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IGF2BP3-mediated translation in cell protrusions promotes cell invasiveness and metastasis of pancreatic cancer.

Taniuchi K, Furihata M, Hanazaki K, Saito M, Saibara T - Oncotarget (2014)

Bottom Line: Specific IGF2BP3-bound transcripts-ARF6 and ARHGEF4-that are preferentially translated in membrane protrusions induce further formation of membrane protrusions; consequently, IGF2BP3 promotes cell invasiveness and tumor metastasis.Our results provide insight into the link between regulation of localized translation in cell protrusions and the invasiveness and metastasis of pancreatic cancers.New therapies that prevent local translation in cell protrusions may hold significant clinical promise.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Kochi Medical School, Kochi University, Nankoku, Kochi , Japan.

ABSTRACT
Pancreatic cancers are aggressive because they are highly invasive and highly metastatic; moreover, effective treatments for aggressive pancreatic cancers are lacking. Here, we report that IGF2BP3 promoted the invasiveness and metastasis of pancreatic cancers through locally translated IGF2BP3-bound transcripts. In neural cells, transcripts sorted into cytoplasmic RNA granules are transported to dendrites and translated in these dendrites, thereby mediating long-term synaptic plasticity; however, such cytoplasmic RNA granules are not known to contribute to the progression of pancreatic cancer. We show evidence that IGF2BP3 and IGF2BP3-bound transcripts are localized in cytoplasmic RNA granules that accumulate in membrane protrusions of pancreatic cancer cells. Specific IGF2BP3-bound transcripts-ARF6 and ARHGEF4-that are preferentially translated in membrane protrusions induce further formation of membrane protrusions; consequently, IGF2BP3 promotes cell invasiveness and tumor metastasis. Our results provide insight into the link between regulation of localized translation in cell protrusions and the invasiveness and metastasis of pancreatic cancers. New therapies that prevent local translation in cell protrusions may hold significant clinical promise.

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IGF2BP3 colocalizes with ARF6 mRNA and ARHGEF4 mRNA(A) Those IGF2BP3-bound transcripts that were identified in the RIP analysis and that are included in GO terms relevant to cell motility, invasiveness, and protrusions are shown. Underlines indicate ARF6 and ARHGEF4. (B) The association between IGF2BP3 and ARF6 mRNA or ARHGEF4 mRNA in S2-013 cells cultured on fibronectin was tested via IGF2BP3-IP or control-IP and subsequent RT-PCR amplification of any ARF6, ARHGEF4, and Ubiquitin C in the immunoprecipitate (right panels). Proteins in immunoprecipitates were examined on western blots probed with antibodies against IGF2BP3 and CD63 (left panels). Rabbit IgG isotype control and anti-CD63 antibodies were used as negative controls for coimmunoprecipitation. (C) Colocalization of IGF2BP3 protein (green), and ARF6 or ARHGEF4 mRNA (red) in S2-013 cells cultured on fibronectin. Ubiquitin C mRNA was used as a negative control for colocalization. Arrows, mRNAs colocalized with IGF2BP3 in cell protrusions. Blue, DAPI staining. Bars, 10 μm.
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Figure 4: IGF2BP3 colocalizes with ARF6 mRNA and ARHGEF4 mRNA(A) Those IGF2BP3-bound transcripts that were identified in the RIP analysis and that are included in GO terms relevant to cell motility, invasiveness, and protrusions are shown. Underlines indicate ARF6 and ARHGEF4. (B) The association between IGF2BP3 and ARF6 mRNA or ARHGEF4 mRNA in S2-013 cells cultured on fibronectin was tested via IGF2BP3-IP or control-IP and subsequent RT-PCR amplification of any ARF6, ARHGEF4, and Ubiquitin C in the immunoprecipitate (right panels). Proteins in immunoprecipitates were examined on western blots probed with antibodies against IGF2BP3 and CD63 (left panels). Rabbit IgG isotype control and anti-CD63 antibodies were used as negative controls for coimmunoprecipitation. (C) Colocalization of IGF2BP3 protein (green), and ARF6 or ARHGEF4 mRNA (red) in S2-013 cells cultured on fibronectin. Ubiquitin C mRNA was used as a negative control for colocalization. Arrows, mRNAs colocalized with IGF2BP3 in cell protrusions. Blue, DAPI staining. Bars, 10 μm.

