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Hypoxic regulation of RIOK3 is a major mechanism for cancer cell invasion and metastasis.

Singleton DC, Rouhi P, Zois CE, Haider S, Li JL, Kessler BM, Cao Y, Harris AL - Oncogene (2014)

Bottom Line: This phenotypic change resulted in reduced cell migration in two-dimensional cultures and inhibition of cell invasion through three-dimensional extracellular matrix.Depletion of RIOK3 in cells resulted in fewer and less organised actin filaments.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK.

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.

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RIOK3 associates with TPM3 and actin in cells. (A) Silver-stained PAGE gel of anti-FLAG immunoprecipitates from MDA-MB-231 cells expressing either RIOK3 or FLAG-RIOK3. Species detected by tandem mass spectrometry only in the FLAG-RIOK3 sample are indicated on the right of the gel by gene symbol. (B) Confocal images of MDA-MB-231 cells in normoxia (Nor) and hypoxia (Hyp) demonstrate colocalisation of RIOK3 and TPM3. Scale bar = 10 μm. Phase contrast (PC) images show changes in cellular appearance. (C) Confocal images of MDA-MB-231 cells demonstrate that RIOK3 associates with actin. In normoxia RIOK3 aggregates are often surrounded by F-actin (arrowheads). In hypoxia RIOK3 aggregates disperse and F-actin organisation changes to form stress fibers and a dense F-actin network adjacent to the leading edge (arrowheads). Scale bar = 20 μm. (D) Magnified images of regions in C. Scale bar = 2 μm. (E) Pixel density of F-actin and RIOK3 stain at the leading edge of the hypoxic cell (from D). F-actin is observed immediately adjacent to the cell edge, followed by an intense band of RIOK3 stain and then a by a wider band of F-actin.
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Figure 4: RIOK3 associates with TPM3 and actin in cells. (A) Silver-stained PAGE gel of anti-FLAG immunoprecipitates from MDA-MB-231 cells expressing either RIOK3 or FLAG-RIOK3. Species detected by tandem mass spectrometry only in the FLAG-RIOK3 sample are indicated on the right of the gel by gene symbol. (B) Confocal images of MDA-MB-231 cells in normoxia (Nor) and hypoxia (Hyp) demonstrate colocalisation of RIOK3 and TPM3. Scale bar = 10 μm. Phase contrast (PC) images show changes in cellular appearance. (C) Confocal images of MDA-MB-231 cells demonstrate that RIOK3 associates with actin. In normoxia RIOK3 aggregates are often surrounded by F-actin (arrowheads). In hypoxia RIOK3 aggregates disperse and F-actin organisation changes to form stress fibers and a dense F-actin network adjacent to the leading edge (arrowheads). Scale bar = 20 μm. (D) Magnified images of regions in C. Scale bar = 2 μm. (E) Pixel density of F-actin and RIOK3 stain at the leading edge of the hypoxic cell (from D). F-actin is observed immediately adjacent to the cell edge, followed by an intense band of RIOK3 stain and then a by a wider band of F-actin.

Mentions: As an alternative approach to understand the basis of RIOK3 function in regulating cell migration, we carried out co-immunoprecipitation studies using FLAG-tagged RIOK3 coupled with tandem mass spectrometry to identify interacting species (Fig 4A). We identified a number of interacting proteins including components of the actin cytoskeleton including actins (ACTG1, ACTA2), tropomyosins (TPM3, TPM4) and tropomodulin 3 (TMOD3). Ribosomal subunits (RPS3, RPS14, RPS16, RPS18, RPS20, RPL27A, RPL30) were also identified, consistent with the reported role of RIOK3 in ribosomal biogenesis.17


Hypoxic regulation of RIOK3 is a major mechanism for cancer cell invasion and metastasis.

Singleton DC, Rouhi P, Zois CE, Haider S, Li JL, Kessler BM, Cao Y, Harris AL - Oncogene (2014)

RIOK3 associates with TPM3 and actin in cells. (A) Silver-stained PAGE gel of anti-FLAG immunoprecipitates from MDA-MB-231 cells expressing either RIOK3 or FLAG-RIOK3. Species detected by tandem mass spectrometry only in the FLAG-RIOK3 sample are indicated on the right of the gel by gene symbol. (B) Confocal images of MDA-MB-231 cells in normoxia (Nor) and hypoxia (Hyp) demonstrate colocalisation of RIOK3 and TPM3. Scale bar = 10 μm. Phase contrast (PC) images show changes in cellular appearance. (C) Confocal images of MDA-MB-231 cells demonstrate that RIOK3 associates with actin. In normoxia RIOK3 aggregates are often surrounded by F-actin (arrowheads). In hypoxia RIOK3 aggregates disperse and F-actin organisation changes to form stress fibers and a dense F-actin network adjacent to the leading edge (arrowheads). Scale bar = 20 μm. (D) Magnified images of regions in C. Scale bar = 2 μm. (E) Pixel density of F-actin and RIOK3 stain at the leading edge of the hypoxic cell (from D). F-actin is observed immediately adjacent to the cell edge, followed by an intense band of RIOK3 stain and then a by a wider band of F-actin.
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Figure 4: RIOK3 associates with TPM3 and actin in cells. (A) Silver-stained PAGE gel of anti-FLAG immunoprecipitates from MDA-MB-231 cells expressing either RIOK3 or FLAG-RIOK3. Species detected by tandem mass spectrometry only in the FLAG-RIOK3 sample are indicated on the right of the gel by gene symbol. (B) Confocal images of MDA-MB-231 cells in normoxia (Nor) and hypoxia (Hyp) demonstrate colocalisation of RIOK3 and TPM3. Scale bar = 10 μm. Phase contrast (PC) images show changes in cellular appearance. (C) Confocal images of MDA-MB-231 cells demonstrate that RIOK3 associates with actin. In normoxia RIOK3 aggregates are often surrounded by F-actin (arrowheads). In hypoxia RIOK3 aggregates disperse and F-actin organisation changes to form stress fibers and a dense F-actin network adjacent to the leading edge (arrowheads). Scale bar = 20 μm. (D) Magnified images of regions in C. Scale bar = 2 μm. (E) Pixel density of F-actin and RIOK3 stain at the leading edge of the hypoxic cell (from D). F-actin is observed immediately adjacent to the cell edge, followed by an intense band of RIOK3 stain and then a by a wider band of F-actin.
Mentions: As an alternative approach to understand the basis of RIOK3 function in regulating cell migration, we carried out co-immunoprecipitation studies using FLAG-tagged RIOK3 coupled with tandem mass spectrometry to identify interacting species (Fig 4A). We identified a number of interacting proteins including components of the actin cytoskeleton including actins (ACTG1, ACTA2), tropomyosins (TPM3, TPM4) and tropomodulin 3 (TMOD3). Ribosomal subunits (RPS3, RPS14, RPS16, RPS18, RPS20, RPL27A, RPL30) were also identified, consistent with the reported role of RIOK3 in ribosomal biogenesis.17

Bottom Line: This phenotypic change resulted in reduced cell migration in two-dimensional cultures and inhibition of cell invasion through three-dimensional extracellular matrix.Depletion of RIOK3 in cells resulted in fewer and less organised actin filaments.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Oncology, Weatherall Institute of Molecular Medicine, John Radcliffe Hospital, University of Oxford, Oxford, UK.

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.

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Related in: MedlinePlus