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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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ARF6 and ARHGEF4 promote cell motility and invasion via forming cell protrusions(A) RNA oligonucleotides were transiently transfected into S2-013 cells; the siRNAs targeted ARF6 (siARF6) or ARHGEF4 (siARHGEF4); the negative control was a scrambled RNA (Scr). Western blot was performed using anti-ARF6 or anti-ARHGEF4 antibody. (B) Oligonucleotides were transiently transfected into S2-013 cells; the siRNA targeted ARF6 (siARF6); the negative control was a scrambled RNA (Scr). S2-013 cells transfected with Scr or siARF6 were incubated on fibronectin, and cells were stained with anti-ARF6 antibody (green) and phalloidin (red). Arrows; peripheral actin structures in cell protrusions of Scr-transfected cells. Blue, DAPI staining. Bars, 10 μm. (C) Oligonucleotides were transiently transfected into S2-013 cells; the siRNA (siARHGEF4) targeted ARHGEF4. Scr- or siAHGEF4- transfected S2-013 cells were incubated on fibronectin, and cells were stained with anti-ARHGEF4 antibody (green) and phalloidin (red). Arrows; peripheral actin structures in cell protrusions of Scr-transfected cells. Blue, DAPI staining. Bars, 10 μm. (D) Quantification of data shown in Figure 7B and 7C, as described in Figure 6B. Columns, mean; bars, SD. *p < 0.001 compared with Scr-transfected controls (Student's t-test). (E, F) Oligonucleotides targeting ARF6 or ARHGEF4 or Scr was transiently transfected into S2-013 or PANC-1cells. The motility (E) and two-chamber invasion assays (F) were performed. Migrating cells in four fields per group were scored. Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.001 compared with Scr-transfected control (Student's t-test).
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Figure 7: ARF6 and ARHGEF4 promote cell motility and invasion via forming cell protrusions(A) RNA oligonucleotides were transiently transfected into S2-013 cells; the siRNAs targeted ARF6 (siARF6) or ARHGEF4 (siARHGEF4); the negative control was a scrambled RNA (Scr). Western blot was performed using anti-ARF6 or anti-ARHGEF4 antibody. (B) Oligonucleotides were transiently transfected into S2-013 cells; the siRNA targeted ARF6 (siARF6); the negative control was a scrambled RNA (Scr). S2-013 cells transfected with Scr or siARF6 were incubated on fibronectin, and cells were stained with anti-ARF6 antibody (green) and phalloidin (red). Arrows; peripheral actin structures in cell protrusions of Scr-transfected cells. Blue, DAPI staining. Bars, 10 μm. (C) Oligonucleotides were transiently transfected into S2-013 cells; the siRNA (siARHGEF4) targeted ARHGEF4. Scr- or siAHGEF4- transfected S2-013 cells were incubated on fibronectin, and cells were stained with anti-ARHGEF4 antibody (green) and phalloidin (red). Arrows; peripheral actin structures in cell protrusions of Scr-transfected cells. Blue, DAPI staining. Bars, 10 μm. (D) Quantification of data shown in Figure 7B and 7C, as described in Figure 6B. Columns, mean; bars, SD. *p < 0.001 compared with Scr-transfected controls (Student's t-test). (E, F) Oligonucleotides targeting ARF6 or ARHGEF4 or Scr was transiently transfected into S2-013 or PANC-1cells. The motility (E) and two-chamber invasion assays (F) were performed. Migrating cells in four fields per group were scored. Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.001 compared with Scr-transfected control (Student's t-test).

Mentions: To determine whether ARF6, ARHGEF4, or both participated in the induction of membrane protrusions, we analyzed peripheral actin structures in membrane ruffles of control-RNAi, ARF6-RNAi, and ARHGEF4-RNAi S2-013 cells cultured on fibronectin. Based on western blot data, 72 h after transfection, expression of ARF6 or ARHGEF4 was markedly higher in control siRNA-transfected S2-013 cells than in ARF6 siRNA-transfected or ARHGEF4 siRNA-transfected, respectively (Figure 7A). Confocal microscopy revealed that ARF6- or ARHGEF4-knockdown in S2-013 decreased peripheral actin structures (Figure 7B for ARF6-knockdown and Figure 7C for ARHGEF4-knockdown). Furthermore, ARF6- or ARHGEF4-knockdown in S2-013 cells significantly inhibited fibronectin-mediated formation of membrane protrusions (Figure 7D). These results indicated that ARF6 and ARHGEF4 played a role in forming these membrane protrusions.


