis were studied by a fluorescence AlamarBlue® assay and flow cytometry, respectively. No difference in Eag1 mRNA expression was observed between the cell lines. However, high Eag1 protein levels were observed in the invasive WPE1-NB26 cells, in contrast to the weak protein expression in RWPE-1 cells. Accordingly, astemizole decreased cell proliferation at nanomolar concentrations only in the invasive WPE1-NB26 cells. Our results suggest that astemizole may have clinical relevance for prostate cancer treatment in patients with high Eag1 protein levels.

Tissue-engineered human 3D model of bladder cancer for invasion study and drug discovery.

The tumour microenvironment is critical to both the initiation and maintenance of tumorigenesis. Reconstitution of the microenvironment is a major challenge for invitro cancer models. Indeed, conventional 2D culture systems cannot replicate the complexity, diversity and dynamic nature of the tumour microenvironment. In this study, we have developed a 3D endotheliazed vesical equivalent by using tissue engineering from primary human cells in which non-invasive or invasive bladder cancer (BCa) cell lines, cultured as compact spheroids, were incorporated. Invasive BCa cells cross the basement membrane and invade the stromal compartment whereas non-invasive BCa cells are confined to the urothelium. Our 3D BCa model could be used as a reliable model for assessing drug responses, potentially reducing or partially replacing animal experiments, and thus should have applications in the identification of novel targets as well as toxicological evaluation of anti-cancer therapies.

The role of LIM and SH3 protein-1 in bladder cancer metastasis.

The LIM and SH3 protein-1 (LASP-1) is a multi-domain protein that is involved in several malignant cancers. The role of LASP-1 in malignant phenotypes including high invasive properties and unrestricted cell proliferation, remain to be elucidated. The present study reported the association of LASP-1 expression with bladder cancer malignancy and its role in cancer cell invasion and proliferation. The immunohistochemical analysis of the expression status of LASP-1 in radical cystectomy specimens from invasive bladder cancer patients revealed that the LASP-1-positive patients demonstrated a decreased survival rate compared with the LASP-1-negative patients. The expression level of LASP-1 was increased in invasive bladder cancer cell lines compared with the non-invasive bladder cancer cell lines. Invasive cancer cells form invadopodia, the filamentous actin-based membrane protrusions that are essential in cancer cell invasion. Knockdown of LASP-1 reduced the ability to form invadopodia, resulting in decreased invasive capacity of the LASP-1 knockdown cells. In addition, knockdown of LASP-1 reduced cell proliferation. These results suggest that LASP-1 is important in invadopodia formation and cell proliferation of bladder cancer cells, promoting the malignant properties and resulting in poor-prognosis.

Functional Assay of Cancer Cell Invasion Potential Based on Mechanotransduction of Focused Ultrasound.

Cancer cells undergo a number of biophysical changes as they transform from an indolent to an aggressive state. These changes, which include altered mechanical and electrical properties, can reveal important diagnostic information about disease status. Here, we introduce a high-throughput, functional technique for assessing cancer cell invasion potential, which works by probing for the mechanically excitable phenotype exhibited by invasive cancer cells. Cells are labeled with fluorescent calcium dye and imaged during stimulation with low-intensity focused ultrasound, a non-contact mechanical stimulus. We show that cells located at the focus of the stimulus exhibit calcium elevation for invasive prostate (PC-3 and DU-145) and bladder (T24/83) cancer cell lines, but not for non-invasive cell lines (BPH-1, PNT1A, and RT112/84). In invasive cells, ultrasound stimulation initiates a calcium wave that propagates from the cells at the transducer focus to other cells, over distances greater than 1 mm. We demonstrate that this wave is mediated by extracellular signaling molecules and can be abolished through inhibition of transient receptor potential channels and inositol trisphosphate receptors, implicating these proteins in the mechanotransduction process. If validated clinically, our technology could provide a means to assess tumor invasion potential in cytology specimens, which is not currently possible. It may therefore have applications in diseases such as bladder cancer, where cytologic diagnosis of tumor invasion could improve clinical decision-making.

