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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

Simultaneous inhibition of Rac1- and Rap1-GTPases inhibited cancer cell migration more effectively than Rac1 or Rap1 alone(A–D) HeLa cells were plated onto 35-mm2 dishes, and incubated for 24 h under normoxic conditions. Cells were then transfected with Rac1-N17, Rap1-N17, or pCDNA3.1 control plasmid (Mock) vectors both alone and in combination, and then incubated for 6 h. A confluent monolayer of HeLa cells was then scratched with a sterile pipet tip, and incubated for 18 h under normoxic conditions. The migration of cells into the space left by the scratch was photographed using a phase-contrast microscope at 200× magnification (A). 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 (B). HeLa cells transfected with Rap1N17 or/and Rac1N17 were subjected to migration assays using a Boyden chamber. Cells were incubated under normoxia or hypoxia for 45 min, followed by reoxygenation for 18 h. Migrated cells were counted 18 h after incubation (C). Rac1 and Rap1 activities were detected by GST-pulldown and western blotting. 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). Data shown are representative of three independent experiments (A–D). Data in bar graph are presented as means ± SD (B and C). *p < 0.05; **p < 0.01 relative to mock-treated control group. #p < 0.05; ##p < 0.01 relative to Rac1N17- or Rap1N17-treated normoxia (B) or hypoxia-reoxygenation (C and D) group. (E) Hypothetical model of Tβ4-mediated Rap1-GTPase signaling in tumor metastasis and cancer cell migration. Rap1 affects tumor metastasis and cancer cell migration directly or indirectly through Rac1 activation. When Rac1 activation is blocked by a Rac1-GTPase specific inhibitor, cancer cell motility, in terms of both metastasis and migration, is maintained by Rap1 activation as a result of Tβ4 expression. Gray lines represent novel mechanisms identified in this study.
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Figure 6: Simultaneous inhibition of Rac1- and Rap1-GTPases inhibited cancer cell migration more effectively than Rac1 or Rap1 alone(A–D) HeLa cells were plated onto 35-mm2 dishes, and incubated for 24 h under normoxic conditions. Cells were then transfected with Rac1-N17, Rap1-N17, or pCDNA3.1 control plasmid (Mock) vectors both alone and in combination, and then incubated for 6 h. A confluent monolayer of HeLa cells was then scratched with a sterile pipet tip, and incubated for 18 h under normoxic conditions. The migration of cells into the space left by the scratch was photographed using a phase-contrast microscope at 200× magnification (A). 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 (B). HeLa cells transfected with Rap1N17 or/and Rac1N17 were subjected to migration assays using a Boyden chamber. Cells were incubated under normoxia or hypoxia for 45 min, followed by reoxygenation for 18 h. Migrated cells were counted 18 h after incubation (C). Rac1 and Rap1 activities were detected by GST-pulldown and western blotting. 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). Data shown are representative of three independent experiments (A–D). Data in bar graph are presented as means ± SD (B and C). *p < 0.05; **p < 0.01 relative to mock-treated control group. #p < 0.05; ##p < 0.01 relative to Rac1N17- or Rap1N17-treated normoxia (B) or hypoxia-reoxygenation (C and D) group. (E) Hypothetical model of Tβ4-mediated Rap1-GTPase signaling in tumor metastasis and cancer cell migration. Rap1 affects tumor metastasis and cancer cell migration directly or indirectly through Rac1 activation. When Rac1 activation is blocked by a Rac1-GTPase specific inhibitor, cancer cell motility, in terms of both metastasis and migration, is maintained by Rap1 activation as a result of Tβ4 expression. Gray lines represent novel mechanisms identified in this study.

Mentions: To assess the effect of compensatory feedback activation of Rap1 on cancer cell migration, we treated cells with Rap1N17 and/or Rac1N17 to inhibit Rap1 and Rac1, respectively. Cell migration was inhibited in both Rap1N17- and Rac1N17-transfected cells, relative to controls. Synergistic effects were seen following co-transfection with both Rap1N17 and Rac1N17, with cell migration significantly lower than that of individual treatments alone (Figure 6A). Cell migration was inhibited by ~25% and 20% in Rap1N17- and Rac1N17-treated cells, respectively, compared to ~50% in Rap1N17 and Rac1N17 co-transfected cells (Figure 6B). Additional inhibitory effects of Rap1N17 or/and Rac1N17 were confirmed using a Boyden chamber under H/R conditions (Figure 6C). In addition, Rap1 and Rac1 activity were inhibited by Rap1N17 and Rac1N17, respectively, under normoxic and H/R conditions. Rap1 activity was increased in Rac1N17-treated cells under normoxic conditions (Figure 6D), which is consistent with the results shown in Figure 4, although the effects of these inhibitors on the individual signaling molecules driving these phenotypes is unknown. Taken together, the data presented here highlight the role of Tβ4-mediated Rap1 activation as an encouraging target for the prevention of cell migration in Rac1-inhibitor-treated cells by preventing compensatory activation of Rap1 (Figure 6E).


