Non-invasive Cells Research Articles

Upregulation of MEK5 by Stat3 promotes breast cancer cell invasion and metastasis.

Mitogen extracellular-signal-regulated kinase kinase 5 (MEK5) plays an important role in promoting cell proliferation and tumorigenesis. The aberrant expression of MEK5 has been reported in various malignant diseases including cancers of breast, prostate, lung, colorectal and brain. However, the function and regulation of MEK5 signaling pathway are ambiguous and remain elusive with respect to its oncogenic roles in various cancers, especially in the regulation of the initiation and progression of cancer invasion and metastasis. Ectopic expression of MEK5 or knockdown of MEK5 by shRNA with invitro cell based models demonstrated the role of MEK5 in regulation of epithelial mesenchymal transition (EMT) and breast cancer invasion and metastasis. Here, we show that MEK5 upregulated by Stat3 promotes breast cancer cell invasion through EMT. Further study demonstrated that Stat3 could bind to promoter region of MEK5 and enhanced MEK5 transcription and expression. In addition, the phosphorylation of MEK5 significantly increased in breast cancer cells corresponding to metastatic capability of breast cancer cells. The depletion of MEK5 by shRNA significantly decreased breast cancer invasion. Ectopic expression of MEK5 could confer non-invasive breast cancer cells to become invasion capable cells. Moreover, the phosphorylation of Erk5, a MEK5-regulated downstream kinase, was also upregulated consistent with the increased level of active MEK5. Our studies provide insights into a molecular mechanism by which MEK5 transcriptionally upregulated by Stat3 augments breast cancer cell EMT, which subsequently enhances cancer cell invasion and metastasis. This finding may suggest that Stat3 and MEK5/Erk5 pathways could be an effective therapeutic target for inhibition of breast cancer invasion and metastasis.

Characterization of Innate Immune Responses of Human Endothelial Cells Induced by Porphyromonas gingivalis and Their Derived Outer Membrane Vesicles.

Atherosclerosis, a chronic inflammatory disease of the blood vessels, is one of the most common causes of morbidity and mortality world-wide. Involvement of Porphyromonas gingivalis in atherosclerosis is supported by observations from epidemiological, clinical, immunological, and molecular studies. Previously we reported that P. gingivalis vesicles have a much higher invasive efficiency than their originating cells. Here, we further compare the role of P. gingivalis cells and their vesicles in expression of chemoattractant proteins including CXCL1, CXCL2, and CXCL8, and adhesive molecules such as E-selectin in human umbilical vein endothelial cells (HUVECs). Both P. gingivalis 33277 cells and vesicles were able to up-regulate expression of these molecules, while the vesicles acted as more potent inducers of the inflammatory response associated with the development of atherosclerosis, consequently resulting in significant monocyte adhesion to a monolayer of HUVECs. Interestingly, we found that elevated expression of CXCL8 and E-selectin in endothelial cells induced by P. gingivalis correlated with the invasive ability of P. gingivalis cells and vesicles. Non-invasive bacterial cells and vesicles had no effect on expression of these genes. This study highlights the potential risk of P. gingivalis cells and vesicles in initiation of atherosclerosis and provides a potential target for the development of novel therapeutics against bacteria-associated atherosclerosis.

MicroRNA and protein profiles in invasive versus non-invasive oral tongue squamous cell carcinoma cells in vitro

Complex molecular pathways regulate cancer invasion. This study overviewed proteins and microRNAs (miRNAs) involved in oral tongue squamous cell carcinoma (OTSCC) invasion. The human highly aggressive OTSCC cell line HSC-3 was examined in a 3D organotypic human leiomyoma model. Non-invasive and invasive cells were laser-captured and protein expression was analyzed using mass spectrometry-based proteomics and miRNA expression by microarray. In functional studies the 3D invasion assay was replicated after silencing candidate miRNAs, miR-498 and miR-940, in invasive OTSCC cell lines (HSC-3 and SCC-15). Cell migration, proliferation and viability were also studied in the silenced cells. In HSC-3 cells, 67 proteins and 53 miRNAs showed significant fold-changes between non-invasive vs. invasive cells. Pathway enrichment analyses allocated “Focal adhesion” and “ECM-receptor interaction” as most important for invasion. Significantly, in HSC-3 cells, miR-498 silencing decreased the invasion area and miR-940 silencing reduced invasion area and depth. Viability, proliferation and migration weren’t significantly affected. In SCC-15 cells, down-regulation of miR-498 significantly reduced invasion and migration. This study shows HSC-3 specific miRNA and protein expression in invasion, and suggests that miR-498 and miR-940 affect invasion in vitro, the process being more influenced by mir-940 silencing in aggressive HSC-3 cells than in the less invasive SCC-15.

