Inhibition of Rho-Kinase Abrogates Migration of Human Transitional Cell Carcinoma Cells: Results of an in vitro Study

Introduction: Migration of cells involves a complex signaling network. The aim of the present study was to elucidate the impact of Rho-kinase (ROK) on G protein-coupled receptor-induced migration of human transitional cell carcinoma cells in an in vitro experimental setting. Materials and Methods: Intracellular calcium concentration ([Ca2+](i)) was measured with the indicator dye Fura-2 in response to lysophosphatidic acid, thrombin and sphingosine-1-phosphate. Phospholipase C activity was determined in myo-[H-3]inositol-(0.5 p,mu Ci/ml) labeled cells. Migration was performed using a Boyden chamber. Transient transfection of a dominant-negative mutant of ROK was done with calcium phosphate. For staining of actin filaments, tetramethylrhodamine isothiocyanate-conjugated phalloidin was used. Results: Lysophosphatidic acid, thrombin and sphingosine-1-phosphate cause increases in [Ca2+](i), cellular responses being accompanied by an enhancement of phospholipase C activity and sensitive to the G(i) inhibitor pertussis toxin. Agonists potently stimulated migration of 124 and J82 cells. Inhibition of Rho proteins by Clostridium difficile toxin B abrogated cell migration. Inhibition of ROK using HA1077 and Y-27632 mimicked the properties of toxin B. Expression of a ROK mutant drastically reduced migration. Conclusions:G protein-coupled receptors potently stimulated cell migration in 124 and J82 cells. Rho proteins and ROK play a pivotal role in this signaling cascade. Rho and ROK may be putative targets for new therapy options in bladder cancer. Copyright (c) 2010 S. Karger AG, Base

Inhibitory role of the small leucine-rich proteoglycan biglycan in bladder cancer.

Urothelial bladder cancer is the ninth most common cancer. Despite surgical and chemotherapeutic treatment the prognosis is still poor once bladder cancer progresses to a muscle-invasive state. Discovery of new diagnostic markers and pathophysiologic effectors might help to contribute to novel diagnostic and therapeutic options. The extracellular matrix microenvironment shaped by the extracellular matrix critically affects tumor cell and stroma cell functions. Therefore, aim of the present study was to assess the possible implication of the small leucine-rich proteoglycan biglycan in progression of human urothelial bladder cancer.For this purpose tumor biopsies of 76 bladder cancer patients with different tumor stages (pTa, pT1-T4) were investigated with respect to biglycan expression and correlated with a long-term (10 years) clinical follow-up. Interestingly, higher biglycan mRNA expression was associated with higher tumor stages and muscle invasiveness. In vitro knock-down of endogenous biglycan in human urothelial carcinoma cells (J82 cells) increased proliferation, whereas addition of recombinant biglycan and overexpression of biglycan inhibited tumor cell proliferation. In line with this growth-inhibitory effect of biglycan, transplantation of J82 cells after knock-down of biglycan resulted in significantly increased growth of subcutaneous xenograft tumors in nude mice in vivo. Furthermore, treatment with two anti-proliferative, multi-receptor tyrosine kinase inhibitors-sunitinib and sorafenib-strongly upregulated biglycan expression. Collectively, the experimental data suggest that high biglycan expression is associated with reduced tumor cell proliferation. In accordance, Kaplan-Meier analysis revealed higher 10-year survival in patients with high biglycan mRNA expression in tumor biopsies.In conclusion, the present data suggest that biglycan is an endogenous inhibitor of bladder cancer cell proliferation that is upregulated in response to anti-proliferative tyrosine kinase inhibitors. In addition, high biglycan expression is associated with favorable prognosis

The impact of the receptor of hyaluronan-mediated motility (RHAMM) on human urothelial transitional cell cancer of the bladder.

