MCAM Expression Research Articles

Study on Inhibitory Effect of MaiMenDong Decoction and WeiJing Decoction Combination with Cisplatin on NCI-A549 Xenograft in Nude Mice and Its Mechanism.

MaiMenDong Decoction and WeiJing Decoction (Jin formula) is a traditional Chinese medication that consists of 8 medicinal plants, which recorded in the classical TCM literature Jin Kui Yao Lue and has been utilized in the treatment of lung diseases for hundreds of years in China. The present study aimed to determine the anti-tumor activity and the underlying mechanisms of Jin formula combined with cisplatin in the treatment of non-small cell lung cancer (NSCLC). Xenograft model of NCI-A549 was established in Balb/c nude mice. Five groups, including normal, MOCK, Jin, cisplatin (DDP), and Jin+DDP were included in the study. We found that Jin formula ameliorated the body weight loss caused by DDP 15 days after drug administration. Moreover, the combination of Jin with DDP enhanced the anti-tumor function of DDP. Microarray analysis showed that Jin suppressed gene expression of certain pathways which regulating cell cycle and apoptosis. Furthermore, DDP mainly decreased the gene expression level of angiogenesis associated factors, such as VEGFA, TGF-β and MMP-1. Moreover, co-treatment with Jin and DDP not only down-regulated Bcl-2 and E2F1, but also decreased the expression of MYC, MET, and MCAM. In addition, co-formula decreased the levels of p-AKT (thr308) and p-PTEN, increased Bax/Bcl-2 value, and resulted in apoptosis of tumor cells. Taken together, Jin+DDP significantly inhibited the growth of A549 cell transplanted solid tumor with slight side effect compared to the treatment by DDP only, and had a better effect than the Jin group. The mechanisms may be mainly associated with inactivation of PI3K/AKT pathway and apoptosis induction.

Long Noncoding RNA uc001pwg.1 Is Downregulated in Neointima in Arteriovenous Fistulas and Mediates the Function of Endothelial Cells Derived from Pluripotent Stem Cells.

Recent studies indicate important roles for long noncoding RNAs (lncRNAs) as essential regulators of gene expression. However, the specific roles of lncRNAs in stenotic lesions of arteriovenous fistula (AVF) failure are still largely unknown. We first analyzed the expression profiles of lncRNAs in human stenosed and nonstenotic uremic veins using RNA-sequencing methodology. A total of 19 lncRNAs were found to be differentially expressed in stenotic lesions. Among these, uc001pwg.1 was one of the most significantly downregulated lncRNAs and enriched in both control vein segments and human umbilical vein endothelial cells (HUVECs). Further studies revealed that uc001pwg.1 overexpression could increase nitric oxide synthase (eNOS) phosphorylation and nitric oxide (NO) production in endothelial cells (ECs) derived from human-induced pluripotent stem cells (HiPSCs). Mechanistically, uc001pwg.1 improves endothelial function via mediating MCAM expression. This study represents the first effort of identifying a novel candidate lncRNA for modulating the function of iPSC-ECs, which may facilitate the improvement of stem cell-based therapies for AVF failure.

Histone Deacetylase Inhibitors As Enhancers of Human Hematopoietic Stem Cell Activity

IntroductionThe identification of compounds which increase the number but also keep or enhance the activity of hematopoietic stem and progenitor cells (HSPCs) could improve the clinical outcome after autologous and allogeneic hematopoietic stem cell transplantation (HSCT). So far, most attempts to increase HSPC numbers ex vivo have been unsuccessful because of either inadequate cell numbers and/or loss of engraftment capacity and HSPC quality during expansion. Executing drug discovery screens in vertebrate systems is generally expensive, technically challenging and time consuming. Therefore, the zebrafish represents a versatile vertebrate model allowing HSPC regulation and development studies during embryogenesis and adulthood.MethodsWe used a semi-automated chemical screen to identify modulators of HSPC activity by transgenic (cmyb:EGFP) zebrafish embryos. Verification of identified histone deacetylase (HDAC) inhibitor candidates was carried out in vitro using human CD34+ HSPCs which were isolated from apharesis samples of healthy donors after mobilization with G-CSF by anti-CD34 coupled magnetic beads. The influence of HDAC inhibitors on HSPC phenotype, gene expression pattern as well as adhesion and migration capacity was analyzed after 5 days of treatment either in single or in co-culture with bone marrow-derived mesenchymal stromal cells (MSCs).ResultsThe HDAC inhibitors valproic acid (VPA), resminostat and entinostat were shown to significantly amplify the number of hematopoietic precursors in a chemical in vivo zebrafish embryo screen (Arulmozhivarman et al. 2016). Treatment of human CD34+ HSPCs with these compounds in vitro resulted in a significantly increased percentage of CD34+CD90+ cells up to 60% compared to controls which showed only 2% of double positive cells as well as in 3-fold higher CD34+ and about 12-fold higher CD34+CD90+ absolute cell numbers. CD34 is a well-known surface marker for human immature HSPCs and in combination with CD90 it defines a potentially pluripotent subpopulation. In a co-culture setting, we found that VPA treated cells showed 2 to 3-fold higher attachment capacity on MSCs compared to the control cells. This finding led us to quantify the adhesive capacity of cells using static adhesion assay and atomic force microscopy based single-cell force spectroscopy (AFM-SCFS). Interestingly, detachment forces of VPA treated HSPCs were 3 times increased on MSCs compared to control cells and a similar phenotype was observed by static adhesion assay. Accordingly, the chemokine-mediated migration of VPA treated HSPCs towards SDF-1/CXCL12 was inhibited. To reveal underlying downstream molecules and mechanisms mediating the modified cellular characteristics, a whole genome expression array was carried out for HSPCs treated with VPA in comparison to untreated controls. Amongst a panel of regulated genes, the melanoma cell adhesion molecule (MCAM/CD146), Notch 3 and its downstream effector Hes-1 as well as the SDF-1 receptor CXCR-4 were found to be significantly changed. Whereas the decreased expression of CXCR4 correlates with the inhibited migration potential of VPA-treated HSPCs and Notch-3/Hes-1 have a known role in normal and malignant hematopoiesis (Gu et al. 2016), the induced expression of MCAM on HSPCs was not described so far. The result was confirmed by flow cytometry which revealed a 40% MCAM-positive cell population when treated with VPA, whereas the control showed only negative cells. Additionally, significant higher transcript levels were detected for MCAM by quantitative real-time PCR in VPA expanded cells. Recently, we described a role of MCAM in MSCs for the hematopoietic support (Stopp et al. 2013). The inducible expression in HSPCs may reflect homotypic interactions which preserve a more immature subpopulation with high stem cell activity.ConclusionWe describe for the first time the ability of the HDAC inhibitors VPA, resminostat and entinostat to efficiently expand CD34+ HSPCs ex vivo especially supporting a CD34+CD90+ subpopulation with potentially high stem cell activity. Moreover, a potential role of MCAM in this context may offer new perspectives of the HSPC expansion ex vivo for the improvement of HSCT. DisclosuresNo relevant conflicts of interest to declare.

