Abstract
Targeting the protein neddylation pathway has become an attractive anticancer strategy; however, the role of death receptor-mediated extrinsic apoptosis during treatment remained to be determined. The activation of extrinsic apoptosis and its role in MLN4924 treatment of human esophageal squamous cell carcinoma (ESCC) were evaluated both in vitro and in vivo The expression of the components of extrinsic apoptotic pathway was determined by immunoblotting analysis and downregulated by siRNA silencing for mechanistic studies. Pharmaceutical or genetic inactivation of neddylation pathway induced death receptor 5 (DR5)-mediated apoptosis and led to the suppression of ESCC in murine models. Mechanistically, neddylation inhibition stabilized activating transcription factor 4 (ATF4), a Cullin-Ring E3 ubiquitin ligases (CRL) substrate. Transcription factor CHOP was subsequently transactivated by ATF4 and further induced the expression of DR5 to activate caspase-8 and induce extrinsic apoptosis. Moreover, the entire neddylation pathway was hyperactivated in ESCC and was negatively associated with patient overall survival. Our findings highlight a critical role of ATF4-CHOP-DR5 axis-mediated extrinsic apoptosis in neddylation-targeted cancer therapy and support the clinical investigation of neddylation inhibitors (e.g., MLN4924) for the treatment of ESCC, a currently treatment-resistant disease with neddylation hyperactivation. Clin Cancer Res; 22(16); 4145-57. ©2016 AACR.
Highlights
Protein posttranslational modifications play a crucial role in the regulation of tumorigenesis and tumor progression
For the first time, we reported that targeting hyperactivated neddylation pathway pharmaceutically or genetically triggered activating transcription factor 4 (ATF4)–CHOP–death receptor 5 (DR5) axis-mediated extrinsic apoptosis to significantly inhibit the growth of human esophageal squamous cell carcinoma (ESCC) tumors
To investigate the activation status of neddylation pathway in esophageal cancer, we firstly examined the expression levels of global protein neddylation by IHC staining of the tissue arrays derived from human ESCC, which contain 95 pairs of primary tumor versus adjacent normal tissues
Summary
To investigate the activation status of neddylation pathway in esophageal cancer, we firstly examined the expression levels of global protein neddylation by IHC staining of the tissue arrays derived from human ESCC, which contain 95 pairs of primary tumor versus adjacent normal tissues. Noxa downregulation via siRNA silencing remarkably suppressed MLN4924induced apoptosis, as evidenced by (i) the decrease of Annexin V– positive (Fig. 5C, left plots) and CASP3-activated (Fig. 5C, right plots) cells; and (ii) the reduction of cleaved CASP3 and PARP (Fig. 5D) These findings highlighted a pivotal role of Noxa transactivation in MLN4924-induced intrinsic apoptosis. Like MLN4924, knockdown of UBA3 and NAE1 upregulated the expression of ATF4, CHOP, and DR5 as well as Noxa (Supplementary Fig. S6D) in both EC1 and Kyse450 cell www.aacrjournals.org lines These results demonstrate that genetic inactivation of neddylation pathway via NAE1/UBA3 siRNA silencing fully recapitulates MLN4924-induced apoptosis activation in ESCC cells. These observations indicated that MLN4924 inhibited esophageal tumor growth both in vitro and in vivo via the ATF4/CHOP/ DR5/CASP8 extrinsic apoptosis and ATF4/Noxa intrinsic apoptosis pathways (Fig. 6E)
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