Identification of the DEAD box RNA helicase DDX3 as a therapeutic target in colorectal cancer.

Cynthia Berlinicke,Farhad Vesuna,Onno Kranenburg,Elsken van der Wall,Justin Brilliant,Venu Raman,Johan G. Offerhaus,Kari Trumpi,Paul J. van Diest,Wendy de Leng,Marise R. Heerma van Voss,Horst Bürger

doi:10.18632/oncotarget.4873

Abstract

Identifying druggable targets in the Wnt-signaling pathway can optimize colorectal cancer treatment. Recent studies have identified a member of the RNA helicase family DDX3 (DDX3X) as a multilevel activator of Wnt signaling in cells without activating mutations in the Wnt-signaling pathway. In this study, we evaluated whether DDX3 plays a role in the constitutively active Wnt pathway that drives colorectal cancer. We determined DDX3 expression levels in 303 colorectal cancers by immunohistochemistry. 39% of tumors overexpressed DDX3. High cytoplasmic DDX3 expression correlated with nuclear β-catenin expression, a marker of activated Wnt signaling. Functionally, we validated this finding in vitro and found that inhibition of DDX3 with siRNA resulted in reduced TCF4-reporter activity and lowered the mRNA expression levels of downstream TCF4-regulated genes. In addition, DDX3 knockdown in colorectal cancer cell lines reduced proliferation and caused a G1 arrest, supporting a potential oncogenic role of DDX3 in colorectal cancer. RK-33 is a small molecule inhibitor designed to bind to the ATP-binding site of DDX3. Treatment of colorectal cancer cell lines and patient-derived 3D cultures with RK-33 inhibited growth and promoted cell death with IC50 values ranging from 2.5 to 8 μM. The highest RK-33 sensitivity was observed in tumors with wild-type APC-status and a mutation in CTNNB1. Based on these results, we conclude that DDX3 has an oncogenic role in colorectal cancer. Inhibition of DDX3 with the small molecule inhibitor RK-33 causes inhibition of Wnt signaling and may therefore be a promising future treatment strategy for a subset of colorectal cancers.

Highlights

Significant advancements have been made in the prevention and treatment of colorectal cancer, this disease still ranks third on the list of causes of cancer related deaths in the United States, which underlines the need for development of new targeted therapies in this field. [1] On a genomic level colorectal cancer is frequently characterized by loss of the tumor suppressor gene p53 and activation of the RAS-RAF signaling pathway, alterations that are common in a multitude of solid tumors
We demonstrated that DDX3 is overexpressed in breast and lung cancers and that targeting DDX3 by RK-33 promotes cell death. [3, 4] This requirement for DDX3 can in part be explained by its involvement in Wnt signaling, as was shown previously by our group and others. [4,5,6] As the majority of colorectal cancers is driven by mutations in the Wnt-signaling pathway, we explored the possible contribution of DDX3 to Wnt-associated colorectal cancer oncogenesis
The clinical relevance of the development of Wnt signaling inhibitors which work in a constitutively activated setting is tremendous, since mutations in the Wnt-signaling pathway are the first genetic alterations in the adenoma-carcinoma sequence, but advanced colorectal cancers with mutations in APC or CTNNB1 remain dependent on upstream Wnt signaling activity. [13, 14] Especially colorectal cancer stem cells rely on Wnt signaling, and potent inhibition may inhibit the resistant tumor initiating cell population

Summary

Introduction

Significant advancements have been made in the prevention and treatment of colorectal cancer, this disease still ranks third on the list of causes of cancer related deaths in the United States, which underlines the need for development of new targeted therapies in this field. [1] On a genomic level colorectal cancer is frequently characterized by loss of the tumor suppressor gene p53 and activation of the RAS-RAF signaling pathway, alterations that are common in a multitude of solid tumors. [2] identifying druggable targets in this pathway would be beneficial for optimizing colorectal cancer treatment. Within this context, we identified a member of the RNA helicase gene family, DDX3, which exhibits oncogenic properties in breast and lung carcinomas. DDX3 was identified as an allosteric activator of CK1ɛ and hereby promotes phosphorylation of the scaffold protein dishevelled, which activates Wnt signaling during Caenorhabditis elegans and Xenopus development and in mammalian HEK293t cells. This function of DDX3 was independent of its RNA-helicase activity. This function of DDX3 was independent of its RNA-helicase activity. [5] In addition, DDX3 was found to regulate the stability of β-catenin protein expression in a helicase-dependent manner through translational regulation of Rac1. [6] In addition, our group identified a direct interaction between DDX3 and β-catenin and its functional role in regulating TCF-4 mediated transcriptional activity in lung cancer cell lines. [4] Notably, DDX3 activity has been linked to Wntsignaling activity by the identification of coinciding CTNNB1 and DDX3X activating mutations in Wnt-type medulloblastomas. [7,8,9]

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