Source of Funding: None

1069CLINICAL SIGNIFICANCE OF SNAIL EXPRESSION IN BLADDERTUMOR AND ITS HYPOXIC REGULATION

Takeo Kosaka*, Eiji Kikuchi, Akira Miyajima, Suguru Shirotake,Takahiro Maeda, Shuji Mikami, Kenjiro Suzuki, Mototsugu Oya,Tokyo, Japan

INTRODUCTION AND OBJECTIVES: Accumulating evidencehas suggested that epithelial &endash; mesenchymal transition (EMT)is a key process in cancer progression. We have recently demonstratedthe prognostic significance of Snail expression in upper urinary tracturothelial carcinoma (UTUC). The aim of this study was to determinethe association between Snail expression and the clinico-pathologicalfeatures of BT and the potential upstream target of Snail, with a specialfocus on a hypoxic microenvironment.

METHODS: A total 64 patients who had been surgically treatedfor invasive BT were included in this study. Median follow-up was 50months and mean patient age was 68.4 years old. The expression ofSnail and E-cadherin was evaluated by Immunohistochemistry (IHC).Three human bladder cancer cell lines T24, 5637, and UMUC-3 wereanalyzed. Western blot and quantitative RT-PCR analysis were per-formed to evaluate the expression of Snail, E-cadherin and vimentinunder normoxic and hypoxic conditios. We used CoCl2 to mimic thehypoxia. Invasive activity under normoxic and hypoxic conditions wasquantified using Matrigel invasion chambers.

RESULTS: IHC analysis revealed the level of Snail expressionin the nucleus was significantly associated with a high grade (p�0.014)or higher pathological stage (p�0.0018) and the presence of lymphnode involvement (p�0.041). The 5-year progression-free survival(PFS) was 52.2% for patients with higher Snail expression compared to86.0% for patients with lower Snail expression (Log-rank p�0.003).Time course analysis under hypoxia revealed increased HIF-1� ex-pression in all cell lines. In T24 cells, hypoxia significantly induced Snailup-regulation (2.61�0.23 fold increase, p�0.01) accompanied by up-regulated vimentin (1.21�0.32 fold increase, p�0.05,). Similar resultswere observed in UMUC-3 cells. In T24 cells, in vitro invasion assayrevealed increased number of migrated cells under hypoxia(1.3�0.2 fold increase, p�0.05), compared with normoxia.

CONCLUSIONS: These results indicated that the contributionof Snail to clinic-pathological behavior was similar between UTUC andinvasive BT. Moreover, they imply that hypoxia triggers and modulatedthe expression of Snail as the center of the multistep process ofinvasion and metastasis of BT.

Source of Funding: None

1070DEVELOPMENT OF A RAPAMYCIN SENSITIVE CELL LINEFROM UROTHELIAL CARCINOMA WITH SARCOMATOIDFEATURES

Edward Diaz*, Christina Ching, Donna Hansel, Cleveland, OH

INTRODUCTION AND OBJECTIVES: Multiple commercial celllines for urothelial carcinoma (UC) exist, but few were developed fromaggressive variants of urothelial carcinoma, such as those with sarco-

matoid or squamous features. There are also very few urothelial celllines that can be sustained in a serum free environment. Recent studiessupport a relationship between regulation of the mammalian target ofrapamycin (mTOR) pathway and carcinogenesis in UC. Our objectivewas to develop a serum independent cell line from an aggressivevariant of urothelial carcinoma, and assess the effect of mTOR inhibi-tion on these cells.

METHODS: The primary specimen was obtained from a 67 yocaucasian female with high grade UC with sarcomatoid features andsquamous differentiation. Cells were immortalized using two rounds ofcell propagation in an immune deficient mouse model. The final tumorwas harvested from the mouse, dissociated, and grown in serum freeconditions supplemented with growth factors. The cells were thentested for expression of cell markers found in aggressive UC (CK7,CK20, p63, p53, and Ker903) using a cell block protocol and immuno-staining. Sensitivity of the cell line to mTOR inhibition was then testedin vivo using a double arm trial of placebo vs rapamycin in mice withestablished tumors.

RESULTS: Cells grew in a monolayer adherent to standardtissue culture plates. Morphology and viability was maintained aftermultiples passages within serum free conditions (Over 20 passages).Cell block revealed approximately 30% staining for p63, 100% stainingfor CK7, 0% staining for CK20, 100% staining for p53, and 40% stainingfor Ker903, confirming the cell line’s human urothelial origin. The cellline demonstrated tumorigenicity in the mouse xenograft model. Singleinjection of 3 million cells resulted in the growth of a tumor with max 1cm dimension after 31 days from inoculation. Tumors derived from thiscell line demonstrated in vivo sensitivity to the mTOR inhibitor, rapa-mycin. Starting average tumor volume was: 231.5 cm3 for rapamycinarm 302.4 cm3 for placebo arm. Average tumor volume after treatmentwas: 336 cm3 for rapamycin arm, and 682 cm3 for placebo arm. Nometastatic disease was present in either treatment group.

CONCLUSIONS: This provides a unique urothelial cancer cellline derived from an aggressive variant of UC and one that can besustained in serum free conditions. Preliminary results in this studysuggest mTOR signaling is important to the proliferative potential of thiscell line, and possibly to this aggressive variant of UC.

Source of Funding: None

1071ANDROGEN RECEPTOR SIGNALS REGULATEUDP-GLUCURONOSYLTRANSFERASES: A POTENTIALMECHANISM OF ANDROGEN-INDUCED BLADDERCARCINOGENESIS

Koji Izumi*, Yichun Zheng, Hiroshi Miyamoto, Rochester, NY

INTRODUCTION AND OBJECTIVES: Men have a substan-tially higher risk of bladder cancer than women. We previously showed,using N-butyl-N(4-hydroxybutyl) nitrosamine (BBN) treated AR knock-out (ARKO) mouse models, that androgens and androgen receptor(AR) played a vital role in bladder carcinogenesis. UDP-glucuronosyl-transferases (UGTs) are major phase II drug metabolism enzymes, andUGT1A subtypes, especially UGT1A1, UGT1A4, and UGT1A9 in hu-mans, were shown to be important in detoxifying bladder carcinogens.The bladder is known to express UGTs, and UGT1A expression isrelatively low in human bladder cancer. The purpose of this study is toinvestigate the relationship between AR signals and UGT1A expres-sion in the bladder.

METHODS: AR-negative bladder cell lines, normal urotheliumSVHUC and urothelial carcinoma 5637, were stably expressed withAR. The expression of UGT1A subtypes in AR-positive and AR-nega-tive bladder cell lines were measured by quantitative real-time PCRfollowing treatment with dihydrotestosterone (DHT) and/or hydroxyflu-tamide (HF). UGT1a expression was also determined in mice with orwithout orchiectomy and in ARKO male mice treated with BBN.

RESULTS: DHT treatment in SVHUC-AR reduced mRNA ex-pression of all the UGT1A subtypes (19–75% decrease), and HFantagonized the effects of DHT. In SVHUC-Vector, DHT and HF

e430 THE JOURNAL OF UROLOGY� Vol. 185, No. 4S, Supplement, Monday, May 16, 2011