High Invasiveness Research Articles

Abstract 3414: Role of FGFR2 in invasion and metastasis through different activation of downstream signaling in gastric cancer

Abstract Activation of fibroblast growth factor receptors (FGFRs), the family of receptor tyrosine kinase (RTK), initiates intracellular signaling networks for key cellular processes. Previous reports show that FGFR2 amplification induces activation of downstream signaling in gastric cancer influencing cell proliferation and survival. In this study, we evaluated activation of FGFRs and related downstream pathways, and also determine the effect of FGFRs in tumor invasion and metastasis in gastric cancer. First, we performed phospho-RTK array in 25 gastric cancer cell lines and found that FGFR2 and FGFR3 were more frequently phosphorylated than FGFR1 or FGFR4 in gastric cancer. Next, we observed genomic amplification status and phosphorylation level of FGFR2 using real-time PCR and Western blot. KATO III and SNU-16 with higher expression of FGFR2 showed high correlation between DNA amplification and phosphorylation status. Then, using invasion assay, cells with higher FGFR3 showed higher invasiveness compare to cells with FGFR2 overexpression. When we observed the activation status of downstream signaling molecules, cells with higher FGFR2 were more dependent on PI3K-AKT-mTOR pathway, while cells with higher FGFR3 were more dependent on ERK pathway. In conclusion, FGFRs have various biological roles through different signaling pathways in gastric cancer, suggesting that FGFRs are the potential molecular targets in gastric cancer. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3414. doi:1538-7445.AM2012-3414

Abstract 1388: Acquired resistance to anti-VEGF therapy in glioblastoma is associated with an inflammatory and mesenchymal phenotype

Abstract Glioblastoma is the most common malignant brain tumor and is characterized by rapid angiogenesis-dependent (re)growth, cell heterogeneity, and extensive local tissue infiltration. Antiangiogenic therapy, among the most promising treatment developments, rapidly reduces vascular permeability and delays glioblastoma progression but the therapy itself ultimately promotes an aggressive treatment-resistant phenotype. The aim of the present study was to identify mechanisms responsible for glioblastoma resistance to antiangiogenic therapy. When mice bearing U87 and NSC11 xenografts treated with bevacizumab became moribund, tumor tissue was extracted to obtain tumor cells with acquired resistance to bevacizumab. U87 and NSC11 cells were dissociated and cultured in serum-containing and stem cell media, respectively. To verify resistance, the resistant cells were expanded in vitro and then re-implanted orthotopically in nude mice. Mice were randomized to receive bevacizumab or vehicle and followed for survival. As anticipated, tumors formed by parental U87 and NSC11 cells were sensitive, whereas tumors obtained at the time of acquired resistance to anti-VEGF therapy were resistant to bevacizumab and did not have prolongation of survival compared to untreated controls. To elucidate the molecular mechanisms underlying resistance to antiangiogenic treatment, we performed gene expression profiling using Affymetrix U133Plus_2.0 arrays comparing untreated and bevacizumab-treated tumors. Comparison of anti-VEGF therapy-resistant cell lines to untreated controls demonstrated an increase in genes associated with a mesenchymal phenotype. Antiangiogenic therapy increased the expression of genes of mesenchymal origin, cellular migration/invasion, and inflammation including chemokine secretion, myeloid cell chemotaxis and multiple markers reflecting a pro-inflammatory environment. Consistent with these data, mice bearing U87 resistant tumors showed significantly greater infiltration of F4/80+ myeloid cells compared to wild-type tumors. Comparing the mesenchymal gene signature from Phillips et al with the gene changes in our experiments using Gene Set Enrichment Analysis (GSEA v2.07, Broad Institute, MIT), we show that NSC11 tumors treated with bevacizumab showed a highly significant correlation to the published mesenchymal gene signature. In addition to the pro-inflammatory changes, invasion-related genes were also upregulated. We found the U87 and NSC11 resistant cells have significantly higher invasion rates in vitro compared with U87 and NSC11 parental cell lines. Our studies identify prominent mechanisms of glioblastoma resistance to antiangiogenic therapy. A better understanding of resistance may provide new strategies to prolong the efficacy of this therapy and accelerate the integration of combination therapies into clinical trials. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1388. doi:1538-7445.AM2012-1388

