Serum and Tissue Profiling in Bladder Cancer Combining Protein and Tissue Arrays

Orenes‐Piñero, Esteban; Barderas, Rodrigo; Rico, Daniel; Casal, J. Ignacio; Pisano, David G.; Navajo, José Alejandro; Algaba, Ferrán; Piulats, Josep María; Sänchez‐Carbayo, Marta

doi:10.1021/pr900273u

Cited by 30 publications

(42 citation statements)

References 48 publications

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“…A bladder cancer tissue microarray (TMA) was constructed at the CNIO as described (59,60) (2,61). Protein expression patterns of ET-1 were assessed using standard avidin-biotin immunoperoxidase procedures and antigen retrieval methods (0.01% citric acid for 15 minutes under microwave treatment) prior to overnight incubation with mouse monoclonal primary antibodies against ET-1 (Zymed, Invitrogen).…”

Section: Methodsmentioning

confidence: 99%

“…Protein expression patterns of ET-1 were assessed using standard avidin-biotin immunoperoxidase procedures and antigen retrieval methods (0.01% citric acid for 15 minutes under microwave treatment) prior to overnight incubation with mouse monoclonal primary antibodies against ET-1 (Zymed, Invitrogen). Sections were then scored as described (59,60). Patient follow-up information allowed the evaluation of protein expression versus DSS.…”

Section: Methodsmentioning

confidence: 99%

“…The consensus value of the 3 or 4 representative cores from each tumor sample arrayed was used for statistical analyses. The association of the expression of ET-1 and COX2 with histopathologic stage and tumor grade was evaluated using the nonparametric Wilcoxon-Mann-Whitney and Kruskal-Wallis tests (59,60). ET-1 and COX2 expression was evaluated as continuous variable based on the number of cells expressing the protein in the cytoplasm.…”

Section: Methodsmentioning

confidence: 99%

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Tumor endothelin-1 enhances metastatic colonization of the lung in mouse xenograft models of bladder cancer

Said¹,

Smith²,

Sänchez‐Carbayo³

et al. 2011

J. Clin. Invest.

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118

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Many patients with advanced bladder cancer develop lethal metastases to the lung. The vasoconstricting protein endothelin-1 (ET-1) has been implicated in this process, although the mechanism(s) by which it promotes metastasis remains unclear. Here, we have evaluated whether tumor ET-1 expression can serve as a biomarker for lung metastasis and whether it is required for metastatic disease. Evaluation of ET-1 mRNA and protein expression in four patient cohorts revealed that levels of ET-1 are higher in patients with muscle-invasive bladder cancers, which are associated with higher incidence of metastasis, and that high ET-1 levels are associated with decreased disease-specific survival. Consistent with its proinflammatory activity, we found that tumorderived ET-1 acts through endothelin-1 receptor A (ET A R) to enhance migration and invasion of both tumor cells and macrophages and induces expression of inflammatory cytokines and proteases. Using human and mouse cancer cells depleted of ET-1 and pharmacologic blockade of ET receptors in lung metastasis models, we found that tumor ET-1 expression and ET A R activity are necessary for metastatic lung colonization and that this process is preceded by and dependent on macrophage infiltration of the lung. In contrast, tumor ET-1 expression and ET A R activity appeared less important in established primary or metastatic tumor growth. These findings strongly suggest that ET A R inhibitors might be more effective as adjuvant therapeutic agents than as initial treatment for advanced primary or metastatic disease.

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

See 1 more Smart Citation

Tumor endothelin-1 enhances metastatic colonization of the lung in mouse xenograft models of bladder cancer

Said¹,

Smith²,

Sänchez‐Carbayo³

et al. 2011

J. Clin. Invest.

Self Cite

118

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“…Protein expression patterns of versican were assessed using 2 tissue microarrays (TMAs) (87,88). Antigen-retrieval methods (0.01% citric acid for 15 minutes under microwave treatment) were utilized prior to incubation with mouse anti-versican monoclonal antibody (LifeSpan BioSciences) at 1:400.…”

Section: Construction and Immunostaining Of Bladder Cancer Tissue Micmentioning

confidence: 99%

“…The secondary antibodies (Vector Laboratories) were biotinylated horse anti-mouse antibodies (1:500 dilution). Diaminobenzidine was utilized as the final chromogen and hematoxylin as the nuclear counterstain (48,87,88). The consensus value of the 3 or 4 representative cores from each tumor sample arrayed was used for statistical analyses.…”

Section: Construction and Immunostaining Of Bladder Cancer Tissue Micmentioning

confidence: 99%

RhoGDI2 suppresses lung metastasis in mice by reducing tumor versican expression and macrophage infiltration

