Abstract 3059: Pseudokinase Trb3 inhibits tumor cell growth and promotes a G2/M phase cell cycle arrest in prostate cancer

INTRODUCTION Mammalian homolog Tribbles (Trbs) is a newly characterized protein family that includes three different isoforms: Trb1, Trb2, and Trb3.Tribbles are serine/threonine kinases lacking catalytic activity, thus their classification as pseudokinases. Despite their catalytic inactivity, Tribbles can interact with different proteins and regulate different biological functions. The most studied tribble family member, Trb3, was reported to play a major role in Drosophila9s ventral furrow formation. Further studies revealed that Trb3 is also involved in diabetes, stress-response, and development. Previously, Trb3 upregulation was detected in certain types of cancer but its function remains unknown. The goal of this study is to gain a better understanding of the biological function of Trb3 in cancer, including the molecular mechanism of action. METHODS To determine the role of Trb3 in tumorigenesis, Trb3 expression was compared in normal, primary and metastatic tissue using cohort GSE-6919. Small hairpin RNA GIPZ construct was used to deplete Trb3 in prostate cancer DU145 cell line. Conversely, Trb3 was ectopically overexpressed in prostate cancer PC-3 cells using CMV-driven Trb3 expression vector. Then subsequent effects on cell proliferation and cell cycle progression were studied. The MTS and Brdu incorporation assays were used to assess cell proliferation. Cell cycle analysis was performed using flow cytometry of propidium iodide stained cells. Western blot analysis was used to identify proteins regulated post silencing or overexpression of Trb3. RESULTS Using the cohort analysis, we identified higher levels of Trb3 in the primary tumor compared to the normal tissue. Silencing of Trb3 in DU145 promoted cell growth and higher colony formation ability. Cell cycle analysis revealed an increase in S phase in Trb3 silenced DU145. Furthermore, the cell proliferation protein Ki67 was increased in DU145 cells with Trb3 silenced. Cyclin D1 levels were reduced. However, active cdc25c, a phosphatase that promotes cell entry into mitosis, was increased. On the other hand, overexpression of Trb3 in prostate cancer PC-3 inhibited tumor cell proliferation, reduced colony formation, and induced cell cycle arrest at G2/M phase. CONCLUSION In this study, we found that knocking down of Trb3 in prostate cancer promotes cell proliferation and colony formation ability by increasing proliferation protein KI67, while reducing cyclin D1 levels. Furthermore, the phosphatase cdc25c is activated resulting in an enhanced entry into mitosis. Conversely, Trb3 overexpression has an antiproliferative effect and inhibits colony formation ability. Trb3 also promotes a G2/M phase arrest in prostate cancer PC-3 cells. Our results suggest that Trb3 is activated in the primary tumor to prevent tumor growth by promoting cell cycle arrest. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 3059. doi:1538-7445.AM2012-3059