Comparative immunohistochemical characteristics of Ki-67, p53, caspase-3 expression in gastric hyperplastic polyps and non-invasive intestinal-type gastric adenocarcinoma

Aim. The problem of early diagnostic of gastric cancer has a tight connection with the issue of premalignant lesions, especially gastric polyps. The aim of this research was to compare the expression levels of Ki-67, p53 and caspase-3 in epithelial cells and stromal cells of gastric hyperplastic polyps on the one hand and tumor cells and stromal cells of non-invasive intestinal-type gastric adenocarcinoma on the other hand. Materials and methods. For this purpose, pathohistological and immunohistochemical studies were used. The biopsies of 10 patients with hyperplastic polyps (the age of the patients ranged from 52 to 73 years) and surgical material of 10 patients with non-invasive (pTis, pT1) intestinal-type gastric adenocarcinoma (the age of patients ranged from 45 to 83 years) were analyzed. Results. It was established that epithelial cells of gastric hyperplastic polyps are characterized by low expression level of Ki-67, p53 and caspase-3, just as stromal cells of hyperplastic polyps are characterized by low expression level of Ki-67 and the medium expression level of caspase-3. Moreover, it has been established that tumor cells of non-invasive intestinal-type gastric adenocarcinoma are characterized by medium expression level of Ki-67, caspase-3 and also low expression level of p53, just as stromal cells of non-invasive adenocarcinoma are characterized by low expression level of Ki-67 and low expression level of caspase-3. The differences between the expression levels of Ki-67, p53 and caspase-3, that were established for hyperplastic polyps on the one hand and non-invasive adenocarcinoma on the other hand, are statistically significant (p<0,05). Conclusion. These data demonstrate, that proliferative activity of non-invasive intestinal-type gastric adenocarcinoma tumor cells is higher than proliferative activity of gastric hyperplastic polyps epithelial cells. Moreover, non-invasive intestinal-type gastric adenocarcinoma differs by higher activity of p53 accumulation, higher level of tumor cells apoptosis and lower level of stromal cells apoptosis.

Pancreatic stellate cell secreted IL-6 stimulates STAT3 dependent invasiveness of pancreatic intraepithelial neoplasia and cancer cells.

Pancreatic ductal adenocarcinoma (PDAC) is a dynamic tumor supported by several stromal elements such as pancreatic stellate cells (PSC). Significant crosstalk exists between PSCs and tumor cells to stimulate oncogenic signaling and malignant progression of PDAC. However, how PSCs activate intercellular signaling in PDAC cells remains to be elucidated. We have previously shown that activated signal transducer and activator of transcription 3 (STAT3) signaling is a key component in the progression of pancreatic neoplasia. We hypothesize that PSC secreted IL-6 activates STAT3 signaling to promote PanIN progression to PDAC. Human PDAC and mouse PanIN cells were treated with PSC-conditioned media (PSC-CM), and phospho- and total-STAT3 levels by immunoblot analysis were determined. IL-6 was quantified in PSC-CM and cell invasion and colony formation assays were performed in the presence or absence of a neutralizing IL-6 antibody and the JAK/STAT3 inhibitor AZD1480. Serum from Ptf1aCre/+;LSL-KrasG12D/+;Tgfbr2flox/flox' (PKT) and LSL-KrasG12D/+; Trp53R172H/+; Pdx1Cre/+ (KPC) mice demonstrated increased levels of IL-6 compared to serum from non-PDAC bearing KC and PK mice. PSC secreted IL-6 activated STAT3 signaling in noninvasive, precursor PanIN cells as well as PDAC cells, resulting in enhanced cell invasion and colony formation in both cell types. There was a significant positive linear correlation between IL-6 concentration and the ratio of phosphorylated STAT3/total STAT3. IL-6 neutralization or STAT3 inhibition attenuated PSC-CM induced activation of STAT3 signaling and tumorigenicity. These data provide evidence that PSCs are directly involved in promoting the progression of PanINs towards invasive carcinoma. This study demonstrates a novel role of PSC secreted IL-6 in transitioning noninvasive pancreatic precursor cells into invasive PDAC through the activation of STAT3 signaling.