Mentions: To investigate whether RNA itself was present in IGF2BP3-containing granules and to identify any IGF2BP3-bound transcripts localized in these granules, we performed RNA immunoprecipitation with anti-IGF2BP3 and extracts from S2-013 cells that had been cultured on fibronectin; we then used next-generation sequencing to identify any mRNAs in the resultant immunoprecipitates (Figure S1A-D). The results of RIP assay are presented as log ratios in Table S1. We identified 2,826 RNAs that were significantly enriched in anti-IGF2BP3 immunoprecipitates relative to rabbit IgG isotype control immunoprecipitates (Table S1). The complete gene list derived from the 2,826 RNAs was uploaded onto the Gene Expression Omnibus Database http://www.ncbi.nlm.nih.gov/geo/ (GEO accession: GSE47597). To gain further insight into the biological functionalities of these IGF2BP3-bound mRNAs, the list of identified genes were subjected to gene ontology (GO) analysis focused on the GO category of “Biological Processes”. A larger number of GO terms matched the gene list (P < 10−5; Table S2), and this GO set was significantly enriched with cellular functions relevant to apoptosis, cell cycle, signal transduction, cell proliferation, cell adhesion, and cell migration. The transcripts that matched any GO term related to both cell migration and cell protrusion are listed in Figure 4A. We used RT-PCR to validate two of transcripts from this list; these IGF2BP3-bound mRNAs were ADP-ribosylation factor 6 (ARF6) and Rho guanine nucleotide exchange factor 4 (ARHGEF4). RT-PCR was performed on complexes immunoprecipitated with anti-IGF2BP3, rabbit IgG isotype control antibody, or anti-CD63 antibody; neither isotype control antibody nor anti-CD63 was expected to immunoprecipitate ARF6 or ARHGEF4 mRNA (Figure 4B). Both transcripts immunoprecipitated with anti-IGF2BP3, but neither transcript immunoprecipitated with isotype control antibody or anti-CD63.


IGF2BP3-mediated translation in cell protrusions promotes cell invasiveness and metastasis of pancreatic cancer.

Taniuchi K, Furihata M, Hanazaki K, Saito M, Saibara T - Oncotarget (2014)

IGF2BP3 colocalizes with ARF6 mRNA and ARHGEF4 mRNA(A) Those IGF2BP3-bound transcripts that were identified in the RIP analysis and that are included in GO terms relevant to cell motility, invasiveness, and protrusions are shown. Underlines indicate ARF6 and ARHGEF4. (B) The association between IGF2BP3 and ARF6 mRNA or ARHGEF4 mRNA in S2-013 cells cultured on fibronectin was tested via IGF2BP3-IP or control-IP and subsequent RT-PCR amplification of any ARF6, ARHGEF4, and Ubiquitin C in the immunoprecipitate (right panels). Proteins in immunoprecipitates were examined on western blots probed with antibodies against IGF2BP3 and CD63 (left panels). Rabbit IgG isotype control and anti-CD63 antibodies were used as negative controls for coimmunoprecipitation. (C) Colocalization of IGF2BP3 protein (green), and ARF6 or ARHGEF4 mRNA (red) in S2-013 cells cultured on fibronectin. Ubiquitin C mRNA was used as a negative control for colocalization. Arrows, mRNAs colocalized with IGF2BP3 in cell protrusions. Blue, DAPI staining. Bars, 10 μm.
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Related In: Results  -  Collection

