Analysis of liver tumor-prone mouse models of the hippo kinase scaffold proteins RASSF1A and SAV1

Abstract

The tumor suppressor gene RASSF1A is epigenetically silenced in most human cancers. As a binding partner of the kinases MST1 and MST2, the mammalian orthologs of the Drosophila Hippo kinase, RASSF1A is a potential regulator of the Hippo tumor suppressor pathway. RASSF1A shares these properties with the scaffold protein SAV1. The role of this pathway in human cancer has remained enigmatic inasmuch as Hippo pathway components are rarely mutated in tumors. Here we show that Rassf1a homozygous knockout mice develop liver tumors. However, heterozygous deletion of Sav1 or codeletion of Rassf1a and Sav1 produced liver tumors with much higher efficiency than single deletion of Rassf1a. Analysis of RASSF1A-binding partners by mass spectrometry identified the Hippo kinases MST1, MST2, and the oncogenic IκB kinase TBK1 as the most enriched RASSF1Ainteracting proteins. The transcriptome of Rassf1a-/- livers was more deregulated than that of Sav1+/- livers, and the transcriptome of Rassf1a-/-, Sav1+/- livers was similar to that of Rassf1a-/- mice. We found that the levels of TBK1 protein were substantially upregulated in livers lacking Rassf1a. Furthermore, transcripts of several b-tubulin isoforms were increased in the Rassf1a-deficient livers presumably reflecting a role of RASSF1A as a microtubule-stabilizing protein. In human liver cancer, RASSF1A frequently undergoes methylation at the promoter but this was not observed for MST1, MST2, or SAV1. Our results suggest a multifactorial role of RASSF1A in suppression of liver carcinogenesis.