3D Traction Forces in Cancer Cell Invasion

Cell invasion through a dense three-dimensional (3D) matrix is believed to depend on the ability of cells to generate traction forces. To quantify the role of cell tractions during invasion in 3D, we present a technique to measure the elastic strain energy stored in the matrix due to traction-induced deformations. The matrix deformations around a cell were measured by tracking the 3D positions of fluorescent beads tightly embedded in the matrix. The bead positions served as nodes for a finite element tessellation. From the strain in each element and the known matrix elasticity, we computed the local strain energy in the matrix surrounding the cell. We applied the technique to measure the strain energy of highly invasive MDA-MB-231 breast carcinoma and A-125 lung carcinoma cells in collagen gels. The results were compared to the strain energy generated by non-invasive MCF-7 breast and A-549 lung carcinoma cells. In all cases, cells locally contracted the matrix. Invasive breast and lung carcinoma cells showed a significantly higher contractility compared to non-invasive cells. Higher contractility, however, was not universally associated with higher invasiveness. For instance, non-invasive A-431 vulva carcinoma cells were the most contractile cells among all cell lines tested. As a universal feature, however, we found that invasive cells assumed an elongated spindle-like morphology as opposed to a more spherical shape of non-invasive cells. Accordingly, the distribution of strain energy density around invasive cells followed patterns of increased complexity and anisotropy. These results suggest that not so much the magnitude of traction generation but their directionality is important for cancer cell invasion.

Quantitative Analysis of the Effect of Cancer Invasiveness and Collagen Concentration on 3D Matrix Remodeling

Extracellular matrix (ECM) remodeling is a key component of cell migration and tumor metastasis, and has been associated with cancer progression. Despite the importance of matrix remodeling, systematic and quantitative studies on the process have largely been lacking. Furthermore, it remains unclear if the disrupted tensional homeostasis characteristic of malignancy is due to initially altered ECM and tissue properties, or to the alteration of the tissue by tumor cells. To explore these questions, we studied matrix remodeling by two different prostate cancer cell lines in a three-dimensional collagen system. Over one week, we monitored structural changes in gels of varying collagen content using confocal reflection microscopy and quantitative image analysis, tracking metrics of fibril fraction, pore size, and fiber length and diameter. Gels that were seeded with no cells (control), LNCaP cells, and DU-145 cells were quantitatively compared. Gels with higher collagen content initially had smaller pore sizes and higher fibril fractions, as expected. However, over time, LNCaP- and DU-145-populated matrices showed different structural properties compared both to each other and to the control gels, with LNCaP cells appearing to favor microenvironments with lower collagen fiber fractions and larger pores than DU-145 cells. We posit that the DU-145 cells' preference for denser matrices is due to their higher invasiveness and proteolytic capabilities. Inhibition of matrix proteases resulted in reduced fibril fractions for high concentration gels seeded with either cell type, supporting our hypothesis. Our novel quantitative results probe the dynamics of gel remodeling in three dimensions and suggest that prostate cancer cells remodel their ECM in a synergistic manner that is dependent on both initial matrix properties as well as their invasiveness.

Spectratyping analysis of the islet-reactive T cell repertoire in diabetic NOD Igμnull mice after polyclonal B cell reconstitution

BackgroundNon Obese Diabetic mice lacking B cells (NOD.Igμnull mice) do not develop diabetes despite their susceptible background. Upon reconstitution of B cells using a chimera approach, animals start developing diabetes at 20 weeks of age.MethodsWe have used the spectratyping technique to follow the T cell receptor (TCR) V beta repertoire of NOD.Igμnull mice following B cell reconstitution. This technique provides an unbiased approach to understand the kinetics of TCR expansion. We have also analyzed the TCR repertoire of reconstituted animals receiving cyclophosphamide treatment and following tissue transplants to identify common aggressive clonotypes.ResultsWe found that B cell reconstitution of NOD.Igμnull mice induces a polyclonal TCR repertoire in the pancreas 10 weeks later, gradually diversifying to encompass most BV families. Interestingly, these clonotypic BV expansions are mainly confined to the pancreas and are absent from pancreatic lymph nodes or spleens. Cyclophosphamide-induced diabetes at 10 weeks post-B cell reconstitution reorganized the predominant TCR repertoires by removing potential regulatory clonotypes (BV1, BV8 and BV11) and increasing the frequency of others (BV4, BV5S2, BV9, BV16-20). These same clonotypes are more frequently present in neonatal pancreatic transplants under the kidney capsule of B-cell reconstituted diabetic NOD.Igμnull mice, suggesting their higher invasiveness. Phenotypic analysis of the pancreas-infiltrating lymphocytes during diabetes onset in B cell reconstituted animals show a predominance of CD19+ B cells with a B:T lymphocyte ratio of 4:1. In contrast, in other lymphoid organs (pancreatic lymph nodes and spleens) analyzed by FACS, the B:T ratio was 1:1. Lymphocytes infiltrating the pancreas secrete large amounts of IL-6 and are of Th1 phenotype after CD3-CD28 stimulation in vitro.ConclusionsDiabetes in NOD.Igμnull mice appears to be caused by a polyclonal repertoire of T cell accumulation in pancreas without much lymphoid organ involvement and is dependent on the help by B cells.