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.

Zinc finger E-box binding homeobox-1 (Zeb1) drives anterograde lysosome trafficking and tumor cell invasion via upregulation of Na+/H+ Exchanger-1 (NHE1).

Tumor cell invasion through the extracellular matrix is facilitated by the secretion of lysosome-associated proteases. As a common mechanism for secretion, lysosomes must first traffic to the cell periphery (anterograde trafficking), consistent with invasive cells often containing lysosomes closer to the plasma membrane compared to non-invasive cells. Epithelial to mesenchymal transition (EMT) is a transcriptionally driven program that promotes an invasive phenotype, and Zeb1 is one transcription factor that activates the mesenchymal gene expression program. The role of lysosome trafficking in EMT-driven invasion has not been previously investigated. We found that cells with increased levels of Zeb1 displayed lysosomes located closer to the cell periphery and demonstrated increased protease secretion and invasion in 3-dimensional (3D) cultures compared to their epithelial counterparts. Additionally, preventing anterograde lysosome trafficking via pharmacological inhibition of Na+/H+ exchanger 1 (NHE1) or shRNA depletion of ADP-ribosylation like protein 8b (Arl8b) reversed the invasive phenotype of mesenchymal cells, thus supporting a role for lysosome positioning in EMT-mediated tumor cell invasion. Immunoblot revealed that expression of Na+/H+ exchanger 1 correlated with Zeb1 expression. Furthermore, we found that the transcription factor Zeb1 binds to the Na+/H+ exchanger 1 promoter, suggesting that Zeb1 directly controls Na+/H+ transcription. Collectively, these results provide insight into a novel mechanism regulating Na+/H+ exchanger 1 expression and support a role for anterograde lysosome trafficking in Zeb1-driven cancer progression. © 2016 Wiley Periodicals, Inc.

Abstract C22: ROCK kinases drive invasive pancreatic tumor growth

Abstract Pancreatic cancer is one of the leading causes of cancer death and despite advances in chemotherapeutic regimens the overall 5-year survival rate remains less than 5%. The actomyosin-regulating ROCK1 and ROCK2 kinases are downstream-targets of the Rho GTPase pathway. They contribute to various processes, such as cell adhesion, motility, proliferation, differentiation and survival, which then influence numerous stages of cancer growth and progression. Interestingly, exome sequencing of pancreatic cancer genomes revealed that 15% of pancreatic cancer patients carry an amplification of the ROCK1 gene (1). Moreover, we found significant increases in ROCK1 and ROCK2 RNA expression in pancreatic cancer datasets obtained using Oncomine. When we further determined the protein expression of ROCK2 in pancreatic ductal adenocarcinoma (PDAC) we found an up-regulation of ROCK2 during disease progression. Higher ROCK2 levels also correlated with less differentiated tumors. A characteristic of advanced stages of pancreatic cancer is a collagen and fibroblast enriched stroma. To analyze the effect of ROCK kinases on cancer cell invasion, we performed 3-dimensional organotypic assays. Our results demonstrate that increased ROCK signaling was sufficient to convert non-invasive PDAC cells into ones capable of invasion into organotypic collagen matrix. Using an RNA sequencing approach, we investigated the effect of ROCK activation on pancreatic cancer cell gene transcription. Differential expression analysis revealed an enrichment of gene sets that are involved in cell-matrix interaction, and we found a higher release of matrix metalloproteinases MMP10 and MMP13 upon ROCK activation. In addition, our organotypic studies revealed extensive tissue remodeling and an accumulation of cleaved collagen bundles at the sites of PDAC cell invasion. Furthermore, collagen degradation and cell invasion into organotypic matrices were significantly reduced by application of a broad-spectrum MMP inhibitor, confirming that ROCK-induced invasion is dependent on MMP activity. Interestingly, our studies also indicated that ROCK-driven invasion of PDAC cells into collagen matrix enabled cell growth. To study the function of ROCK kinase signaling in pancreatic cancer in vivo, we expressed conditionally active ROCK2 in a mouse model of PDAC. We found that an increase of ROCK activity in pancreatic cancer cells accelerated PDAC progression, which resulted in reduced survival. Contrary, the administration of a ROCK inhibitor during tumor progression had a beneficial effect on survival. In summary, our results suggest that targeting ROCK kinases should be considered for chemotherapy of invasive pancreatic cancer. (1) Biankin et al. Nature. 2012 Nov 15;491(7424):399-405 Citation Format: Nicola Rath, Shereen Kadir, Jennifer P. Morton, Andreia V. Pinho, Lena Helbig, Linda Julian, Ewan J. McGhee, Gabriela Kalna, Alexei Vazquez, Kurt I. Anderson, Ilse Rooman, Michael S. Samuel, Michael F. Olson. ROCK kinases drive invasive pancreatic tumor growth. [abstract]. In: Proceedings of the AACR Special Conference: Function of Tumor Microenvironment in Cancer Progression; 2016 Jan 7–10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2016;76(15 Suppl):Abstract nr C22.

