Objective: Clusterin (CLU) is implicated in regulating clear renal cell carcinoma (CRCC) progression and metastasis, yet the mechanisms are not elucidated. In the present study, we explored the potential role of CLU in CRCC metastasis. Methods: Levels of CLU mRNA and CLU protein were measured by RT-PCR and immunohistochemistry analysis in 22 CRCC with metastasis and 22 without metastasis and 22 samples of normal kidney tissue. After CLU silencing and re-expression, the migration and invasion in vitro and in vivo of Caki-2 cells were determined by wound healing assay, transwell migration assay and pulmonary nodule assay, respectively. The expression of pERK1/2 and MMP-9 were detected by RT-PCR and Western blot assay. Results: We found a significant increase of CLU and CLU mRNA expression in CRCC, and the expression of CLU is strongly correlated in patients with metastatic disease. We discovered that CLU-rich Caki-2 cells displayed higher invasive ability which prompted us to investigate if CLU silencing could reduce the migration and invasion in Caki-2 cells. Compared with the vector-transfected cells, CLU knocked-down (CLUi) cells showed reduced migration and invasion in vitro, as well as decreased metastatic potential in experimental metastasis. Re-expression of CLU in CLUi cells restored the invasive phenotypes. We found that MMP-9 was downregulated in CLUi cells. We also discovered that levels of activated ERK1/2 correlated with the rich expression of CLU and MMP-9. Conclusion: Our data suggest that CLU may regulate aggressive behavior of human CRCC cells through modulating ERK1/2 signaling and MMP-9 expression.

Keywords:
Clear renal cell carcinoma, Metastasis, Clusterin
1.
Lanigan D: Prognostic factors in renal cell carcinoma. Br J Urol 1995;75:565-571.
2.
Rosenberg ME, Silkensen J: Clusterin: physiologic and pathophysiologic considerations. Int J Biochem Cell Biol 1995,27:633-645.
3.
Lau SH, Sham JS, Xie D, Tzang CH, Tang D, Ma N, Hu L, Wang Y, Wen JM, Xiao G, Zhang WM, Lau GK, Yang M, Guan XY: Clusterin plays an important role in hepatocellular carcinoma metastasis. Oncogene 2006;25:1242-1250.
4.
Yom CK, Woo HY, Min SY, Kang SY, Kim HS: Clusterin overexpression and relapse-free survival in breast cancer. Anticancer Res 2009;29:3909-3912.
5.
Chen Q, Wang Z, Zhang K, Liu X, Cao W, Zhang L, Zhang S, Yan B, Wang Y, Xia C: Clusterin confers gemcitabine resistance in pancreatic cancer. World J Surg Oncol 2011;9:59.
6.
Andersen CL, Schepeler T, Thorsen K, Birkenkamp-Demtröder K, Mansilla F, Aaltonen LA, Laurberg S, Ørntoft TF: Clusterin expression in normal mucosa and colorectal cancer. Mol Cell Proteomics 2007;6:1039-1048.
7.
Xie D, Lau SH, Sham JS, Wu QL, Fang Y, Liang LZ, Che LH, Zeng YX, Guan XY: Up-regulated expression of cytoplasmic clusterin in human ovarian carcinoma. Cancer 2005;103:277-283.
8.
Rizzi F, Bettuzzi S: Clusterin (CLU) and prostate cancer. Adv Cancer Res 2009;105:1-19.
9.
Miyake H, Gleave M, Kamidono S, Hara I: Overexpression of clusterin in transitional cell carcinoma of the bladder is related to disease progression and recurrence. Urology 2002;59:150-154.
10.
Miyake H, Hara S, Arakawa S, Kamidono S, Hara I: Over expression of clusterin is an independent prognostic factor for nonpapillary renal cell carcinoma. J Urol 2002;167:703-706.
11.
Busam KJ, Kucukgol D, Eastlake-Wade S, Frosina D, Delgado R, Jungbluth AA: Clusterin expression in primary and metastatic melanoma. J Cutan Pathol 2006;33:619-623.
12.
Bi J, Guo AL, Lai YR, Li B, Zhong JM, Wu HQ, Xie Z, He YL, Lv ZL, Lau SH, Wang Q, Huang XH, Zhang LJ, Wen JM, Guan XY: Overexpression of clusterin correlates with tumor progression, metastasis in gastric cancer: a study on tissue microarrays. Neoplasma 2010;57:191-197.
13.
Miyake H, Gleave ME, Arakawa S, Kamidono S, Hara I: Introducing the clusterin gene into human renal cell carcinoma cells enhances their metastatic potential. J Urol 2002;167:2203-2208.
14.