Abstract 5791: 3D tissue engineering bladder model for cancer invasion study

Abstract INTRODUCTION: Our understanding of the biological processes involved in bladder cancer (BCa) is greatly limited by the models currently available. In fact, the combination of in vitro and in vivo models of BCa has failed to elucidate all the fundamental aspects of the disease. The eighth most commonly diagnosed cancer in Western societies, BCa has become a growing public health concern, and more realistic models are needed to reveal the mechanisms involved in tumor initiation and progression. METHODS: Bladder substitutes have been constructed by tissue engineering with healthy human fibroblasts and urothelial cells, using the self-assembly method. Meanwhile, spheroids have been produced from non-invasive (RT4) and invasive (T24) BCa cell lines expressing DsRed fluorescent protein. The invasive potential of these spheroids was characterized in a type-I collagen gel (2.5mg/mL). Then, the spheroids were implanted on the surface of bladder substitutes, after which their development was followed by fluorescence microscopy. RESULTS: Both of the cancer cell lines used were able to form compact spheroids and grow on bladder equivalents. The invasive behaviour of spheroids varied depending on the nature of the cells used. The non-invasive RT4 cell line was unable to cross the basal lamina whereas the invasive T24 cell line was able to do so. CONCLUSION: The establishment of such a model for studying cancer biology in a physiological environment will help bridge the gap between overly simple cell culture models and more complex transgenic mice models. This new model offers a unique opportunity to study separately the players involved in the development of BCa and thus represents a powerful tool for the mechanistic analysis of this complex pathology. Citation Format: Cassandra Ringuette Goulet, Geneviève Bernard, Stéphane Chabaud, Frédéric Pouliot, Stéphane Bolduc. 3D tissue engineering bladder model for cancer invasion study [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5791. doi:10.1158/1538-7445.AM2017-5791

UBE2J2 promotes hepatocellular carcinoma cell epithelial-mesenchymal transition and invasion in vitro.

Ubiquitin-conjugating enzyme E2 J2 (UBE2J2) is an ubiquitin proteasome component that responds to proteotoxic stress. We found that UBE2J2 was highly expressed in cellular protrusions of HCCLM3 metastatic hepatocellular carcinoma (HC) cells. Immunohistochemical analyses showed that UBE2J2 was expressed at higher levels in HC patient tissues than in corresponding non-tumor tissues. Because cellular protrusions are important for cell invasion, we hypothesized that UBE2J2 promotes HC cell invasion. We used chip-based surface plasmon resonance (SPR) to assess possible mechanisms of UBE2J2-regulated HCCLM3 cell invasion. We found that p-EGFR interacted with UBE2J2, and this finding was confirmed by co-immunoprecipitation analysis. UBE2J2 overexpression activated endothelial-mesenchymal transition in the non-invasive SMMC7721 HC cell line, and promoted invasion. UBE2J2 silencing reduced HCCLM3 cell invasion and endocytosis, and downregulated p-EGFR expression. p-EGFR inhibition by lapatinib reduced UBE2J2-promoted cell invasion, suggesting p-EGFR is important for UBE2J2-mediated HCCLM3 cell invasion. These findings demonstrate that endocytosis by HC cells is closely related to invasion, and may provide new anti-HC therapeutic targets. UBE2J2 may also be a novel biomarker for clinical HC diagnosis.

MP48-06 3D TISSUE ENGINEERING BLADDER MODEL FOR CANCER INVASION STUDY

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology II1 Apr 2017MP48-06 3D TISSUE ENGINEERING BLADDER MODEL FOR CANCER INVASION STUDY Cassandra R Goulet, Geneviéve Bernard, Stéphane Chabaud, Frédéric Pouliot, and Stéphane Bolduc Cassandra R GouletCassandra R Goulet More articles by this author , Geneviéve BernardGeneviéve Bernard More articles by this author , Stéphane ChabaudStéphane Chabaud More articles by this author , Frédéric PouliotFrédéric Pouliot More articles by this author , and Stéphane BolducStéphane Bolduc More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2017.02.1487AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Our understanding of the biological processes involved in bladder cancer (BCa) is greatly limited by the models currently available. In fact, the combination of in vitro and in vivo models of BCa has failed to elucidate all the fundamental aspects of the disease. The eighth most commonly diagnosed cancer in Western societies, BCa has become a growing public health concern, and more realistic models are needed to reveal the mechanisms involved in tumor initiation and progression. METHODS Bladder substitutes have been constructed by tissue engineering with healthy human fibroblasts and urothelial cells, using the self-assembly method. Meanwhile, spheroids have been produced from non-invasive (RT4) and invasive (T24) BCa cell lines expressing DsRed fluorescent protein. The invasive potential of these spheroids was characterized in a type-I collagen gel (2.5mg/mL). Then, the spheroids were implanted on the surface of bladder substitutes, after which their development was followed by fluorescence microscopy. RESULTS Both of the cancer cell lines used were able to form compact spheroids and grow on bladder equivalents. The invasive behaviour of spheroids varied depending on the nature of the cells used. The non-invasive RT4 cell line was unable to cross the basal lamina whereas the invasive T24 cell line was able to do so. CONCLUSIONS The establishment of such a model for studying cancer biology in a physiological environment will help bridge the gap between overly simple cell culture models and more complex transgenic mice models. This new model offers a unique opportunity to study separately the players involved in the development of BCa and thus represents a powerful tool for the mechanistic analysis of this complex pathology. © 2017FiguresReferencesRelatedDetails Volume 197Issue 4SApril 2017Page: e639 Advertisement Copyright & Permissions© 2017MetricsAuthor Information Cassandra R Goulet More articles by this author Geneviéve Bernard More articles by this author Stéphane Chabaud More articles by this author Frédéric Pouliot More articles by this author Stéphane Bolduc More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

