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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

Thymosin beta 4 (Tβ4) expression is correlated with Rac1- and Rap1-GTPase activation, lung metastasis, and cell migration in hypoxia-reoxygenation (H/R)-experienced cancer cells(A–C) B16F10 cells were cultivated in vitro in log phase and transfected with Tβ4-TALEN. Tβ4 expression in Tβ4-TALEN-treated B16F10 cells was detected by RT-PCR (A, top) or realtime PCR (A, bottom). Five 7-week-old C57BL/6 wild-type mice were injected with 2 × 105 B16F10 control or Tβ4-TALEN-treated cells via tail-vein injection. All mice were sacrificed 14 d after tumor injection. Lung metastasis was shown in the photograph (B). The degree of lung metastasis was assessed by counting tumor colonies under a light dissection microscope (C). (D and E) HeLa cells were subjected to reoxygenation for 30 or 60 min following incubation in a hypoxia chamber for 45 min. RNA was isolated, and Tβ4 transcript levels were measured by RT-PCR (D, top, upper) or realtime PCR (D, bottom). Tβ4 protein levels were detected by western blotting (D, top, lower). Rac1 and Rap1 activities were examined using a GST-pulldown assay targeting the RBD domain, and visualized by western blotting (E). (F) HeLa cells were transfected with scrambled control siRNA or Tβ4-siRNA, respectively and incubated for 24 h prior to incubation under hypoxia (45 min) and reoxygenation (60 min) conditions. Rac1 and Rap1 activities were detected by GST-pulldown and western blotting. (G and H) HeLa cells transfected with scrambled control siRNA or Tβ4-siRNA were plated on 35-mm2 dishes and incubated under normoxic conditions for 24 h. A confluent monolayer of HeLa cells was then scratched with a sterile pipet tip, and incubated in normoxia or hypoxia for 45 min, followed by reoxygenation for 18 h. 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 each time point 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). Data in bar graph are presented as means ± SD (A, C, D, and 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 (D–F). *p < 0.05; **p < 0.01 relative to the control (A–H).
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Figure 1: Thymosin beta 4 (Tβ4) expression is correlated with Rac1- and Rap1-GTPase activation, lung metastasis, and cell migration in hypoxia-reoxygenation (H/R)-experienced cancer cells(A–C) B16F10 cells were cultivated in vitro in log phase and transfected with Tβ4-TALEN. Tβ4 expression in Tβ4-TALEN-treated B16F10 cells was detected by RT-PCR (A, top) or realtime PCR (A, bottom). Five 7-week-old C57BL/6 wild-type mice were injected with 2 × 105 B16F10 control or Tβ4-TALEN-treated cells via tail-vein injection. All mice were sacrificed 14 d after tumor injection. Lung metastasis was shown in the photograph (B). The degree of lung metastasis was assessed by counting tumor colonies under a light dissection microscope (C). (D and E) HeLa cells were subjected to reoxygenation for 30 or 60 min following incubation in a hypoxia chamber for 45 min. RNA was isolated, and Tβ4 transcript levels were measured by RT-PCR (D, top, upper) or realtime PCR (D, bottom). Tβ4 protein levels were detected by western blotting (D, top, lower). Rac1 and Rap1 activities were examined using a GST-pulldown assay targeting the RBD domain, and visualized by western blotting (E). (F) HeLa cells were transfected with scrambled control siRNA or Tβ4-siRNA, respectively and incubated for 24 h prior to incubation under hypoxia (45 min) and reoxygenation (60 min) conditions. Rac1 and Rap1 activities were detected by GST-pulldown and western blotting. (G and H) HeLa cells transfected with scrambled control siRNA or Tβ4-siRNA were plated on 35-mm2 dishes and incubated under normoxic conditions for 24 h. A confluent monolayer of HeLa cells was then scratched with a sterile pipet tip, and incubated in normoxia or hypoxia for 45 min, followed by reoxygenation for 18 h. 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 each time point 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). Data in bar graph are presented as means ± SD (A, C, D, and 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 (D–F). *p < 0.05; **p < 0.01 relative to the control (A–H).

Mentions: To examine the possibility that Tβ4 interacts with Rap1, we performed a yeast two-hybrid screen using the RalGDS-Ras binding domain (RBD), which binds active Rap1-GTP, as a bait. The initial screen revealed 86 colonies that exhibited strong blue signals (Supplementary Figure 1), suggesting that Tβ4 may be a regulator of Rap1 GTPase activation. Furthermore, as Rap1 and Rac together regulate the secretion of sAPPalpha [24], we examined the effect of Tβ4 on lung metastasis in vivo. B16F10 cells were transfected with a transcription activator-like effector nuclease (TALEN) targeting Tβ4, effectively suppressing Tβ4 expression using RT-PCR (Figure 1A, top) and realtime PCR (Figure 1A, bottom). These cells were then injected into the tail vein of C57BL/6 mice. Tumor metastasis was found to be reduced in mice injected with the Tβ4-TALEN-transfected cells relative to control cells (Figure 1B and 1C), suggesting that tumor metastasis may be associated with Tβ4 signaling pathways.