Enrichment and function research of large cell lung cancer stem cell-like cells

背景与目的目前国内外还没有确切的、得到公认的肺癌干细胞的筛选标记分子、指标和方法，常用方法为通过流式细胞技术，借鉴其他肿瘤干细胞分选标记来分选肺癌干细胞，但其筛选特异性低、工作量巨大。本研究采用无血清悬浮培养法富集肺癌干细胞，对肺癌干细胞的筛选方法进行探索。方法采用添加生长因子的无血清培养基对人大细胞肺癌细胞株L9981进行悬浮培养，获得肺癌细胞球体。对含血清培养贴壁L9981细胞和无血清培养成球后的L9981细胞，通过显微镜下观察比较二者的生物学形态，应用Vi-cell细胞活力分析仪计数细胞并绘制生长曲线比较二者的增殖能力，通过Transwell实验研究它们的侵袭能力差异，并通过接种裸鼠观察二者在体内的成瘤性来研究肺癌细胞球体的生物学功能。结果与血清贴壁培养L9981细胞相比，无血清培养L9981细胞成球形生长，贴壁L9981和L9981球体细胞的倍增时间分别为（56.05±1.95）h和（33.00±1.44）h，球体细胞的侵袭和成瘤能力分别为贴壁L9981细胞的5倍和20倍。结论通过无血清悬浮培养的L9981细胞可以形成肺癌球体细胞群，L9981球体细胞的侵袭和成瘤能力均明显高于贴壁L9981细胞，显示L9981球体细胞中富集了肺癌干细胞样的细胞。无血清悬浮培养肺癌球体细胞可作为富集肺癌干细胞样细胞的一种候选方法。

The importance of quantifying propagule pressure to understand invasion: an examination of riparian forest invasibility

The widely held belief that riparian communities are highly invasible to exotic plants is based primarily on comparisons of the extent of invasion in riparian and upland communities. However, because differences in the extent of invasion may simply result from variation in propagule supply among recipient environments, true comparisons of invasibility require that both invasion success and propagule pressure are quantified. In this study, we quantified propagule pressure in order to compare the invasibility of riparian and upland forests and assess the accuracy of using a community's level of invasion as a surrogate for its invasibility. We found the extent of invasion to be a poor proxy for invasibility. The higher level of invasion in the studied riparian forests resulted from greater propagule availability rather than higher invasibility. Furthermore, failure to account for propagule pressure may confound our understanding of general invasion theories. Ecological theory suggests that species-rich communities should be less invasible. However, we found significant relationships between species diversity and invasion extent, but no diversity-invasibility relationship was detected for any species. Our results demonstrate that using a community's level of invasion as a surrogate for its invasibility can confound our understanding of invasibility and its determinants.

Phenotypic Changes in a Laboratory-Derived Ertapenem-Resistant Escherichia coli Strain

The aim of this study was to identify phenotypic changes in a laboratory-derived strain of ertapenem-resistant Escherichia coli (Ec-ERT) when compared to its susceptible parent strain (Ec-WT). In both strains, we assessed both the effects of ertapenem via time-kill curves and the occurrence of cross resistance with other beta-lactams. The strains were compared based on growth pattern, biochemical-physiological profile and changes in the subproteome using 2D-DIGE followed by MALDI-TOF/TOF MS. To assess virulence, we employed a murine model of intraperitoneal infection in which we investigated the invasiveness of both strains. Growth persistence of the laboratory-derived resistant strain was observed via the time-kill curve assay, but cross resistance was not observed for other beta-lactams. We also observed a slower growth rate and changes in the biochemical and physiological characteristics of the drug-resistant bacteria. In the resistant strain, a total of 51 protein spots were increased in abundance relative to the wild-type strain, including an outer membrane protein A, which is related to bacterial virulence. The mouse infection assay showed a higher invasiveness of the Ec-ERT strain in relation to the Ec-WT strain. In conclusion, the alterations driven by ertapenem in E. coli reinforce the idea that antimicrobial agents may interfere in several aspects of bacterial cell biology, with possible implications for host-bacteria interactions.

The Role of Raf Kinase Inhibitor Protein in Rheumatoid Fibroblast-like Synoviocytes Invasiveness and Cytokine and Matrix Metalloproteinase Expression

Fibroblast-like synoviocytes (FLS) play an important role in the pathogenesis of rheumatoid arthritis. Raf kinase inhibitor protein (RKIP) negatively regulates the Raf/MEK/ERK and NF-κB pathway. The role of RKIP in rheumatoid FLS is unknown. The purpose of the present study was to investigate the function of RKIP in rheumatoid FLS. Rheumatoid FLS were transfected with either RKIP-expressing plasmids or RKIP small interfering RNA (siRNA). RKIP protein was detected in rheumatoid synovial tissue (ST) and FLS. RKIP overexpression significantly decreased IL-6 mRNA expression in TNF-α-stimulated rheumatoid FLS. RKIP overexpression also showed a decreased trend in IL-8, MMP-1, and MMP-3 mRNA expression in TNF-α-stimulated rheumatoid FLS. RKIP silencing resulted in significantly increased MMP-1 and MMP-3 mRNA expression in TNF-α-stimulated rheumatoid FLS. RKIP silencing also increased IL-6 and IL-8 mRNA expression in TNF-α-stimulated rheumatoid FLS, but this increase did not reach statistical significance. TNF-α-induced ERK and NF-κB activation was suppressed in FLS with RKIP overexpression. RKIP silencing resulted in a significantly higher invasion index in TNF-α-stimulated rheumatoid FLS compared to controls. These results suggest that RKIP might be a potential therapeutic target for rheumatoid arthritis.