MCAM/CD146 promotes tamoxifen resistance in breast cancer cells through induction of epithelial–mesenchymal transition, decreased ERα expression and AKT activation

Tamoxifen resistance presents a prominent clinical challenge in endocrine therapy for hormone sensitive breast cancer. However, the underlying mechanisms that contribute to tamoxifen resistance are not fully understood. In this study, we established a tamoxifen resistant MCF-7 cell line (MCF-7-Tam-R) by continuously incubating MCF-7 cells with 4-OH-tamoxifen. We found that melanoma cell adhesion molecule (MCAM/CD146), a unique epithelial-to-mesenchymal transition (EMT) inducer, was significantly up-regulated at both mRNA and protein levels in MCF-7-Tam-R cells compared to parental MCF-7 cells. Mechanistic research demonstrated that MCAM promotes tamoxifen resistance by transcriptionally suppressing ERα expression and activating the AKT pathway, followed by induction of EMT. Elevated MCAM expression was inversely correlated with recurrence-free and distant metastasis-free survival in a cohort of 4142 patients with breast cancer derived from a public database, particularly in the subgroup only treated with tamoxifen. These results demonstrate a novel function of MCAM in conferring tamoxifen resistance in breast cancer. Targeting MCAM might be a promising therapeutic strategy to overcome tamoxifen resistance in breast cancer patients.

Abstract 1681: Inhibition of tumor growth in vivo through neutralization of the interaction of MCAM with laminin alpha 4

Abstract Melanoma cell adhesion molecule (MCAM/CD146) is highly expressed on a subset of many tumor types including melanoma and glioblastoma, and expression of MCAM correlates with enhanced cellular invasion, increased metastasis, and poor patient prognosis. The expression of MCAM on tumor cells confers unique migratory and adhesion capacity, which, if specifically targeted, could result in interruption of multiple biological processes required for tumor growth and spread. Although a role for MCAM in tumor progression has long been appreciated, the unidentified ligand for MCAM precluded the development of targeted approaches to inhibit extracellular interactions mediated by MCAM. Recently, we identified the MCAM ligand as laminin alpha 4. Laminin alpha 4 is present within the extracellular matrix of the tumor in addition to the vasculature throughout the body, providing an adhesive substrate for MCAM expressing tumor cells both within the tumor microenvironment and the blood vessels. The presence of laminin alpha 4 within the tumor and the vasculature may allow for a biological interaction by which MCAM mediated adhesion could be involved in both the homotypic aggregation required for tumor growth, in addition to the vascular adhesion required for the processes of metastasis and angiotropism. We generated monoclonal antibodies that bind to the epitope required for MCAM interactions with laminin alpha 4. Such antibodies entirely abrogate interactions of the tumor cell with laminin alpha 4 in vitro. Furthermore, we characterized the inhibition of tumor growth in vivo mediated by neutralization of MCAM/laminin alpha 4 interactions. Notably, tumor growth was delayed by blockade of MCAM, but required inhibition of both the MCAM expressed by the tumor, as well as vascular MCAM. We further characterize the unique pattern of MCAM expression on tumor cells within the perivascular extracellular matrix in human tumors, which suggest that these interactions are involved in human disease pathogenesis. Thus, antibodies neutralizing the interaction between MCAM and laminin alpha 4 have potential to inhibit a critical molecular interaction used by myriad tumor types. Citation Format: Ken Flanagan, Lauri W. Li, Carlos Lorenzana, Stephen J. Tam, Yue Liu, Philip J. Dolan, Lana Alexander, Josh Salmans, Robin M. Barbour, Jeffrey N. Higaki, Tarlochan Nijjar, Wagner Zago, Ted A. Yednock, Gene Kinney. Inhibition of tumor growth in vivo through neutralization of the interaction of MCAM with laminin alpha 4. [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 1681. doi:10.1158/1538-7445.AM2015-1681

ZBTB7A Suppresses Melanoma Metastasis by Transcriptionally Repressing MCAM.