Impact by pancreatic stellate cells on epithelial-mesenchymal transition and pancreatic cancer cell invasion: Adding a third dimension in vitro

Pancreatic cancer is associated with a highly abundant stroma and low-grade inflammation. In the local tumour microenvironment, elevated glucose levels, the presence of tumour-associated stellate cells and macrophages are hypothesised to promote the tumour progression and invasion. The present study investigated the influence by the microenvironment on pancreatic cancer cell invasion in vitro. After co-culture with tumour-associated pancreatic stellate cells (TPSCs), pancreatic cancer cells displayed up to 8-fold reduction in levels of epithelial-mesenchymal transition (EMT) markers E-cadherin and ZO-1, while β-catenin and vimentin levels were increased. A 3D organotypic model showed that TPSCs stimulated pancreatic cancer cell invasion, both as single cell (PANC-1) and cohort (MIAPaCa-2) invasion. The combined presence of TPSCs and M2-like macrophages induced invasion of the non-invasive BxPC-3 cells. High glucose conditions further enhanced changes in EMT markers as well as the cancer cell invasion. In summary, co-culture with TPSCs induced molecular changes associated with EMT in pancreatic cancer cells, regardless of differentiation status, and the organotypic model demonstrated the influence of microenvironmental factors, such as glucose, stellate cells and macrophages, on pancreatic cancer cell invasion.

Systematic Analysis of AU-Rich Element Expression in Cancer Reveals Common Functional Clusters Regulated by Key RNA-Binding Proteins.

Defects in AU-rich elements (ARE)-mediated posttranscriptional control can lead to several abnormal processes that underlie carcinogenesis. Here, we performed a systematic analysis of ARE-mRNA expression across multiple cancer types. First, the ARE database (ARED) was intersected with The Cancer Genome Atlas databases and others. A large set of ARE-mRNAs was over-represented in cancer and, unlike non-ARE-mRNAs, correlated with the reversed balance in the expression of the RNA-binding proteins tristetraprolin (TTP, ZFP36) and HuR (ELAVL1). Serial statistical and functional enrichment clustering identified a cluster of 11 overexpressed ARE-mRNAs (CDC6, KIF11, PRC1, NEK2, NCAPG, CENPA, NUF2, KIF18A, CENPE, PBK, TOP2A) that negatively correlated with TTP/HuR mRNA ratios and was involved in the mitotic cell cycle. This cluster was upregulated in a number of solid cancers. Experimentally, we demonstrated that the ARE-mRNA cluster is upregulated in a number of tumor breast cell lines when compared with noninvasive and normal-like breast cancer cells. RNA-IP demonstrated the association of the ARE-mRNAs with TTP and HuR. Experimental modulation of TTP or HuR expression led to changes in the mitosis ARE-mRNAs. Posttranscriptional reporter assays confirmed the functionality of AREs. Moreover, TTP augmented mitotic cell-cycle arrest as demonstrated by flow cytometry and histone H3 phosphorylation. We found that poor breast cancer patient survival was significantly associated with low TTP/HuR mRNA ratios and correlated with high levels of the mitotic ARE-mRNA signature. These results significantly broaden the role of AREs and their binding proteins in cancer, and demonstrate that TTP induces an antimitotic pathway that is diminished in cancer. Cancer Res; 76(14); 4068-80. ©2016 AACR.

