ABSTRACT
Hypoxia is a common feature of locally advanced breast cancers that is associated with increased metastasis and poorer survival. Stabilisation of hypoxia-inducible factor-1α (HIF1α) in tumours causes transcriptional changes in numerous genes that function at distinct stages of the metastatic cascade. We demonstrate that expression of RIOK3 (RIght Open reading frame kinase 3) was increased during hypoxic exposure in an HIF1α-dependent manner. RIOK3 was localised to distinct cytoplasmic aggregates in normoxic cells and underwent redistribution to the leading edge of the cell in hypoxia with a corresponding change in the organisation of the actin cytoskeleton. Depletion of RIOK3 expression caused MDA-MB-231 to become elongated and this morphological change was due to a loss of protraction at the trailing edge of the cell. This phenotypic change resulted in reduced cell migration in two-dimensional cultures and inhibition of cell invasion through three-dimensional extracellular matrix. Proteomic analysis identified interactions of RIOK3 with actin and several actin-binding factors including tropomyosins (TPM3 and TPM4) and tropomodulin 3. Depletion of RIOK3 in cells resulted in fewer and less organised actin filaments. Analysis of these filaments showed reduced association of TPM3, particularly during hypoxia, suggesting that RIOK3 regulates actin filament specialisation. RIOK3 depletion reduced the dissemination of MDA-MB-231 cells in both a zebrafish model of systemic metastasis and a mouse model of pulmonary metastasis. These findings demonstrate that RIOK3 is necessary for maintaining actin cytoskeletal organisation required for migration and invasion, biological processes that are necessary for hypoxia-driven metastasis.
Subject(s)
Actin Cytoskeleton/genetics , Breast Neoplasms/genetics , Neoplasm Invasiveness/genetics , Protein Serine-Threonine Kinases/biosynthesis , Animals , Breast Neoplasms/pathology , Cell Hypoxia/genetics , Cell Line, Tumor , Cell Movement/genetics , Gene Expression Regulation, Neoplastic , Humans , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Mice , Neoplasm Invasiveness/pathology , Neoplasm Metastasis , Protein Serine-Threonine Kinases/genetics , Tropomyosin/genetics , ZebrafishABSTRACT
Conditionally replicating adenoviruses (CRAd) 'armed' with prodrug-activating genes have the potential to augment the efficacy of virotherapy. An Escherichia coli nitroreductase (NTR) gene (nfsB) was introduced into the E3B region of the systemically active CRAd ONYX-411, to produce ONYX-411(NTR), which had single agent oncolytic activity equivalent to unarmed virus in vitro and in vivo. A fluorogenic probe (SN 29884) developed to monitor NTR expression revealed robust, durable NTR expression in ONYX-411(NTR) infected neoplastic but not primary human cell lines. NTR expression occurred >24 h post-infection in parallel with fiber and was sensitive to ara-C indicating transcriptional linkage to viral replication. A novel NTR prodrug, the 3,5-dinitrobenzamide-2-bromomustard SN 27686, was shown to be more dose potent and selective than CB 1954 and provided a superior bystander effect in 3D multicellular layer cultures. Its water-soluble phosphate ester SN 28343 was substantially more active than CB 1954 against xenografts containing a minority of stable NTR-expressing cells. A single intravenous dose of ONYX-411(NTR) (10(8) PFU) to nude mice bearing large H1299 xenografts (>350 mm(3)) resulted in tumor-specific NTR expression which increased over time. Despite extensive viral spread by day 14, this conservative virus dose and schedule was unable to control such well-established tumors. However, subsequent administration of SN 28343 resulted in the majority of mice (62.5%) being tumor-free on day 120.