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Figure 4: IGF2BP3 colocalizes with ARF6 mRNA and ARHGEF4 mRNA(A) Those IGF2BP3-bound transcripts that were identified in the RIP analysis and that are included in GO terms relevant to cell motility, invasiveness, and protrusions are shown. Underlines indicate ARF6 and ARHGEF4. (B) The association between IGF2BP3 and ARF6 mRNA or ARHGEF4 mRNA in S2-013 cells cultured on fibronectin was tested via IGF2BP3-IP or control-IP and subsequent RT-PCR amplification of any ARF6, ARHGEF4, and Ubiquitin C in the immunoprecipitate (right panels). Proteins in immunoprecipitates were examined on western blots probed with antibodies against IGF2BP3 and CD63 (left panels). Rabbit IgG isotype control and anti-CD63 antibodies were used as negative controls for coimmunoprecipitation. (C) Colocalization of IGF2BP3 protein (green), and ARF6 or ARHGEF4 mRNA (red) in S2-013 cells cultured on fibronectin. Ubiquitin C mRNA was used as a negative control for colocalization. Arrows, mRNAs colocalized with IGF2BP3 in cell protrusions. Blue, DAPI staining. Bars, 10 μm.
Mentions: To investigate whether RNA itself was present in IGF2BP3-containing granules and to identify any IGF2BP3-bound transcripts localized in these granules, we performed RNA immunoprecipitation with anti-IGF2BP3 and extracts from S2-013 cells that had been cultured on fibronectin; we then used next-generation sequencing to identify any mRNAs in the resultant immunoprecipitates (Figure S1A-D). The results of RIP assay are presented as log ratios in Table S1. We identified 2,826 RNAs that were significantly enriched in anti-IGF2BP3 immunoprecipitates relative to rabbit IgG isotype control immunoprecipitates (Table S1). The complete gene list derived from the 2,826 RNAs was uploaded onto the Gene Expression Omnibus Database http://www.ncbi.nlm.nih.gov/geo/ (GEO accession: GSE47597). To gain further insight into the biological functionalities of these IGF2BP3-bound mRNAs, the list of identified genes were subjected to gene ontology (GO) analysis focused on the GO category of “Biological Processes”. A larger number of GO terms matched the gene list (P < 10−5; Table S2), and this GO set was significantly enriched with cellular functions relevant to apoptosis, cell cycle, signal transduction, cell proliferation, cell adhesion, and cell migration. The transcripts that matched any GO term related to both cell migration and cell protrusion are listed in Figure 4A. We used RT-PCR to validate two of transcripts from this list; these IGF2BP3-bound mRNAs were ADP-ribosylation factor 6 (ARF6) and Rho guanine nucleotide exchange factor 4 (ARHGEF4). RT-PCR was performed on complexes immunoprecipitated with anti-IGF2BP3, rabbit IgG isotype control antibody, or anti-CD63 antibody; neither isotype control antibody nor anti-CD63 was expected to immunoprecipitate ARF6 or ARHGEF4 mRNA (Figure 4B). Both transcripts immunoprecipitated with anti-IGF2BP3, but neither transcript immunoprecipitated with isotype control antibody or anti-CD63.

Bottom Line: Specific IGF2BP3-bound transcripts-ARF6 and ARHGEF4-that are preferentially translated in membrane protrusions induce further formation of membrane protrusions; consequently, IGF2BP3 promotes cell invasiveness and tumor metastasis.Our results provide insight into the link between regulation of localized translation in cell protrusions and the invasiveness and metastasis of pancreatic cancers.New therapies that prevent local translation in cell protrusions may hold significant clinical promise.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, Kochi Medical School, Kochi University, Nankoku, Kochi , Japan.

ABSTRACT
Pancreatic cancers are aggressive because they are highly invasive and highly metastatic; moreover, effective treatments for aggressive pancreatic cancers are lacking. Here, we report that IGF2BP3 promoted the invasiveness and metastasis of pancreatic cancers through locally translated IGF2BP3-bound transcripts. In neural cells, transcripts sorted into cytoplasmic RNA granules are transported to dendrites and translated in these dendrites, thereby mediating long-term synaptic plasticity; however, such cytoplasmic RNA granules are not known to contribute to the progression of pancreatic cancer. We show evidence that IGF2BP3 and IGF2BP3-bound transcripts are localized in cytoplasmic RNA granules that accumulate in membrane protrusions of pancreatic cancer cells. Specific IGF2BP3-bound transcripts-ARF6 and ARHGEF4-that are preferentially translated in membrane protrusions induce further formation of membrane protrusions; consequently, IGF2BP3 promotes cell invasiveness and tumor metastasis. Our results provide insight into the link between regulation of localized translation in cell protrusions and the invasiveness and metastasis of pancreatic cancers. New therapies that prevent local translation in cell protrusions may hold significant clinical promise.

Show MeSH
Related in: MedlinePlus