The tumor suppressor Rassf1a prevents mouse liver tumorigenesis and regulates levels of the oncogenic kinase Tbk1 and of beta tubulin gene expression in the liver

The tumor suppressor gene RASSF1A (Ras association domain family protein 1A) coding for a microtubule stabilizing protein is epigenetically silenced in most human cancers. As a binding partner of the kinases MST1 and MST2, the mammalian orthologues 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 because Hippo pathway components are rarely mutated. Rassf1a homozygous knockout mice developed liver tumors. However, heterozygous deletion of Sav1 or co-deletion 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 IkB kinase TBK1 as the most significantly enriched RASSF1A-interacting 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, and at the transcript level, factors regulating Tbk1 stability, including Usp2 and Dtx4, were also dysregulated. Furthermore, transcripts of several beta tubulin isoforms were increased in the Rassf1a-deficient liver genotypes presumably reflecting a role of Rassf1a as a tubulin-binding and microtubule-stabilizing protein. Our data suggest a multifactorial role of Rassf1a in suppression of liver carcinogenesis. Analysis of gene expression in liver of wildtype 129 mice, Rassf1a-/- mice (RA), Sav1+/- mice (SA) and Rassf1a-/- Sav1+/- mice (RA_SA)抑癌基因RASSF1A（Ras关联结构域家族蛋白1A）编码一种微管稳定蛋白，在绝大多数人类癌症中发生表观遗传沉默。作为果蝇Hippo激酶的哺乳动物同源物——MST1与MST2激酶的结合伴侣，RASSF1A是Hippo抑癌通路的潜在调控因子，该特性与支架蛋白SAV1相符。由于Hippo通路组分极少发生突变，该通路在人类癌症中的作用始终成谜。Rassf1a纯合敲除小鼠可罹患肝脏肿瘤；然而，仅Sav1杂合缺失，或共同缺失Rassf1a与Sav1，其肝脏肿瘤的发生效率远高于仅单敲除Rassf1a的小鼠。通过质谱技术分析RASSF1A的结合伴侣，研究鉴定出Hippo激酶MST1、MST2以及致癌性IκB激酶TBK1为与RASSF1A互作最显著富集的蛋白。Rassf1a纯合敲除小鼠肝脏的转录组紊乱程度高于Sav1杂合缺失小鼠肝脏，而Rassf1a纯合敲除且Sav1杂合缺失小鼠的肝脏转录组则与单纯Rassf1a纯合敲除小鼠相似。我们发现，在缺失Rassf1a的肝脏中，TBK1蛋白的表达水平显著上调；在转录层面，调控TBK1稳定性的因子（包括Usp2与Dtx4）也出现异常表达。此外，在Rassf1a缺陷型肝脏基因型中，多种β微管蛋白同工型的转录本水平升高，这或许印证了Rassf1a作为微管结合并稳定微管的蛋白的功能。本研究结果表明，Rassf1a在抑制肝脏癌变过程中发挥多因素调控作用。本研究对野生型129小鼠、Rassf1a纯合敲除小鼠（RA）、Sav1杂合缺失小鼠（SA）以及Rassf1a纯合敲除且Sav1杂合缺失小鼠（RA_SA）的肝脏基因表达展开了分析。