A comprehensive evaluation of Hippo pathway silencing in sarcomas

Abstract

// Nicole M. Merritt 1 , Colleen A. Fullenkamp 1 , Sarah L. Hall 1 , Qining Qian 2 , Chandni Desai 1 , Jon Thomason 1 , Allyn M. Lambertz 1 , Adam J. Dupuy 3 , Benjamin Darbro 2 and Munir R. Tanas 1 1 Department of Pathology, University of Iowa, Iowa City, IA, USA 2 Department of Pediatrics, University of Iowa, Iowa City, IA, USA 3 Department of Anatomy and Cell Biology, University of Iowa, Iowa City, IA, USA Correspondence to: Munir R. Tanas, email: munir-tanas@uiowa.edu Keywords: Hippo pathway; sarcoma; TAZ; YAP; epigenetics Abbreviations: TAZ: transcriptional coactivator with PDZ-binding motif; YAP: yes associated protein; AZA: 5-azacytidine; TSA: Trichostatin A Received: April 25, 2018      Accepted: July 12, 2018      Published: August 03, 2018 ABSTRACT TAZ and YAP are transcriptional coactivators negatively regulated by the Hippo pathway that have emerged as key oncoproteins in several cancers including sarcomas. We hypothesized that loss of expression of the Hippo kinases might be a mechanism of activating TAZ and YAP. By immunohistochemistry, TAZ/YAP activated clinical sarcoma samples demonstrated loss of MST1 (47%), MST2 (26%), LATS1 (19%), and LATS2 (27%). Western blot similarly demonstrated loss of MST1 (58%), MST2 (25%), and LATS2 (17%). Treatment with MG132 demonstrated an accumulation of MST2 in 25% of sarcoma cell lines, indicating that proteosomal degradation regulates MST2 expression. qRT-PCR in sarcoma cell lines demonstrated loss of expression of the Hippo kinases at the RNA level, most pronounced in MST1 (42%) and MST2 (25%). 5-azacytidine treatment in sarcoma cell lines modestly reversed expression of predominantly MST1 (8%) and MST2 (17%), indicating CpG island hypermethylation can silence expression of MST1 and MST2 . Trichostatin A treatment reversed expression of MST1 (58%) and MST2 (67%), indicating histone deacetylation also plays a role in silencing expression of MST1 and MST2 . Loss of expression of the Hippo kinases is frequent in sarcomas and is due to a variety of mechanisms including regulation at the post-translational level and epigenetic silencing.