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Tumor Biology 5: Tumor and Metastasis Suppressors and Mechanisms

Regulation of expression of RKIP prostate cancer metastasis suppressor gene.

Meghan M. Brennan, Zheng Fu, Patrick Lester, Lauren P. Wallner and Evan T. Keller
Meghan M. Brennan
University of Michigan, Ann Arbor, MI and Mayo Clinic, Rochester, MN
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Zheng Fu
University of Michigan, Ann Arbor, MI and Mayo Clinic, Rochester, MN
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Patrick Lester
University of Michigan, Ann Arbor, MI and Mayo Clinic, Rochester, MN
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Lauren P. Wallner
University of Michigan, Ann Arbor, MI and Mayo Clinic, Rochester, MN
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Evan T. Keller
University of Michigan, Ann Arbor, MI and Mayo Clinic, Rochester, MN
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DOI:  Published May 2005
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Proc Amer Assoc Cancer Res, Volume 46, 2005

Abstract

321

Raf kinase inhibitor protein (RKIP) is a metastasis suppressor gene (MSG) whose expression is down-regulated through unknown mechanisms in prostate cancer (CaP) metastases. CaP cancer is often treated with androgen-deprivation, which has been shown to increase expression of IL-6, a cytokine known to activate the androgen receptor. IL-6 expression is also increased by the transcription factor NF-κB, for which there is increasing evidence of significance in CaP progression. Furthermore, NF-κB can be stimulated by TNF-α. Accordingly, the goal of this study was to determine if androgen, IL-6, or TNF-α regulate RKIP expression. In these studies, protein levels were measured using western blot and mRNA levels using real time RT-PCR with results being normalized to β-microglobulin. Androgen-dependent LNCaP cells, androgen-independent C4-2B cells or immortalized prostate epithelial RWPE cells were initially treated with 0-10nM dihydrotestosterone (DHT) or vehicle (V) for 24 and 48 hours. DHT increased RKIP mRNA expression by approximately 114% compared to V in RWPE cells and decreased its expression by approximately 93% and 57% in LNCaP and C4-2B cells, respectively. DHT increased RKIP protein expression in RWPE cells by 78% and decreased its expression in LNCaP cells by 45%; whereas, no change was seen in C4-2B cells. The cells were next treated with V or IL-6 (0-50ng) for 6 hours and RKIP expression was measured and normalized to β-microglobulin. Cells were again treated with 0-50ng IL-6 for 48 hours. IL-6 decreased RKIP mRNA expression compared to V by approximately 22% and 52% in RWPE and LNCaP cells, respectively and increased RKIP expression in C4-2B cells by 29%. IL-6 decreased RKIP protein expression in LNCaP cells by 40% but it was not changed in the other cell lines. The cells were then treated with 0-10ng/ml TNF-α for 24 and 48 hours. TNF-α increased RKIP mRNA expression in RWPE and LNCaP cells by 47% and 259%, respectively, and decreased it in C4-2B cells by 46%. At the protein level RKIP expression decreased by 26% in C4-2B and increased by 36% and 18% in RWPE and LNCaP, respectively. To determine the in vivo relevance of these findings, mice were orchiectomized and RKIP expression in various tissues was measured by RT-PCR. Orchiectomy suppressed RKIP expression in prostate, muscle and lung and had no effect on RKIP expression in brain, stomach and spleen. These data demonstrate that RKIP regulation is complex and differs between noncancerous and cancer cell lines as well as between androgen-dependent and androgen-independent cell lines.

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Cancer Research: 65 (9 Supplement)
May 2005
Volume 65, Issue 9 Supplement
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Regulation of expression of RKIP prostate cancer metastasis suppressor gene.
Meghan M. Brennan, Zheng Fu, Patrick Lester, Lauren P. Wallner and Evan T. Keller
Cancer Res May 1 2005 (65) (9 Supplement) 75;

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Regulation of expression of RKIP prostate cancer metastasis suppressor gene.
Meghan M. Brennan, Zheng Fu, Patrick Lester, Lauren P. Wallner and Evan T. Keller
Cancer Res May 1 2005 (65) (9 Supplement) 75;
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