Hyaluronan (HA) is a carbohydrate of the extracellular matrix with tumor promoting effects in a variety of cancers. The present study addressed the role of HA matrix for progression and prognosis of human bladder cancer by studying the expression and function of HA-related genes.Tissue samples of 120 patients with different stages of transitional cell bladder cancer, who underwent surgical treatment for bladder cancer at the University Hospital of Essen were analysed. mRNA-expression levels of HA synthases (HAS1-3) and HA-receptors (RHAMM and CD44) were evaluated by real time RT-PCR in comparison to healthy bladder tissue as control. In uni- and multivariate cox proportional hazard survival regression analysis, the impact of the gene expression levels on survival was assessed. In vitro knock-down of RHAMM, CD44 and HAS isoenzymes was achieved by siRNA and lentiviral shRNA in J82 bladder cancer cells. Transfected cells were analysed in vitro with regard to proliferation, cell cycle and apoptosis. J82 cells after knock-down of RHAMM were xenografted into male nu/nu athymic mice to monitor tumor progression in vivo.In invasive tumor stages RHAMM-, HAS1 and HAS2 mRNA-expression levels were elevated whereas HAS3v1 was reduced as compared to non-invasive tumors. Subsequently, Kaplan-Meier analysis revealed reduced bladder cancer specific survival in patients with high RHAMM mRNA and low HAS3v1 expression. Elevated RHAMM in invasive tumors was confirmed by RHAMM immunohistochemistry. Furthermore, multivariate analysis revealed that only RHAMM expression was associated with poor prognosis independent from other survival factors (HR=2.389, 95% CI 1.227-4.651, p=0.01). Lentiviral RHAMM knock-down revealed reduced J82 cell proliferation in vitro and reduced xenograft tumor growth in vivo.The data suggest that RHAMM plays a crucial role in mediating progression of muscle-invasive bladder cancer and recommends RHAMM for further evaluation as a prognostic marker or therapeutic target in bladder cancer therapy

Rap2B-Dependent Stimulation of Phospholipase C-ɛ by Epidermal Growth Factor Receptor Mediated by c-Src Phosphorylation of RasGRP3

Receptor tyrosine kinase regulation of phospholipase C-ɛ (PLC-ɛ), which is under the control of Ras-like and Rho GTPases, was studied with HEK-293 cells endogenously expressing PLC-coupled epidermal growth factor (EGF) receptors. PLC and Ca(2+) signaling by the EGF receptor, which activated both PLC-γ1 and PLC-ɛ, was specifically suppressed by inactivation of Ras-related GTPases with clostridial toxins and expression of dominant-negative Rap2B. EGF induced rapid and sustained GTP loading of Rap2B, binding of Rap2B to PLC-ɛ, and Rap2B-dependent translocation of PLC-ɛ to the plasma membrane. GTP loading of Rap2B by EGF was inhibited by chelation of intracellular Ca(2+) and expression of lipase-inactive PLC-γ1 but not of PLC-ɛ. Expression of RasGRP3, a Ca(2+)/diacylglycerol-regulated guanine nucleotide exchange factor for Ras-like GTPases, but not expression of various other exchange factors enhanced GTP loading of Rap2B and PLC/Ca(2+) signaling by the EGF receptor. EGF induced tyrosine phosphorylation of RasGRP3, but not RasGRP1, apparently caused by c-Src; inhibition of c-Src interfered with EGF-induced Rap2B activation and PLC stimulation. Collectively, these data suggest that the EGF receptor triggers activation of Rap2B via PLC-γ1 activation and tyrosine phosphorylation of RasGRP3 by c-Src, finally resulting in stimulation of PLC-ɛ

Second-Line Treatment of Advanced Urothelial Cancer with Paclitaxel and Everolimus in a German Phase II Trial (AUO Trial AB 35/09)

