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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

The Rac1-GTPase inhibitor NSC23766 inhibits cancer cell migration under hypoxia-reoxygenation conditions in vitro(A–E) HeLa cells were treated with NSC23766 (40 μM) or DMSO, and incubated under normoxic or hypoxic conditions for 45 min, followed by reoxygenation for 60 min. Rac1 activity was detected by GST-pulldown and western blotting (A). A confluent monolayer of HeLa cells was then scratched with a sterile pipet tip, and incubated for 18 h under normoxic conditions. Migration of cells into the space left by the scratch was photographed using a phase contrast microscope at 200× magnification (B). The empty area remaining at 18 h was quantified using NIH image analysis software (Image J, version 1.62), and compared to that of the 0-h time point (C). RNA was isolated from cells, and Tβ4 transcript levels measured by RT-PCR (D, top) or realtime PCR (D, bottom). Rap1 activity was detected by GST-pulldown and western blotting (E). 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, (E). Data in bar graph are presented as means ± SD (C and D). *p < 0.05; **p < 0.01 relative to NSC23766-untreated normoxia control. #p < 0.05; ##p < 0.01 relative to NSC23766-untreated hypoxia-reoxygenation group (A–E). (F) NSC23766-treated HeLa cells were subjected to a cell migration assay using a Boyden chamber. Migrated cells were counted at 6, 12, and 18 h after incubation. Data are presented as means ± SD. **p < 0.01 vs. control at the 0-h time point. ##p < 0.01 relative to NSC23766-untreated group at each time point. (G and H) HeLa cells were then transfected with Rac1N17 or control plasmid vectors for 6 h. A confluent monolayer of HeLa cells was scratched with a sterile pipet tip and incubated for 45 min under normoxia or hypoxia, 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 18 h was quantified using NIH image analysis software (Image J, version 1.62), and compared to that of the 0-h time point. Data in bar graph are presented as means ± SD. **p < 0.01 relative to NSC23766-untreated normoxia control. &p < 0.05 relative to Rac1N17-untreated hypoxia-reoxygenation group (H). Data shown are representative of three independent experiments (A–H).
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Figure 5: The Rac1-GTPase inhibitor NSC23766 inhibits cancer cell migration under hypoxia-reoxygenation conditions in vitro(A–E) HeLa cells were treated with NSC23766 (40 μM) or DMSO, and incubated under normoxic or hypoxic conditions for 45 min, followed by reoxygenation for 60 min. Rac1 activity was detected by GST-pulldown and western blotting (A). A confluent monolayer of HeLa cells was then scratched with a sterile pipet tip, and incubated for 18 h under normoxic conditions. Migration of cells into the space left by the scratch was photographed using a phase contrast microscope at 200× magnification (B). The empty area remaining at 18 h was quantified using NIH image analysis software (Image J, version 1.62), and compared to that of the 0-h time point (C). RNA was isolated from cells, and Tβ4 transcript levels measured by RT-PCR (D, top) or realtime PCR (D, bottom). Rap1 activity was detected by GST-pulldown and western blotting (E). 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, (E). Data in bar graph are presented as means ± SD (C and D). *p < 0.05; **p < 0.01 relative to NSC23766-untreated normoxia control. #p < 0.05; ##p < 0.01 relative to NSC23766-untreated hypoxia-reoxygenation group (A–E). (F) NSC23766-treated HeLa cells were subjected to a cell migration assay using a Boyden chamber. Migrated cells were counted at 6, 12, and 18 h after incubation. Data are presented as means ± SD. **p < 0.01 vs. control at the 0-h time point. ##p < 0.01 relative to NSC23766-untreated group at each time point. (G and H) HeLa cells were then transfected with Rac1N17 or control plasmid vectors for 6 h. A confluent monolayer of HeLa cells was scratched with a sterile pipet tip and incubated for 45 min under normoxia or hypoxia, 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 18 h was quantified using NIH image analysis software (Image J, version 1.62), and compared to that of the 0-h time point. Data in bar graph are presented as means ± SD. **p < 0.01 relative to NSC23766-untreated normoxia control. &p < 0.05 relative to Rac1N17-untreated hypoxia-reoxygenation group (H). Data shown are representative of three independent experiments (A–H).

Mentions: To re-affirm the effect of Rac1 inhibition on cancer cell migration, H/R-experienced cells were incubated with NSC23766. Rac1 activity was increased under H/R conditions, but was effectively inhibited by NSC23766 (Figure 5A). Cancer cell migration was also decreased following NSC23766 treatment under both normoxic and H/R conditions (Figure 5B). NSC23766 inhibited cancer cell migration by ~30% under normoxic and ~20% under H/R conditions, relative to untreated controls (Figure 5C). However, the migration of H/R-experienced NSC23766-treated cells was comparable to that of untreated controls maintained under normoxic conditions. As before, this incomplete inhibition of caner cell migration may be the result of compensatory activation of other molecules associated with cancer cell migration.


