Abstract A39: H3K9 methyltransferases G9a/Glp link the chromatin regulation of lung adenocarcinoma tumor propagating cells and lung bronchiolaveolar stem cells
Authors: Rowbotham Samuel P. , Kim Carla F. 
Published: May 2016

Abstract
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 determined to test if lung stem cells and lung cancer stem cell-like tumor propagating cells (TPCs) shared mechanisms of chromatin regulation and if these contributed to the TPC cell fate. We have previously demonstrated that the stem cell marker Sca-1 enriches for mouse bronchioalveolar stem cells (BASCs) and cancer stem celll-like lung adenocarcinoma cells with enhanced metastatic and tumor propagation abilities (TPCs).
A FACS screen of lung 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.
Depletion of G9a throughout tumorigenesis causes significant increases in tumor burden and in a Lox-stop-Lox-Kras; p53-flox mouse model of lung adenocarcinoma. Tumors generated with a Cre/shG9a lentivirus had a significantly higher tumor burden, tumor grade (P&lt;0.0001) than those with a control Cre/shLuciferase virus, and gave rise to significantly more metastases (47% vs 0%, P=0.0035). FACS Analysis demonstrated that the shG9a tumors had a significantly expanded Sca-1+ CD24+ TPC fraction (27.7% vs 15.6%, P=0.019), and shG9a Metastases were even further enriched in TPCs (63.5% + 8.3%). This suggests that G9a depletion drives tumor cells towards the more tumorigenic TPC cell fate and this may be responsible for the greatly accelerated disease progression in these mice. 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&lt;0.0005). BASC cultures also show increases in bronchiolar-lineage organoids (P&lt;0.0005), implying that cell fate decisions may regulated by H3K9me1/2.
These findings demonstrate that common means of epigenetic regulation exist between mouse lung stem cells and lung TPCs, and suggests that lung TPCs may adopt these whilst promoting disease progression. 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.
Citation Format: Samuel P. Rowbotham, Carla F. Kim. H3K9 methyltransferases G9a/Glp link the chromatin regulation of lung adenocarcinoma tumor propagating cells and lung bronchiolaveolar stem cells. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr A39.