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S. Rowbotham
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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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P1.04-059 - The Role of Histone Methyltransferases G9a/Glp in Mouse Lung Tumor Propagating Cells and Lung Stem Cells (ID 1543)
09:30 - 09:30 | Author(s): S. Rowbotham
- Abstract
Background:
Proper epigenetic control of transcription is essential for stem cell homeostasis and is frequently disrupted in cancer, but this has not been well investigated in lung biology or lung disease. We have previously demonstrated that the stem cell marker Sca-1 enriches for mouse bronchioalveolar stem cells (BASCs) and lung adenocarcinoma cells with enhanced metastatic and tumor propagation abilities (TPCs).
Methods:
I performed a small chemical library screen using a Kras; p53-flox driven mouse lung adenocarcinoma cell line, CK1750 with high expression of the stem cell markers Sca-1 and CD24. Chemically treated cels were seperated and screened by FACS analysis for changes in the surface antigens Sca-1 and CD24. I treated Sca-1 low expressing mouse lung adenocarcinoma cell lines TM1 and TnM2 with either 1 uM UNC0638 or DMSO for 4 days. 100,000 cells per mouse were injected intravenously. After 3-4 Weeks mice were sacrificed and lungs and other organs were analyzed for tumor formation. Treated cells were also plated in 3D organoid culture and grown for 14 days. After this matrigel plugs were fixed and counted for organoid formation. I isolated BASCs and AT2 cells from 6 week old mice by FACS and plated cells in 3D organoid co-culture. Cells were grown with either 250 nM UNC0638 or DMSO for 21 days. At this point plugs were fixed, sectioned and organoids were stained for lung lineage markers SPC and CC10 and organoids expressing each were scored.
Results:
A FACS screen of adenocarcinoma cell lines revealed that UNC0638, an inhibitor of H3K9me1/2 methyltransferases G9a and Glp enriches for high Sca-1 expressing, TPC-like cells. Gene expression analysis of primary adenocarcinomas shows that G9a/Glp are down-regulated in Sca-1+ metastatic TPCs. Furthermore, analysis of 400+ early stage patient lung adenocarcinomas reveals that low G9a expression and high expression of KDM3A, an H3K9me1/2 demethylase, significantly correlate with worse survival (P=0.008, P=0.002). This implies that dysregulation of H3K9me1/2 is also a significant factor in human disease. Interestingly, G9a/Glp inhibition of adenocarcinoma cells prior to transplantation increases Sca-1+ cells but does not increase recipient lung tumor burden. Instead, significantly more tumors are found in non-lung locations (58% vs. 13%, P=0.02). Whilst inhibition does not affect cell proliferation or migration, colony forming efficiency in 3D organoid culture is significantly increased (1.1% vs 0.3%, P=0.04). This suggests that altered stem-like properties such as tumor initiation may underlie the more tumorigenic phenotypes of H3K9me1/2 low, Sca-1+ TPCs. H3K9me1/2 also regulates the behavior of lung stem cells. G9a/Glp inhibition of BASCs or alveolar type 2 cells in 3D co-culture assays increases both Sca-1+ cells and undifferentiated organoids, and significantly decreases alveolar-lineage organoids (P<0.0005). BASC cultures also show increases in bronchiolar-lineage organoids (P<0.0005), implying that cell fate decisions may regulated by H3K9me1/2.
Conclusion:
These findings suggest that common mechanisms of epigenetic regulation exist between mouse lung stem cells and lung TPCs. Determining the precise mechanisms of this regulation will be important for our understanding of lung biology and disease, with potential implications for the diagnosis and treatment of human adenocarcinoma.