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)

Thymosin beta 4 (Tβ4) expression is correlated with Rac1- and Rap1-GTPase activation, lung metastasis, and cell migration in hypoxia-reoxygenation (H/R)-experienced cancer cells(A–C) B16F10 cells were cultivated in vitro in log phase and transfected with Tβ4-TALEN. Tβ4 expression in Tβ4-TALEN-treated B16F10 cells was detected by RT-PCR (A, top) or realtime PCR (A, bottom). Five 7-week-old C57BL/6 wild-type mice were injected with 2 × 105 B16F10 control or Tβ4-TALEN-treated cells via tail-vein injection. All mice were sacrificed 14 d after tumor injection. Lung metastasis was shown in the photograph (B). The degree of lung metastasis was assessed by counting tumor colonies under a light dissection microscope (C). (D and E) HeLa cells were subjected to reoxygenation for 30 or 60 min following incubation in a hypoxia chamber for 45 min. RNA was isolated, and Tβ4 transcript levels were measured by RT-PCR (D, top, upper) or realtime PCR (D, bottom). Tβ4 protein levels were detected by western blotting (D, top, lower). Rac1 and Rap1 activities were examined using a GST-pulldown assay targeting the RBD domain, and visualized by western blotting (E). (F) HeLa cells were transfected with scrambled control siRNA or Tβ4-siRNA, respectively and incubated for 24 h prior to incubation under hypoxia (45 min) and reoxygenation (60 min) conditions. Rac1 and Rap1 activities were detected by GST-pulldown and western blotting. (G and H) HeLa cells transfected with scrambled control siRNA or Tβ4-siRNA were plated on 35-mm2 dishes and incubated under normoxic conditions for 24 h. A confluent monolayer of HeLa cells was then scratched with a sterile pipet tip, and incubated in normoxia or hypoxia for 45 min, followed by reoxygenation for 18 h. 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 each time point 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). Data in bar graph are presented as means ± SD (A, C, D, and 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 (D–F). *p < 0.05; **p < 0.01 relative to the control (A–H).
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Related In: Results  -  Collection

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Figure 1: Thymosin beta 4 (Tβ4) expression is correlated with Rac1- and Rap1-GTPase activation, lung metastasis, and cell migration in hypoxia-reoxygenation (H/R)-experienced cancer cells(A–C) B16F10 cells were cultivated in vitro in log phase and transfected with Tβ4-TALEN. Tβ4 expression in Tβ4-TALEN-treated B16F10 cells was detected by RT-PCR (A, top) or realtime PCR (A, bottom). Five 7-week-old C57BL/6 wild-type mice were injected with 2 × 105 B16F10 control or Tβ4-TALEN-treated cells via tail-vein injection. All mice were sacrificed 14 d after tumor injection. Lung metastasis was shown in the photograph (B). The degree of lung metastasis was assessed by counting tumor colonies under a light dissection microscope (C). (D and E) HeLa cells were subjected to reoxygenation for 30 or 60 min following incubation in a hypoxia chamber for 45 min. RNA was isolated, and Tβ4 transcript levels were measured by RT-PCR (D, top, upper) or realtime PCR (D, bottom). Tβ4 protein levels were detected by western blotting (D, top, lower). Rac1 and Rap1 activities were examined using a GST-pulldown assay targeting the RBD domain, and visualized by western blotting (E). (F) HeLa cells were transfected with scrambled control siRNA or Tβ4-siRNA, respectively and incubated for 24 h prior to incubation under hypoxia (45 min) and reoxygenation (60 min) conditions. Rac1 and Rap1 activities were detected by GST-pulldown and western blotting. (G and H) HeLa cells transfected with scrambled control siRNA or Tβ4-siRNA were plated on 35-mm2 dishes and incubated under normoxic conditions for 24 h. A confluent monolayer of HeLa cells was then scratched with a sterile pipet tip, and incubated in normoxia or hypoxia for 45 min, followed by reoxygenation for 18 h. 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 each time point 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). Data in bar graph are presented as means ± SD (A, C, D, and 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 (D–F). *p < 0.05; **p < 0.01 relative to the control (A–H).
Mentions: To examine the possibility that Tβ4 interacts with Rap1, we performed a yeast two-hybrid screen using the RalGDS-Ras binding domain (RBD), which binds active Rap1-GTP, as a bait. The initial screen revealed 86 colonies that exhibited strong blue signals (Supplementary Figure 1), suggesting that Tβ4 may be a regulator of Rap1 GTPase activation. Furthermore, as Rap1 and Rac together regulate the secretion of sAPPalpha [24], we examined the effect of Tβ4 on lung metastasis in vivo. B16F10 cells were transfected with a transcription activator-like effector nuclease (TALEN) targeting Tβ4, effectively suppressing Tβ4 expression using RT-PCR (Figure 1A, top) and realtime PCR (Figure 1A, bottom). These cells were then injected into the tail vein of C57BL/6 mice. Tumor metastasis was found to be reduced in mice injected with the Tβ4-TALEN-transfected cells relative to control cells (Figure 1B and 1C), suggesting that tumor metastasis may be associated with Tβ4 signaling pathways.

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