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Y. Kim
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P1.02 - Biology/Pathology (ID 614)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 10/16/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P1.02-071 - SFN Stabilizes Oncoproteins through Binding with SKP1 to Block SCF<Sup>FBW7</Sup> Ubiquiting Ligase (ID 9121)
09:30 - 16:00 | Author(s): Y. Kim
- Abstract
Background:
Lung adenocarcinoma is the most common subtype of non-small cell lung cancer (NSCLC) and accounts for about 50% of them. Although EGFR or EML4-ALK has been identified as oncogenic driver mutation and translocation for advanced adenocarcinoma, trigger or mechanism of its early progression is still unclear. Previously, we revealed that stratifin (SFN, 14-3-3 sigma) has tissue-specific functions and regulate cell cycle progression in a positive manner in lung adenocarcinoma (Shiba-Ishii A et al. Mol Cancer 2015). Moreover, S-phase kinase-associated protein 1 (SKP 1) which is an adaptor part of SCF-type E3 ubiquitin ligase complex including SCF[FBW7], SCF[SKP2] and SCF[β][-TRCP] was identified as one of the SFN binding protein by pull-down assay and LC-MS/MS analysis. The aim of this study is to analyze the molecular mechanism of tumor progression in lung adenocarcinoma associated with SFN binding with SKP1. We have hypothesized that SFN binds with SKP1 among various SCF complexes and specifically blocks SCF[FBW7] function to ubiquitinate oncoproteins such as cyclin E1, c-Myc, c-Jun, and notch 1.
Method:
Endogenous interaction of SKP1 and SFN or FBW7 was examined by co-immunoprecipitation using A549, lung adenocarcinoma cells. We performed ubiquitination assay under the treatment of proteosome inhibitor, MG132 to induce accumulation of ubiquitinated oncoproteins after siRNA-SFN transfection. Moreover, to investigate whether SFN regulates the localization of SKP1, we performed immunofluorescence staining of A549 after siRNA-SFN treatment.
Result:
We found that SKP1 interacted with SFN and FBW7, respectively in lung adenocarcinoma cells. The binding activity of FBW7 with SKP1 increased after suppression of SFN, indicating that SFN and FBW7 might competitively bind with SKP1. Moreover, knockdown of SFN led to reduction of oncoproteins such as cyclin E1, c-Myc, c-Jun and notch 1 and showed accumulation of poly-ubiquitinated oncoproteins relative to the control by blocking proteosome degradation. However, p27[Kip1] (substrate of SCF[SKP2]) and IKB (substrate of SCF[β][-TRCP]) showed no expression change after knockdown of SFN. While SKP1 mainly localized in cytoplasm of A549, knockdown of SFN induced translocation of SKP1 to nucleus.
Conclusion:
SFN induces the stabilization of oncoproteins by blocking SCF[FBW7 ]ubiquitin ligase in lung adenocarcinoma and associated with its malignant progression. SFN will be a promising theraputic target for lung adenocarcinoma.
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P2.02 - Biology/Pathology (ID 616)
- Event: WCLC 2017
- Type: Poster Session with Presenters Present
- Track: Biology/Pathology
- Presentations: 1
- Moderators:
- Coordinates: 10/17/2017, 09:30 - 16:00, Exhibit Hall (Hall B + C)
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P2.02-055 - Stratifin Regulates Stabilization of Receptor Tyrosine Kinases via Activation of Ubiquitin-Specific Protease 8 in Lung Adenocarcinoma (ID 8354)
09:30 - 16:00 | Author(s): Y. Kim
- Abstract
Background:
Receptor tyrosine kinases (RTKs) such as epidermal growth factor receptor (EGFR) and hepatocyte growth factor receptor (MET) are the best-known therapeutic targets in lung adenocarcinoma. Previously, we have revealed that stratifin (SFN, 14-3-3 sigma) acts as a novel oncogene, accelerating tumor initiation and progression of lung adenocarcinoma and interacts with ubiquitin-specific protease 8 (USP8) (IJC 2011, Mol Cancer 2015). USP8 is one of the deubiquitination enzymes that stabilize specific protein substrates by removing ubiquitin from the proteins, and is known to target receptor tyrosine kinases (RTKs). In this study, we investigated the molecular mechanism underlying the binding of SFN to USP8 in lung adenocarcinoma cells, as the role of this interaction in RTK stabilization was considered a promising avenue for identifying a useful therapeutic target for lung adenocarcinoma.
Method:
Expressions of USP8 and SFN in human lung adenocarcinoma tissues (n=193) were examined by immunohistochemistry and statistically analyzed with clinicopathological features of patients. Functional analysis of USP8 and SFN such as cellular proliferation assay, apoptosis assay, and wound healing assay was examined after siRNA-USP8 or SFN transfection. Regulation mechanism of USP8 and SFN on RTKs stabilization was demonstrated using co-immunoprecipitation, western blot analysis, and immunofluorescence.
Result:
USP8 specifically bound to SFN in lung adenocarcinoma cells. Both USP8 and SFN showed higher expression in human lung adenocarcinoma than in normal lung tissue, and their expression was mutually correlated. Expression of SFN, but not that of USP8, was significantly associated with histological subtype, pathological stage, and patient’s prognosis. In vitro, USP8 binds SFN at the early- and late-endosome in immortalized adenocarcinoma in situ (AIS) cells. Moreover, USP8 or SFN knockdown led to down-regulation of tumor cell proliferation, RTK expression, and expression of downstream factors including AKT and STAT3, as well as accumulation of ubiquitinated RTKs leading to lysosomal degradation. Additionally, transfection with mutant USP8 and mutant SFN, which are unable to interact each other, reduced the expression of RTKs and their downstream factors, indicating that interaction with SFN is important for USP8-mediated stabilization of RTKs via deubiquitination.
Conclusion:
RTKs are regulated by ubiquitin-lysosome system, and aberrant stabilization of RTKs contributes to the proliferative activity of many human cancers, including NSCLC. Here, we demonstrate SFN induces aberrant activation of USP8 and subsequently protects RTKs from lysosomal degradation, resulting in hyperactivation of these signaling pathways. SFN may be central to the development of a useful therapeutic strategy for both early and advanced lung adenocarcinomas.
