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Hypoxia/reoxygenation-experienced cancer cell migration and metastasis are regulated by Rap1- and Rac1-GTPase activation via the expression of thymosin beta-4.

Lee JW, Ryu YK, Ji YH, Kang JH, Moon EY - Oncotarget (2015)

Bottom Line: Inhibition of Tβ4 expression using transcription activator-like effector nucleases (TALEN) significantly decreased lung metastasis of B16F10 cells.Rap1-regulated Rac1 activity was decreased by a dominant negative Rap1 (Rap1N17), and increased by 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (CPT), a Rap1 activator.These data suggest that a combination therapy targeting both Rap1 and Rac1 activity may be an effective method of inhibiting tumor metastasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience and Biotechnology, Sejong University, Seoul 143-747, Korea.

ABSTRACT
Signaling by small guanosine triphosphatases (GTPase), Rap1/Rac1, is one of the major pathways controlling cancer cell migration and tumor metastasis. Thymosin beta-4 (Tβ4), an actin-sequestering protein, has been shown to increase migration of cancer cells. Episodes of hypoxia and re-oxygenation (H/R) are an important phenomenon in tumor microenvironment (TME). We investigated whether Tβ4 could play as an intermediary to crosstalk between Rac1- and Rap1- GTPase activation under hypoxia/reoxygenation (H/R) conditions. Inhibition of Tβ4 expression using transcription activator-like effector nucleases (TALEN) significantly decreased lung metastasis of B16F10 cells. Rac1 and Rap1 activity, as well as cancer cell migration, increased following induction of Tβ4 expression in normoxia- or H/R-experienced cells, but were barely detectable in Tβ4-depleted cells. Rap1-regulated Rac1 activity was decreased by a dominant negative Rap1 (Rap1N17), and increased by 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (CPT), a Rap1 activator. In contrast, a Rac1-specific inhibitor, NSC23766, and dominant negative Rac1 (Rac1N17) enhanced Tβ4 expression and aberrant Rap1 activity. While NSC23766 and Rac1N17 incompletely inhibited tumor metastasis in vivo, and H/R-experienced cancer cell migration in vitro, more efficient attenuation of cancer cell migration was accomplished by simultaneous inactivation of Rap1 and Rac1 with Rap1N17 and Rac1N17, respectively. These data suggest that a combination therapy targeting both Rap1 and Rac1 activity may be an effective method of inhibiting tumor metastasis.

No MeSH data available.


Related in: MedlinePlus

Cancer cell migration is dependent on Tβ4-mediated activation of Rap1- and Rac1-GTPases(A–C) HeLa cells were transfected with pCMV-Tβ4 plasmid for 24 h. Tβ4 transcript levels were measured by RT-PCR (A, upper) or realtime PCR (B). Tβ4 protein levels were determined by western blotting (A, lower). Rac1 and Rap1 activity were detected by GST-pulldown and western blotting, as described in the Materials and methods (C). (D–F) Cells were transfected with scrambled control siRNA or Tβ4-siRNA, and incubated for 24 h, after which RNA was isolated. Tβ4 transcript levels were measured by RT-PCR (D, upper) or realtime PCR (E). Tβ4 protein levels were detected by western blotting (D, lower). Rac1 and Rap1 activities were detected by GST-pulldown and western blotting (F). (G and H) HeLa cells were plated on 35-mm2 dishes and incubated under normoxic conditions for 24 h, and then transfected with either control vector (mock) or pCMV-Tβ4 plasmids for 18 h. A confluent monolayer of HeLa cells was then scratched with a sterile pipet tip, and incubated for 12 h under normoxic conditions. Migration of cells into the space left by the scratch was photographed using a phase-contrast microscope at 200× magnification (G). The empty area remaining at 12 h was quantified using NIH image analysis software (version 1.62), and compared to that of the 0-h time point (H) Data shown are representative of three independent experiments (A–H). Band intensities were normalized relative to controls using NIH image analysis software (Image J, version 1.62). Fold changes relative to the control are indicated under each band (A, C, D, and F). Data in bar graph are presented as means ± SD (B, E, and H). *p < 0.05; **p < 0.01 relative to the control (A–H).
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Figure 2: Cancer cell migration is dependent on Tβ4-mediated activation of Rap1- and Rac1-GTPases(A–C) HeLa cells were transfected with pCMV-Tβ4 plasmid for 24 h. Tβ4 transcript levels were measured by RT-PCR (A, upper) or realtime PCR (B). Tβ4 protein levels were determined by western blotting (A, lower). Rac1 and Rap1 activity were detected by GST-pulldown and western blotting, as described in the Materials and methods (C). (D–F) Cells were transfected with scrambled control siRNA or Tβ4-siRNA, and incubated for 24 h, after which RNA was isolated. Tβ4 transcript levels were measured by RT-PCR (D, upper) or realtime PCR (E). Tβ4 protein levels were detected by western blotting (D, lower). Rac1 and Rap1 activities were detected by GST-pulldown and western blotting (F). (G and H) HeLa cells were plated on 35-mm2 dishes and incubated under normoxic conditions for 24 h, and then transfected with either control vector (mock) or pCMV-Tβ4 plasmids for 18 h. A confluent monolayer of HeLa cells was then scratched with a sterile pipet tip, and incubated for 12 h under normoxic conditions. Migration of cells into the space left by the scratch was photographed using a phase-contrast microscope at 200× magnification (G). The empty area remaining at 12 h was quantified using NIH image analysis software (version 1.62), and compared to that of the 0-h time point (H) Data shown are representative of three independent experiments (A–H). Band intensities were normalized relative to controls using NIH image analysis software (Image J, version 1.62). Fold changes relative to the control are indicated under each band (A, C, D, and F). Data in bar graph are presented as means ± SD (B, E, and H). *p < 0.05; **p < 0.01 relative to the control (A–H).