Abstract SY23-02: RTK endocytosis and other feedback mechanisms in cancer

Abstract The view of signal transduction pathways as a set of parallel pipelines connecting transmembrane receptor tyrosine kinases (RTKs) and the genome has given way, over the last decade, to the notion of layered networks, highly rich in internal connections. This paradigm is well exemplified by the RTK subfamily called ERBB, namely receptors for growth factors of the EGF (epidermal growth factor) and neuregulin groups: whereas ERBB-1 (also called EGFR) binds seven different growth factors and ERBB-4 binds several isoforms of neuregulins, ERBB-2/HER2 binds no known growth factor and the kinase activity of ErbB-3/HER3 is almost dead. Because both EGFR and HER2 frequently display aberrant expression and mutant forms in human tumors, their mode of action and regulation are of interest. Like other autonomous bi-stable systems, the ERBB network is regulated by feedback loops, both positive and negative. For example, one outcome of activation by growth factors is the transcriptional induction of EGF-like molecules (positive feedback). On the other hand, once activated the receptors are rapidly endocytosed and degraded in lysosomes (negative feedback). According to recent studies, negative feedback loops are often defective in tumors, whereas positive loops are frequently enhanced. In general, negative feedback regulation may be divided into two temporal phases: the immediate phase (approximately 20 minutes from stimulation) comprises posttranslational protein modifications, such as protein phosphorylation and ubiquitylation, as well as intracellular translocations of enzymes and adaptor proteins, including receptor endocytosis. The late phase of feedback regulation harnesses the transcriptional machinery, and it comprises newly synthesized proteins (e.g., phosphatases of the mitogen-activated protein kinases, MKPs), which restrain signaling. Interestingly, microRNAs complement the roles played by mRNA synthesis: multiple microRNAs undergo rapid turnover within minutes after growth factor stimulation, while another set is newly synthesized. In addition to the immediately down-regulated microRNAs (ID-miRs) and E3 ubiquitin ligases like CBL and Nedd4 family members, the first phase of negative RTK regulation entails tyrosine phosphatases like DEP-1, which dephosphorylates EGFR, and de-ubiquitinating enzymes (DUBs). E3 ligases, in concert with ubiquitin-binding proteins, which nucleate four different endosomal sorting complexes called ESCRTs, coordinate receptor sorting for either lysosomal degradation or recycling to the plasma membrane. In line with their negative regulatory functions, certain components of ESCRTs have been implicated in tumor suppression, and, on the other hand, inactive forms of the RTK-specific E3 ligase, namely CBL, are oncogenic. In analogy with regulators of the endocytic pathway, the group of immediate early genes (IEGs; e.g., FOS and JUN), which is induced by growth factors within the first 30 minutes, is endowed with an oncogenic potential. Likewise, tumors of epithelial and brain origins deviate from the physiological tight regulation of the IEGs: both ID-miRs and several RNA- and DNA-binding proteins, collectively termed delayed early genes (DEGs; e.g., the RNA-binding protein ZFP-36) shape the pulse like profile of IEG induction, but transformed cells escape this type of regulation. In summary, the emerging landscape of feedback regulation in malignant cells proposes multi-site manipulation of systems control: RTKs are often derailed to a recycling route by means of hyperactive RAB-coupling proteins (e.g., RAB11FIP1) and other aberrations, while both the ID-miRs and the DEGs are frequently down-regulated in tumors. Collectively, these aberrations enhance cytoplasmic signaling and augment expression of oncogenic transcription factors, which result in accelerated cell proliferation and higher invasiveness. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr SY23-02. doi:10.1158/1538-7445.AM2011-SY23-02

Abstract 5173: DKK4 activates non-canonical JNK signaling pathway while inhibiting the Wnt-canonical pathway in human renal cell carcinoma

