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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 promotes cell motility and invasion in PDAC cell culture(A) Effect of IGF2BP3-siRNA in S2-013 cells. Western blots probed with anti-IGF2BP3 antibody show two S2-013 IGF2BP3-RNAi clones (siIGF-1-2) transfected with siRNA targeting IGF2BP3 and two scrambled control-RNAi clones (Scr-1-2). (B) Confluent cell monolayers of control-RNAi S2-013 cells or IGF2BP3-RNAi S2-013 cells were wounded (upper panels). Cells that migrated into an initially cell-free wound were counted; specifically, cells in four defined areas per group per experiment were counted (lower panel). Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.001 compared to Scr-1 or Scr-2 (Student's t-test). (C) Control-RNAi or IGF2BP3-RNAi S2-013 cells were seeded into two-chamber motility chambers. Migrating cells in four fields per group were counted. Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.001 compared to Scr-1 or Scr-2 (Student's t-test). (D) Control-RNAi or IGF2BP3-RNAi S2-013 cells were seeded into Matrigel invasion chambers. Invading cells in four fields per group were counted. Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.001 compared to Scr-1 or Scr-2 (Student's t-test). (E) The mock control vector or myc-tagged IGF2BP3-rescue construct was transiently transfected into control-RNAi and IGF2BP3-RNAi cells; 48 h later, the two-chamber invasion assay was performed. Western blots probed with anti-IGF2BP3 antibody are shown in left panels. Closed arrow head, endogenous IGF2BP3; open arrow head, exogenous IGF2BP3. Invading cells in four fields per group were counted (right panels). Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.005 compared with corresponding siIGF-1 or siIGF-2 transfected mock vector (Student's t-test). (F) Hematoxylin and eosin staining of paraffin sections from xenograft, pancreatic tumors derived from control-RNAi S2-013 cells or IGF2BP3-RNAi S2-013 cells. Control-RNAi cells aggressively invaded surrounding pancreatic tissue in mice. IGF2BP3-RNAi cells were evident only at the capsular interface of tumor with the pancreas. R, retroperitoneal soft-tissue; P, peritoneal wall; N, normal pancreatic tissue. Original magnification: × 200.
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Figure 2: IGF2BP3 promotes cell motility and invasion in PDAC cell culture(A) Effect of IGF2BP3-siRNA in S2-013 cells. Western blots probed with anti-IGF2BP3 antibody show two S2-013 IGF2BP3-RNAi clones (siIGF-1-2) transfected with siRNA targeting IGF2BP3 and two scrambled control-RNAi clones (Scr-1-2). (B) Confluent cell monolayers of control-RNAi S2-013 cells or IGF2BP3-RNAi S2-013 cells were wounded (upper panels). Cells that migrated into an initially cell-free wound were counted; specifically, cells in four defined areas per group per experiment were counted (lower panel). Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.001 compared to Scr-1 or Scr-2 (Student's t-test). (C) Control-RNAi or IGF2BP3-RNAi S2-013 cells were seeded into two-chamber motility chambers. Migrating cells in four fields per group were counted. Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.001 compared to Scr-1 or Scr-2 (Student's t-test). (D) Control-RNAi or IGF2BP3-RNAi S2-013 cells were seeded into Matrigel invasion chambers. Invading cells in four fields per group were counted. Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.001 compared to Scr-1 or Scr-2 (Student's t-test). (E) The mock control vector or myc-tagged IGF2BP3-rescue construct was transiently transfected into control-RNAi and IGF2BP3-RNAi cells; 48 h later, the two-chamber invasion assay was performed. Western blots probed with anti-IGF2BP3 antibody are shown in left panels. Closed arrow head, endogenous IGF2BP3; open arrow head, exogenous IGF2BP3. Invading cells in four fields per group were counted (right panels). Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.005 compared with corresponding siIGF-1 or siIGF-2 transfected mock vector (Student's t-test). (F) Hematoxylin and eosin staining of paraffin sections from xenograft, pancreatic tumors derived from control-RNAi S2-013 cells or IGF2BP3-RNAi S2-013 cells. Control-RNAi cells aggressively invaded surrounding pancreatic tissue in mice. IGF2BP3-RNAi cells were evident only at the capsular interface of tumor with the pancreas. R, retroperitoneal soft-tissue; P, peritoneal wall; N, normal pancreatic tissue. Original magnification: × 200.

Mentions: To investigate whether IGF2BP3 affected cell motility and invasion, IGF2BP3 expression in S2-013 cells was suppressed by vector-based expression of an IGF2BP3- siRNA. To achieve substantial suppression of IGF2BP3, we established cell clones subject to RNA interference (RNAi) by expressing an IGF2BP3-siRNA. IGF2BP3 knockdown was confirmed on immunoblots (Figure 2A). Suppression of IGF2BP3 expression in S2-013 cells did not affect cell growth in an in vitro MTT assay (data not shown), but it did inhibit cell motility into a wounded area of confluent cultures (Figure 2B). In trans-well motility assays, motility of S2-013 cells was significantly lower in IGF2BP3-knockdown cells than in control cells (Figure 2C). In two-chamber invasion assays, IGF2BP3-RNAi S2-013 cells were significantly less invasive than the control-RNAi S2-013 cells (Figure 2D). We found that transfection of an IGF2BP3-rescue construct into IGF2BP3-RNAi S2-013 cells abrogated the changes to cell invasiveness caused by the IGF2BP3-RNAi (Figure 2E).