P034 Detection of soluble HLA antigens (sHLA) in blood for early detection of non-small cell lung carcinoma (NSCLC)

Aim Non-small and small cell lung cancer (NSCLC and SCLC) is the leading cause of cancer-related deaths worldwide. Early detection and appropriate intervention are critical for successful treatment. Current screening methods cannot clearly distinguish between benign and malignant nodules. The human histocompatibility antigens (HLA), which control the immune response, can be crucial in escape from tumor immune surveillance resulting in tumor progression and metastasis. Increased HLA levels in circulation of cancer patients have been detected in the past. Therefore, the aim was to determine circulating HLA antigens could serve as potential biomarkers to distinguish between benign and malignant lung cancer nodules. Methods HLA class I (ABC) and class II (DRB1 and DQ) antigen levels were analyzed in cell lines and conditioned media of epithelial H358 and mesenchymal H1703. Then, serum samples from 25 patients with benign nodules (non-invasive cells) and 25 patients with malignant nodule (Stage I NSCLC-with invasive cells) were analyzed for HLA-class I (ABC), HLA-class II (DRB1, DQA1, DQ, DMB) antigens in their serum. Equal amounts of total proteins from each group were used for comparison of HLA antigen levels. Western Blot with Chemiluminiscence detection system was used for evaluating HLA antigen levels in samples. Results A significantly higher level of HLA class I antigens was observed in conditioned medium from the invasive cell line compared to conditioned medium from non-invasive cell line ( p p Conclusion In-vitro studies using non-invasive and invasive cell lines showed significantly increased levels of HLA-class I in conditioned medium obtained from the invasive cell line compared to the non-invasive cell line. The Mann–Whitney Rank Sum Test showed significant differences in the serum levels of HLA-ABC, DQA1, HLA-DQ, HLA-DRB1, HLA-DMB, between patients with benign nodule and Stage I NSCLC patients. These preliminary data suggest that levels of HLA class I and class II antigens could act as potential biomarkers to diagnose early stages of NSCLC.

Validation of a network-based strategy for the optimization of combinatorial target selection in breast cancer therapy: siRNA knockdown of network targets in MDA-MB-231 cells as an in vitro model for inhibition of tumor development.

Network-based strategies provided by systems biology are attractive tools for cancer therapy. Modulation of cancer networks by anticancer drugs may alter the response of malignant cells and/or drive network re-organization into the inhibition of cancer progression. Previously, using systems biology approach and cancer signaling networks, we identified top-5 highly expressed and connected proteins (HSP90AB1, CSNK2B, TK1, YWHAB and VIM) in the invasive MDA-MB-231 breast cancer cell line. Here, we have knocked down the expression of these proteins, individually or together using siRNAs. The transfected cell lines were assessed for in vitro cell growth, colony formation, migration and invasion relative to control transfected MDA-MB-231, the non-invasive MCF-7 breast carcinoma cell line and the non-tumoral mammary epithelial cell line MCF-10A. The knockdown of the top-5 upregulated connectivity hubs successfully inhibited the in vitro proliferation, colony formation, anchorage independence, migration and invasion in MDA-MB-231 cells; with minimal effects in the control transfected MDA-MB-231 cells or MCF-7 and MCF-10A cells. The in vitro validation of bioinformatics predictions regarding optimized multi-target selection for therapy suggests that protein expression levels together with protein-protein interaction network analysis may provide an optimized combinatorial target selection for a highly effective anti-metastatic precision therapy in triple-negative breast cancer. This approach increases the ability to identify not only druggable hubs as essential targets for cancer survival, but also interactions most susceptible to synergistic drug action. The data provided in this report constitute a preliminary step toward the personalized clinical application of our strategy to optimize the therapeutic use of anti-cancer drugs.