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)

ARF6 and ARHGEF4 promote cell motility and invasion via forming cell protrusions(A) RNA oligonucleotides were transiently transfected into S2-013 cells; the siRNAs targeted ARF6 (siARF6) or ARHGEF4 (siARHGEF4); the negative control was a scrambled RNA (Scr). Western blot was performed using anti-ARF6 or anti-ARHGEF4 antibody. (B) Oligonucleotides were transiently transfected into S2-013 cells; the siRNA targeted ARF6 (siARF6); the negative control was a scrambled RNA (Scr). S2-013 cells transfected with Scr or siARF6 were incubated on fibronectin, and cells were stained with anti-ARF6 antibody (green) and phalloidin (red). Arrows; peripheral actin structures in cell protrusions of Scr-transfected cells. Blue, DAPI staining. Bars, 10 μm. (C) Oligonucleotides were transiently transfected into S2-013 cells; the siRNA (siARHGEF4) targeted ARHGEF4. Scr- or siAHGEF4- transfected S2-013 cells were incubated on fibronectin, and cells were stained with anti-ARHGEF4 antibody (green) and phalloidin (red). Arrows; peripheral actin structures in cell protrusions of Scr-transfected cells. Blue, DAPI staining. Bars, 10 μm. (D) Quantification of data shown in Figure 7B and 7C, as described in Figure 6B. Columns, mean; bars, SD. *p < 0.001 compared with Scr-transfected controls (Student's t-test). (E, F) Oligonucleotides targeting ARF6 or ARHGEF4 or Scr was transiently transfected into S2-013 or PANC-1cells. The motility (E) and two-chamber invasion assays (F) were performed. Migrating cells in four fields per group were scored. Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.001 compared with Scr-transfected control (Student's t-test).
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Figure 7: ARF6 and ARHGEF4 promote cell motility and invasion via forming cell protrusions(A) RNA oligonucleotides were transiently transfected into S2-013 cells; the siRNAs targeted ARF6 (siARF6) or ARHGEF4 (siARHGEF4); the negative control was a scrambled RNA (Scr). Western blot was performed using anti-ARF6 or anti-ARHGEF4 antibody. (B) Oligonucleotides were transiently transfected into S2-013 cells; the siRNA targeted ARF6 (siARF6); the negative control was a scrambled RNA (Scr). S2-013 cells transfected with Scr or siARF6 were incubated on fibronectin, and cells were stained with anti-ARF6 antibody (green) and phalloidin (red). Arrows; peripheral actin structures in cell protrusions of Scr-transfected cells. Blue, DAPI staining. Bars, 10 μm. (C) Oligonucleotides were transiently transfected into S2-013 cells; the siRNA (siARHGEF4) targeted ARHGEF4. Scr- or siAHGEF4- transfected S2-013 cells were incubated on fibronectin, and cells were stained with anti-ARHGEF4 antibody (green) and phalloidin (red). Arrows; peripheral actin structures in cell protrusions of Scr-transfected cells. Blue, DAPI staining. Bars, 10 μm. (D) Quantification of data shown in Figure 7B and 7C, as described in Figure 6B. Columns, mean; bars, SD. *p < 0.001 compared with Scr-transfected controls (Student's t-test). (E, F) Oligonucleotides targeting ARF6 or ARHGEF4 or Scr was transiently transfected into S2-013 or PANC-1cells. The motility (E) and two-chamber invasion assays (F) were performed. Migrating cells in four fields per group were scored. Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.001 compared with Scr-transfected control (Student's t-test).
Mentions: To determine whether ARF6, ARHGEF4, or both participated in the induction of membrane protrusions, we analyzed peripheral actin structures in membrane ruffles of control-RNAi, ARF6-RNAi, and ARHGEF4-RNAi S2-013 cells cultured on fibronectin. Based on western blot data, 72 h after transfection, expression of ARF6 or ARHGEF4 was markedly higher in control siRNA-transfected S2-013 cells than in ARF6 siRNA-transfected or ARHGEF4 siRNA-transfected, respectively (Figure 7A). Confocal microscopy revealed that ARF6- or ARHGEF4-knockdown in S2-013 decreased peripheral actin structures (Figure 7B for ARF6-knockdown and Figure 7C for ARHGEF4-knockdown). Furthermore, ARF6- or ARHGEF4-knockdown in S2-013 cells significantly inhibited fibronectin-mediated formation of membrane protrusions (Figure 7D). These results indicated that ARF6 and ARHGEF4 played a role in forming these membrane protrusions.

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