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 promotes cell motility and invasion in PDAC cell culture(A) Effect of IGF2BP3-siRNA in S2-013 cells. Western blots probed with anti-IGF2BP3 antibody show two S2-013 IGF2BP3-RNAi clones (siIGF-1-2) transfected with siRNA targeting IGF2BP3 and two scrambled control-RNAi clones (Scr-1-2). (B) Confluent cell monolayers of control-RNAi S2-013 cells or IGF2BP3-RNAi S2-013 cells were wounded (upper panels). Cells that migrated into an initially cell-free wound were counted; specifically, cells in four defined areas per group per experiment were counted (lower panel). Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.001 compared to Scr-1 or Scr-2 (Student's t-test). (C) Control-RNAi or IGF2BP3-RNAi S2-013 cells were seeded into two-chamber motility chambers. Migrating cells in four fields per group were counted. Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.001 compared to Scr-1 or Scr-2 (Student's t-test). (D) Control-RNAi or IGF2BP3-RNAi S2-013 cells were seeded into Matrigel invasion chambers. Invading cells in four fields per group were counted. Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.001 compared to Scr-1 or Scr-2 (Student's t-test). (E) The mock control vector or myc-tagged IGF2BP3-rescue construct was transiently transfected into control-RNAi and IGF2BP3-RNAi cells; 48 h later, the two-chamber invasion assay was performed. Western blots probed with anti-IGF2BP3 antibody are shown in left panels. Closed arrow head, endogenous IGF2BP3; open arrow head, exogenous IGF2BP3. Invading cells in four fields per group were counted (right panels). Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.005 compared with corresponding siIGF-1 or siIGF-2 transfected mock vector (Student's t-test). (F) Hematoxylin and eosin staining of paraffin sections from xenograft, pancreatic tumors derived from control-RNAi S2-013 cells or IGF2BP3-RNAi S2-013 cells. Control-RNAi cells aggressively invaded surrounding pancreatic tissue in mice. IGF2BP3-RNAi cells were evident only at the capsular interface of tumor with the pancreas. R, retroperitoneal soft-tissue; P, peritoneal wall; N, normal pancreatic tissue. Original magnification: × 200.
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Figure 2: IGF2BP3 promotes cell motility and invasion in PDAC cell culture(A) Effect of IGF2BP3-siRNA in S2-013 cells. Western blots probed with anti-IGF2BP3 antibody show two S2-013 IGF2BP3-RNAi clones (siIGF-1-2) transfected with siRNA targeting IGF2BP3 and two scrambled control-RNAi clones (Scr-1-2). (B) Confluent cell monolayers of control-RNAi S2-013 cells or IGF2BP3-RNAi S2-013 cells were wounded (upper panels). Cells that migrated into an initially cell-free wound were counted; specifically, cells in four defined areas per group per experiment were counted (lower panel). Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.001 compared to Scr-1 or Scr-2 (Student's t-test). (C) Control-RNAi or IGF2BP3-RNAi S2-013 cells were seeded into two-chamber motility chambers. Migrating cells in four fields per group were counted. Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.001 compared to Scr-1 or Scr-2 (Student's t-test). (D) Control-RNAi or IGF2BP3-RNAi S2-013 cells were seeded into Matrigel invasion chambers. Invading cells in four fields per group were counted. Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.001 compared to Scr-1 or Scr-2 (Student's t-test). (E) The mock control vector or myc-tagged IGF2BP3-rescue construct was transiently transfected into control-RNAi and IGF2BP3-RNAi cells; 48 h later, the two-chamber invasion assay was performed. Western blots probed with anti-IGF2BP3 antibody are shown in left panels. Closed arrow head, endogenous IGF2BP3; open arrow head, exogenous IGF2BP3. Invading cells in four fields per group were counted (right panels). Data derive from three independent experiments. Columns, mean; bars, SD. *p < 0.005 compared with corresponding siIGF-1 or siIGF-2 transfected mock vector (Student's t-test). (F) Hematoxylin and eosin staining of paraffin sections from xenograft, pancreatic tumors derived from control-RNAi S2-013 cells or IGF2BP3-RNAi S2-013 cells. Control-RNAi cells aggressively invaded surrounding pancreatic tissue in mice. IGF2BP3-RNAi cells were evident only at the capsular interface of tumor with the pancreas. R, retroperitoneal soft-tissue; P, peritoneal wall; N, normal pancreatic tissue. Original magnification: × 200.
Mentions: To investigate whether IGF2BP3 affected cell motility and invasion, IGF2BP3 expression in S2-013 cells was suppressed by vector-based expression of an IGF2BP3- siRNA. To achieve substantial suppression of IGF2BP3, we established cell clones subject to RNA interference (RNAi) by expressing an IGF2BP3-siRNA. IGF2BP3 knockdown was confirmed on immunoblots (Figure 2A). Suppression of IGF2BP3 expression in S2-013 cells did not affect cell growth in an in vitro MTT assay (data not shown), but it did inhibit cell motility into a wounded area of confluent cultures (Figure 2B). In trans-well motility assays, motility of S2-013 cells was significantly lower in IGF2BP3-knockdown cells than in control cells (Figure 2C). In two-chamber invasion assays, IGF2BP3-RNAi S2-013 cells were significantly less invasive than the control-RNAi S2-013 cells (Figure 2D). We found that transfection of an IGF2BP3-rescue construct into IGF2BP3-RNAi S2-013 cells abrogated the changes to cell invasiveness caused by the IGF2BP3-RNAi (Figure 2E).

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