Wolfgang Jaeger

63 Erythropoietin accelerates the regeneration of ureteral function in a murine model of obstructive uropathy

Purpose: Unilateral ureteral obstruction halts ureteral peristalsis, and may cause pain and lead to infection. Ureteral ability to recover after obstruction removal remains unclear. Erythropoietin has protective effects in nonhematopoietic organs and restores peristalsis in hypocontractile intestinal smooth muscle cells. We investigated the role of erythropoietin in ureteral smooth muscle function and its therapeutic value for unilateral ureteral obstruction. Materials and Methods: Unilateral ureteral obstruction was created for 24, 48 and 72 hours in 22 mice per group using a nontraumatic microclip via laparotomy. We determined erythropoietin, erythropoietin receptor and b-common receptor expression in obstructed and unobstructed ureters by reverse transcriptasepolymerase chain reaction and immunohistochemistry. Ten mice per group received 20 IU erythropoietin for 4 days and controls received saline. Hydronephrosis regression after obstruction removal was assessed by ultrasound. Peristalsis was determined microscopically before and after obstruction removal. Results: Erythropoietin, erythropoietin receptor and b-common receptor were expressed in the unobstructed and obstructed ureters of untreated mice. Erythropoietin mRNA was up-regulated in response to obstruction and erythropoietin expression was identified in ureteral smooth muscle. After obstruction removal hydronephrosis and ureteral dysfunction correlated with obstruction duration. Hydronephrosis resolution and ureteral peristalsis restoration were significantly accelerated in erythropoietin treated mice compared to controls. Conclusions: Erythropoietin treatment significantly promoted functional recovery of the ureter after obstruction removal. Erythropoietin may be a helpful strategy for ureteral motility recovery and hydronephrosis resolution in ureteral obstruction.

50 Silencing Notch-1 and Notch-3 promotes epithelial-to-mesenchymal transition (EMT) and self-renewal potential in human bladder cancer

INTRODUCTION AND OBJECTIVES: Beside its regulatory functions during development, the Notch pathway plays an eminent role in the tumorigenesis of malignancies. There are four different Notch receptors, which demonstrate context-dependent activity and can work antagonistically to each other. We have previously described a role for Notch-2 in promoting EMT and invasion in bladder cancer (BCA). Our objective here was to elucidate the role of Notch-1 and -3 in EMT and invasion, hypothesizing that both promote the epithelial, less invasive phenotype. METHODS: Immunohistochemistry was performed on a tissue microarray (TMA) of 104 tumors from patients with muscle invasive BCA. Notch-1 and -3 expression was assessed in a panel of bladder cancer cell lines by Western blot (WB) and correlated to previously established EMT status (based on E-cadherin and ZEB1) and invasive ability. Notch-1 and -3 were silenced by lentiviral shRNA vector in 3 cell lines with high constitutive Notch-1 and -3 expression (UM-UC1, UMUC6 and UM-UC15). Protein and mRNA expression of genes relevant to EMT, invasion and stemness were profiled by WB, real-time PCR, immunofluorescence and FACS. Proliferation, migration and invasion were measured by crystal violet, scratch, and Boyden chamber assays, respectively. Self-renewal potential was assessed by clonogenic assay and spheroid formation in MammoCultTM medium. RESULTS: High expression of Notch-3 receptor was associated with lower pathological stage (p 0.05) and absence of lymphovascular invasion (p 0.05) in the cystectomy specimens. Notch-1 expression did not correlate with pathologic variables. WB revealed expression of Notch-1 and -3 in epithelial bladder cancer cell lines only. Silencing of Notch-1 or Notch-3 in the selected epithelial cell lines promoted EMT at the mRNA and protein levels. Accordingly, phenotypic changes were seen in the form of enhanced migration and invasion but unaltered proliferation. In addition, colony and spheroid formation was increased after specific Notch-3 silencing. The simultaneous knock down of Notch-1 and -3 showed no additive effect. CONCLUSIONS: Our results provide evidence that Notch-1 and Notch-3 are involved in invasion and progression of BCA by maintaining an epithelial phenotype and inhibiting EMT and self-renewal potential. This is the opposite of what we have seen previously with Notch-2. Beside verification of our observations in metastatic xenograft models, further investigation is needed to elucidate the molecular mechanisms of these alterations.

Abstract 1494: Silencing Notch-1 and Notch-3 promotes epithelial-to-mesenchymal transition (EMT) and self-renewal potential in human bladder cancer.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Background: Beside its regulatory functions during development, the Notch pathway plays an eminent role in the tumorigenesis of malignancies. There are four different Notch receptors, which demonstrate context-dependent activity and can work antagonistically to each other. We have previously described a role for Notch-2 in promoting EMT and invasion in bladder cancer (BCA). Our objective here was to elucidate the role of Notch-1 and -3 in EMT and invasion, hypothesizing that both promote the epithelial, less invasive phenotype. Methods: Immunohistochemistry was performed on a tissue microarray (TMA) of 104 tumors from patients with muscle invasive BCA. Notch-1 and -3 expression was assessed in a panel of bladder cancer cell lines by Western blot (WB) and correlated to previously established EMT status (based on E-cadherin and ZEB1) and invasive ability. Notch-1 and -3 were silenced by lentiviral shRNA vector in 3 cell lines with high constitutive Notch-1 and -3 expression (UM-UC1, UM-UC6 and UM-UC15). Protein and mRNA expression of genes relevant to EMT, invasion and stemness were profiled by WB, real-time PCR, immunofluorescence and FACS. Proliferation, migration and invasion were measured by crystal violet, scratch, and Boyden chamber assays, respectively. Self-renewal potential was assessed by clonogenic assay and spheroid formation in MammoCult™ medium. Results: High expression of Notch-3 receptor was associated with lower pathological stage (p<0.05) and absence of lymphovascular invasion (p<0.05) in the cystectomy specimens. Notch-1 expression did not correlate with pathologic variables. WB revealed expression of Notch-1 and -3 in epithelial bladder cancer cell lines only. Silencing of Notch-1 or Notch-3 in the selected epithelial cell lines promoted EMT at the mRNA and protein levels. Accordingly, phenotypic changes were seen in the form of enhanced migration and invasion but unaltered proliferation. In addition, colony and spheroid formation was increased after specific Notch-3 silencing. The simultaneous knock down of Notch-1 and -3 showed no additive effect. Conclusion: Our results provide evidence that Notch-1 and Notch-3 are involved in invasion and progression of BCA by maintaining an epithelial phenotype and inhibiting EMT and self-renewal potential. This is the opposite of what we have seen previously with Notch-2. Beside verification of our observations in metastatic xenograft models, further investigation is needed to elucidate the molecular mechanisms of these alterations. Citation Format: Wolfgang Jaeger, Tetsutaro Hayashi, Shannon Awrey, Kilian Gust, Thomas Cordonnier, Na Li, Ralph Buttyan, Estelle Li, Ladan Fazli, Theodorus Van Der Kwast, Bas W. G. Van Rhijn, Peter C. Black. Silencing Notch-1 and Notch-3 promotes epithelial-to-mesenchymal transition (EMT) and self-renewal potential in human bladder cancer. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1494. doi:10.1158/1538-7445.AM2013-1494