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E2F-1 lacking the transcriptional activity domain induces autophagy.

ABSTRACT: The transcription factor E2F-1 plays a crucial role in the control of cell proliferation. E2F-1 has tumor suppressive properties by inducing apoptosis and autophagy. In this study, E2F-1 and its truncated form (E2Ftr), lacking the transactivation domain (TAD), were compared for their ability to induce autophagy. In Gaussia luciferase-based assays, both E2F-1 and E2Ftr induced the proteolytic cleavage of the autophagic marker LC3. In addition, LC3 and autophagy protein 5 (Atg5) were upregulated by E2F-1 and E2Ftr. Likewise, both E2F proteins induced a punctate pattern of GFP-tagged LC3, indicating autophagosome formation. The presence of double-membrane autophagic vesicles induced by E2F-1 and E2Ftr was confirmed by transmission electron microscopy (TEM). The application of z-VAD-fmk, a caspase inhibitor, partially blocked both E2F-1 and E2Ftr-mediated cytotoxicity. Moreover, Atg5 (-/-) cells were more resistant to the E2F-1 or E2Ftr-induced cell killing effect than Atg5 wt cells. The TAD of E2F-1 is not essential for induction of autophagy; apoptosis and autophagy cooperate for an efficient cancer cell killing effect induced by E2F-1 or E2Ftr. E2Ftr-induced autophagy is a promising approach to destroy tumors that are resistant to conventional treatments.

SUBMITTER: Garcia-Garcia A

PROVIDER: S-EPMC3462036 | biostudies-literature | 2012 Sep

REPOSITORIES: biostudies-literature

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E2F-1 lacking the transcriptional activity domain induces autophagy.

Garcia-Garcia Aracely A Rodriguez-Rocha Humberto H Tseng Michael T MT Montes de Oca-Luna Roberto R Zhou H Sam HS McMasters Kelly M KM Gomez-Gutierrez Jorge G JG

Cancer biology & therapy 20120724 11

The transcription factor E2F-1 plays a crucial role in the control of cell proliferation. E2F-1 has tumor suppressive properties by inducing apoptosis and autophagy. In this study, E2F-1 and its truncated form (E2Ftr), lacking the transactivation domain (TAD), were compared for their ability to induce autophagy. In Gaussia luciferase-based assays, both E2F-1 and E2Ftr induced the proteolytic cleavage of the autophagic marker LC3. In addition, LC3 and autophagy protein 5 (Atg5) were upregulated b ...[more]

PMID: 22825328

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Project description:BackgroundNRP/B, a family member of the BTB/Kelch repeat proteins, is implicated in neuronal and cancer development, as well as the regulation of oxidative stress responses in breast and brain cancer. Our previous studies indicate that the NRP/B-BTB/POZ domain is involved in the dimerization of NRP/B and in a complex formation with the tumor suppressor, retinoblastoma protein. Although much evidence supports the potential role of NRP/B as a tumor suppressor, the molecular mechanisms of NRP/B action on E2F transcription factors have not been elucidated.MethodsThree-dimensional modeling of NRP/B was used to generate point mutations in the BTB/Kelch domains. Tet-on inducible NRP/B expression was established. The NRP/B deficient breast cancer cell line, MDA-MB-231, was generated using lentiviral shNRP/B to evaluate the effect of NRP/B on cell proliferation, invasion and migration. Immunoprecipitation was performed to verify the interaction of NRP/B with E2F and histone deacetylase (HDAC-1), and the expression level of NRP/B protein was analyzed by Western blot analysis. Changes in cell cycle were determined by flow cytometry. Transcriptional activities of E2F transcription factors were measured by chloramphenicol acetyltransferase (CAT) activity.ResultsEctopic overexpression of NRP/B demonstrated that the NRP/B-BTB/POZ domain plays a critical role in E2F-mediated transcriptional activity. Point mutations within the BTB/POZ domain restored E2-promoter activity inhibited by NRP/B. Loss of NRP/B enhanced the proliferation and migration of breast cancer cells. Endogenous NRP/B interacted with E2F and HDAC1. Treatement with an HDAC inhibitor, trichostatin A (TSA), abolished the NRP/B-mediated suppression of E2-promoter activity. Gain or loss of NRP/B in HeLa cells confirmed the transcriptional repressive capability of NRP/B on the E2F target genes, Cyclin E and HsORC (Homo sapiens Origin Recognition Complex).ConclusionsThe present study shows that NRP/B acts as a transcriptional repressor by interacting with the co-repressors, HDAC1, providing new insight into the molecular mechanisms of NRP/B on tumor suppression.

Project description:Despite significant progress in diagnostics and therapeutics, over 50 thousand patients die from colorectal cancer annually. Hence, there is urgent need for new lines of treatment. Triptolide, a natural compound isolated from the Chinese herb Tripterygium wilfordii, is effective against multiple cancers. We have synthesized a water soluble analog of triptolide, named Minnelide, which is currently in phase I trial against pancreatic cancer. The aims of the current study were to evaluate whether triptolide/Minnelide is effective against colorectal cancer and to elucidate the mechanism by which triptolide induces cell death in colorectal cancer. Efficacy of Minnelide was evaluated in subcutaneous xenograft and liver metastasis model of colorectal cancer. For mechanistic studies, colon cancer cell lines HCT116 and HT29 were treated with triptolide and the effect on viability, caspase activation, annexin positivity, lactate dehydrogenase release, and cell cycle progression was evaluated. Effect of triptolide on E2F transcriptional activity, mRNA levels of E2F-dependent genes, E2F1- retinoblastoma protein (Rb) binding, and proteins levels of regulator of G1-S transition was also measured. DNA binding of E2F1 was evaluated by chromatin immunoprecipitation assay. Triptolide decreased colon cancer cell viability in a dose- and time-dependent fashion. Minnelide markedly inhibited the growth of colon cancer in the xenograft and liver metastasis model of colon cancer and more than doubles the median survival of animals with liver metastases from colon cancer. Mechanistically, we demonstrate that at low concentrations triptolide induces apoptotic cell death but at higher concentrations it induces cell cycle arrest. Our data suggest that triptolide is able to induce G1 cell cycle arrest by inhibiting transcriptional activation of E2F1. Our data also show that triptolide downregulates E2F activity by potentially modulating events downstream of DNA binding. Therefore, we conclude that Triptolide and Minnelide are effective against colon cancer in multiple pre-clinical models.

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E2F-1 lacking the transcriptional activity domain induces autophagy.

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E2F-1 lacking the transcriptional activity domain induces autophagy.

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