Park, Jae Myung

492 Background: Beclin 1 interacts with its cofactor UVRAG to promote autophagy, a cellular degradation process induced by cellular stress. While cells with defective autophagy are prone to genomic instability, it is unknown whether Beclin 1 and/or UVRAG can regulate DNA damage/repair. Methods: Colorectal cancer cell lines (HT-29, DLD1) with Beclin 1 or UVRAG siRNA were irradiated ± 5-FU. DNA damage (pATM, pH2Ax, 53BP1, RAD51), cell death (annexin V, caspase-3, clonogenic survival), and centrosome number (γ-tubulin staining), were determined. Beclin 1 protein expression was analyzed in pretreatment endoscopic biopsies from 96 rectal carcinoma patients who received radiation + 5-FU followed by surgery. Results: Beclin 1 or UVRAG knockdown was shown to enhance DNA double-strand breaks (DSBs) and cell death induced by radiation ± 5-FU. Suppression of either gene increased the percentage of irradiated cells with nuclear foci expressing 53BP1 (nonhomologous end joining), but not RAD51 (homologous recombination). Beclin 1 was shown to regulate UVRAG, and a UVRAG deletion mutant defective in Beclin 1 binding decreased protection against radiation-induced DSBs. Knockdown of Beclin 1 or UVRAG increased centrosome number in irradiated cells. While autophagy inhibition by ATG5 or LC3 siRNA enhanced radiation-induced DSBs, they did not alter centrosome number suggesting independence from autophagy. In rectal carcinomas, high vs low level Beclin 1 expression was detected in 56 (58.3%) and 40 (41.7%) tumors, respectively. Pre-treatment Beclin 1 was not associated with clinicopathological variables. However, high vs low Beclin 1 predicted a significantly reduced pathological response (macroscopic vs. microscopic or no residual tumor) to chemoradiation (high: 14.2% vs. low: 40%; p = 0.017). High vs low Beclin 1 was independently associated with poorer overall survival (HR, 4.30; 95% CI, 1.42-16.47; p = 0.009). Conclusions: Beclin 1 and UVRAG confer protection against radiation-induced DNA damage and maintain centrosome stability. The ability of Beclin 1 overexpression to confer resistance to chemoradiation, suggests its potential utility as a predictive biomarker in rectal cancer.

Abstract Background: Autophagy and apoptosis regulate cancer cell viability in response to cytotoxic stress; however, the functional relationship between these processes is unclear. During autophagy, the multifunctional p62/sequestosome 1 protein shuttles ubiquitinated proteins to the lysosome. Previously, we found that ectopic p62 can promote chemotherapy-induced apoptosis. Since Bcl-2 can regulate both autophagy and apoptosis, we determined whether ABT-263-induced apoptosis is regulated by p62. Methods: Human colorectal cancer cell lines (HCT116, DLD1) were incubated with ABT-263 (0-2 μM) and caspase cleavage (immunoblotting), caspase-8 activation (luminescence), apoptosis (annexin V labeling), and autophagy (localization of GFP-LC3 by confocal microscopy) were analyzed. Caspase-8 was suppressed by siRNA or using caspase-8-deficient Jurkat cells. P62 mutants were generated by site-directed mutagenesis. Caspase-8 self aggregation and colocalization with p62 were studied in cells with lentiviral mCherry-p62, transfected with a non-cleavable caspase-8 fused with fluorescent Venus proteins (BiFC). Results: We demonstrate that ABT-263 induces autophagy and a caspase-8-mediated apoptosis, shown using caspase-8 siRNA or caspase-8-deficient cells. Regulation of this process by p62 was demonstrated by the ability of ectopic p62 expression to enhance apoptosis and caspase-8 activation that were disrupted by mutations at p62 functional domains. In contrast, p62 shRNA was shown to attenuate ABT-263-induced apoptosis. ABT-263 treatment enhanced binding between caspase-8 and p62, and triggered caspase-8 self aggregation that colocalized with p62, shown by Biomolecular Fluorescence Complementation (BiFC) assay using confocal microscopy. Consistent with ectopic p62, accumulation of p62 by the lysosomal autophagy inhibitors, chloroquine or bafilomycin A1, enhanced ABT-263-mediated caspase cleavage. Conclusion: p62 can regulate caspase-8 self aggregation/activation by ABT-263 to enable crosstalk between autophagy and apoptosis. Citation Format: Shengbing Huang, Jae Myung Park, Chunrong Yu, Frank A. Sinicrope. p62/sequestosome 1 regulates caspase-8 activation by the Bcl-2 antagonist ABT-263 to enable crosstalk between autophagy and apoptosis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1733. doi:10.1158/1538-7445.AM2013-1733

Abstract Background. Autophagy maintains energy metabolism and enables tumor cell survival during cellular stress. Beclin 1 is involved in autophagosome formation during which membrane-bound LC3 binds to p62/sequestosome 1. We reported that ectopic p62 can promote 5-fluorouracil-induced cell death (JBC 286:40002-12, 2011). It is unknown whether these autophagy markers can predict clinical outcome. Materials & Methods. Beclin 1, LC3B, and p62 proteins were analyzed by immunohistochemistry in tissue microarrays containing stage II and III colon carcinomas (N=151) from patients receiving 5-FU or surgery alone. The mean weighted score (intensity x extent) was dichotomized for survival analysis using Cox models. Results. Increased levels of cytoplasmic LC3, Beclin 1, and p62 expression were detected in 21%, 14%, and 68% of cancers, respectively, and were not associated with tumor site, stage or grade. By multivariable analysis, increased LC3 was significantly associated with worse disease-free survival (DFS) (p=0.022), but not overall survival (OS) [p=0.069](Table). Beclin 1 was not prognostic, but increased p62 was significantly associated with improved DFS (p=0.038), but not OS (p= 0.176)[Table]. When restricted to 5-FU-treated patients, increased LC3 was associated with worse DFS [HR= 2.38 (1.19, 4.75, p=0.020), elevated Beclin 1 predicted worse OS [HR=2.83 (1.27, 6.34), p = 0.019], and p62 was not prognostic. Conclusion. LC3 and Beclin 1 overexpression were independently associated with adverse outcome. Increased p62 was associated with better outcome consistent with its ability to promote cell death. These data support strategies to inhibit autophagy for therapeutic advantage. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 4112. doi:1538-7445.AM2012-4112