Isorhapontigenin (ISO) Inhibits Invasive Bladder Cancer Formation In Vivo and Human Bladder Cancer Invasion In Vitro by Targeting STAT1/FOXO1 Axis.

Although our most recent studies have identified Isorhapontigenin (ISO), a novel derivative of stilbene that isolated from a Chinese herb Gnetum cleistostachyum, for its inhibition of human bladder cancer growth, nothing is known whether ISO possesses an inhibitory effect on bladder cancer invasion. Thus, we addressed this important question in current study and discovered that ISO treatment could inhibit mouse-invasive bladder cancer development following bladder carcinogen N-butyl-N-(4-hydroxybutyl) nitrosamine (BBN) exposure in vivo We also found that ISO suppressed human bladder cancer cell invasion accompanied by upregulation of the forkhead box class O 1 (FOXO1) mRNA transcription in vitro Accordingly, FOXO1 was profoundly downregulated in human bladder cancer tissues and was negatively correlated with bladder cancer invasion. Forced expression of FOXO1 specifically suppressed high-grade human bladder cancer cell invasion, whereas knockdown of FOXO1 promoted noninvasive bladder cancer cells becoming invasive bladder cancer cells. Moreover, knockout of FOXO1 significantly increased bladder cancer cell invasion and abolished the ISO inhibition of invasion in human bladder cancer cells. Further studies showed that the inhibition of Signal transducer and activator of transcription 1 (STAT1) phosphorylation at Tyr701 was crucial for ISO upregulation of FOXO1 transcription. Furthermore, this study revealed that metalloproteinase-2 (MMP-2) was a FOXO1 downstream effector, which was also supported by data obtained from mouse model of ISO inhibition BBN-induced mouse-invasive bladder cancer formation. These findings not only provide a novel insight into the understanding of mechanism of bladder cancer's propensity to invasion, but also identify a new role and mechanisms underlying the natural compound ISO that specifically suppresses such bladder cancer invasion through targeting the STAT1-FOXO1-MMP-2 axis. Cancer Prev Res; 9(7); 567-80. ©2016 AACR.

Liprin-α1 is a regulator of vimentin intermediate filament network in the cancer cell adhesion machinery.

PPFIA1 is located at the 11q13 region, which is one of the most commonly amplified regions in several epithelial cancers including head and neck squamous cell carcinoma and breast carcinoma. Considering the location of PPFIA1 in this amplicon, we examined whether protein encoded by PPFIA1, liprin-α1, possesses oncogenic properties in relevant carcinoma cell lines. Our results indicate that liprin-α1 localizes to different adhesion and cytoskeletal structures to regulate vimentin intermediate filament network, thereby altering the invasion and growth properties of the cancer cells. In non-invasive cells liprin-α1 promotes expansive growth behavior with limited invasive capacity, whereas in invasive cells liprin-α1 has significant impact on mesenchymal cancer cell invasion in three-dimensional collagen. Current results identify liprin-α1 as a novel regulator of the tumor cell intermediate filaments with differential oncogenic properties in actively proliferating or motile cells.

Na+/H+ exchanger NHE1 regulation modulates metastatic potential and epithelial-mesenchymal transition of triple-negative breast cancer cells.