The excessive metastatic propensity of melanoma makes it the most deadly form of skin cancer, yet the underlying mechanism of metastasis remains elusive. Here, mining of cancer genome datasets discovered a frequent loss of chromosome 19p13.3 and associated downregulation of the zinc finger transcription factor ZBTB7A in metastatic melanoma. Functional assessment of ZBTB7A-regulated genes identified MCAM, which encodes an adhesion protein key to melanoma metastasis. Using an integrated approach, it is demonstrated that ZBTB7A directly binds to the promoter and transcriptionally represses the expression of MCAM, establishing ZBTB7A as a bona fide transcriptional repressor of MCAM. Consistently, downregulation of ZBTB7A results in marked upregulation of MCAM and enhanced melanoma cell invasion and metastasis. An inverse correlation of ZBTB7A and MCAM expression in association with melanoma metastasis is further validated with data from analysis of human melanoma specimens. Together, these results uncover a previously unrecognized role of ZBTB7A in negative regulation of melanoma metastasis and have important clinical implications.

Melanoma cells homing to the brain: an in vitro model.

We developed an in vitro contact through-feet blood brain barrier (BBB) model built using type IV collagen, rat astrocytes, and human umbilical vein endothelial cells (HUVECs) cocultured through Transwell porous polycarbonate membrane. The contact between astrocytes and HUVECs was demonstrated by electron microscopy: astrocytes endfeet pass through the 8.0 μm pores inducing HUVECs to assume a cerebral phenotype. Using this model we evaluated transmigration of melanoma cells from two different patients (M1 and M2) selected among seven melanoma primary cultures. M2 cells showed a statistically significant higher capability to pass across the in vitro BBB model, compared to M1. Expression of adhesion molecules was evaluated by flow cytometry: a statistically significant increased expression of MCAM, αvβ3, and CD49b was detected in M1. PCR array data showed that M2 had a higher expression of several matrix metalloproteinase proteins (MMPs) compared to M1. Specifically, data suggest that MMP2 and MMP9 could be directly involved in BBB permeability and that brain invasion by melanoma cells could be related to the overexpression of many MMPs. Future studies will be necessary to deepen the mechanisms of central nervous system invasion.

VLA-4 blockade promotes differential routes into human CNS involving PSGL-1 rolling of T cells and MCAM-adhesion of TH17 cells

Focus of this study was the characterization of human T-cell blood–brain-barrier migration and corresponding molecular trafficking signatures. We examined peripheral blood and cerebrospinal fluid immune cells from patients under long-term anti-VLA-4/natalizumab therapy (LTNT), as well as CNS specimens. LTNT patients' cerebrospinal fluid T cells exhibited healthy central-/effector-memory ratios, but lacked CD49d and showed enhanced MCAM expression. LTNT lead to an increase of PSGL-1 expression on peripheral T cells. While VCAM-1 (VLA-4-receptor) was expressed at all CNS barriers, P-selectin (PSGL-1-receptor) was mainly detected at the choroid plexus. Accordingly, in vitro experiments under physiological flow conditions using primary human endothelial cells and LTNT patients' T cells showed increased PSGL-1-mediated rolling and residual adhesion even under VLA-4-blockade. Adhesion of MCAM+/TH17 cells was not affected by VLA-4-blocking alone, but was abrogated when both VLA-4 and MCAM were inhibited. Consistent with these data, MCAM+ cells were detected in white matter lesions, and in gray matter of MS patients. Our data indicate that lymphocyte trafficking into the CNS under VLA-4 blockade can occur by employing the alternative adhesion molecules, PSGL-1 and MCAM, the latter representing an exclusive pathway for TH17 cells to migrate over the blood–brain barrier.

Is quantitative real time polymerase chain reaction MCAM transcript assay really suitable for prognostic and predictive management of melanoma patients?