Objective: The efficacy of second-line treatment after failure of platinum-based chemotherapy in patients with advanced urothelial cancer is limited. Based on encouraging preclinical and clinical phase I data, we evaluated the safety and efficacy of the combination of paclitaxel and everolimus in these patients. Methods: In this trial, patients having failed to respond to prior platinum-based combination treatment of urothelial cancer were treated with paclitaxel (175 mg/m2 i.v., 3-weekly) and the mTOR-inhibitor everolimus (10 mg p.o., once daily). The patients were treated until tumor progression or until a maximum of 6 cycles was completed. A one-stage design was used to evaluate the objective response rate (ORR) as the primary endpoint. Results: A total of 27 patients (67% male; median age 63 years) were enrolled. The most frequent grade III/IV toxicities were anemia (28%), peripheral neuropathy (28%), and fatigue (24%). No treatment-related deaths were reported. Complete and partial remissions were observed in 0/24 and 3/24 patients eligible for efficacy analysis, respectively (ORR 13%). Progression-free survival was 2.9 months [95% confidence interval (95% CI) 1.9-4.2], and the median overall survival was 5.6 months (95% CI 4.8-10.2). Conclusion: The combination of paclitaxel and everolimus has not achieved the expected efficacy in second-line treatment of urothelial cancer and should not be further explored

Increased proliferation of tumor cells in response to BGN knock-down in vivo.

<p>The xenograft tumors were harvested after 7 weeks and processed for immunostaining and morphological analysis. BGN immunostaining and image analysis revealed decreased BGN expression compared to scrambled control (<b>A</b>, <b>B</b>). The area fraction of BGN positive staining was 25.3% in controls versus 10.8% in xenograft tumors derived from shBGN cells, n=6, **<i>p</i>=0.0087 (<b>C</b>). Next, the proliferative index was assessed using the Ki67 antigen. Compared to control (<b>D</b>) the percentage of the area fraction of Ki67 positive cells was significantly elevated in tumors composed of shBGN transduced J82 cells (<b>E, F</b>; 7.2% vs. 16.7%, n=6, **<i>p</i>=0.005).(<b>G</b>-<b>I</b>) The mean microvessel density (MVD) as determined by CD31 immunostaining was not affected in the xenograft tumors, n=6.</p

Accelerated growth of xenograft tumors in response to BGN knock-down.

<p>After knock-down of BGN in human J82 cells <i>in </i><i>vitro</i> the transduced cells were injected bilaterally into the flanks of nude mice and tumor growth was monitored over a period of 7 weeks. As a result the tumor volume was significantly higher after application of J82 cells transduced with shRNA targeting BGN compared to scrambled control (375 mm² vs. 204 mm²); n=6, ***<i>p</i><0.001. </p

BGN accumulation increases with tumor stage.

<p><b>A</b> - <b>C</b>, BGN immunostaining was performed on paraffin-embedded tissue sections. (<b>A</b>), Ta; (<b>B</b>), T2; (<b>C</b>), T4. (<b>D</b>), quantitative image analysis of BGN in non-invasive tumors (Ta, T1) and muscle-invasive tumor stages (T2-T4). (<b>E</b>), correlation of BGN positive area fraction with tumor staging. n=5 patient biopsies of each tumor stage, ***<i>p</i><0.0001, **<i>p</i>=0.0025.</p

Cancer-specific survival of patients with bladder cancer was discriminated by the extent of BGN mRNA.

<p>The 76 bladder cancer patients (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0080084#pone-0080084-g001" target="_blank">Figure 1</a>) were divided into two groups (BGN high and BGN low) and subjected to Kaplan-Meier survival analysis. For optimal separation of the groups with respect to relative BGN-expression variable cut points were evaluated and log rank test p-values were recorded. Optimal separation of two groups corresponding to the lowest p-value (<i>p</i>=0.044) was found at a relative BGN-expression value of 0.09.</p

BGN expression is up-regulated after treatment of J82 cells with the multi kinase inhibitors sorafenib and sunitinib.

<p>J82 were treated (24 h) with tyrosine kinase inhibitors (sorafenib, 10 µM and sunitinib, 1 µM). (<b>A</b>) BGN mRNA expression as determined by RT-qPCR. (<b>B</b>), BGN secretion into the cell culture supernatant was determined by immunoblotting after chondroitinase ABC treatment and normalized to the β-tubulin signal of the cell layer; n=4, **<i>p</i><0.01. </p