In triple-negative breast cancer (TNBC), the high recurrence rate, increased invasion and aggressive metastatic formation dictate patient survival. We previously demonstrated a critical role for the Na+/H+ exchanger isoform 1 (NHE1) in controlling metastasis of triple-negative cells. Here, we investigated the effect of changes to three regulatory loci of NHE1. Two via the Ras/Raf/ERK/p90RSK pathway: p90RSK/14-3-3 (S703A) and ERK1/2 (S766,770,771A, SSSA) and a third via a calmodulin-binding domain (K641,R643,645,647E, 1K3R4E). MDA-MB-231 cells with a mutation at the p90RSK site (S703A-NHE1) changed from a wild-type mesenchymal morphology to a smaller epithelial-like phenotype with a loss of expression of mesenchymal marker vimentin. S703A cells also had reduced metastatic potential and markedly decreased rates of migration, invasion, spheroid growth, anchorage-dependent and soft agar colony formation. Similarly, BI-D1870, a specific inhibitor of p90RSK, significantly inhibited the metastatic potential of highly invasive MDA-MB-231 and moderately invasive MDA-MB-468 TNBC cells, but was minimally effective in non-invasive Hs578T TNBC cells. In contrast, invasion and spheroid growth were unaffected in cells containing NHE1 with mutations interfering with its activation by ERK1/2 (SSSA), though rates of migration and colony formation were reduced. Cells with a constitutive activation of NHE1 via the 1K3R4E mutation exhibited higher rates of migration, invasion, and spheroid growth. Taken together, our data demonstrate the critical role of NHE1 in metastasis, and suggest a novel link between NHE1 and the expression and cytosolic organization of vimentin, a key factor in epithelial-mesenchymal transition, that is dependent on p90RSK/14-3-3-mediated activation of the exchanger.

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 ...

Abstract A39: Epigenetic upregulation of lncRNA HOTAIR expression in three-dimensional culture of triple-negative breast cancer cells

Abstract lncRNA HOX transcript antisense RNA (HOTAIR) represses gene expression via recruitment of the chromatin modifying complex polycomb repressive complex 2 (PRC2) and its overexpression mediates invasion and metastasis in breast cancer. In an apparent paradox we observed silencing of HOTAIR expression in MDA-MB231 cells, an invasive and metastatic triple-negative breast cancer cell line in two dimensional monolayer culture (2-D). This observation prompted us to question limitations of 2-D culture as a model system to investigate the role of HOTAIR in breast cancer. Thus we examined HOTAIR expression in reconstituted basement membrane matrix Matrigel-based three dimensional organotypic culture (rBM 3-D) because MDA-MB231 cells undergo stellate morphogenesis, a phenomenon that is characteristic of invasive cancer cells. In MDA-MB231 cells HOTAIR expression in rBM 3-D culture and the grafted tumors exhibited ~130-fold and ~60-fold over than that in 2-D culture, respectively. Induction of HOTAIR in rBM 3-D culture involved transcription from an uncharacterized promoter located in the 1st intron of the HOXC11 gene. However HOXC11 did not exhibit difference in its expression between 2-D and rBM 3-D cultures. Induction of HOTAIR in rBM 3-D culture was congruent to a wide spread increase in expression of the protein coding HOX genes. Moreover stellate morphogenesis was abrogated by the HOTAIR-specific siRNA and replaced by mass morphogenesis that is characteristic of non-invasive cells. To validate induction of HOTAIR as a response to extracellular matrix (ECM), we knocked down a major ECM receptor integrin α2 using lentiviral vectors expressing integrin α2-specific shRNA. As expected knockdown of integrin α2 resulted in a substantial decrease of HOTAIR expression. Because the HOX genes are tightly regulated by CpG methylation, we compared CpG methylation of the promoters of HOTAIR and HOXA9 in 2-D and rBM 3-D cultures. Indeed the promoters of HOTAIR and HOXA9 exhibited lower CpG methylation in rBM 3-D culture than that in 2-D culture. Moreover 5-Azacytidinewe up-regulated the expression of HOTAIR and HOXA9 in 2-D culture of MDA-mb231 cells. Moreover induction of HOTAIR was associated with increased binding of BRD4 to the HOTAIR promoter region located in the HOXC11 1st intron. The BRD4-specific siRNA substantially reduced the expression of HOTAIR in rBM 3-D culture. Taken together our findings suggest that ECM up-regulate the expression of HOTAIR via reduced CpG methylation and increased BRD4 binding in a human triple-negative breast cancer cell line. Note: This abstract was not presented at the conference. Citation Format: Miao Li, Xi Li, Bin Shan. Epigenetic upregulation of lncRNA HOTAIR expression in three-dimensional culture of triple-negative breast cancer cells. [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer: Mechanisms to Medicines ; 2015 Dec 4-7; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2016;76(6 Suppl):Abstract nr A39.