Abstract Introduction and Objective: Renal cell carcinoma (RCC) is the third leading cause of death among urological tumors. Wnt/beta-catenin signaling, which plays an important role in cancer, includes beta-catenin dependent and beta-catenin independent pathways. The functional significance of several wnt antagonists have been reported in renal cancer. However the functional significance of Wnt antagonist DICKKOPF-4 (DKK4) has not been investigated in RCC. Recently DKK4 function has been reported to have two opposing functions (oncogenic or tumor suppressor) in colon cancer. As far as we know, there have been no reports regarding DKK4 and renal cancer. Therefore we focused on the role of DKK4 in renal cancer. Methods: We analyzed the expression of DKK4 in renal cancer tissues and adjacent normal kidney tissues by immunohistochemistry. To assess the function of DKK4, we established stable DKK4 transfected A-498 cells and performed functional analyses including TCF/LEF reporter assay, cell viability, colony formation, apoptosis, cell cycle, invasive capability, wound healing capability and in vivo tumor growth. Results: DKK4 expression was significantly higher in cancer compared to adjacent normal kidney tissues. The relative TCF/LEF activity was significantly lower in DKK4 transfected cells compared to empty vector, and nuclear beta-catenin expression was decreased in DKK4 transfectants. Also beta-catenin downstream effector proteins, cyclinD1 and c-Myc, showed decreased expression in DKK4 transfectants. This result suggests that DKK4 may be involved in the beta-catenin dependent pathway and that DKK4 is a beta-catenin dependent pathway inhibitor. However, higher invasiveness and migration was observed in stably transfected DKK4 cells and DKK4 promoted tumor growth in vitro and in vivo. To investigate the oncogenic function in DKK4 transfectants, we focused on the Wnt non-canonical/JNK signaling pathway and phosphorylation of JNK. We found that expression and phosphorylation of c-Jun were increased in DKK4 transfected A-498 cells. MMP-2 expression was also significantly increased in DKK4 stable trasnfectants. These results suggest that DKK4 promotes the non-canonical pathway by activating JNK, resulting in up-regulation of c-Jun expression and phosphorylation, and increasing MMP-2 expression. Conclusions: This is the first report documenting that DKK4 expression is higher in renal cancer tissues compared to matched normal kidney tissues. We examined the role of DKK4 in the canonical and non-canonical Wnt pathways and found that DKK4 inhibited the canonical pathway. Though DKK4 did not induce apoptosis it promoted renal cancer cell proliferation, invasion, and migration via the non-canonical JNK pathway, which also increased MMP-2 expression. Thus the current findings contribute important information about the role of DKK4 in renal cancer cells. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 5173. doi:10.1158/1538-7445.AM2011-5173

Abstract 2355: Full-length wild-type CD99 increases the migration and invasiveness of human malignant glioma cells

Abstract The malignant glioma is the most common primary human brain tumor, and its migration and invasiveness away from the primary tumor mass is considered a leading cause of tumor recurrence and treatment failure. Recently, gene expression profiling revealed CD99, which is a transmembrane glycoprotein, is more expressed in malignant glioma than in normal brain. It has been implicated in cell adhesion and migration in various cells, even if the function of this gene is not understood clearly. This gene has two isoforms of wild-type and splicing variant. We revealed that only wild-type CD99 is expressed in human glioma cells and tissues. Through MTS, colony-forming, and anoikis assays, we found that wild-type CD99 in glioma cells has enough resistance for anoikis and ability to survive and grow in anchorage independence, as is opposed to previous reports in other tumors. Using tissue microarray, we validated that CD99 demonstrates higher expression in gliomas compared to non-neoplastic brain. We inhibited CD99 expression by siRNA and demonstrated decreased glioma cell migration and invasion. In contrast, when CD99 was over-expressed in glioma cells, we observed enhancement of cell migration and invasiveness. Orthotopic brain tumor model with nude mice harboring CD99 overexpressing glioma cells results in significant decreased survival rate, compared to control mice. Moreover, higher invasiveness is observed in overexpressing mice by pathological review. Taken together, our findings suggest that CD99 play an important role in the migration and invasion of human gliomas. CD99 could be a target to inhibit migration and invasion especially in CD99-expressing gliomas. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 2355. doi:10.1158/1538-7445.AM2011-2355

Abstract 1634: Targeting L1 cell adhesion molecule using lentivirus-mediated short hairpin RNA interference reverses aggressiveness of oral squamous cell carcinoma

Abstract Oral cancer consistently ranks as one of the ten most frequently diagnosed cancers in the world. Despite multidisciplinary treatment with surgery, chemotherapy, and radiation, more than 50% of patients with oral squamous cell carcinoma (OSCC) receiving therapy will eventually develop recurrent and metastatic disease, which carries a particularly poor prognosis. Characterization of the specific molecular alternations associated with OSCC cell growth, invasion, and metastasis can advance our understanding of the molecular mechanisms underlying cancer induction and progression as well provide a basis for the development of new and effective therapeutic treatments for cancer patients. The L1 cell adhesion molecule (L1CAM) has been implicated in tumor progression of many types of cancers, but its role in OSCC has not been investigated. In the present study, we demonstrated overexpression of L1CAM in OSCC cells, but not in normal keratinocytes, using both clinical specimens and cell lines. This overexpression demonstrated a strong correlation with less differentiation and a higher invasion potential of cancer cells, supporting the significance of L1CAM in human OSCC tumor progression. Targeting L1CAM gene expression in SCC4 cells overexpressing L1CAM using a lentivirusmediated small hairpin RNA (shRNA) led to a significant reduction in cell proliferation in vitro via retardation of cell cycle at the G1 phase. In addition, shRNA knockdown of L1CAM strongly attenuated the migration and invasion of SCC4 cells, and this was also observed to parallel increased E-cadherin levels and decreased levels vimentin, fibronectin, and Snail-family transcription factors, indicating that L1CAM expression was related to the epithelial-mesenchymal transition. Furthermore, while mice receiving orthotopically placed control SCC4 cells died within 40 days due to invasive tumor growth and regional lymph node metastasis, prolonged animal survival and complete suppression of tumor progression was observed in mice implanted with L1CAM-deficent SCC4 cells, further substantiating the fundamental importance of L1CAM in OSCC pathophysiology. Our findings suggested that L1CAM is a critical mediator of tumor progression in OSCC, and targeting L1CAM using lentivirus-mediated shRNA may be a useful molecular pharmaceutical approach for the treatment of advanced OSCC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 1634. doi:10.1158/1538-7445.AM2011-1634