Cho NH, Shim HS, Rha SY, Kang SH, Hong SH, Choi YD, Hong SJ, Cho SH: Increased expression of matrix metalloproteinase 9 correlates with poor prognostic variables in renal cell carcinoma. Eur Urol 2003;44:560-566.
15.
Stetler-Stevenson WG, Hewitt R, Corcoran M: Matrix metalloproteinases and tumor invasion: from correlation and causality to the clinic. Semin Cancer Biol 1996;7:147-154.
16.
Inamoto T, Azuma H, Sakamoto T, Kiyama S, Ubai T, Kotake Y, Watanabe M, Katsuoka Y: Invasive ability of human renal cell carcinoma cell line Caki-2 is accelerated by gamma-aminobutyric acid, via sustained activation of ERK1/2 inducible matrix metalloproteinases. Cancer Invest 2007;25:574-583.
17.
Hong S, Park KK, Magae J, Ando K, Lee TS, Kwon TK, Kwak JY, Kim CH, Chang YC: Ascochlorin inhibits matrix metalloproteinase-9 expression by suppressing activator protein-1-mediated gene expression through the ERK1/2 signaling pathway. J Biol Chem 2005;280:25202-25209.
18.
Shim YJ, Kang BH, Jeon HS, Park IS, Lee KU, Lee IK, Park GH, Lee KM, Schedin P, Min BH: Clusterin induces matrix metalloproteinase-9 expression via ERK1/2 and PI3K/Akt/NF-κB pathways in monocytes/macrophages. J Leukoc Biol 2011;90:761-769.
19.
Shin YJ, Kang SW, Jeong SY, Shim YJ, Kim YH, Kim BM, Kee SH, Park JJ, Park IS, Min BH: Clusterin enhances proliferation of primary astrocytes through extracellular signal-regulated kinase activation. Neuroreport 2006;17:1871-1875
20.
Zellweger T, Miyake H, July LV, Akbari M, Kiyama S, Gleave ME: Chemosensitization of human renal cell cancer using antisense oligonucleotides targeting the antiapoptotic gene clusterin. Neoplasia 2001;3:360-367.
21.
Trougakos IP, So A, Jansen B, Gleave ME, Gonos ES: Silencing expression of the clusterin/apolipoprotein j gene in human cancer cells using small interfering RNA induces spontaneous apoptosis, reduced growth ability, and cell sensitization to genotoxic and oxidative stress. Cancer Res 2004;64:1834-1842.
22.
Scaltriti M, Bettuzzi S, Sharrard RM, Caporali A, Caccamo AE, Maitland NJ: Clusterin in both malignant and nonmalignant prostate epithelial cells induces cell cycle arrest and apoptosis. Br J Cancer 2004;91:1842-1850.
23.
Miyake H, Hara S, Arakawa S, Kamidono S, Hara I: Overexpression of clusterin is an independent prognostic factor for nonpapillary renal cell carcinoma. J Urol 2002;167:703-706.
24.
Shannan B, Seifert M, Leskov K, Willis J, Boothman D, Tilgen W, Reichrath J: Challenge and promise: roles for clusterin in pathogenesis, progression and therapy of cancer. Cell Death Differ 2006;13:12-19.
25.
Zellweger T, Miyake H, Cooper S, Chi K, Conklin BS, Monia B, Gleave ME: Antitumor activity of antisense clusterin oligonucleotides is improved in vitro and in vivo by incorporation of 2-o-(2-methoxy) ethyl chemistry. J Pharmacol Exp Ther 2001;298:934-940.
26.
Harborth J, Elbashir SM, Bechert K, Tuschl T, Weber K: Identification of essential genes in cultured mammalian cells using small interfering RNAs. J Cell Sci 2001;114:4557-4565.
27.
Zucker S, Lysik RM, Zarrabi MH, Moll U: M(r) 92,000 type IV collagenase is increased in plasma of patients with colon cancer and breast cancer. Cancer Res 1993;53:140-146.
28.
Lakka SS, Gondi CS, Yanamandra N, Dinh DH, Olivero WC, Gujrati M, Rao JS: Synergistic down-regulation of urokinase plasminogen activator receptor and matrix metalloproteinase-9 in SNB19 glioblastoma cells efficiently inhibits glioma cell invasion, angiogenesis, and tumor growth. Cancer Res 2003;63:2454-2461.
29.
Bernhard EJ, Gruber SB, Muschel RJ: Direct evidence linking expression of matrix metalloproteinase 9 (92-kDa gelatinase/collagenase) to the metastatic phenotype in transformed rat embryo cells. Proc Natl Acad Sci USA 1994;91:4293-4297.
30.
Wortzel I, Seger R: The ERK cascade: distinct functions within various subcellular organelles. Genes Cancer 2011;2:195-209.
© 2013 S. Karger AG, Basel
2013
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