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X. Sun
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P2.03a - Poster Session with Presenters Present (ID 464)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Advanced NSCLC
- Presentations: 1
- Moderators:
- Coordinates: 12/06/2016, 14:30 - 15:45, Hall B (Poster Area)
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P2.03a-059 - LCL161 Increases Paclitaxel-Induced Apoptosis by Degrading cIAP1 and cIAP2 in NSCLC (ID 5282)
14:30 - 14:30 | Author(s): X. Sun
- Abstract
Background:
LCL161, a novel Smac mimetic, is known to have anti-tumor activity and improve chemosensitivity in various cancers. However, the function and mechanisms of the combination of LCL161 and paclitaxel in non-small cell lung cancer (NSCLC) remain unknown.
Methods:
Cellular inhibitor of apoptotic protein 1 and 2 (cIAP1 and cIAP2) expression in NSCLC tissues and adjacent non-tumor tissues were assessed by immunohistochemistry. The correlations between cIAP1,2 expression and clinicopathological characteristics, prognosis were analyzed. Cell viability and apoptosis were measured by MTT assays and Flow cytometry. Western blot and co-immunoprecipitation assay were performed to measure the protein expression and interaction in NF-κB pathway. siRNA-mediated gene silencing and caspases activity assays were applied to demonstrate the role and mechanisms of cIAP1,2 and RIP1 in lung cancer cell apoptosis. Mouse xenograft NSCLC models were used in vivo to determine the therapeutic efficacy of LCL161 alone or in combination with paclitaxel.
Results:
The expression of cIAP1 and cIAP2 in Non-small cell lung cancer (NSCLC) tumors was significantly higher than that in adjacent normal tissues. cIAP1 was highly expressed in patients with late TNM stage NSCLC and a poor prognosis. Positivity for cIAP1 and cIAP2 was an independent prognostic factor that indicated a poorer prognosis in NSCLC patients. LCL161, an IAP inhibitor, cooperated with paclitaxel to reduce cell viability and induce apoptosis in NSCLC cells. Molecular studies revealed that paclitaxel increased TNFα expression, thereby leading to the recruitment of various factors and the formation of the TRADD-TRAF2-RIP1-cIAP complex. LCL161 degraded cIAP1 and cIAP2, releasing RIP1 from the complex. Subsequently, RIP1 was stabilized and bound to caspase-8 and FADD, thereby forming the caspase-8/RIP1/FADD complex, which activated caspase-8, caspase-3 and ultimately lead to apoptosis. In nude mouse xenograft experiments, the combination of LCL161 and paclitaxel degraded cIAP1,2, activated caspase-3 and inhibited tumor growth with few toxic effects.
Conclusion:
Thus, LCL161 could be a useful agent for the treatment of NSCLC in combination with paclitaxel.
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P3.01 - Poster Session with Presenters Present (ID 469)
- Event: WCLC 2016
- Type: Poster Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 12/07/2016, 14:30 - 15:45, Hall B (Poster Area)
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P3.01-063 - XIAP Inhibits Mature Smac Induced Apoptosis by Degrading It through Ubiquitination in NSCLC (ID 5287)
14:30 - 14:30 | Author(s): X. Sun
- Abstract
Background:
X-linked inhibitor of apoptosis protein (XIAP) and second mitochondrial-derived activator of caspase (Smac) are two important prognostic biomarkers for cancer. They are negatively correlated in many types of cancers. However, their relationship is still unknown in lung cancer.
Methods:
RT-PCR and Western blot were performed to explore the correlation between Smac and XIAP at the level of mRNA and protein in NSCLC patients. Full-length XIAP, Smac and mature Smac were generated by PCR and cloned into pcDNA3.0-Flag/Myc. The location of mature Smac and full-length Smac was detected by immunofluorescence. MTT assay and Flow cytometry were detected the cell viability and apoptosis of transfected cells. Caspase-3 activity was masured by Caspase-3 activity assay. Co-immunoprecipitation assay was done to reveal the direct relation between XIAP and Smac. Nude mouse xenograft experiment further proved the relation and the function of Smac and XIAP in vivo.
Results:
In this study, we found that there was a negative correlation between Smac and XIAP at the level of protein but not mRNA in NSCLC patients. However, XIAP overexpression had no effect on degrading endogenous Smac in lung cancer cell lines. Therefore, we constructed plasmids with full length of Smac (fSmac) and mature Smac (mSmac) which located in cytoplasm instead of original mitochondrial location, and confirmed by immunofluorescence. Subsequently, we found that mSmac rather than fSmac was degraded by XIAP and inhibited cell viability. CHX chase assay and ubiquitin assay were performed to illustrate XIAP degraded mSmac through ubiquitin pathway. Overexpression of XIAP partially reverted apoptotic induction and cell viability inhibition by mSmac, which was due to inhibiting caspases-3 activation. In nude mouse xenograft experiments, mSmac inhibited Ki67 expression and slowed down lung cancer growth, while XIAP partially reversed the effect of mSmac by degrading it.
Conclusion:
In conclusion, XIAP inhibits mature Smac-induced apoptosis by degrading it through ubiquitination in NSCLC.