Genetically modified adipose tissue−derived mesenchymal stem cells overexpressing CXCR4 display increased motility, invasiveness, and homing to bone marrow of NOD/SCID mice

This study evaluates usefulness of CXCR4 overexpression via retroviral transduction in adipose tissue-derived mesenchymal stem cells (AT-MSCs) as a strategy to increase their migration and engraftment ability. AT-MSCs were isolated from lipoaspirates from human healthy donors with liberase 3. Cells were transduced with retroviral vector carrying either CXCR4 or green fluorescent protein (GFP) complementary DNA, and neo-resistant colonies were selected and used in experiments. Chemotaxis, invasion through Matrigel, motor activity, gene expression, osteodifferentiation potential, and engraftment into bone marrow of nonobese diabetic/severe combined immunodeficient mice were analyzed for CXCR4-overexpressing cells and GFP-control cells. Approximately 90% of retrovirus-transduced AT-MSCs expressed CXCR4 or GFP and maintained their ability to differentiate into osteocytes. CXCR4-transduced AT-MSCs displayed enhanced migration and higher invasiveness toward SDF-1 gradient. The upregulation of CXCR4 led to phosphorylation of mitogen-activated protein and AKT kinases and an increase in metalloproteinase expression after SDF-1 stimulation. The transplantation of CXCR4-transduced AT-MSCs into nonobese diabetic/severe combined immunodeficient mice led to increased engraftment into bone marrow in comparison to GFP-transduced AT-MSCs. Adipose tissue is one of the alternative sources of MSCs to bone marrow. We showed that AT-MSCs overexpressing CXCR4 preserve their ability for osteodifferentiation. Enhanced migration and engraftment of the transduced AT-MSCs into bone marrow indicate the usefulness of this strategy in overcoming low engraftment of MSCs in clinical approaches of cellular therapies for bone disorders and can represent a powerful tool in regenerative medicine and gene therapies. Thus, these cells may be used as an alternative to bone marrow-derived MSCs.