Mentions: To re-examine whether Rac1 and Rap1 activity was dependent on Tβ4 expression under normoxic conditions, we modified Tβ4 expression in HeLa cells using a pCMV-Tβ4 plasmid or Tβ4-siRNA. Overexpression of Tβ4 via the transfection of HeLa cells with pCMV-Tβ4 (Figure 2A and 2B) led to increased activity of both Rac1 and Rap1 relative to that of empty vector controls (Figure 2C). In contrast, when cells were treated with Tβ4-siRNA, Tβ4 expression was inhibited significantly (Figure 2D and 2E). The Tβ4 knockdown inhibited Rac1 and Rap1 activity (Figure 2F).


Hypoxia/reoxygenation-experienced cancer cell migration and metastasis are regulated by Rap1- and Rac1-GTPase activation via the expression of thymosin beta-4.

Lee JW, Ryu YK, Ji YH, Kang JH, Moon EY - Oncotarget (2015)

Cancer cell migration is dependent on Tβ4-mediated activation of Rap1- and Rac1-GTPases(A–C) HeLa cells were transfected with pCMV-Tβ4 plasmid for 24 h. Tβ4 transcript levels were measured by RT-PCR (A, upper) or realtime PCR (B). Tβ4 protein levels were determined by western blotting (A, lower). Rac1 and Rap1 activity were detected by GST-pulldown and western blotting, as described in the Materials and methods (C). (D–F) Cells were transfected with scrambled control siRNA or Tβ4-siRNA, and incubated for 24 h, after which RNA was isolated. Tβ4 transcript levels were measured by RT-PCR (D, upper) or realtime PCR (E). Tβ4 protein levels were detected by western blotting (D, lower). Rac1 and Rap1 activities were detected by GST-pulldown and western blotting (F). (G and H) HeLa cells were plated on 35-mm2 dishes and incubated under normoxic conditions for 24 h, and then transfected with either control vector (mock) or pCMV-Tβ4 plasmids for 18 h. A confluent monolayer of HeLa cells was then scratched with a sterile pipet tip, and incubated for 12 h under normoxic conditions. Migration of cells into the space left by the scratch was photographed using a phase-contrast microscope at 200× magnification (G). The empty area remaining at 12 h was quantified using NIH image analysis software (version 1.62), and compared to that of the 0-h time point (H) Data shown are representative of three independent experiments (A–H). Band intensities were normalized relative to controls using NIH image analysis software (Image J, version 1.62). Fold changes relative to the control are indicated under each band (A, C, D, and F). Data in bar graph are presented as means ± SD (B, E, and H). *p < 0.05; **p < 0.01 relative to the control (A–H).
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4496400&req=5

