Background: Sox9 plays critical roles in the specification and differentiation of numerous progenitor and differentiated cell types during embryonic and fetal development. Sox9 is overexpressed in 40 - 50% of lung adenocarcinomas and associated with poor prognosis in lung cancer patients. We set out to identify the upstream pathways that regulate Sox9 expression in lung cancer as well as the role of Sox9 in lung adenocarcinoma progression. Several developmental and stem cell pathways are known induce Sox9 transcription during carcinogenesis, including the TGB-β, Wnt/β-catenin, Sonic Hedgehog, and NF-κB signaling pathways. SOX9 has also been shown to be a transcriptional target of the Notch pathway during mouse development, although the binding sites for Notch within the mouse SOX9 promoter are not conserved in humans.

Results: We mined gene expression data from three publicly available datasets and found that Hes1, a known Notch target gene, is co-expressed with Sox9 in lung adenocarcinoma. We validated the data by identifying significant overlap in Sox9 and Hes1 protein expression levels in a human lung cancer tissue microarray. Furthermore, Sox9 mRNA and protein levels were upregulated over 100-fold as early as 14 days after Notch1 induction in the Notch1-induced mouse model of lung cancer, suggesting that Sox9 overexpression is an early event during lung cancer development. Through a series of in vitro assays, we determined that Sox9 is immediately downstream of Notch1, but not Notch3, in lung adenocarcinoma cell lines. By ChIP and luciferase reporter assays, we located the previously unidentified human RBPjk binding site, the principle effector of canonical Notch1 signaling, immediately upstream of the SOX9 transcriptional start site. We also examined TGF-β, a known inducer of epithelial-to-mesenchymal transition (EMT) in lung cancer. We determined that induction of Sox9 expression by Notch1 is independent of TGF-β signaling, that TGF-β upregulates both Sox9 and Notch1 expression, and that TGF-β and Notch1 cooperate in their regulation of Sox9 expression. Sox9 overexpression led to an induced EMT phenotype, characterized by increased cell invasion/migration, and EMT-related gene expression changes. Sox9 mRNA expression correlated with EMT-like gene expression signatures in several gene expression microarray datasets, and Sox9 protein levels were negatively correlated with expression of the epithelial marker, E-cadherin, in a lung cancer tissue microarray. Our data also demonstrate that Notch1-induced EMT in lung adenocarcinoma is mediated, at least partly, through Sox9.

Conclusion: These results establish Sox9 as a key Notch1 target gene mediating Notch1- and TGF-β- induced EMT, leading to poor survival in lung adenocarcinoma.

Citation Format: Kathleen Capaccione, Xuehui Hong, Katherine M. Morgan, Wenyu Liu, Thaddeus Allen, J. Michael Bishop, Sharon R. Pine. Sox9 mediates Notch pathway-induced epithelial-mesenchymal transition (EMT) in lung adenocarcinoma. [abstract]. In: Proceedings of the AACR-IASLC Joint Conference on Molecular Origins of Lung Cancer; 2014 Jan 6-9; San Diego, CA. Philadelphia (PA): AACR; Clin Cancer Res 2014;20(2Suppl):Abstract nr B17.