Calcitonin Receptor-Zonula Occludens-1 Interaction Is Critical for Calcitonin-Stimulated Prostate Cancer Metastasis

The role of neuroendocrine peptide calcitonin (CT) and its receptor (CTR) in epithelial cancer progression is an emerging concept with great clinical potential. Expression of CT and CTR is frequently elevated in prostate cancers (PCs) and activation of CT–CTR axis in non-invasive PC cells induces an invasive phenotype. Here we show by yeast-two hybrid screens that CTR associates with the tight junction protein Zonula Occludens-1 (ZO-1) via the interaction between the type 1 PDZ motif at the carboxy-terminus of CTR and the PDZ3 domain of ZO-1. Mutation of either the CTR C-PDZ-binding motif or the ZO-1-PDZ3 domain did not affect binding of CTR with its ligand or G-protein-mediated signaling but abrogated destabilizing actions of CT on tight junctions and formation of distant metastases by orthotopically implanted PC cells in nude mice, indicating that these PDZ domain interactions were pathologically relevant. Further, we observed CTR-ZO-1 interactions in PC specimens by proximity ligation immunohistochemistry, and identified that the number of interactions in metastatic PC specimens was several-fold larger than in non-metastatic PC. Our results for the first time demonstrate a mechanism by which PDZ-mediated interaction between CTR and ZO1 is required for CT-stimulated metastasis of prostate cancer. Since many receptors contain PDZ-binding motifs, this would suggest that PDZ-binding motif-adaptor protein interactions constitute a common mechanism for cancer metastasis.

Implications of Epithelial&amp;ndash;Stromal Interaction 1 in Diseases Associated with Inflammatory Signaling

Epithelial–stromal interaction 1 (EPSTI1) was initially identified as an induced gene in breast cancer epithelial cells by cocultured stromal fibroblasts. This discovery led to further investigation and understanding of the role of EPSTI1 in cancer. Aberrant elevation of EPSTI1 occurs primarily in invasive breast cancer epithelial cells. Forced overexpression of EPSTI1 in noninvasive cancer cells can substitute for the stromal fibroblasts. EPSTI1 was further implicated in cancer by our most recent study that identified it as one of the few most upregulated genes in human breast cancer by Kruppel-like factor 8 (KLF8), a pro-cancerous transcription factor in many cancer types. Our study also demonstrated that EPSTI1 interacts with valosin-containing protein to promote the degradation of nuclear factor κ-light-chain-enhancer of activated B cells (NF-κB) inhibitor alpha, leading to the activation and nuclear translocation of NF- κB. Additionally, EPSTI1 was shown to inhibit apoptosis by inactivating caspase 8. Studies on hepatitis C and E viruses have indicated that EPSTI1 plays a role in inhibition of the viral replication by promoting the expression of protein kinase R or protein kinase RNA-activated, a viral response gene, suggesting a role of EPSTI1 in immune response. Interestingly, in addition to transducing stromal signals, EPSTI1 has been implicated in immune privilege and autoimmune diseases, including systemic lupus erythematosus, rheumatoid arthritis, and histiocytic necrotizing lymphadenitis. This review seeks to comb EPSTI1-related studies as it was cloned a dozen years ago with a particular focus on the mechanisms of its regulation and signaling, as well as its potential roles in the diseases.

A20 inhibits the motility of HCC cells induced by TNF-α.

Metastasis of hepatocellular carcinoma (HCC) can be facilitated by TNF-α, a prototypical inflammatory cytokine in the HCC microenvironment. A20 is a negative regulator of NF-κB signaling pathway. In the present study we ask whether A20 plays a role in HCC metastasis. We found that A20 expression was downregulated in the invasive cells of microvascular invasions (MVI) compared with the noninvasive cells in 89 tissue samples from patients with HCC by immunochemistry methods. Overexpression of A20 in HCC cell lines inhibited their motility induced by TNF-α. Furthermore, the overexpression of A20 inhibited epithelial-mesenchymal transition (EMT), FAK activation and RAC1 activity. By contrast, knockdown of A20 in one HCC cell line results in the converse. In addition, the overexpression of A20 restrained the formation of MVI in HCC xenograft in nude mice treated with TNF-α. All the results suggested that A20 functioned as a negative regulator in motility of HCC cells induced by TNF-α.