Abstract 5084: Real-time, quantitative cellular analysis of migration and invasion

Abstract Chemotactic-driven cell migration is an important biological process impacting many areas of life science research including immunology, cancer and developmental biology. The dominant high-throughput solution for measuring cellular chemotaxis in vitro is the Boyden-chamber. However, traditional Boyden chamber products have inherent liabilities including: a) the inability to directly visualize the cell migration process, b) a requirement of tens of thousands of cells per well, c) the need to label cells for quantitation, d) a relative insensitivity to surface integrin signaling, and e) high random migration artifacts. Microfluidic solutions have sought to eliminate some of these deficiencies, specifically by providing the ability to visualize the cell migration process, but at the expense of relatively low throughput and cumbersome quantitation methods. Using a live-cell imaging approach and real-time image analysis quantitation, we have developed a new technology which eliminates most of the deficiencies of existing Boyden chambers and most importantly, provides direct visualization of both migration and invasion as it occurs. Integrated metrics precisely quantify the chemotactic response using significantly fewer cells (500-5000 cells per well) in a 96-well format. We exemplify this novel image based technique by studying the migration and invasion of both non-tumor and tumor-derived cell lines. Using Fetal bovine serum (FBS) as a chemoattractant, HT-1080 and NIH-3T3 cells were evaluated for their ability to directionally migrate across a coated surface or invade through a basement membrane extract by embedding the cells in an extracellular matrix prior to seeding. Qualitatively, we observed significant morphological differences between migrating and invading cells, the latter adopting a mesenchymal morphology reminiscent of cells that have undergone epithelial-mesenchymal transition (EMT) and exhibiting thinner, spindle-like morphology as they invade through the matrix. Using both cell count and cell area metrics, we observed a significant difference between migration and invasion kinetics of the relatively non-invasive cell line, NIH-3T3, and the highly invasive human fibrosarcoma derived cell line, HT-1080. Moreover, we observed significant and selective inhibition of HT-1080 cell invasion in the presence of GM6001, a broad inhibitor of Matrix Metalloproteinases (MMPs) with an IC50 of 25.7 nM. This new, real-time, quantitative cellular analysis technique illustrates a novel method to simultaneously study migration and invasion of tumor cells. Citation Format: Meagan Roddy, John Rauch, Lindy O’Clair, Daniel M. Appledorn. Real-time, quantitative cellular analysis of migration and invasion. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5084.

Abstract 5082: A quantitative and multi-parameter flow cytometry assay to simultaneously assess cell migration and immunophenotype

Abstract Cell migration occurs during physiological and pathological processes, which include wound healing and tumor metastasis. In vitro migration assays are necessary to understand the mechanism underlying cell migration and also to identify inhibitory or stimulatory molecules. The Boyden chamber assay is commonly used to measure cell motility in vitro where the number of cells that migrate through the transwell membrane is quantitated manually using an inverted microscope. Alternatively, cells can be detached from the membrane and stained with a fluorescent probe, prior to counting cells using a plate reader. These conventional assays are limited by an inability to simultaneously characterize individual cells while monitoring migration. To address this, we have developed a multi-parameter flow cytometry-based migration assay for the quantitative measurement of cell migration and simultaneous immunophenotypic analysis of the migrated cells. Invasive HT-1080 and non-invasive MCF-7 cancer cell lines were plated in the upper layer of a cell permeable membrane, as per the standard Boyden chamber assay. BD Horizon™ Fixable Viability Stain (FVS) was used to determine the optimal detachment conditions providing maximum yield with minimal impact on cell viability. Migrated cells were also co-stained with multiple conjugated antibodies to assess cell surface marker expression. Cells were analyzed on BD Accuri™ flow cytometer for rapid quantitation of migrated cells and analysis of up to 4 parameters. Alternatively, BD FACSCelesta™ flow cytometer was used for higher parameter analysis. Migrated cells were identified based on light scatter properties and viability staining with FVS, while immunophenotype was assessed in live cells. Our results demonstrate that this flow cytometric assay can be used to rapidly quantify changes in the migratory ability of different cell lines in the presence of inhibitors or stimulators. Additionally, we were able to assess the expression of hallmark cancer cell markers (CD44, CD24, CD326 (EpCAM)) before and after migration. The ability to quantify the number of migrating cells in response to specific stimuli and to simultaneously define cell immunophenotype represents a significant advancement, as compared to conventional cell motility assays. Our results demonstrate that this novel approach allows for a deeper analysis of cell migration that could enable complex drug discovery studies and the discovery of new cell signatures associated with metastatic progression. Citation Format: Mirko Corselli, Xiao Wang, Lissette Wilensky, Aaron Middlebrook, Smita Ghanekar, Jacob Rabenstein, Nil Emre. A quantitative and multi-parameter flow cytometry assay to simultaneously assess cell migration and immunophenotype. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 5082.

