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An ARF/CtBP2 complex regulates BH3-only gene expression and p53-independent apoptosis.

ABSTRACT: The alternative reading frame (ARF) tumor suppressor exerts both p53-dependent and p53-independent functions. The corepressor C-terminal binding protein (CtBP) interacts with ARF, resulting in proteasome-mediated degradation of CtBP. ARF can induce apoptosis in p53-null colon cancer cells, in a manner dependent on ARF interaction with CtBP. Bik was uniquely identified in an apoptotic gene array as coordinately upregulated in colon cancer cells after either CtBP2 knockdown or ARF overexpression. Validating the array findings, ARF induced Bik mRNA and protein expression, and this activity required an intact CtBP binding domain. Apoptosis induced by CtBP deficiency was substantially impaired when Bik expression was simultaneously silenced. An analysis of the Bik promoter revealed binding sites for the CtBP-interacting basic Kruppel-like factor (BKLF). A Bik promoter luciferase reporter was repressed by BKLF and CtBP2, and ARF reversed CtBP-associated repression. Chromatin immunoprecipitation analyses showed that CtBP was recruited to the Bik promoter largely by BKLF. Expression profiling of BH3-only gene expression in ARF-expressing or CtBP-deficient cells revealed that Bik was uniquely regulated by ARF/CtBP in colon cancer cells, whereas additional BH3-only proteins (Bim, Bmf) showed CtBP-dependent repression in osteosarcoma cells. ARF antagonism of CtBP repression of Bik and other BH3-only genes may have a critical role in ARF-induced p53-independent apoptosis and tumor suppression.

SUBMITTER: Kovi RC

PROVIDER: S-EPMC2924672 | biostudies-literature | 2010 Mar

REPOSITORIES: biostudies-literature

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An ARF/CtBP2 complex regulates BH3-only gene expression and p53-independent apoptosis.

Kovi R C RC Paliwal S S Pande S S Grossman S R SR

Cell death and differentiation 20091002 3

The alternative reading frame (ARF) tumor suppressor exerts both p53-dependent and p53-independent functions. The corepressor C-terminal binding protein (CtBP) interacts with ARF, resulting in proteasome-mediated degradation of CtBP. ARF can induce apoptosis in p53-null colon cancer cells, in a manner dependent on ARF interaction with CtBP. Bik was uniquely identified in an apoptotic gene array as coordinately upregulated in colon cancer cells after either CtBP2 knockdown or ARF overexpression. ...[more]

PMID: 19798104

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Project description:Despite the recent advancement in treating melanoma, options are still limited for patients without BRAF mutations or in relapse from current treatments. BH3 mimetics against members of the BCL-2 family have gained excitement with the recent success in hematological malignancies. However, single drug BH3 mimetic therapy in melanoma has limited effectiveness due to escape by the anti-apoptotic protein MCL-1 and/or survival of melanoma-initiating cells (MICs). We tested the efficacy of the BH3 mimetic combination of A-1210477 (an MCL-1 inhibitor) and ABT-263 (a BCL-2/BCL-XL/BCL-W inhibitor) in killing melanoma, especially MICs. We also sought to better define Dynamin-Related Protein 1 (DRP-1)'s role in melanoma; DRP-1 is known to interact with members of the BCL-2 family and is a possible therapeutic target for melanoma treatment. We used multiple assays (cell viability, apoptosis, bright field, immunoblot, and sphere formation), as well as the CRISPR/Cas9 genome-editing techniques. For clinical relevance, we employed patient samples of different mutation status, including some relapsed from current treatments such as anti-PD-1 immunotherapy. We found the BH3 mimetic combination kill both the MICs and non-MICs (bulk of melanoma) in all cell lines and patient samples irrespective of the mutation status or relapsed state (p?<?0.05). Unexpectedly, the major pro-apoptotic proteins, NOXA and BIM, are not necessary for the combination-induced cell death. Furthermore, the combination impedes the activation of DRP-1, and inhibition of DRP-1 further enhances apoptosis (p?<?0.05). DRP-1 effects in melanoma differ from those seen in other cancer cells. These results provide new insights into BCL-2 family's regulation of the apoptotic pathway in melanoma, and suggest that inhibiting the major anti-apoptotic proteins is sufficient to induce cell death even without involvement from major pro-apoptotic proteins. Importantly, our study also indicates that DRP-1 inhibition is a promising adjuvant for BH3 mimetics in melanoma treatment.

Dataset Information

An ARF/CtBP2 complex regulates BH3-only gene expression and p53-independent apoptosis.

Publications

An ARF/CtBP2 complex regulates BH3-only gene expression and p53-independent apoptosis.

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