Epitelial-to-mesenchimal transition and invasion are upmodulated by tumor-expressed granzyme B and inhibited by docosahexaenoic acid in human colorectal cancer cells.

BackgroundGranzyme B (GrB) is a serine protease, traditionally known as expressed by cytotoxic lymphocytes to induce target cell apoptosis. However, it is emerging that GrB, being also produced by a variety of normal and neoplastic cells and potentially acting on multiple targets, might represent a powerful regulator of a wide range of fundamental biological processes. We have previously shown that GrB is expressed in urothelial carcinoma tissues and its expression is associated to both pathological tumor spreading and EMT. We have also shown that docosahexaenoic acid (DHA), a dietary ω-3 polyunsaturated fatty acid with anti-tumor activity, while inhibiting urothelial and pancreatic carcinoma cell invasion also inhibited their GrB expression in vitro. In this study, we characterized a panel of colorectal carcinoma (CRC) cells, with different invasive capabilities, for GrB expression and for the contribution of GrB to their EMT and invasive phenotype. In addition, we investigated the effect of DHA on CRC cell-associated GrB expression, EMT and invasion.MethodsThe expression levels of GrB and EMT-related markers were evaluated by Western blotting. GrB knockdown was performed by Stealth RNAi small interfering RNA silencing and ectopic GrB expression by transfection of human GrB vector. Cell invasion was determined by the BioCoat Matrigel invasion chamber test.ResultsGrB was produced in 57.1 % CRC cell lines and 100 % CRC-derived Cancer Stem Cells. Although GrB was constitutive expressed in both invasive and noninvasive CRC cells, GrB depletion in invasive CRC cells downmodulated their invasion in vitro, suggesting a contribution of GrB to CRC invasiveness. GrB loss or gain of function downmodulated or upmodulated EMT, respectively, according to the analysis of cancer cell expression of three EMT biomarkers (Snail1, E-cadherin, N-cadherin). Moreover, TGF-β1-driven EMT was associated to the enhancement of GrB expression in CRC cell lines, and GrB depletion led to downmodulation of TGF-β1-driven EMT. In addition, DHA inhibited GrB expression, EMT and invasion in CRC cells in vitro.ConclusionsThese findings present a novel role for GrB as upmodulator of EMT in CRC cells. Moreover, these results support the use of DHA, a dietary compound without toxic effects, as adjuvant in CRC therapy.

Non-invasive glucagon-like peptide-1 receptor imaging in pancreas with 18F-Al labeled Cys39-exendin-4

PurposeGlucagon-like peptide-1 receptor (GLP-1R) is abundantly expressed on beta cells and may be an ideal target for the pancreas imaging. Monitoring the GLP-1R of pancreas could be benefit for understanding the pathophysiology of diabetes. In the present study, 18F-Al labeled exendin-4 analog, 18F-Al-NOTA-MAL-Cys39-exendin-4, was evaluated for PET imaging GLP-1R in the pancreas. MethodsThe targeting of 18F-Al labeled exendin-4 analog was examined in healthy and streptozotocin induced diabetic rats. Rats were injected with 18F-Al-NOTA-MAL-Cys39-exendin-4 and microPET imaging was performed at 1 h postinjection, followed by ex vivo biodistribution. GLP-1R expression in pancreas was determined through post mortern examinations. ResultsThe pancreas of healthy rats was readily visualized after administration of 18F-Al-NOTA-MAL-Cys39-exendin-4, whereas the pancreas of diabetic rats, as well as those from rats co-injected with excess of unlabeled peptides, was barely visible by microPET. At 60 min postinjection, the pancreatic uptakes were 1.02 ± 0.15%ID/g and 0.23 ± 0.05%ID/g in healthy and diabetic rats respectively. Under block, the pancreatic uptakes of non-diabetic rats reduced to 0.21 ± 0.07%ID/g at the same time point. Biodistribution data and IHC staining confirmed the findings of the microPET imaging. ConclusionThe favorable preclinical data indicated that 18F-Al-NOTA-MAL-Cys39-exendin-4may be suitable for non-invasive monitoring functional pancreatic beta cells.