MP45-01 EXOSOMAL MIRNAS: KEY REGULATORS OF CELL-CELL COMMUNICATION BETWEEN BLADDER CANCER CELLS AND TUMOR MICROENVIRONMENT

You have accessJournal of UrologyBladder Cancer: Basic Research & Pathophysiology I1 Apr 2016MP45-01 EXOSOMAL MIRNAS: KEY REGULATORS OF CELL-CELL COMMUNICATION BETWEEN BLADDER CANCER CELLS AND TUMOR MICROENVIRONMENT Sophie Baumgart, Joana Heinzelmann, Elmar Krause, Marie Stampe Ostenfeld, Arndt Hartmann, Michael Stöckle, and Kerstin Junker Sophie BaumgartSophie Baumgart More articles by this author , Joana HeinzelmannJoana Heinzelmann More articles by this author , Elmar KrauseElmar Krause More articles by this author , Marie Stampe OstenfeldMarie Stampe Ostenfeld More articles by this author , Arndt HartmannArndt Hartmann More articles by this author , Michael StöckleMichael Stöckle More articles by this author , and Kerstin JunkerKerstin Junker More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2016.02.278AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Interaction of tumor cells and tumor microenvironment (TME) plays an important role in tumor progression. It was shown that microRNAs (miRNAs) packed in exosomes (EV) can affect cell-cell communication at the site of origin (TME) as well as distant sites. One aim of the project is the identification of a specific miRNA expression pattern from tumor-derived EV of different urinary bladder cancer (UBC) cell lines in correlation to their invasive potential. Afterwards, the functional role of specific exosomal miRNAs on cells of the TME (e.g. naive fibroblasts) will be analyzed. METHODS EV were isolated from invasive (T-24,253J-BV, J82) and non-invasive (RT-112, 5637) UBC cell lines and characterized by Western Blot analysis, Nano Particle Tracking Analysis and electron microscopy. MiRNA expression analysis in UBC cells and their EV was performed by using miRNA microarray and qPCR. Uptake of PKH26-labeled UBC-EV by immortalized fibroblasts was shown by laser scanning microscopy. EV-mediated miRNA transfer between cancer cells and fibroblasts was verified by transfection of donor UBC cells with the C. elegans-specific miRNA, cel-miR-39, EV isolation and RNAse treatment, transfer to recipient fibroblasts (TAFs and naive fibroblasts), and miRNA-specific qPCR analysis using totalRNA from the recipient fibroblasts. RESULTS Electron microscopy images revealed the typical size and vesicular structure of EV. The size measurement revealed a mean size of EV among 61-81nm. Isolated EV exhibited a high amount of exosomal markers (CD81, syntenin) and less presence of contamination marker calreticulin. 16 miRNAs were identified distinguishing invasive UBC cells from non-invasive cells. EV secreted by invasive UBC cells are characterized by a specific miRNA signature of 25 miRNAs compared to EV from non-invasive UBC cells. 6 significantly different expressed miRNAs clearly separate both invasive and non-invasive UBC cells and their secreted EV. Labeled EV of UBC cells were taken up by naive fibroblasts. After co-cultivation of EV obtained from transfected UBC cells and naive fibroblasts, high expression of cel-miR-39 was detected in naive fibroblasts. CONCLUSIONS EV secreted by UBC cells exhibit a specific miRNA signature depending on the invasive potential of the originating cells. For the first time we demonstrated the EV-mediated transfer of miRNAs between UBC cells and naive fibroblasts. Furthermore, EV of UBC cells can be taken up by fibroblasts. These results emphasize the role of exosomal miRNAs for the interaction between tumor cells and the tumor microenvironment. Further studies have to show the functional relevance of selected exosomal miRNAs. © 2016FiguresReferencesRelatedDetails Volume 195Issue 4SApril 2016Page: e607 Advertisement Copyright & Permissions© 2016MetricsAuthor Information Sophie Baumgart More articles by this author Joana Heinzelmann More articles by this author Elmar Krause More articles by this author Marie Stampe Ostenfeld More articles by this author Arndt Hartmann More articles by this author Michael Stöckle More articles by this author Kerstin Junker More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Glycolysis and the pentose phosphate pathway are differentially associated with the dichotomous regulation of glioblastoma cell migration versus proliferation.

