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A. Shiba
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MINI 35 - Biology (ID 161)
- Event: WCLC 2015
- Type: Mini Oral
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:T.A. Boyle, M.G. Kris
- Coordinates: 9/09/2015, 18:30 - 20:00, Mile High Ballroom 2c-3c
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MINI35.12 - Stratifin Accelerates Progression of Lung Adenocarcinoma at an Early Stage (ID 506)
19:30 - 19:35 | Author(s): A. Shiba
- Abstract
- Presentation
Background:
Adenocarcinoma in situ (AIS) of the lung has an extremely favorable prognosis. However, early but invasive adenocarcinoma (eIA) sometimes has a fatal outcome. We had previously compared the expression profiles of AIS with those of eIA showing lymph node metastasis or a fatal outcome, and found that stratifin (SFN, 14-3-3 sigma) was a differentially expressed gene related to cell proliferation (Aya Shiba-Ishii, IJC. 2011). We also found that SFN expression was totally suppressed in normal lung tissue, whereas demethylation of its promoter triggered aberrant SFN overexpression in eIAs in a p53-independent manner (Aya Shiba-Ishii, AJP. 2012). SFN has been linked to cancer most directly, possibly having tissue-specific functions and regulating progression of the cell cycle. Here, we performed an in vivo study to clarify the role of SFN in progression of lung adenocarcinoma.
Methods:
We induced stable knockdown of SFN using two individual shRNAs (shSFN). To evaluate the oncogenic activity of SFN, we injected A549-shSFN intrabronchially or intravenously into SCID mice. Additionally, we generated SFN-transgenic mice (Tg-SPC-SFN[+/-]) showing lung-specific expression of human SFN (hSFN) under the control of a tissue-specific enhancer, the SPC promoter. In order to observe the tumorigenic activity of SFN, Tg-SPC-SFN[+/-] and WT ICR mice were intraperitoneally administered 4 mg 4-(methylnitrosamino)-1-(3-pyridyl)-1-butanone (NNK, a pulmonary carcinogen) or saline as a control, and tumorigenicity was assessed for 20 weeks. Lungs of representative mice were periodically examined using animal CT.
Results:
Although control A549 cells formed advanced tumors in the lungs of SCID mice after intrabronchial and/or intravenous injection, we also found pleural dissemination in the control group (in 75% after intravenous injection and in 25% after intrabronchial injection). However, A549-shSFN did not form any tumors. Next, we confirmed the lung-specific expression hSFN in Tg-SPC-SFN[+/-] using RT-PCR and IHC. In a chemical carcinogenesis experiment, animal CT revealed several pulmonary tumors in some Tg-SPC-SFN[+/-] from 15 weeks after NNK administration, and at 20 weeks 47.8% of Tg-SPC-SFN[+/-] (11/23) had developed lung tumors, whereas only 11.1% of WT ICR (3/27) had done so (statistically significant). Surprisingly, two of seven Tg-SPC-SFN[+/-] mice (28.6%) developed tumors even though they were not administered NNK. All of the tumors that developed in Tg-SPC-SFN[+/-] lung expressed hSFN abundantly.
Conclusion:
Here, we showed that suppression of SFN expression in lung adenocarcinoma A549 cells was significantly reduced in terms of not only lung tumor formation but also metastatic potential. Additionally, it was found that Tg-SPC-SFN[+/-] mice developed lung tumors at a significantly higher rate than control mice after NNK administration. Interestingly, several Tg-SPC-SFN[+/-] mice developed lung tumors without carcinogen. Because these tumors showed high hSFN expression, SFN was thought to facilitate not only tumor progression but also tumor initiation, and to work as an oncogene. Soda et al. found that 100% of Tg-EML4-ALK mice developed hundreds of adenocarcinoma nodules in both lungs within a few weeks after birth (Nature 2007). Although the oncogenic activity of SFN is weaker than that of EML4-ALK fusion kinase, SFN might also have the potential to initiate peripheral-type lung adenocarcinoma.
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P1.04 - Poster Session/ Biology, Pathology, and Molecular Testing (ID 233)
- Event: WCLC 2015
- Type: Poster
- Track: Biology, Pathology, and Molecular Testing
- Presentations: 1
- Moderators:
- Coordinates: 9/07/2015, 09:30 - 17:00, Exhibit Hall (Hall B+C)
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P1.04-071 - Inhibition of EGFR Lysosomal Degradation in Lung Adenocarcinoma by Ubiquitin-Specific Protease 8 and Stratifin (ID 475)
09:30 - 09:30 | Author(s): A. Shiba
- Abstract
Background:
The epidermal growth factor receptor (EGFR) is one of the best-known targets of therapy for non-small cell lung cancer (NSCLC). Our purpose was to investigate whether ubiquitin-specific protease 8 (USP8) and stratifin (14-3-3σ or SFN) inhibit or stimulate lysosomal degradation of EGFR in lung adenocarcinoma.
Methods:
Using Western blotting and immunofluorescence analysis, we examined the effect of USP8 or SFN knockdown by siRNA and overexpression of USP8. Expressions of USP8 and SFN in normal and tumorous lung tissue were examined by Western blotting and immunohistochemistry.
Results:
USP8 or SFN knockdown led to downregulation of cellular proliferation, receptor tyrosine kinases such as EGFR and proto-oncogenes (c-Met), and downstream signaling pathways such as the AKT, ERK, and STAT3 pathways, whereas it upregulated the accumulation of EGFR at lysosomes for degradation. However, overexpression of USP8 led to an increase of EGFR and downstream signaling after EGF stimulation. Moreover, USP8 and SFN expressions were increased in the tumorous lung tissue in comparison with normal lung tissue from the same patient.
Conclusion:
USP8 and SFN inhibit ubiquitination of EGFR for lysosomal degradation in lung adenocarcinoma cells, suggesting that USP8 and SFN could be potential therapeutic targets for NSCLC.