Said¹,

Sänchez‐Carbayo²,

Smith³

et al. 2012

J. Clin. Invest.

Self Cite

130

111

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Half of patients with muscle-invasive bladder cancer develop metastatic disease, and this is responsible for most of the deaths from this cancer. Low expression of RhoGTP dissociation inhibitor 2 (RhoGDI2; also known as ARHGDIB and Ly-GDI) is associated with metastatic disease in patients with muscle-invasive bladder cancer. Moreover, a reduction in metastasis is observed upon reexpression of RhoGDI2 in xenograft models of metastatic cancer. Here, we show that RhoGDI2 suppresses lung metastasis in mouse models by reducing the expression of isoforms V1 and V3 of the proteoglycan versican (VCAN; also known as chondroitin sulfate proteoglycan 2 [CSPG2]). In addition, we found that high versican levels portended poor prognosis in patients with bladder cancer. The functional importance of tumor expression of versican in promoting metastasis was established in in vitro and in vivo studies in mice that implicated a role for the chemokine CCL2 (also known as MCP1) and macrophages. Further analysis indicated that RhoGDI2 suppressed metastasis by altering inflammation in the tumor microenvironment. In summary, we demonstrate what we believe to be a new mechanism of metastasis suppression that works by reducing host responses that promote metastatic colonization of the lung. Therapeutic targeting of these interactions may provide a novel adjuvant strategy for delaying the appearance of clinical metastasis in patients. IntroductionOne-half of patients with muscle-invasive (MI) urothelial cancer (UC) of the bladder develop distant metastases, even after radical surgery of the primary tumors. We identified RhoGTP dissociation inhibitor 2 (RhoGDI2; also known as ARHGDIB and Ly-GDI, and abbreviated herein as GDI2) as an invasion and metastasis suppressor in human bladder cancer cell lines (1) and have shown that its expression is inversely associated with clinical outcome after treatment of MI tumors (2). Independently, in comparative gene expression profiling of invasive bladder cancer cell lines and human MI UC samples, we identified versican (VCAN; also known as chondroitin sulfate proteoglycan 2, [CSPG2]) as highly expressed in invasive and metastatic cancers (3).Versican is a highly conserved structural component of the ECM that is involved in neuronal development (4-8), the inflammatory phase of pulmonary-vascular diseases, atherosclerosis (9-12), and the invasive and metastatic signatures of many cancers (13-25). Four isoforms or spliced variants have been reported for versican, and the roles of V0, V1, and V3 and to a lesser extent V2 isoforms are recognized in cancer, vascular disease, and neuronal development (detailed in refs. 8, 26, 27, and the references cited therein). These isoforms contribute to proliferative, adhesive, and migratory states of tumor cells and modulate their interactions with stroma in the tumor microenvironment (26,28,29).Versican expression is regulated by cytokines, chemokines, and hypoxia (6, 7, 9-12, 21, 26, 29-36) via transcription factors such as TCF-4, SP-1, AP-1, and p53, which have bin...

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Systematic quantification of peptides/proteins in urine using selected reaction monitoring

et al. 2011

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The evaluation of biomarkers in bodily fluids necessitates the development of robust methods to quantify proteins in a complex background, using large sets of samples. The ability to multiplex numerous analytes in a single assay expedites the process. Liquid chromatography-mass spectrometry (LC-MS) analyses performed in selected reaction monitoring (SRM) in conjunction with stable isotope dilution MS present an effective way to detect and quantify biomarker candidates in bodily fluids. The strategy presented involves an initial qualification of predefined sets of proteins in urine. The technique was applied to detect and quantify peptides in urine samples as surrogates for a few endogenous proteins. Multiplexed assays were developed to analyze proteins associated with bladder cancer; a few exogenous proteins were added as internal standards. The sample preparation and the analytical protocols were optimized to ensure reproducibility, analytical precision, and quantification limits in the low nanogram per milliliter range. Analyses were performed using known amounts of isotopically labeled peptides. Systematic replication of the measurements indicated intra-assay and inter-assay variability, with CVs in the range of 10%. The differences measured for two targeted proteins were correlated with their level of expression in the corresponding tumors using immunohistochemistry.

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Serum and Tissue Profiling in Bladder Cancer Combining Protein and Tissue Arrays

Cited by 30 publications

References 48 publications

Tumor endothelin-1 enhances metastatic colonization of the lung in mouse xenograft models of bladder cancer

Tumor endothelin-1 enhances metastatic colonization of the lung in mouse xenograft models of bladder cancer

RhoGDI2 suppresses lung metastasis in mice by reducing tumor versican expression and macrophage infiltration

Systematic quantification of peptides/proteins in urine using selected reaction monitoring

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