The dichotomy between glioblastoma cell migration and proliferation is regulated by various parameters including oxygen tension. In glioblastoma stem-like cells, hypoxia induces downregulation of pentose phosphate pathway (PPP) enzymes and a flux shift towards glycolysis. We investigated whether the 2 parallel glucose metabolic pathways are intrinsically linked with cell function and whether these pathways are mechanistically involved in regulating functional programs. Enzyme expression, migration, and proliferation under hypoxia were studied in multiple cell types. Rapidly and slowly dividing or migrating glioblastoma cells were separated, and enzyme profiles were compared. Glucose-6-phosphate dehydrogenase (G6PD) and Aldolase C (ALDOC), the most strongly inversely regulated PPP and glycolysis enzymes, were knocked down by short hairpin RNA. Hypoxia caused downregulation of PPP enzymes and upregulation of glycolysis enzymes in a broad spectrum of cancer and nonneoplastic cells and consistently stimulated migration while reducing proliferation. PPP enzyme expression was increased in rapidly dividing glioblastoma cells, whereas glycolysis enzymes were decreased. Conversely, glycolysis enzymes were elevated in migrating cells, whereas PPP enzymes were diminished. Knockdown of G6PD reduced glioblastoma cell proliferation, whereas ALDOC knockdown decreased migration. Enzyme inhibitors had similar effects. G6PD knockdown in a highly proliferative but noninvasive glioblastoma cell line resulted in prolonged survival of mice with intracerebral xenografts, whereas ALDOC knockdown shortened survival. In a highly invasive glioblastoma xenograft model, tumor burden was unchanged by either knockdown. Cell function and metabolic state are coupled independently of hypoxia, and glucose metabolic pathways are causatively involved in regulating "go or grow" cellular programs.

Genomic profiling of invasive melanoma cell lines by array comparative genomic hybridization.

Malignant melanoma is one of the most aggressive human cancers. Invasion of cells is the first step in metastasis, resulting in cell migration through tissue compartments. We aimed to evaluate genomic alterations specifically associated with the invasive characteristics of melanoma cells. Matrigel invasion assays were used to determine the invasive properties of cell lines that originated from primary melanomas. Array comparative genomic hybridization analyses were carried out to define the chromosome copy number alterations (CNAs). Several recurrent CNAs were identified by array comparative genomic hybridization that affected melanoma-related genes. Invasive primary cell lines showed high frequencies of CNAs, including the loss of 7q and gain of 12q chromosomal regions targeting PTPN12, ADAM22, FZD1, TFPI2, GNG11, COL1A2, SMURF1, VGF, RELN and GLIPR1 genes. Gain of the GDNF (5p13.1), GPAA1, PLEC and SHARPIN (8q24.3) genes was significantly more frequent in invasive cell lines compared with the noninvasive ones. Importantly, copy number gains of these genes were also found in cell lines that originated from metastases, suggesting their role in melanoma metastasis formation. The present study describes genomic differences between invasive and noninvasive melanoma cell lines that may contribute toward the aggressive phenotype of human melanoma cells.

Macrophage migration inhibitory factor - a therapeutic target in gallbladder cancer

BackgroundPoor prognosis in gallbladder cancer is due to late presentation of the disease, lack of reliable biomarkers for early diagnosis and limited targeted therapies. Early diagnostic markers and novel therapeutic targets can significantly improve clinical management of gallbladder cancer.MethodsProteomic analysis of four gallbladder cancer cell lines based on the invasive property (non-invasive to highly invasive) was carried out using the isobaric tags for relative and absolute quantitation labeling-based quantitative proteomic approach. The expression of macrophage migration inhibitory factor was analysed in gallbladder adenocarcinoma tissues using immunohistochemistry. In vitro cellular assays were carried out in a panel of gallbladder cancer cell lines using MIF inhibitors, ISO-1 and 4-IPP or its specific siRNA.ResultsThe quantitative proteomic experiment led to the identification of 3,653 proteins, among which 654 were found to be overexpressed and 387 were downregulated in the invasive cell lines (OCUG-1, NOZ and GB-d1) compared to the non-invasive cell line, TGBC24TKB. Among these, macrophage migration inhibitory factor (MIF) was observed to be highly overexpressed in two of the invasive cell lines. MIF is a pleiotropic proinflammatory cytokine that plays a causative role in multiple diseases, including cancer. MIF has been reported to play a central role in tumor cell proliferation and invasion in several cancers. Immunohistochemical labeling of tumor tissue microarrays for MIF expression revealed that it was overexpressed in 21 of 29 gallbladder adenocarcinoma cases. Silencing/inhibition of MIF using siRNA and/or MIF antagonists resulted in a significant decrease in cell viability, colony forming ability and invasive property of the gallbladder cancer cells.ConclusionsOur findings support the role of MIF in tumor aggressiveness and suggest its potential application as a therapeutic target for gallbladder cancer.Electronic supplementary materialThe online version of this article (doi:10.1186/s12885-015-1855-z) contains supplementary material, which is available to authorized users.

RECK is not an independent prognostic marker for breast cancer.