109 DKK4 ACTIVATES NON-CANONICAL JNK SIGNALING PATHWAY WHILE INHIBITING THE WNT-CANONICAL PATHWAY IN HUMAN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research1 Apr 2011109 DKK4 ACTIVATES NON-CANONICAL JNK SIGNALING PATHWAY WHILE INHIBITING THE WNT-CANONICAL PATHWAY IN HUMAN RENAL CELL CARCINOMA Hiroshi Hirata, Koji Ueno, Koichi Nakajima, Laura Tabatabai, Yuji Hinoda, Nobuhisa Ishii, Peter Carroll, and Rajvir Dahiya Hiroshi HirataHiroshi Hirata San Francisco, CA More articles by this author , Koji UenoKoji Ueno San Francisco, CA More articles by this author , Koichi NakajimaKoichi Nakajima Ohtaku, Japan More articles by this author , Laura TabatabaiLaura Tabatabai San Francisco, CA More articles by this author , Yuji HinodaYuji Hinoda Ube, Japan More articles by this author , Nobuhisa IshiiNobuhisa Ishii Ohtaku, Japan More articles by this author , Peter CarrollPeter Carroll San Francisco, CA More articles by this author , and Rajvir DahiyaRajvir Dahiya San Francisco, CA More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2011.02.175AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES Renal cell carcinoma (RCC) is the third leading cause of death among urological tumors. Although the rate of detection of incidental RCC has increased with improved diagnostic techniques, metastatic lesions are still found at diagnosis in about 30% of RCC patients.Wnt/beta-catenin signaling, which is one of important cancer pathways, includes beta-catenin dependent and independent pathways. The functional significance of some Wnt antagonists has been reported in renal cancer. However the functional significance of Wnt antagonist DKK4 has not been investigated in RCC. Recently DKK4 function has been reported to have two opposing functions (oncogenic /tumor suppressor) in colon cancer by different research groups. As far as we know, there have been no reports regarding DKK4 and RCC. METHODS We initially found that the expression of DKK4 was significantly higher in renal cancer tissues compared to adjacent normal kidney tissues. To assess the function of DKK4, we established stable DKK4 transfected cells and performed functional analyses including TCF/LEF reporter assay. RESULTS The relative TCF/LEF activity was significantly lower in DKK4 transfected cells compared to empty vector, and nuclear beta-catenin expression was decreased in DKK4 transfectants. Also beta-catenin downstream effector proteins, cyclinD1 and c-Myc, showed decreased expression in DKK4 transfectants. This result indicates that DKK4 is a beta-catenin dependent pathway inhibitor. However, higher invasiveness and migration was observed in stably transfected DKK4 cells and DKK4 promoted tumor growth in vitro and in vivo. To investigate the oncogenic function in DKK4 transfectants, we focused on the Wnt non-canonical/JNK signaling pathway. We found that phosphorylation of JNK, expression and phosphorylation of c-Jun were increased in DKK4 transfected A-498 cells. MMP-2 expression was also significantly increased in DKK4 stable transfectants. CONCLUSIONS This is the first report documenting that DKK4 expression is higher in renal cancer tissues compared to matched normal kidney tissues. DKK4 inhibited the canonical pathway. Though DKK4 did not induce apoptosis it promoted renal cancer cell proliferation, invasion, and migration via the non-canonical JNK pathway, which also increased MMP-2 expression. Thus the current findings contribute significant information about the role of DKK4 in renal cancer cells. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e46 Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Hiroshi Hirata San Francisco, CA More articles by this author Koji Ueno San Francisco, CA More articles by this author Koichi Nakajima Ohtaku, Japan More articles by this author Laura Tabatabai San Francisco, CA More articles by this author Yuji Hinoda Ube, Japan More articles by this author Nobuhisa Ishii Ohtaku, Japan More articles by this author Peter Carroll San Francisco, CA More articles by this author Rajvir Dahiya San Francisco, CA More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Plant invasions in the landscape

Biological invasions and changes in land-use are two components of global change affecting biodiversity worldwide. There is overriding evidence that invasions can dramatically change the landscape and that particular land-use types facilitate invasions. Still, these issues have not formally percolated into risk analysis of biological invasions, and only recently has the influence of the surrounding landscape on invasive species spread started to be considered. In this paper we review the literature on the influence of the surrounding landscape on the local level of plant invasions (i.e., abundance and richness of alien plants in plant communities). Our review confirms that there are more alien plant species and they are more abundant at fragment edges than in the interior of fragments. The decline on the level of invasion towards the interior of fragments is sharp. To a lesser extent, there is higher invasion in small isolated fragments than in large connected patches. However, despite their relevance, the influence of connectivity and shape of the fragments have been scarcely explored. Besides the fact that a site has more invaders if surrounded by a human-dominated landscape than by a natural one, the past history and the configuration of that landscape are also important. Invasion within land-uses is often associated with the historical legacy of changes in land-use, indicating that current land-uses might represent an invasion credit to future invasions. Accurate accounts of the invasion process and effective conservation programs will depend on such considerations.

Enhanced enzymatic activity of Candida species responsible for oral candidiasis in renal transplant recipients

Renal transplant recipients (RTRs) are regarded to be predisposed to oral candidiasis. This study was undertaken to evaluate the activity of hydrolytic enzymes in strains causing oral candidiasis in RTR. A total of 123 Candida albicans and 10 Candida krusei strains were isolated from 200 RTRs (39 RTRs suffered from symptomatic candidiasis, the remaining patients had no clinical symptoms of infection). All fungi were identified based on routine mycological procedures. Because of a small number of non-albicans strains, only C. albicans isolates were compared for enzymatic activity. The activity of 19 hydrolytic enzymes was assessed by API ZYM(®) test. The usage of mycophenolate mofetil was connected with higher ratio of clinically apparent oral candidiasis compared to immunosuppressive regimens without this drug (74.4% vs. 46.8%, respectively, P < 0.01). Candida albicans from RTRs showed higher enzymatic activity compared with strains from immunocompetent patients. Only two enzymes were found to be more active in C. albicans causing symptomatic candidiasis in RTRs (cystine arylamidase: P = 0.001, and α-fucosidase: P = 0.01) compared with saprophytic strains. Atrophic candidiasis showed higher activity of esterase lipase (C8) and α-mannosidase compared with the pseudomembraneous type. We suggest that the enhanced enzymatic activity is responsible for higher invasiveness of Candida residing in the oral cavity of RTRs.

