Abstract 862: Inhibition of Hippo tumor suppressors MST1/2 slows prostate cancer cell proliferation and invasion

Abstract

Abstract Serine/threonine kinase 3 (STK3) and its paralog STK4, encode MST2 and MST1 kinases respectively, which are essential members of the highly conserved Hippo Tumor suppressor pathway which regulate Yes 1 Associated protein (YAP1). In normal cells YAP1 activation is beneficial for organ and tissue regeneration, with overgrowth being controlled by MST1/2. In cancer cells, MST1/2 play a role in cancer cell proliferation, differentiation and apoptosis by inhibiting YAP1, yet frequent deregulation of MST1/2 leads to hyper-activation of YAP1 in various cancers. Th current dogma is that STK3/4 are tumor suppressor genes. Yet, STK3, but not STK4 in prostate cancer (PC) is frequently amplified. The goal of this study was to determine if STK3 and/or STK4 gene products MST1/2 play a role in PC growth and/or metastasis. Approach. To assess if MST1 and/or MST2 play a role in PC proliferation, pharmacological and genetic studies were carried out across varying PC cell lines in vitro. XMU-MP-1 a narrow spectrum kinase inhibitor of MST1/2 was used across five distinct PC cell lines. We utilized 2D proliferation and 3D PC cell spheroids to assess effects through live cell image using Incuctye S3. Invasion assays were performed using a 96 well scratch wound migration with or without matrigel with PC3 and DU145 cells. We queried cbioPortal for PC data sets to look at incidence of STK3 and STK4 copy number alterations. Knockdowns of STK3 and STK4 genes using shRNAs to observe phenotypic differences and growth inhibition. Knockdown of STK3/4 pathways were verified through western blot. Results. Proliferation experiments 5 cell lines found that inhibition of STK3/4 with XMU-MP-1 significantly slows 2D and 3D spheroid PC cell growth. IC50 scores ranged from ~500nM to 5uM. Additionally, STK3/4 inhibition in the extremely invasive PC3 and DU145 cell lines significantly inhibited cell migration and matrigel invasion. PC data sets showed that the STK3 gene is frequently amplified in PC tumors which is contrary to an expected tumor suppressive role. In contrast, STK4 was not found to be amplified suggesting variation in STK3 versus STK4 roles. Depletion of STK3 and STK4 individually by shRNA knockdown also inhibited cell growth PC cells. Phenotypic markers of apoptosis and cell death could be seen 72 hours post viral transduction with shRNA knockdown in 22Rv1 and PC3 PC cells. The shRNA knockdowns showed deceased phosphorylated MOB kinase activator 1(MOB1) a downstream phospho-target of MST1/2. However, mixed results were obtained for subsequent YAP phosphorylation. Conclusions. Our results demonstrate that STK3/4 are essential for PC cell proliferation and migration/invasion. Similar effects were observed by both inhibition of MST1/2 either directly through drug treatment or STK3/4 gene depletion by shRNA knockdown. Whether these paralogs have unique or overlapping effects and if they can be targeted for PC therapy remains to be elucidated. Citation Format: Amelia Schirmer, Alexis B. Stokes, Erick J. Maravilla, Weiwei Fu, Everardo Macias. Inhibition of Hippo tumor suppressors MST1/2 slows prostate cancer cell proliferation and invasion [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 862.