BackgroundThe REversion-inducing Cysteine-rich protein with Kazal motif (RECK) is a well-known inhibitor of matrix metalloproteinases (MMPs) and cellular invasion. Although high expression levels of RECK have already been correlated with a better clinical outcome for several tumor types, its main function, as well as its potential prognostic value for breast cancer patients, remain unclear.MethodsThe RECK expression profile was investigated in a panel of human breast cell lines with distinct aggressiveness potential. RECK functional analysis was undertaken using RNA interference methodology. RECK protein levels were also analyzed in 1040 cases of breast cancer using immunohistochemistry and tissue microarrays (TMAs). The association between RECK expression and different clinico-pathological parameters, as well as the overall (OS) and disease-free (DFS) survival rates, were evaluated.ResultsHigher RECK protein expression levels were detected in more aggressive breast cancer cell lines (T4-2, MDA-MB-231 and Hs578T) than in non-invasive (MCF-7 and T47D) and non-tumorigenic (S1) cell lines. Indeed, silencing RECK in MDA-MB-231 cells resulted in elevated levels of pro-MMP-9 and increased invasion compared with scrambled (control) cells, without any effect on cell proliferation. Surprisingly, by RECK immunoreactivity analysis on TMAs, we found no association between RECK positivity and survival (OS and DFS) in breast cancer patients. Even considering the different tumor subtypes (luminal A, luminal B, Her2 type and basal-like) or lymph node status, RECK remained ineffective for predicting the disease outcome. Moreover, by multivariate Cox regression analysis, we found that RECK has no prognostic impact for OS and DFS, relative to standard clinical variables.ConclusionsAlthough it continues to serve as an invasion and MMP inhibitor in breast cancer, RECK expression analysis is not useful for prognosis of these patients.

Keratinocytes have the metastatic touch

Notch inhibitors may prevent a metastatic transition in melanoma.

Abstract 5182: Characterization of miRNA expression pattern from in-vitro obtained exosomes of different urinary bladder cancer cell lines

Abstract Introduction & objectives: Interaction of tumor cells and the tumor microenvironment (TME) plays an important role in tumor development and progression. It was shown that microRNAs (miRNAs), packed in exosomes, can affect cell-cell communication at the site of origin as well as the TME. The aim of the project is the identification of a specific miRNA expression pattern from in-vitro obtained tumor-derived exosomes of different UBC cell lines in correlation to their malignant potential. Furthermore, we want to analyze the effect of these exosomal miRNAs on tumor-associated fibroblasts (TAFs). Materials & methods: Exosomes were isolated from invasive (T-24,253J-BV, J82) and non-invasive (RT-112, 5637) UBC cell lines. The number and size of vesicles were measured by NTA. The vesicles were examined for exosomal and contamination markers by Western blotting. TotalRNA was isolated from the exosomes upon treatment with RNase. MiRNA expression pattern was analyzed from exosomes secreted by invasive and non-invasive UBC cells using miRNA microarray. The validation of significantly differently expressed miRNAs was performed by using qPCR. Exosome-mediated miRNA transfer between cancer cells and TAFs was verified by 1) transfection of donor UBC cells with the C. elegans-specific miRNA, cel-miR-39, 2) Exosome isolation and RNAse treatment, 3) Transfer to recipient TAFs, and 4) miRNA-specific qRT-PCR analysis using totalRNA from the recipient TAFs. Results: The isolated exosomes from UBC cells exhibited a high amount of exosomal markers (CD63, CD81, syntenin). 16 miRNAs were identified, which distinguishes invasive UBC cells from non-invasive cells. Exosomes secreted by invasive UBC cells are characterized by a specific miRNA signature of 25 miRNAs compared to exosomes from non-invasive UBC cells. The validation of differently expressed miRNAs is ongoing. After successful transfection of RT-112 and T-24 with cel-miR-39, cel-miR-39 was detected in RT-112 and T-24 exosomes as well as in recipient TAFs cultivated in the presence of these exosomes. Conclusion: Exosomes secreted by UBC cells exhibit a specific miRNA signature depending on the invasive potential of the originating cells. We could proof an exosome-mediated transfer of miRNAs between tumor cells and TAF. These results emphasize the role of exosomal miRNAs for the interaction between tumor cells and the tumor microenvironment. Further studies have to show the functional relevance of selected exosomal miRNAs. Citation Format: Sophie Baumgart, Joana Heinzelmann, Michael Stoeckle, Marie Stampe Ostenfeld, Kerstin Junker. Characterization of miRNA expression pattern from in-vitro obtained exosomes of different urinary bladder cancer cell lines. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5182. doi:10.1158/1538-7445.AM2015-5182

