** 0.01; *** 0.005. increase in liver size and the development of liver tumor15,16,17. Deregulation of Hpo pathway leads to the stabilization and nuclear sequestration of YAP. Nuclear YAP binds to and activates the transcription factors TEADs (the TEAD/TEF family transcription factors) and eventually turns on the expression of downstream genes including and reduced cell proliferation and impaired acinar formation ability19. TEAD4 alone also promoted anchorage-independent growth in MCF10A cells23. These observations indicate that TEADs Pf4 may also play an important role in human cancers. Vestigial-like (VGLL) proteins have recently emerged as a new group of TEAD-interacting partners participating in tumorigenesis. VGLL proteins are transcriptional cofactors that are named after transcriptional co-activator Vestigial (Vg), the grasp regulator of wing development. There are 4 VGLL proteins in mammals, named VGLL1-4. These proteins bear no sequence similarity except for the TDU domain name (the TEAD-interacting domain name)24,25,26,27,28. Previous studies showed that VGLL1 promotes cell proliferation and exhibits high expression in basal-like breast cancer29,30. Similarly, is usually amplified in soft tissue sarcoma and inhibition of results in decreased cell proliferation and migration31. Different from other members in VGLL family, VGLL4 contains an extra TDU domain name and is considered to be functionally different. For example, VGLL4 can promote apoptosis via negatively regulating inhibitor of apoptosis proteins (IAPs)32. However, the exact role of VGLL4 in cancers, especially in lung cancer, and whether and how VGLL4 is usually involved in the Hpo pathway are not clear. Here, we found that VGLL4 is usually consistently downregulated in both murine and human lung ADC specimens. Our data further proved that VGLL4 functions as a suppressor of lung cancer growth and progression via direct competition with YAP in forming the complex with TEADs through two TDU domains. Results VGLL4 is usually downregulated in both mouse and human lung ADC specimens To study the potential role of VGLL4 in lung cancer, we first examined the expression level of VGLL4 in mRNA relative to mouse normal lungs (Physique 1A). Through immunohistochemistry (IHC) study, we further showed that VGLL4 protein levels decreased in mouse lung ADCs (Physique 1B). Moreover, VGLL4 displayed a more diffused cytoplasmic staining in lung ADCs, but a predominant nuclear staining was seen in normal lungs (Physique 1B). Open in a separate window Physique 1 VGLL4 is usually lowly expressed in both human and mouse lung adenocarcinomas. (A) Real-time PCR quantification of mRNA levels in mouse 0.05. (B) H&E and Vgll4 immunohistochemical staining on mouse mRNA levels in human lung adenocarcinoma and paired pathologically normal lungs by real-time PCR. The values were presented as log10 ratio of the expression of human lung adenocarcinomas vs normal lung specimens. (D) Immunohistochemical staining of VGLL4 on human lung adenocarcinoma and normal lungs. Scale bar, 500 m (top) and 50 m (bottom). (E) Statistical analysis of nuclear VGLL4 staining in human lung adenocarcinoma and normal lung specimens. ADC, adenocarcinoma; NL, normal lung. We further examined the expression status of VGLL4 in human lung ADC specimens. Interestingly, we found that almost all the human lung ADC samples (29 of 30) had a relatively lower expression in comparison with paired pathologically normal lungs (Physique 1C). IHC studies in a cohort made up of 27 normal lungs and 77 lung ADCs showed that 92.6% of patients (25 out of 27) exhibited high nuclear VGLL4 expression in their normal lungs, whereas only 22.1% of patients (17 out of 77) had high nuclear expression of VGLL4 in their lung ADCs. The GF 109203X pattern of VGLL4 GF 109203X nuclear expression was statistically different between normal lungs and lung ADCs ( 0.01) (Physique GF 109203X 1D and ?and1E).1E). Taken together, these results indicated that VGLL4 is usually consistently expressed at lower level in lung ADC compared with normal lung tissues. VGLL4 suppresses lung tumor cell growth in a series of lung cancer cell lines and found that A549 and CRL-5872 had relatively low but detectable level of VGLL4 expression (Supplementary information, Physique S1). We then overexpressed VGLL4 in these two cell lines. Our data showed that ectopic VGLL4 expression significantly inhibited the proliferation of these two cell lines (Physique 2A and ?and2B2B and Supplementary information, Physique S2). Cell cycle analysis showed that both A549 and CRL-5872 cells with VGLL4 overexpression displayed a reduced cell ratio in G2/M phase (Physique 2C and ?and2D).2D). We further showed that VGLL4 overexpression inhibited both the anchorage-independent cell growth in soft agar (Physique 2E.