Metastatic lung cancer is one of the most lethal forms of cancer and molecular pathways driving metastasis Rabbit Polyclonal to FGFR1 Oncogene Partner. are still not clearly elucidated. factor Zeb1 and is elevated in mesenchymal-like metastatic lung cancer cells. Foxf2 expression induced robust EMT migration invasion and metastasis in lung cancer cells whereas Foxf2 inhibition significantly repressed these phenotypes. We also demonstrated that Foxf2 transcriptionally represses E-Cadherin and miR-200 independent of Zeb1 to form a double negative feedback loop. We therefore identified a novel mechanism whereby the miR-200 family and the miR-183~96~182 cluster inhibit lung cancer invasion and metastasis by targeting Foxf2. metastatic potencies 344 or control 344SQ-GFP induced cells were subcutaneously implanted into syngeneic mice. The primary tumor sizes for both the control and the Foxf2 expressing cells were Cot inhibitor-2 comparable consistent with no significant difference in cellular proliferation between the tumor types as evident from Ki67 staining (Supplementary Fig. 4D). However the mice with 344SQ-Foxf2 tumors demonstrated a ~3-fold increase in the number of metastatic lung nodules compared to the control cells (Fig. 3I) within just 4 weeks. This was confirmed by haematoxylin and eosin staining of lung sections from the groups (Fig. 3J). These results establish Foxf2 as a potent suppressor of the epithelial phenotype which arrests cells in a hyper-invasive state producing rapid metastasis. Foxf2 knockdown suppresses invasion and metastasis To study the converse effect we stably knocked down Foxf2 expression in mesenchymal mouse and human cells by Cot inhibitor-2 shRNA vectors. Foxf2 knockdown in mouse mesenchymal and metastatic 344SQ cells (344SQ-Foxf2-shE) did not result in an apparent change in cell morphology (data not shown) cell proliferation (Supplementary Fig. 4C) or expression of the EMT markers (Fig. 4A-B) but significantly suppressed cellular migration and invasion in Boyden chambers (Fig. 4C and Supplementary Fig. 3L). Similarly in human H157 cells knockdown of FOXF2 (H157-FOXF2-sh5) did not alter the expression of EMT genes (Fig. 4D-E) but produced significant inhibition of migration and invasion compared to vector controls (Fig. 4F and Supplementary Fig. 3M). To test whether down-regulation of Foxf2 expression could alter the metastatic potencies the 344SQ-Foxf2-shE (knockdown) and the 344SQ-pGIPZ-NS (control) cells were injected subcutaneously in syngeneic mice. Both groups formed comparable sized tumors at 8 weeks with only a slight increase in proliferating cells in the primary tumors formed by the knockdown cells compared to the controls when assayed by Ki-67 staining (Fig. 4G and Supplementary Fig. 4D). In contrast the Foxf2 knockdown cells exhibited significant repression of lung metastasis (Fig. Cot inhibitor-2 4G) which was confirmed by haematoxylin and eosin stained lung sections (Fig. 4H). These results confirm that inhibition of Foxf2 expression could significantly reduce the migratory and invasive capabilities of metastatic cells abrogating metastasis. Interestingly by manipulating the levels of Foxf2 in the same (344SQ) cell line we could control the metastatic phenotype of the cells highlighting the importance of Foxf2 as a metastasis regulator. Fig. 4 Foxf2 knockdown leads to decreased invasion and metastasis Foxf2 induces rapid repression of E-cadherin and miR-200 independent of Zeb1 Foxf2 expression induces a strong EMT-like phenotype with increased migration invasion and metastasis which is associated with a robust inhibition of E-Cadherin and up-regulation of Zeb1. To understand whether these two changes are a direct and acute consequence of Foxf2 expression we performed a time course assay to determine the changes in expression of these two markers upon induction of Foxf2 in 393P cells. Upon Foxf2 induction E-Cadherin was transcriptionally repressed as early as 4 hours (50% at RNA level) and reached its maximum by 48 hours (more than 95% by mRNA and protein level (60%)) whereas Zeb1 protein levels were not Cot inhibitor-2 considerably elevated (14%) until 48 hours (Fig. 5A-B). Induction of GFP (control vector) did not induce any marker changes (Supplementary Fig. 5A-B). Since the miR-200 family is important regulator of the epithelial phenotype in lung cancer23 we examined whether they are regulated by Foxf2. We observed that mature miR-200a and miR-200b but not miR-200c were significantly repressed (70%) within 48 hours of Foxf2 induction (Fig. 5C) whereas no.