Funding sources: no external funding. Conflicts of interest: none declared. Dear Editor, We have read with great interest the paper ‘Markers of circulating tumour cells in the peripheral blood of patients with melanoma correlate with disease recurrence and progression’, in which Reid et al.1 tried to establish the molecular phenotype of circulating melanoma cells (mCTCs) in order to identify possible biomarkers useful for disease staging, recurrence and prognosis. Although we agree with these authors regarding the use of a multimarker panel for the quantitative real‐time polymerase chain reaction (qRT‐PCR) analysis of the mCTC phenotype, above all when used in the context of laboratory clinical diagnostics, we would like to focus on the need of a stringent validation of each molecular biomarker before its inclusion in a panel for diagnostic purposes. In particular, we focused on the MCAM transcript because it is already known both as a marker of melanoma progression2 and as a more accurate prognostic marker compared with overall clinicopathological characteristics.3 4 In this context, we underline that the most interesting findings provided by Reid et al.1 are in relation to the correlation between MCAM expression and increased tumour burden: these data seem to suggest that MCAM is a suitable marker for monitoring response to therapy, being a surrogate marker of ineffective eradication of circulating melanoma cells and, subsequently, of negative outcome. For these reasons, the authors speculate that MCAM expression could be useful for: (i) monitoring treatment resistance or risk of relapse; (ii) identifying a subset of patients with metastatic disease who respond poorly to conventional systemic treatments and may benefit from an alternative treatment regimen.1

Putative mesenchymal stem cells isolated from adult human ovaries.

The purpose of this study was to show that healthy adult human ovaries can be a source of cells showing typical MSCs characteristics under in vitro conditions. The cells, which were isolated from ovarian cortex tissue and named putative ovarian mesenchymal stem cells (PO-MSCs), were compared to bone marrow-derived MSCs (BM-MSCs) and to adult human dermal fibroblasts (HDFs). The results of a gene expression analysis using the Human Mesenchymal Stem Cell RT² Profiler™ PCR Array revealed that PO-MSCs were different than fibroblasts. They expressed most of the analyzed genes as BM-MSCs, although some genes were differentially expressed. However, the heterogeneity of PO-MSCs samples was revealed. The PO-MSCs expressed the characteristic genes related to MSCs, such as CD105, CD44, CD90, M-CAM, CD73 and VCAM1. In addition, the expression of markers CD44, CD90, M-CAM and STRO-1 was confirmed in PO-MSCs using immunocytochemistry. The PO-MSCs showed multipotent character, since they were able to differentiate into the cells of adipogenic, osteogenic, neural and pancreatic lineage. Healthy adult human ovaries can harbour an interesting population of cells showing typical MSCs characteristics under in vitro conditions and for this reason we named these cells putative MSCs. These cells express genes encoding main MSCs markers and have an interesting differential potential. Based on these results, we propose PO-MSCs as a novel type of MSCs which share some similarities with BM-MSCs. Nevertheless they show distinct and specific characteristics and are not fibroblasts.

Blood MUC-18/MCAM expression in patients with melanoma: a suitable marker of poor outcome

Funding sources: none. Conflicts of interest: none declared. Madam, Multiple marker reverse transcription–polymerase chain reaction (RT‐PCR) assay has been established as the most reliable and sensitive approach for identifying circulating melanoma cells in the peripheral blood of patients with melanoma by the detection of one or more melanoma‐associated markers of differentiation (MAMs).1 Their predictive value in melanoma progression, recurrence or response to therapy has recently been investigated.2 We read with interest the paper entitled ‘Markers of circulating tumour cells in the peripheral blood of patients with melanoma correlate with disease recurrence and progression’ by Reid et al.3 preceded by the Commentary ‘The challenge of developing useful blood‐based biomarkers in melanoma’ by Sullivan,4 both published in a recent issue of BJD. Surprisingly, neither Reid et al. nor Sullivan cited the article that we published in the BJD in 2009 reporting the results of our investigation on a similar issue entitled ‘Melanoma‐associated markers expression in blood: MUC‐18 is associated with advanced stages in melanoma patients’.5

Phenotype, donor age and gender affect function of human bone marrow-derived mesenchymal stromal cells

BackgroundMesenchymal stromal cells (MSCs) are attractive for cell-based therapies ranging from regenerative medicine and tissue engineering to immunomodulation. However, clinical efficacy is variable and it is unclear how the phenotypes defining bone marrow (BM)-derived MSCs as well as donor characteristics affect their functional properties.MethodsBM-MSCs were isolated from 53 (25 female, 28 male; age: 13 to 80 years) donors and analyzed by: (1) phenotype using flow cytometry and cell size measurement; (2) in vitro growth kinetics using population doubling time; (3) colony formation capacity and telomerase activity; and (4) function by in vitro differentiation capacity, suppression of T cell proliferation, cytokines and trophic factors secretion, and hormone and growth factor receptor expression. Additionally, expression of Oct4, Nanog, Prdm14 and SOX2 mRNA was compared to pluripotent stem cells.ResultsBM-MSCs from younger donors showed increased expression of MCAM, VCAM-1, ALCAM, PDGFRβ, PDL-1, Thy1 and CD71, and led to lower IL-6 production when co-cultured with activated T cells. Female BM-MSCs showed increased expression of IFN-γR1 and IL-6β, and were more potent in T cell proliferation suppression. High-clonogenic BM-MSCs were smaller, divided more rapidly and were more frequent in BM-MSC preparations from younger female donors. CD10, β1integrin, HCAM, CD71, VCAM-1, IFN-γR1, MCAM, ALCAM, LNGFR and HLA ABC were correlated to BM-MSC preparations with high clonogenic potential and expression of IFN-γR1, MCAM and HLA ABC was associated with rapid growth of BM-MSCs. The mesodermal differentiation capacity of BM-MSCs was unaffected by donor age or gender but was affected by phenotype (CD10, IFN-γR1, GD2). BM-MSCs from female and male donors expressed androgen receptor and FGFR3, and secreted VEGF-A, HGF, LIF, Angiopoietin-1, basic fibroblast growth factor (bFGF) and NGFB. HGF secretion correlated negatively to the expression of CD71, CD140b and Galectin 1. The expression of Oct4, Nanog and Prdm14 mRNA in BM-MSCs was much lower compared to pluripotent stem cells and was not related to donor age or gender. Prdm14 mRNA expression correlated positively to the clonogenic potential of BM-MSCs.ConclusionsBy identifying donor-related effects and assigning phenotypes of BM-MSC preparations to functional properties, we provide useful tools for assay development and production for clinical applications of BM-MSC preparations.