Figure 2: Cancer cell migration is dependent on Tβ4-mediated activation of Rap1- and Rac1-GTPases(A–C) HeLa cells were transfected with pCMV-Tβ4 plasmid for 24 h. Tβ4 transcript levels were measured by RT-PCR (A, upper) or realtime PCR (B). Tβ4 protein levels were determined by western blotting (A, lower). Rac1 and Rap1 activity were detected by GST-pulldown and western blotting, as described in the Materials and methods (C). (D–F) Cells were transfected with scrambled control siRNA or Tβ4-siRNA, and incubated for 24 h, after which RNA was isolated. Tβ4 transcript levels were measured by RT-PCR (D, upper) or realtime PCR (E). Tβ4 protein levels were detected by western blotting (D, lower). Rac1 and Rap1 activities were detected by GST-pulldown and western blotting (F). (G and H) HeLa cells were plated on 35-mm2 dishes and incubated under normoxic conditions for 24 h, and then transfected with either control vector (mock) or pCMV-Tβ4 plasmids for 18 h. A confluent monolayer of HeLa cells was then scratched with a sterile pipet tip, and incubated for 12 h under normoxic conditions. Migration of cells into the space left by the scratch was photographed using a phase-contrast microscope at 200× magnification (G). The empty area remaining at 12 h was quantified using NIH image analysis software (version 1.62), and compared to that of the 0-h time point (H) Data shown are representative of three independent experiments (A–H). Band intensities were normalized relative to controls using NIH image analysis software (Image J, version 1.62). Fold changes relative to the control are indicated under each band (A, C, D, and F). Data in bar graph are presented as means ± SD (B, E, and H). *p < 0.05; **p < 0.01 relative to the control (A–H).
Mentions: To re-examine whether Rac1 and Rap1 activity was dependent on Tβ4 expression under normoxic conditions, we modified Tβ4 expression in HeLa cells using a pCMV-Tβ4 plasmid or Tβ4-siRNA. Overexpression of Tβ4 via the transfection of HeLa cells with pCMV-Tβ4 (Figure 2A and 2B) led to increased activity of both Rac1 and Rap1 relative to that of empty vector controls (Figure 2C). In contrast, when cells were treated with Tβ4-siRNA, Tβ4 expression was inhibited significantly (Figure 2D and 2E). The Tβ4 knockdown inhibited Rac1 and Rap1 activity (Figure 2F).

Bottom Line: Inhibition of Tβ4 expression using transcription activator-like effector nucleases (TALEN) significantly decreased lung metastasis of B16F10 cells.Rap1-regulated Rac1 activity was decreased by a dominant negative Rap1 (Rap1N17), and increased by 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (CPT), a Rap1 activator.These data suggest that a combination therapy targeting both Rap1 and Rac1 activity may be an effective method of inhibiting tumor metastasis.

View Article: PubMed Central - PubMed

Affiliation: Department of Bioscience and Biotechnology, Sejong University, Seoul 143-747, Korea.

ABSTRACT
Signaling by small guanosine triphosphatases (GTPase), Rap1/Rac1, is one of the major pathways controlling cancer cell migration and tumor metastasis. Thymosin beta-4 (Tβ4), an actin-sequestering protein, has been shown to increase migration of cancer cells. Episodes of hypoxia and re-oxygenation (H/R) are an important phenomenon in tumor microenvironment (TME). We investigated whether Tβ4 could play as an intermediary to crosstalk between Rac1- and Rap1- GTPase activation under hypoxia/reoxygenation (H/R) conditions. Inhibition of Tβ4 expression using transcription activator-like effector nucleases (TALEN) significantly decreased lung metastasis of B16F10 cells. Rac1 and Rap1 activity, as well as cancer cell migration, increased following induction of Tβ4 expression in normoxia- or H/R-experienced cells, but were barely detectable in Tβ4-depleted cells. Rap1-regulated Rac1 activity was decreased by a dominant negative Rap1 (Rap1N17), and increased by 8-(4-chloro-phenylthio)-2'-O-methyladenosine-3',5'-cyclic monophosphate (CPT), a Rap1 activator. In contrast, a Rac1-specific inhibitor, NSC23766, and dominant negative Rac1 (Rac1N17) enhanced Tβ4 expression and aberrant Rap1 activity. While NSC23766 and Rac1N17 incompletely inhibited tumor metastasis in vivo, and H/R-experienced cancer cell migration in vitro, more efficient attenuation of cancer cell migration was accomplished by simultaneous inactivation of Rap1 and Rac1 with Rap1N17 and Rac1N17, respectively. These data suggest that a combination therapy targeting both Rap1 and Rac1 activity may be an effective method of inhibiting tumor metastasis.

No MeSH data available.


Related in: MedlinePlus