The Hippo pathway controls organ size and is involved in both cell proliferation as well as in apoptosis. In cancer, the signalling via Hippo is deregulated and promotes tumor growth. The proto-oncogene YAP1 is the downstream transcriptional co-activator of this pathway with its function depending on the binding to several transcription factors such as TEAD, SMAD and TP73. The tumor suppressor gene RASSF1A is one of the main regulators of the Hippo signalling; however, RASSF1A is epigenetically silenced in cancer. In the literature, there are indications for the role of RASSF1A in the regulation of the pro-apoptotic function of the Hippo pathway. The aim of this study was to analyse the regulatory role of RASSF1A on YAP1 target genes. Therefore, a YAP1-inducible cell line was generated and further characterised after induction of YAP1 and co-expression with RASSF1A.An important observation in this study was the oncogenic potential of YAP1. The induction of YAP1 promotes cell proliferation by activation of pro-proliferative genes and by the transcriptional repression of tumor suppressor genes such as TP53, RASSF1A, BAX, CDKN1A and BBC3. Expression screenings by microarray revealed novel potential YAP1 target genes that are regulated by RASSF1A. In addition, microscopy and flow cytometry data showed that the expression of RASSF1A triggers the nuclear translocation of YAP1 inducing nuclear deformation, cell cycle arrest and apoptosis. In addition, it was observed that RASSF1A and YAP1 repress the expression of growth-associated genes and growth factors such as MDM2. Further analysis indicated that RASSF1A neutralizes the oncogenic function of YAP1 by activation of the YAP1 target genes ANKRD1, AJUBA, BAX and CDKN1A. RASSF1A activates Hippo signalling via the SARAH (Sav/Rassf/Hpo) domain and, together with YAP1, regulates the expression of the target gene ANKRD1. The further characterisation of ANKRD1 indicated its potential function as tumor suppressor gene. ANKRD1 inhibits cell growth and is silenced by promoter methylation in lung and in prostate cancer cell lines. At the protein level, ANKRD1 stabilises TP53 via reduction of the level of MDM2, which results in the transcriptional activation of CDKN1A and BAX. Previous reports suggested ANKRD1 as co- activator of TP53. In this study, the knockdown of ANKRD1 by siRNA and promoter assays corroborated the obtained findings and the data from the literature. The data from this work suggest a novel mechanism of RASSF1A and YAP1 interaction, namely to regulate TP53 and the G1/S cell cycle transition via the Hippo pathway and ANKRD1. The inactivation of RASSF1A by aberrant promoter methylation results in the deregulation of the signalling, which promotes cell proliferation by the action of the oncogene YAP1.
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