MCAM expression is associated with poor prognosis in non-small cell lung cancer

MCAM has been recently identified as a biomarker for epithelial-mesenchymal transition (EMT) and is potentially involved in metastasis of cancer. The current study aimed at investigating the expression of MCAM in non-small-cell lung cancer (NSCLC) and its clinico-pathological significance. A follow-up analysis was performed on 118 patients with NSCLC resected by lobectomy or pneumectomy with systematic lymph node dissection. All patients were followed for 6-60 months. Immunostaining of tissue sections from primary tumors and their lymph node metastasis was performed and evaluated using monoclonal antibody against MCAM, E-cadherin, and vimentin. Correlations were investigated between MCAM immunostaining in primary tumors and E-cadherin, vimentin immunostaining, lymph node metastasis, and survival. MCAM protein expression was found in 46.61 % of squamous cell carcinomas and 37.47 % of adenocarcinomas; MCAM expression positively correlated with vimentin, but inversely with E-cadherin (both P values <0.05). There were significant correlations between the MCAM immunostaining score in primary tumors and in their lymph node metastasis (P = 0.03). According to the Kaplan-Meier survival estimate, the level of MCAM expression in primary tumors was a statistically significant prognostic factor (P < 0.05). MCAM expression in surgically treated NSCLC is clearly associated with lymph node metastasis and poor prognosis.

Markers of circulating tumour cells in the peripheral blood of patients with melanoma correlate with disease recurrence and progression

Multimarker quantitative real-time polymerase chain reaction (qRT-PCR) represents an effective method for detecting circulating tumour cells in the peripheral blood of patients with melanoma. To investigate whether the phenotype of circulating melanoma cells represents a useful indicator of disease stage, recurrence and treatment efficacy. Peripheral blood was collected from 230 patients with melanoma and 152 healthy controls over a period of 3years and 9months. Clinical data and blood samples were collected from patients with primary melanoma (early stages, 0-II, n=154) and metastatic melanoma (late stages, III-IV, n=76). Each specimen was examined by qRT-PCR analysis for the expression of five markers: MLANA, ABCB5, TGFβ2, PAX3d and MCAM. In total, 212 of the patients with melanoma (92%) expressed markers in their peripheral blood. Two markers, MLANA and ABCB5, had the greatest prognostic value, and were identified as statistically significant among patients who experienced disease recurrence within our study period, being expressed in 45% (MLANA) and 49% (ABCB5) of patients with recurrence (P=0·001 and P=0·031, respectively). For patients administered nonsurgical treatments, MCAM expression correlated with poor treatment outcome. Circulating tumour cells were detectable at all stages of disease and long after surgical treatment, even when patients were considered disease free. Specifically, expression of ABCB5 and MLANA had significant prognostic value in inferring disease recurrence, while MCAM expression was associated with poor patient outcome after treatment, confirming multimarker qRT-PCR as a potential technique for monitoring disease status.

Abstract 1124: IGFBP-4 activates the Wnt/beta-catenin signaling pathway and induces M-CAM expression in human renal cell carcinoma

Abstract Introduction and Objective : The Wnt/beta-catenin signaling pathway is inactivated by Wnt antagonists in most cancers. IGFBP-4 is an antagonist of the Wnt/beta-catenin signaling pathway, however its function is not currently understood in renal cell carcinoma (RCC). Methods : The expression levels of IGFBP-4 protein in kidney tissue microarray were examined by immunostaining using antibody to IGFBP-4. The IGFBP-4 mRNA expression level was analyzed in a normal kidney cell line (HK-2), primary renal cancer cell lines (A498, 769-P) and metastatic renal cancer cell lines (ACHN, Hs891.T). To examine the function of IGFBP4, MTS, matrigel invasion, colony formation and wound healing assays were performed using 769-P mock and stable IGFBP-4 transfected cells. To screen for metastatic related genes in IGFBP-4 stable cells, cDNA PCR array was performed with a Human RT2 Profiler PCR Array. We investigated mRNA expression of the MMP family and measured Tcf transcriptional activity to monitor the Wnt/beta-catenin dependent pathway in 769-P mock and IGFBP-4 stable transfected cells. To analyze the role of IGFBP-4 in metastatic RCC cell lines, IGFBP-4 mRNA was knocked down using a siRNA technique. Results : We found that the expression of IGFBP-4 was significantly lower in primary RCC and higher in metastatic RCC compared to normal human kidney tissues. To assess the function of IGFBP4, we established IGFBP4 transfectants (primary renal cancer cell line) and performed functional analyses including Tcf reporter assays, cell viability, invasive capability, mortality, and in vivo tumor growth. Interestingly IGFBP-4 transfectants promoted cell growth (in vitro and in vivo), invasion, and motility in 769-P cancer cells. Tcf transcriptional activity was significantly increased in IGFBP-4 transfectants compared to mock cells and beta-catenin expression was increased. Also the beta-catenin downstream effector, MT1-MMP showed increased expression in IGFBP4 transfectants. Additionally IGFBP4 induced the expression of M-CAM, a marker of tumor progression. In order to assess the role of IGFBP4 in metastatic renal cancer, IGFBP-4 mRNA in a metastatic renal cancer cell line (ACHN) was knocked-down using a siRNA technique. Cell growth and motility was decreased in si-IGFBP4 transfected ACHN cells compared to cells transfected with control siRNA. Conclusions : This is the first report documenting that IGFBP-4 expression in RCC activates cell growth, metastasis, Wnt/beta-catenin signaling and may be involved in RCC metastasis. 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 1124. doi:10.1158/1538-7445.AM2011-1124

