Epithelial-mesenchymal transition (EMT), a critical process of cancer invasion and metastasis, is associated with stemness property of cancer cells. Though Oct4 and Nanog are homebox transcription factors essential to the self-renewal of stem cells and are expressed in several cancers, the role of Oct4/Nanog signaling in tumorigenesis is still elusive. Here microarray and quantitative real-time PCR analysis showed a parallel, elevated expression of Oct4 and Nanog in lung adenocarcinoma (LAC). Ectopic expressions of Oct4 and Nanog in LACs increased the percentage of CD133-expressing subpopulation and sphere formation, enhanced drug resistance, and promoted EMT. Ectopic expressions of Oct4 and Nanog activated Slug and enhanced the tumorinitiating capability of LAC. Furthermore, double knockdown of Oct4 and Nanog suppressed the expression of Slug, reversed the EMT process, blocked the tumorigenic and metastatic ability, and greatly improved the mean survival time of transplanted immunocompromised mice. The immunohistochemical analysis demonstrated that expressions of Oct4, Nanog, and Slug were present in high-grade LAC, and triple positivity of Oct4/Nanog/ Slug indicated a worse prognostic value of LAC patients. Our results support the notion that the Oct4/Nanog signaling controls epithelial-mesenchymal transdifferentiation, regulates tumor-initiating ability, and promotes metastasis of LAC. Cancer Res; 70(24); 10433-44. Ó2010 AACR.

The hypoxic tumor microenvironment induces epithelial-mesenchymal transition (EMT) in tumor cells and increases tumor cell malignancy. Previous studies indicated that malfunction of Wnt signaling is observed in some lung cancer patients. Athough crosstalk between hypoxia and Wnt signaling in tumor cells has recently been revealed, the detailed underlying mechanisms have not been well defined. In the present study, we demonstrated that hypoxia in lung adenocarcinoma cells can enhance Wnt signaling activity by stabilizing β-catenin and altering its localization into the nucleus. Overexpression of HIF-2α increased β-catenin expression, promoted cell mobility, and induced morphological changes to a greater degree than HIF-1α overexpression. Knockdown of HIF-2α decreased β-catenin expression and inhibited hypoxia-induced cell mobility. Moreover, we identified that phosphorylational activation of AKT1 by hypoxia and HIF-2α was required for Wnt activation upon hypoxia treatment. Downregulation of HIF-2α and β-catenin reduced colony formation when cells were exposed to long-term hypoxia treatment. Taken together, our data support that hypoxia activates PI3K/AKT as well as Wnt signaling in a HIF-2α-dependent manner, thus elevating the resistance of lung cancer cells to chronic hypoxia-induced stress.

We previously demonstrated that the epidermal growth factor receptor (EGFR) up-regulated miR-7 to promote tumor growth during lung cancer oncogenesis. Several lines of evidence have suggested that alterations in chromatin remodeling components contribute to cancer initiation and progression. In this study, we identified SMARCD1 (SWI/SNF-related, matrixassociated, actin-dependent regulator of chromatin, subfamily d, member 1) as a novel target gene of miR-7. miR-7 expression reduced SMARCD1 protein expression in lung cancer cell lines. We used luciferase reporters carrying wild type or mutated 3UTR of SMARCD1 and found that miR-7 blocked SMARCD1 expression by binding to two seed regions in the 3UTR of SMARCD1 and down-regulated SMARCD1 mRNA expression. Additionally, upon chemotherapy drug treatment, miR-7 downregulated p53-dependent apoptosis-related gene BAX (BCL2-associated X protein) and p21 expression by interfering with the interaction between SMARCD1 and p53, thereby reducing caspase3 cleavage and the downstream apoptosis cascades. We found that although SMARCD1 sensitized lung cancer cells to chemotherapy drug-induced apoptosis, miR-7 enhanced the drug resistance potential of lung cancer cells against chemotherapy drugs. SMARCD1 was down-regulated in patients with non-small cell lung cancer and lung adenocarcinoma cell lines, and SMARCD1 and miR-7 expression levels were negatively correlated in clinical samples. Our investigation into the involvement of the EGFR-regulated microRNA pathway in the SWI/ SNF chromatin remodeling complex suggests that EGFRmediated miR-7 suppresses the coupling of the chromatin remodeling factor SMARCD1 with p53, resulting in increased chemo-resistance of lung cancer cells.