Abstract 173: SOX4 interacts with EZH2 and HDAC3 to suppress microRNA-31 in invasive esophageal cancer cells

Abstract Tumor metastasis is responsible for 90% of cancer-related deaths. A strong link between microRNA dysregulation and human cancers has recently been established. However, the molecular mechanisms through which microRNAs regulate metastasis and cancer progression remain unclear. We analyzed the reciprocal expression regulation of miR-31 and SOX4 in esophageal squamous and adenocarcinoma cell lines by qRT-PCR and Western Blotting using overexpression and shRNA knock-down approaches. Here we report that SOX4 promotes esophageal tumor cell proliferation and invasion by silencing miR-31 via activation and stabilization of a co-repressor complex with EZH2 and HDAC3. We demonstrate that miR-31 is significantly decreased in invasive esophageal cancer cells compared to non-invasive cell lines, while upregulation of miR-31 inhibits growth, migration and invasion of esophageal adenocarcinoma (EAC) and squamous cell carcinoma (ESCC) cell lines. miR-31, in turn, targets SOX4 for degradation by directly binding to its 3′-UTR. Additionally, miR-31 regulates EZH2 and HDAC3 indirectly. SOX4, EZH2 and HDAC3 levels inversely correlate with miR-31 expression in ESCC cell lines. Ectopic expression of miR-31 in ESCC and EAC cell lines leads to down regulation of SOX4, EZH2 and HDAC3. Conversely, pharmacologic and genetic inhibition of SOX4 and EZH2 restore miR-31 expression. We show that SOX4, EZH2 and HDAC3 form a co-repressor complex that binds to the miR-31 promoter, repressing miR-31 through an epigenetic mark by H3K27me3 and by histone acetylation. Clinically, when compared to normal adjacent tissues, esophageal tumor samples show upregulation of SOX4, EZH2, and HDAC3, and EZH2 expression is significantly increased in metastatic ESCC tissues. Thus, we identified a novel molecular mechanism by which the SOX4, EZH2 and miR-31 circuit promotes tumor progression and potential therapeutic targets for invasive esophageal carcinomas. Citation Format: Rainelli B. Koumangoye, Thomas Andl, Kenneth J. Taubenslag, Steven T. Zilberman, Chase Taylor, Claudia D. Andl. SOX4 interacts with EZH2 and HDAC3 to suppress microRNA-31 in invasive esophageal cancer cells. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 173. doi:10.1158/1538-7445.AM2015-173

Abstract 2906: Role of autophagy in Wnt5A-mediated melanoma invasion and metastasis

Abstract Melanoma is the most aggressive type of skin cancer and the leading cause of death from skin disease. With increasing incidence of the disease, it is crucial to further investigate the cellular mechanisms and molecular pathways that lead to invasion, metastasis, and drug resistance. Recent studies have revealed that high autophagy correlates with melanoma tumor aggressiveness and poor survival in clinical samples, as it is a common mechanism of resistance to therapy. Autophagy inhibition leads to reduced levels in Wnt5A in a breast cancer model, suggesting a cross-talk of Wnt5A and autophagy in cancer. β catenin, a mediator of canonical Wnt signaling, has been shown to act as a negative regulator of both basal and induced autophagy in a colorectal cancer model. Our laboratory showed that high levels of Wnt5A correlate with increased invasion and metastasis in melanoma and that Wnt5A downregulates β catenin; therefore, we hypothesize that autophagy might drive invasion and metastasis in aggressive melanoma through the regulation of Wnt signaling. To study the role of autophagy in Wnt5A-mediated melanoma invasion, we inhibited autophagy in highly invasive melanoma cells using lentivirus-mediated shATG5 knockdown or hydroxychloroquine and evaluated the effects of autophagy inhibition on Wnt5A expression, β catenin, and invasion using 3D spheroid models. Analysis of autophagy flux confirms that highly invasive melanoma cells have high autophagy compared to non-invasive cell lines. The inhibition of autophagy by hydroxychloroquine or shATG5 both resulted in significant decrease in invasion. By western blot analysis, we also observed a decrease in Wnt5A in these cells. These results demonstrate that autophagy inhibition in highly invasive melanoma leads to decrease in invasion in a 3D model that mimics the tumor microenvironment of melanoma. Further dissection of the molecular mechanisms that are involved in this process will enable the identification of novel autophagy targets in aggressive melanoma. Citation Format: Abibatou Ndoye, Anna Budina, Marie Webster, Amanpreet Kaur, Reeti Behera, Maureen Murphy, Ashani T. Weeraratna. Role of autophagy in Wnt5A-mediated melanoma invasion and metastasis. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 2906. doi:10.1158/1538-7445.AM2015-2906