Abstract 530: Evaluation of in vitro invasion of primary vs recurrent glioblastoma multiforme cells using organotypic rodent brain slice culture

Abstract Glioblastoma multiforme (GBM) is the most common primary malignant tumor of the adult central nervous system. The highly lethal nature of this tumor partly derives from the acquisition of an invasive phenotype, which allows the tumor cells to infiltrate the surrounding brain tissues. In fact the tumor cells, leading to the recurrence of the primary neoplasm highly contribute to the lack of success in eradicating this disease. In order to measure the migrating ability of GBM cells and the potential difference in infiltration capacity of primary versus recurrent GBM cells, we developed and validated an organotypic rodent brain slice assay and visualized the fluorescently labeled cells using confocal laser scanning microscopy. The invasion capacity has been quantified applying an innovative image analysis method using a panel of primary and recurrent glioma cell lines derived from patient biopsies and the commercially available GBM cell lines, DBTRG-05MG (recurrent) and T98G (primary). For validation purposes, cytochalasin D, a potent inhibitor of actin polymerization and hence migration and invasion, was used. The results revealed clear differences between primary and recurrent GBM cells: They differ both in their migrating capacity on the surface of the brain slice as well as by their ability to invade into the brain slice. A small population of recurrent GBM cells seems to acquire high infiltrative capacity allowing the invasion of brain tissue to greater distance than the primary GBM cells. Higher invasiveness may be an important characteristic of recurrent tumours, adding to our understanding of their worse clinical evolution and responsiveness to therapy. Note: This abstract was not presented at the AACR 101st Annual Meeting 2010 because the presenter was unable to attend. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 530.

Stage-Specific Expressions of GLUT-1, GLUT-3 and GLUT-12 in Estrogen-Induced Breast Cancer.

Abstract Increased glucose uptake and glycolysis are main metabolic characteristics of malignant cells. The glucose transporter-1 (GLUT-1), GLUT-3 and recently GLUT-12 have been shown in breast cancer cells in which estrogen play a critical role on its regulation and also associated with poor prognosis. However. the stage-specific GLUT regulation during the process of mammary carcinogenesis is unclear. In the present study we are aiming to study the GLUT expression pattern in an in vitro-in vivo model of estrogen induced carcinogenesis in which the human breast epithelial cell line MCF-10F is considered the normal counterpart E2 the transformed, C5 the invasive and T4 the tumorigenic stage. The gene expression of GLUTs was correlated with the ductulogenic pattern in a collagen matrix and the invasive properties using the Boyden chamber The RT-PCR data showed that the GLUT1 expression was downregulated in MCF10F after the treatment with 17β-estradiol (E2) and in the invasive cell type (C5), but not in the tumor cells (T4), which had no changes compared to MCF10F. C5 and T4 cells showed the highest rate of GLUT-3 expression. These cells were also found to be associated with loss of ductulogenesis, solid mass formation and higher invasion capacity. Whereas, GLUT-12 is downregulated in invasive and tumorigenic cells. We conclude that the estrogen-induced malignant transformation is associated with remarkably GLUT-3 expression, GLUT-1 re-expression at further stages, as well as GLUT-12 downregulation for transformed MCF-10F cells. (This work was supported by grant R21 ES015894from the NIEHS) Citation Information: Cancer Res 2009;69(24 Suppl):Abstract nr 5152.

Effect of id1 and id2 proteins (inhibitors of dna binding) expression in human colorectal cell lines

e22179 Background: Id1 and Id2 proteins are negative regulators of basic helix-loop-helix transcription factors, a family of components in the transcriptional network regulating cell growth and differentiation. Id1 and Id2 are overexpressed in human colorectal carcinoma and might play a critical role in the malignant cell phenotype. Methods: Two cell-lines were use for the analysis. NCM460: immortal non-tumoral cells from colon mucosa, and Caco-2: epithelial cells from colorectal carcinoma. Cell-lines were transfected with different constructs based on pLXSN vector (Clontech) to overexpress or inhibit the expression of the Id1 and Id2 genes, and then cultured for the analysis. Quantification of Id1 and Id2 expression were assessed by RT-PCR and Western-blot, respectively. A gene expression profile using oligonucleotide microarrays (Agilent Technologies) was performed in each cell line. Results: NCM460 cells and Caco-2 cells overexpressing Id1 (NCM460-Id1 and Caco-2-Id1) showed a statistically significant higher proliferation, invasion and migration, compared with both normal and Id1 inhibited cells. Id2 overexpression was related with a higher invasion capability only in NCM460 cells, without significant effect on proliferation and migration. NCM460-Id1 cells showed overexpression of PTMA, TYMS and LMNB1 genes, whereas HOXB8, LRP1, MMP17, NDRG1, MAP3K10, SCGb3A1 and BBC3 were upregulated. PTP4A1 and DKK1 were overexpressed in Caco-2-Id1 cells, and NDRG1, LRP1, FMOD and ENO1 were repressed. Conclusions: Id-1 expression is associated with proliferation, migration and invasion in both normal and tumoral colonic cell lines, and it is related with regulation of several genes implicated in colorectal cancer phenotype. Id proteins might be a potential target for the development of new antineoplastic agents. No significant financial relationships to disclose.