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Open Access 01-12-2015 | Research article

Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity

Authors: Hsuan-Heng Yeh, Yu-Fen Tseng, Yu-Chiao Hsu, Sheng-Hui Lan, Shan-Ying Wu, Giri Raghavaraju, Da-En Cheng, Ying-Ray Lee, Tsuey-Yu Chang, Nan-Haw Chow, Wen-Chun Hung, Hsiao-Sheng Liu

Published in: BMC Cancer | Issue 1/2015

Abstract

Background

Mutant Ras plays multiple functions in tumorigenesis including tumor formation and metastasis. Reversion-inducing cysteine-rich protein with Kazal motifs (RECK), a metastasis inhibitor gene, suppresses matrix metalloproteinase (MMP) activity in the metastatic cascade. Clarifying the relationship between Ras and RECK and understanding the underlying molecular mechanism may lead to the development of better treatment for Ras-related tumors.

Methods

Suppression subtractive hybridization PCR (SSH PCR) was conducted to identify Ha-ras^val12 up-regulated genes in bladder cancer cells. Stable cell lines of human breast cancer (MCF-7-ras) and mouse NIH3T3 fibroblasts (7–4) harboring the inducible Ha-ras^val12 oncogene, which could be induced by isopropylthio-β-D-galactoside (IPTG), were used to clarify the relationship between Ras and the up-regulated genes. Chromatin immunoprecipitation (ChIP) assay, DNA affinity precipitation assay (DAPA) and RECK reporter gene assay were utilized to confirm the complex formation and binding with promoters.

Results

Retinoblastoma binding protein-7 (RbAp46) was identified and confirmed as a Ha-ras^val12 up-regulated gene. RbAp46 could bind with histone deacetylase (HDAC1) and Sp1, followed by binding to RECK promoter at the Sp1 site resulting in repression of RECK expression. High expression of Ras protein accompanied with high RbAp46 and low RECK expression were detected in 75% (3/4) of the clinical bladder cancer tumor tissues compared to the adjacent normal parts. Ras induced RbAp46 expression increases invasion of the bladder cancer T24 cells and MMP-9 activity was increased, which was confirmed by specific lentiviral shRNAs inhibitors against Ras and RbAp46. Similarly, knockdown of RbAp46 expression in the stable NIH3T3 cells “7-4” by shRNA decreased Ras-related lung metastasis using a xenograft nude mice model.

Conclusions

We confirmed that RbAp46 is a Ha-ras^val12 up-regulated gene and binds with HDAC1 and Sp1. Furthermore, RbAp46 binds to the RECK promoter at the Sp1 site via recruitment by Sp1. RECK is subsequently activated, leading to increased MMP9 activity, which may lead to increased metastasis in vivo. Our findings of Ras upregulation of RbAp46 may lead to revealing a novel mechanism of Ras-related tumor cell metastasis.

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Title: Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity
Authors: Hsuan-Heng Yeh
Yu-Fen Tseng
Yu-Chiao Hsu
Sheng-Hui Lan
Shan-Ying Wu
Giri Raghavaraju
Da-En Cheng
Ying-Ray Lee
Tsuey-Yu Chang
Nan-Haw Chow
Wen-Chun Hung
Hsiao-Sheng Liu
Publication date: 01-12-2015
Publisher: BioMed Central
Published in: BMC Cancer / Issue 1/2015
Electronic ISSN: 1471-2407
DOI: https://doi.org/10.1186/s12885-015-1155-7

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