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)

The Rac1-GTPase inhibitor NSC23766 inhibits cancer cell migration under hypoxia-reoxygenation conditions in vitro(A–E) HeLa cells were treated with NSC23766 (40 μM) or DMSO, and incubated under normoxic or hypoxic conditions for 45 min, followed by reoxygenation for 60 min. Rac1 activity was detected by GST-pulldown and western blotting (A). A confluent monolayer of HeLa cells was then scratched with a sterile pipet tip, and incubated for 18 h under normoxic conditions. Migration of cells into the space left by the scratch was photographed using a phase contrast microscope at 200× magnification (B). The empty area remaining at 18 h was quantified using NIH image analysis software (Image J, version 1.62), and compared to that of the 0-h time point (C). RNA was isolated from cells, and Tβ4 transcript levels measured by RT-PCR (D, top) or realtime PCR (D, bottom). Rap1 activity was detected by GST-pulldown and western blotting (E). 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, (E). Data in bar graph are presented as means ± SD (C and D). *p < 0.05; **p < 0.01 relative to NSC23766-untreated normoxia control. #p < 0.05; ##p < 0.01 relative to NSC23766-untreated hypoxia-reoxygenation group (A–E). (F) NSC23766-treated HeLa cells were subjected to a cell migration assay using a Boyden chamber. Migrated cells were counted at 6, 12, and 18 h after incubation. Data are presented as means ± SD. **p < 0.01 vs. control at the 0-h time point. ##p < 0.01 relative to NSC23766-untreated group at each time point. (G and H) HeLa cells were then transfected with Rac1N17 or control plasmid vectors for 6 h. A confluent monolayer of HeLa cells was scratched with a sterile pipet tip and incubated for 45 min under normoxia or hypoxia, 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 18 h was quantified using NIH image analysis software (Image J, version 1.62), and compared to that of the 0-h time point. Data in bar graph are presented as means ± SD. **p < 0.01 relative to NSC23766-untreated normoxia control. &p < 0.05 relative to Rac1N17-untreated hypoxia-reoxygenation group (H). Data shown are representative of three independent experiments (A–H).
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Figure 5: The Rac1-GTPase inhibitor NSC23766 inhibits cancer cell migration under hypoxia-reoxygenation conditions in vitro(A–E) HeLa cells were treated with NSC23766 (40 μM) or DMSO, and incubated under normoxic or hypoxic conditions for 45 min, followed by reoxygenation for 60 min. Rac1 activity was detected by GST-pulldown and western blotting (A). A confluent monolayer of HeLa cells was then scratched with a sterile pipet tip, and incubated for 18 h under normoxic conditions. Migration of cells into the space left by the scratch was photographed using a phase contrast microscope at 200× magnification (B). The empty area remaining at 18 h was quantified using NIH image analysis software (Image J, version 1.62), and compared to that of the 0-h time point (C). RNA was isolated from cells, and Tβ4 transcript levels measured by RT-PCR (D, top) or realtime PCR (D, bottom). Rap1 activity was detected by GST-pulldown and western blotting (E). 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, (E). Data in bar graph are presented as means ± SD (C and D). *p < 0.05; **p < 0.01 relative to NSC23766-untreated normoxia control. #p < 0.05; ##p < 0.01 relative to NSC23766-untreated hypoxia-reoxygenation group (A–E). (F) NSC23766-treated HeLa cells were subjected to a cell migration assay using a Boyden chamber. Migrated cells were counted at 6, 12, and 18 h after incubation. Data are presented as means ± SD. **p < 0.01 vs. control at the 0-h time point. ##p < 0.01 relative to NSC23766-untreated group at each time point. (G and H) HeLa cells were then transfected with Rac1N17 or control plasmid vectors for 6 h. A confluent monolayer of HeLa cells was scratched with a sterile pipet tip and incubated for 45 min under normoxia or hypoxia, 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 18 h was quantified using NIH image analysis software (Image J, version 1.62), and compared to that of the 0-h time point. Data in bar graph are presented as means ± SD. **p < 0.01 relative to NSC23766-untreated normoxia control. &p < 0.05 relative to Rac1N17-untreated hypoxia-reoxygenation group (H). Data shown are representative of three independent experiments (A–H).
Mentions: To re-affirm the effect of Rac1 inhibition on cancer cell migration, H/R-experienced cells were incubated with NSC23766. Rac1 activity was increased under H/R conditions, but was effectively inhibited by NSC23766 (Figure 5A). Cancer cell migration was also decreased following NSC23766 treatment under both normoxic and H/R conditions (Figure 5B). NSC23766 inhibited cancer cell migration by ~30% under normoxic and ~20% under H/R conditions, relative to untreated controls (Figure 5C). However, the migration of H/R-experienced NSC23766-treated cells was comparable to that of untreated controls maintained under normoxic conditions. As before, this incomplete inhibition of caner cell migration may be the result of compensatory activation of other molecules associated with cancer cell migration.

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