223 High aldo-keto reductase 1C1 expression in metastatic bladder cancer cells associated with invasive potential and drug resistance

Abstract

INTRODUCTION AND OBJECTIVES: The objective of this study were to investigate the genetic changes responsible for bladder cancer metastasis, using orthotopic metastatic mouse model, and to find new therapeutic targets of invasive bladder cancer. METHODS: Human UM-UC-3 bladder cancer cells were generated to express firefly luciferase 2 and red fluorescent protein tdTomato (UM-UC-3-tdTomato-luc2). Subsequently, we developed an orthotopic xenograft nude mouse model of bladder cancer by inoculating UM-UC-3-tdTomato-luc2 cells through a urethral catheter into the mouse bladder. We then performed bioluminescent imaging (BLI) to noninvasively monitor the growth of bladder tumors in their orthotopic sites. Two mice underwent autopsy after confirmation of lung or liver metastases using BLI. The primary and metastatic tumors were excised and plated on tissue culture dishes to generate bladder, lung, liver, and bone sublines. To examine the role of metastasis-related genes in bladder cancer, we then performed comparative microarray analyses of gene expression, and analyzed gene expression changes in bladder vs. lung, bladder vs. liver and bladder vs. bone. RESULTS: Among the 8 genes commonly up-regulated in metastatic sublines compared to bladder cells by our microarray analysis, the expression of aldo-keto reductase 1C1 (AKR1C1) was especially up-regulated. Furthermore, AKR1C1 was significantly upregulated in the 3 metastatic sublines by qRT-PCR and immunoblot analysis. In clinical samples, we observed increased expression of AKR1C1 in human metastatic tissues compared with the corresponding primary bladder cancer tissues by qRT-PCR and immunohistochemistry analysis. Knockdown of AKR1C1 using small interfering RNA downregulated expression levels of Rac1, phosphorylated Src, and phosphorylated FAK, accompanied by reduced invasive ability. Overexpression of AKR1C isoforms, including AKR1C1, has been demonstrated to be associated with drug resistance in various cancers. UM-CU-3 metastatic cells were more resistant to cisplatin than were UM-UC-3 wild-type cells, and AKR1C1 inhibitor flufenamic acid reversed cisplatin resistance in these cells. CONCLUSIONS: We found that AKR1C1 was up-regulated both in metastatic bladder cancer cells using an orthotopic mouse model and in metastatic sites of human surgical specimens. Our results demonstrate the role of AKR1C1 in regulating bladder cancer metastasis and drug resistance; therefore, AKR1C1 is a potential target for effective treatment of invasive bladder cancer.