IGFBP‐4 activates the Wnt/beta‐catenin signaling pathway and induces M‐CAM expression in human renal cell carcinoma

The Wnt/β-catenin signaling pathway is inactivated by Wnt antagonists in most cancers and IGFBP-4 is an antagonist of the Wnt/ β-catenin signaling pathway. However, the function of IGFBP-4 is not currently understood in renal cell carcinoma (RCC). We initially found that the expression of IGFBP-4 was significantly lower in primary RCC and higher in metastatic RCC compared to normal human kidney tissues. To assess the function of IGFBP4, we established IGFBP4 transfectants (primary renal cancer cell line) and performed functional analyses including Tcf reporter assays, cell viability, invasive capability, mortality, and in vivo tumor growth. Interestingly IGFBP-4 transfectants promoted cell growth (in vitro and in vivo), invasion, and motility in primary renal cancer. Tcf transcriptional activity was significantly increased in IGFBP-4 transfectants compared to mock cells and β-catenin expression was increased. Also the β-catenin downstream effector, MT1-MMP showed increased expression in IGFBP4 transfectants. Additionally IGFBP4 induced the expression of M-CAM, a marker of tumor progression. In order to assess the role of IGFBP4 in metastatic renal cancer, IGFBP-4 mRNA in a metastatic renal cancer cell lines (ACHN) was knocked-down using a siRNA technique. The cell growth and motility was decreased in si-IGFBP4 transfected ACHN cells compared to cells transfected with control siRNA. Tcf activity in ACHN cells was also decreased with si-IGFBP-4 transfection. This is a first report documenting that IGFBP-4 expression in RCC activates cell growth, metastasis, Wnt/beta-catenin signaling and may be involved in RCC metastasis.

108 IGFBP-4 ACTIVATES THE WNT/BETA-CATENIN SIGNALING PATHWAY AND INDUCES M-CAM EXPRESSION IN HUMAN RENAL CELL CARCINOMA

You have accessJournal of UrologyKidney Cancer: Basic Research1 Apr 2011108 IGFBP-4 ACTIVATES THE WNT/BETA-CATENIN SIGNALING PATHWAY AND INDUCES M-CAM EXPRESSION IN HUMAN RENAL CELL CARCINOMA Koji Ueno, Hiroshi Hirata, Z. Laura Tabatabai, Yuji Hinoda, Peter R. Carroll, and Rajvir Dahiya Koji UenoKoji Ueno San Francisco, CA More articles by this author , Hiroshi HirataHiroshi Hirata San Francisco, CA More articles by this author , Z. Laura TabatabaiZ. Laura Tabatabai San Francisco, CA More articles by this author , Yuji HinodaYuji Hinoda Ube, Japan More articles by this author , Peter R. CarrollPeter R. 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.2011.02.174AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The Wnt/beta-catenin signaling pathway is inactivated by Wnt antagonists in most cancers. IGFBP-4 is an antagonist of the Wnt/beta-catenin signaling pathway, however its function is not currently understood in renal cell carcinoma (RCC). METHODS The expression levels of IGFBP-4 protein in kidney tissue microarray were examined by immunostaining using antibody to IGFBP-4. The IGFBP-4 mRNA expression level was analyzed in a normal kidney cell line (HK-2), primary renal cancer cell lines (A498, 769-P) and metastatic renal cancer cell lines (ACHN, Hs891.T). To examine the function of IGFBP4, MTS, matrigel invasion, colony formation and wound healing assays were performed using 769-P mock and stable IGFBP-4 transfected cells. To screen for metastatic related genes in IGFBP-4 stable cells, cDNA PCR array was performed with a Human RT2 Profiler PCR Array. We investigated mRNA expression of the MMP family and observed Tcf transcriptional activity to monitor the Wnt/beta-catenin dependent pathway in 769-P mock and IGFBP-4 stable transfected cells. To analyze the role of IGFBP-4 on metastatic RCC cell lines, IGFBP-4 mRNA was knocked down using a siRNA technique. RESULTS We initially found that the expression of IGFBP-4 was significantly lower in primary RCC and higher in metastatic RCC compared to normal human kidney tissues. To assess the function of IGFBP4, we established IGFBP4 transfectants (primary renal cancer cell line) and performed functional analyses including Tcf reporter assays, cell viability, invasive capability, mortality, and in vivo tumor growth. Interestingly IGFBP-4 transfectants promoted cell growth (in vitro and in vivo), invasion, and motility in primary renal cancer. Tcf transcriptional activity was significantly increased in IGFBP-4 transfectants compared to mock cells and beta-catenin expression was increased. Also the beta-catenin downstream effector, MT1-MMP showed increased expression in IGFBP4 transfectants. Additionally IGFBP4 induced the expression of M-CAM, a marker of tumor progression. In order to assess the role of IGFBP4 in metastatic renal cancer, IGFBP-4 mRNA in a metastatic renal cancer cell lines (ACHN) was knocked-down using a siRNA technique. The cell growth and motility was decreased in si-IGFBP4 transfected ACHN cells compared to cells transfected with control siRNA. CONCLUSIONS This is a first report documenting that IGFBP-4 expression in RCC activates cell growth, metastasis, Wnt/beta-catenin signaling and may be involved in RCC metastasis. © 2011 by American Urological Association Education and Research, Inc.FiguresReferencesRelatedDetails Volume 185Issue 4SApril 2011Page: e46 Peer Review Report Advertisement Copyright & Permissions© 2011 by American Urological Association Education and Research, Inc.MetricsAuthor Information Koji Ueno San Francisco, CA More articles by this author Hiroshi Hirata San Francisco, CA More articles by this author Z. Laura Tabatabai San Francisco, CA More articles by this author Yuji Hinoda Ube, Japan More articles by this author Peter R. 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 ...

