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

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

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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-rasval12 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-rasval12 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-rasval12 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-rasval12 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.

Original languageEnglish
Article number172
JournalBMC Cancer
Volume15
Issue number1
DOIs
Publication statusPublished - 2015 Mar 25

Fingerprint

Up-Regulation
Urinary Bladder Neoplasms
Neoplasm Metastasis
Lung
Genes
Retinoblastoma-Binding Protein 7
Histone Deacetylase 1
Galactosides
ras Proteins
Neoplasms
Chromatin Immunoprecipitation
Matrix Metalloproteinase 9
Matrix Metalloproteinases
Reporter Genes
Oncogenes
Heterografts
Nude Mice
Small Interfering RNA
Cysteine
Carcinogenesis

All Science Journal Classification (ASJC) codes

  • Oncology
  • Genetics
  • Cancer Research

Cite this

Yeh, Hsuan Heng ; Tseng, Yu Fen ; Hsu, Yu Chiao ; Lan, Sheng Hui ; Wu, Shan Ying ; Raghavaraju, Giri ; Cheng, Da En ; Lee, Ying Ray ; Chang, Tsuey Yu ; Chow, Nan-Haw ; Hung, Wen Chun ; Liu, Hsiao-Sheng. / Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity. In: BMC Cancer. 2015 ; Vol. 15, No. 1.
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title = "Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity",
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-rasval12 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-rasval12 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-rasval12 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-rasval12 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.",
author = "Yeh, {Hsuan Heng} and Tseng, {Yu Fen} and Hsu, {Yu Chiao} and Lan, {Sheng Hui} and Wu, {Shan Ying} and Giri Raghavaraju and Cheng, {Da En} and Lee, {Ying Ray} and Chang, {Tsuey Yu} and Nan-Haw Chow and Hung, {Wen Chun} and Hsiao-Sheng Liu",
year = "2015",
month = "3",
day = "25",
doi = "10.1186/s12885-015-1155-7",
language = "English",
volume = "15",
journal = "BMC Cancer",
issn = "1471-2407",
publisher = "BioMed Central",
number = "1",

}

Yeh, HH, Tseng, YF, Hsu, YC, Lan, SH, Wu, SY, Raghavaraju, G, Cheng, DE, Lee, YR, Chang, TY, Chow, N-H, Hung, WC & Liu, H-S 2015, 'Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity', BMC Cancer, vol. 15, no. 1, 172. https://doi.org/10.1186/s12885-015-1155-7

Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity. / Yeh, Hsuan Heng; Tseng, Yu Fen; Hsu, Yu Chiao; Lan, Sheng Hui; Wu, Shan Ying; Raghavaraju, Giri; Cheng, Da En; Lee, Ying Ray; Chang, Tsuey Yu; Chow, Nan-Haw; Hung, Wen Chun; Liu, Hsiao-Sheng.

In: BMC Cancer, Vol. 15, No. 1, 172, 25.03.2015.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Ras induces experimental lung metastasis through up-regulation of RbAp46 to suppress RECK promoter activity

AU - Yeh, Hsuan Heng

AU - Tseng, Yu Fen

AU - Hsu, Yu Chiao

AU - Lan, Sheng Hui

AU - Wu, Shan Ying

AU - Raghavaraju, Giri

AU - Cheng, Da En

AU - Lee, Ying Ray

AU - Chang, Tsuey Yu

AU - Chow, Nan-Haw

AU - Hung, Wen Chun

AU - Liu, Hsiao-Sheng

PY - 2015/3/25

Y1 - 2015/3/25

N2 - 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-rasval12 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-rasval12 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-rasval12 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-rasval12 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.

AB - 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-rasval12 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-rasval12 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-rasval12 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-rasval12 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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