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Elevated TIMP-1 expression is associated with a prometastatic phenotype, disease relapse, and poor survival in neuroblastoma.

ABSTRACT: Approximately two-thirds of patients with neuroblastoma are found to have metastatic disease at time of diagnosis with frequent skeletal, lymph node, central nervous system, and liver involvement. Using a serial in vivo splenic injection model, we have isolated an aggressive subclone (BE(2)-C/LM2) from MYCN-amplified neuroblastomas that demonstrate an enhanced propensity to develop metastatic liver lesions. BE(2)-C/LM2 subclone cells demonstrate increased adherent, soft agar colony and tumorsphere growth in vitro. Components of the tumor microenvironment regulate cancer progression, via networks of cytokines and growth factors. Cytokine array analysis identified increased TIMP-1 in the plasma of mice injected with BE(2)-C/LM2 subclone cells, leading us to hypothesize that TIMP-1 may play a role in our observed prometastatic phenotype. Immunoblotting and ELISA demonstrated enhanced endogenous TIMP-1 expression in our isolated neuroblastoma subclone. Silencing endogenous TIMP-1 successfully blocked in vitro proliferation, soft agar colony formation and tumorsphere formation by BE(2)-C/LM2 cells. Stable RNA interference of endogenous TIMP-1 failed to reverse the prometastatic phenotype of our BE(2)-C/LM2 subclone in our liver metastasis model, suggesting that endogenous TIMP-1 levels may not be an essential component of this in vivo behavior. Notably, tissue microarray analysis and Kaplan-Meier by gene expression demonstrates that elevated TIMP-1 expression is correlated with increased disease relapse and mortality in patients with neuroblastoma. Taken together, our study identifies TIMP-1 as a novel soluble factor that is associated with a prometastatic phenotype in our in vivo model and adverse outcomes in patients with neuroblastoma.

SUBMITTER: Paul P

PROVIDER: S-EPMC5669914 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:BackgroundTissue inhibitor of metalloproteinases-1 (TIMP-1) is a multifunctional protein that can directly regulate apoptosis and metastasis. In this study, we investigated the functional and molecular mechanisms by which TIMP-1 influences triple-negative breast cancer (TNBC).MethodsThe expression level of TIMP-1 in breast cancer tissues was analyzed using the ONCOMINE microarray database. The overall survival of patients with distinct molecular subtypes of breast cancer stratified by TIMP-1 expression levels was evaluated using Kaplan-Meier analysis. Bisulfate sequencing PCR (BSP) was used to analyze the methylation status of the TIMP-1 promoter. Real-time-PCR (RT-PCR), Western blot and ELISA assays were used to evaluate gene and protein expression in cell lines and human tissue specimens. In addition, TIMP-1 function was analyzed using a series of in vitro and in vivo assays with cells in which TIMP-1 was inhibited using RNAi or neutralizing antibodies.ResultsWe found that serum TIMP-1 levels were strongly enhanced in patients with TNBC and that elevated TIMP-1 levels were associated with a poor prognosis in TNBC. However, TIMP-1 levels were not significantly associated with overall survival in other subtypes of breast cancer or in the overall population of breast cancer patients. We also report the first evidence that the TIMP-1 promoter is hypomethylated in TNBC cell lines compared with non-TNBC cell lines, suggesting that aberrant TIMP-1 expression in TNBC results from reduced DNA methylation. RNAi-mediated silencing of TIMP-1 in TNBC cells induced cell cycle arrest at the G1 phase and reduced cyclin D1 expression. In addition, mechanistic analyses revealed that the p-Akt and p-NF-κB signaling pathways, but not the GSK-3β and MAPK1/2 pathways, are associated with TIMP-1 overexpression in TNBC cells. Moreover, neutralizing antibodies against TIMP-1 significantly decreased the rate of tumor growth in vivo.ConclusionsOur findings suggest that TIMP-1 is a biomarker indicative of a poor prognosis in TNBC patients and that targeting TIMP-1 may provide an attractive therapeutic intervention specifically for triple-negative breast cancer patients.

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Elevated TIMP-1 expression is associated with a prometastatic phenotype, disease relapse, and poor survival in neuroblastoma.

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