Deletion of the thrombin cleavage domain of osteopontin mediates breast cancer cell adhesion, proteolytic activity, tumorgenicity, and metastasis

BackgroundOsteopontin (OPN) is a secreted phosphoprotein often overexpressed at high levels in the blood and primary tumors of breast cancer patients. OPN contains two integrin-binding sites and a thrombin cleavage domain located in close proximity to each other.MethodsTo study the role of the thrombin cleavage site of OPN, MDA-MB-468 human breast cancer cells were stably transfected with either wildtype OPN (468-OPN), mutant OPN lacking the thrombin cleavage domain (468-ΔTC) or an empty vector (468-CON) and assessed for in vitro and in vivo functional differences in malignant/metastatic behavior.ResultsAll three cell lines were found to equivalently express thrombin, tissue factor, CD44, αvβ5 integrin and β1 integrin. Relative to 468-OPN and 468-CON cells, 468-ΔTC cells expressing OPN with a deleted thrombin cleavage domain demonstrated decreased cell adhesion (p < 0.001), decreased mRNA expression of MCAM, maspin and TRAIL (p < 0.01), and increased uPA expression and activity (p < 0.01) in vitro. Furthermore, injection of 468-ΔTC cells into the mammary fat pad of nude mice resulted in decreased primary tumor latency time (p < 0.01) and increased primary tumor growth and lymph node metastatic burden (p < 0.001) compared to 468-OPN and 468-CON cells.ConclusionsThe results presented here suggest that expression of thrombin-uncleavable OPN imparts an early tumor formation advantage as well as a metastatic advantage for breast cancer cells, possibly due to increased proteolytic activity and decreased adhesion and apoptosis. Clarification of the mechanisms responsible for these observations and the translation of this knowledge into the clinic could ultimately provide new therapeutic opportunities for combating breast cancer.

Abstract PL03-02: Some cancers follow a stem cell model, while other cancers have common tumorigenic cells with little hierarchical organization

