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Co-immunoprecipitation Assay Using Endogenous Nuclear Proteins from Cells Cultured Under Hypoxic Conditions
Published on: August 2, 2018
The alternative reading frame tumor suppressor antagonizes hypoxia-induced cancer cell migration via interaction with
Seema Paliwal1, Ramesh C Kovi, Bharath Nath
1Department of Cancer Biology, University of Massachusetts Medical School, 364 Plantation Street, Worcester, MA 01605, USA.
Abstract:
The alternative reading frame (ARF) tumor suppressor exerts both p53-dependent and p53-independent activities critical to the prevention of cancer in mice and humans. Recent evidence from mouse models suggests that when p53 is absent, further loss of ARF can widen the tumor spectrum, and potentiate invasion and metastasis. A major target of the p53-independent activity of ARF is the COOH-terminal binding protein (CtBP) family of metabolically regulated transcriptional corepressors, which are degraded upon acute exposure to the ARF protein. CtBPs are activated under conditions of metabolic stress, such as hypoxia, to repress epithelial and proapoptotic genes, and can mediate hypoxia-induced migration of cancer cells. The possibility that ARF could suppress tumor cell migration as part of its p53-independent activities was thus explored. Small-interfering RNA (siRNA)-mediated knockdown of ARF in human lung carcinoma cells led to increased cell migration, especially during hypoxia, and this effect was blocked by concomitant treatment with CtBP2 siRNA. Introduction of ARF into p53 and ARF-null human colon cancer cells inhibited hypoxia-induced migration. Furthermore, overexpression of CtBP2 in ARF-expressing cells enhanced cell migration, and an ARF mutant defective in CtBP-family binding was impaired in its ability to inhibit cell migration induced by CtBP2. ARF depletion or CtBP2 overexpression was associated with decreased PTEN expression and activation of the phosphatidylinositol 3-kinase pathway, and a phosphatidylinositol 3-kinase inhibitor blocked CtBP2-mediated cell migration. Thus, ARF can suppress cell migration by antagonizing CtBP2 and the phosphatidylinositol 3-kinase pathway, and these data may explain the increased aggressiveness of ARF-null tumors in mouse models.
Insights
The alternative reading frame (ARF) tumor suppressor inhibits cancer cell migration by targeting COOH-terminal binding protein 2 (CtBP2) and the phosphatidylinositol 3-kinase pathway, independent of p53. This explains why ARF-deficient tumors are more aggressive.
Area of Science:
- Oncology
- Molecular Biology
- Cancer Research
Background:
- The alternative reading frame (ARF) tumor suppressor has critical p53-dependent and p53-independent roles in cancer prevention.
- Loss of ARF, particularly in p53-deficient contexts, can promote tumor invasion and metastasis.
- A key target of ARF's p53-independent activity is the COOH-terminal binding protein (CtBP) family, which regulates gene expression under metabolic stress.
Purpose of the Study:
- To investigate the role of ARF in suppressing tumor cell migration through its p53-independent activities.
- To explore the mechanism by which ARF antagonizes CtBP-mediated cell migration.
Main Methods:
- Small-interfering RNA (siRNA) was used to knockdown ARF and CtBP2 in human lung carcinoma cells.
- ARF was introduced into p53 and ARF-null human colon cancer cells.
- CtBP2 was overexpressed in ARF-expressing cells, and ARF mutants defective in CtBP binding were analyzed.
- PTEN expression, phosphatidylinositol 3-kinase (PI3K) pathway activation, and the effect of PI3K inhibitors were assessed.
Main Results:
- ARF knockdown increased cell migration, especially under hypoxia, an effect blocked by CtBP2 siRNA.
- ARF introduction inhibited hypoxia-induced migration in cancer cells.
- CtBP2 overexpression enhanced cell migration, while an ARF mutant impaired in CtBP binding showed reduced inhibition of migration.
- ARF depletion or CtBP2 overexpression led to decreased PTEN expression and PI3K pathway activation; PI3K inhibition blocked CtBP2-mediated migration.
Conclusions:
- ARF suppresses tumor cell migration by antagonizing CtBP2 and the phosphatidylinositol 3-kinase pathway in a p53-independent manner.
- These findings provide a molecular explanation for the increased aggressiveness of ARF-null tumors observed in preclinical models.
- Targeting the ARF-CtBP2-PI3K axis could offer new therapeutic strategies for aggressive cancers.
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