Abstract In our studies of hematopoietic malignancies, acute (Nature 441:475) and chronic myeloid leukemia (Cancer Cell 16:137) appear to follow a cancer stem cell model. In both cases, leukemogenic cells are rare, phenotypically distinct from the vast majority of other leukemia cells, and hierarchically organized. However, tumorigenic capacity is a common attribute of cells from a variety of other human and mouse cancers we have studied, including melanoma. Many cancers are not driven by rare cancer stem cells. We recently showed that the optimization of xenotransplantation assay conditions, including the use of more highly immunocompromised NOD/SCID IL2Rγnull mice, can increase the detection of tumorigenic cells in some cancers by orders-of-magnitude (Nature 456:593). This indicated that tumorigenic cells are common in some human cancers, and that to accurately estimate the frequency of cancer cells with tumorigenic potential it is critical to optimize assay conditions. In this regard, the characterization of human cancer stem cells is no different from the characterization of normal human hematopoietic stem cells, which also depended on the use of the most permissive possible xenotransplantation assay conditions, including the use of highly immunocompromised NOD/SCID IL2Rγnull mice, rather than NOD/SCID mice (which retain natural killer and other innate immune cells that reject some transplanted human cells). Optimization of xenotransplantation assay conditions revealed that cells with tumorigenic potential are common in human melanoma (at least 25% of cells from stage 3 and 4 melanomas have the potential to form tumors after transplantation), suggesting that melanoma growth and progression are unlikely to be driven by rare cancer stem cells. We have also been unable to find any markers that can distinguish tumorigenic from non-tumorigenic melanoma cells, suggesting that melanoma lacks the obvious hierarchical organization observed in other cancers that follow a stem cell model, such as leukemia. Despite observing extensive phenotypic heterogeneity among melanoma cells, we have been unable to detect any differences in tumorigenic capacity among melanoma cells from multiple patients that differed in the expression of ABCB5, CD133, CD166, L1-CAM, CD49f, A2B5, p75, CD44, CD54, CD29, MCAM, HNK1, CD49b, CD49d, E-Cadherin, N-Cadherin, or c-kit. When transplanted into NOD/SCID IL2Rγnull (NOG) mice, only 10 melanoma cells from either the positive or negative fractions of each of these markers readily formed tumors. We therefore remain unable to find any evidence that human melanoma cells are segregated into a hierarchy of intrinsically distinct tumorigenic and non-tumorigenic fractions. Moreover, all fractions of melanoma cells appear to be able to recapitulate the phenotypic diversity of the tumors from which they are drawn. For example, irrespective of whether melanomas arose from CD133+ or CD133- melanoma cells, they contained similar proportions of CD133+ and CD133- cells (Cell 138:822). This suggests that the capacity to recapitulate the phenotypic diversity of primary tumors is not necessarily unique to cancer stem cells but rather that many cells can form phenotypically diverse progeny in certain cancers. Finally, we observed significant differences in the growth rates of tumors from different patients, but these differences were not associated with differences in the frequency of cells with tumorigenic potential. This suggests there are likely to be biologically and clinically important differences among melanoma cells that are not explained by the cancer stem cell model. The available data suggest that many melanoma cells are capable of contributing to disease progression and that it will not be possible to cure melanoma by targeting rare populations of cancer stem cells. Preliminary studies of additional mouse and human cancers suggest that melanoma is not unusual in having common tumorigenic cells. Overall, some cancers appear to be driven by minority populations of cancer stem cells, while other cancers appear to have common tumorigenic cells with little evidence of hierarchical organization. It will be critical to determine which cancers follow a stem cell model and which do not so that efforts to identify and target stem cells can be appropriately focused. Citation Format: Elsa Quintana, Mark Shackleton, Sean J. Morrison. Some cancers follow a stem cell model, while other cancers have common tumorigenic cells with little hierarchical organization [abstract]. In: Proceedings of the AACR 101st Annual Meeting 2010; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr PL03-02

Abstract 2924: Genome-wide microarray platforms uncover novel hypermethylated genes in an oral squamous cell carcinoma case-control study: A phase I preclinical biomarker development study

Abstract Introduction: Panels of hypermethylated genes have been proposed as diagnostic markers in primary head and neck squamous cell carcinoma using a pharmacological unmasking expression microarray approach. Such experiments are performed in cancer cell lines, mostly with poor relevance when extrapolating to primary cancers. Objective: To overcome this lack of relevance we used two unbiased genome-wide DNA methylation platforms that represent 28K CpG Islands and the promoter regions of 14K-17K genes to identify hypermethylated genes in Oral Squamous Cell Carcinoma (OSCC), which also exhibited downregulated expression in the Gene Expression Omnibus (GEO) repository. Methods: DNA isolated from normal, premalignant lesions and squamous cell carcinoma lesions of the oral cavity was: a) bisulfite treated and hybridized to Infinium arrays; b) enriched with Methylated DNA Immunoprecipitation (MeDIP) and hybridized to Nimblegen 385K tiling arrays. Bioinformatics strategies were used for background correction, array normalization and data analysis of differentially methylated genomic regions between tumor and normal tissue. Genes identified as hypermethylated in cancer with methylation platforms were cross-validated against expression profiles published in the GEO public data repository. Quantitative Methylation Specific PCR (QMSP) was then used to validate candidate genes that were both, hypermethylated and downregulated in cancer. Results: The two unbiased microarray platforms were successful in identifying three novel genes hypermethylated and down regulated in OSCC: MCAM, EDNRB and CALCA. EDNRB, MCAM and CALCA expression was downregulated in HNSCC when compared to normal tissue. A twofold or higher downregulated expression was seen for EDNRB (59%) than for MCAM (55%) and CALCA (55%). The sensitivity of each gene was higher for EDNRB (82%) than for MCAM (60%) and CALCA (76%). The specificity was also higher for EDNRB (86%) than for CALCA (71%), and MCAM (57%). KIF1a had both high sensitivity (82%) and specificity (86%) but there was no expression data in HNSCC available for this gene in the GEO repository. The Receiver Characteristic Operator Curves (ROC) for individual genes revealed area under the curve values (AUC) for: EDNRB (0.88), CALCA (0.85), KIF1a (0.84), and MCAM (0.61). Methylation of three genes was found in 71% of tumor and 0% of normal tissue. Methylation of two genes was found in 82% of tumor and 29% of normal tissue. Conclusion: We successfully demonstrated that unbiased genome-wide methylation arrays paired with publicly available expression data identify panels of relevant hypermethylated genes in primary OSCC. MCAM, KIF1a, EDNRB and CALCA were identified as hypermethylated in OSSC tumor tissue, when compared to normal tissue. These results should be validated in a Phase II Biomarker Development Study. 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 2924.