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)

Simultaneous inhibition of Rac1- and Rap1-GTPases inhibited cancer cell migration more effectively than Rac1 or Rap1 alone(A–D) HeLa cells were plated onto 35-mm2 dishes, and incubated for 24 h under normoxic conditions. Cells were then transfected with Rac1-N17, Rap1-N17, or pCDNA3.1 control plasmid (Mock) vectors both alone and in combination, and then incubated for 6 h. A confluent monolayer of HeLa cells was then scratched with a sterile pipet tip, and incubated for 18 h under normoxic conditions. The migration of cells into the space left by the scratch was photographed using a phase-contrast microscope at 200× magnification (A). 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 (B). HeLa cells transfected with Rap1N17 or/and Rac1N17 were subjected to migration assays using a Boyden chamber. Cells were incubated under normoxia or hypoxia for 45 min, followed by reoxygenation for 18 h. Migrated cells were counted 18 h after incubation (C). Rac1 and Rap1 activities were detected by GST-pulldown and western blotting. 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). Data shown are representative of three independent experiments (A–D). Data in bar graph are presented as means ± SD (B and C). *p < 0.05; **p < 0.01 relative to mock-treated control group. #p < 0.05; ##p < 0.01 relative to Rac1N17- or Rap1N17-treated normoxia (B) or hypoxia-reoxygenation (C and D) group. (E) Hypothetical model of Tβ4-mediated Rap1-GTPase signaling in tumor metastasis and cancer cell migration. Rap1 affects tumor metastasis and cancer cell migration directly or indirectly through Rac1 activation. When Rac1 activation is blocked by a Rac1-GTPase specific inhibitor, cancer cell motility, in terms of both metastasis and migration, is maintained by Rap1 activation as a result of Tβ4 expression. Gray lines represent novel mechanisms identified in this study.
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Figure 6: Simultaneous inhibition of Rac1- and Rap1-GTPases inhibited cancer cell migration more effectively than Rac1 or Rap1 alone(A–D) HeLa cells were plated onto 35-mm2 dishes, and incubated for 24 h under normoxic conditions. Cells were then transfected with Rac1-N17, Rap1-N17, or pCDNA3.1 control plasmid (Mock) vectors both alone and in combination, and then incubated for 6 h. A confluent monolayer of HeLa cells was then scratched with a sterile pipet tip, and incubated for 18 h under normoxic conditions. The migration of cells into the space left by the scratch was photographed using a phase-contrast microscope at 200× magnification (A). 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 (B). HeLa cells transfected with Rap1N17 or/and Rac1N17 were subjected to migration assays using a Boyden chamber. Cells were incubated under normoxia or hypoxia for 45 min, followed by reoxygenation for 18 h. Migrated cells were counted 18 h after incubation (C). Rac1 and Rap1 activities were detected by GST-pulldown and western blotting. 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). Data shown are representative of three independent experiments (A–D). Data in bar graph are presented as means ± SD (B and C). *p < 0.05; **p < 0.01 relative to mock-treated control group. #p < 0.05; ##p < 0.01 relative to Rac1N17- or Rap1N17-treated normoxia (B) or hypoxia-reoxygenation (C and D) group. (E) Hypothetical model of Tβ4-mediated Rap1-GTPase signaling in tumor metastasis and cancer cell migration. Rap1 affects tumor metastasis and cancer cell migration directly or indirectly through Rac1 activation. When Rac1 activation is blocked by a Rac1-GTPase specific inhibitor, cancer cell motility, in terms of both metastasis and migration, is maintained by Rap1 activation as a result of Tβ4 expression. Gray lines represent novel mechanisms identified in this study.
Mentions: To assess the effect of compensatory feedback activation of Rap1 on cancer cell migration, we treated cells with Rap1N17 and/or Rac1N17 to inhibit Rap1 and Rac1, respectively. Cell migration was inhibited in both Rap1N17- and Rac1N17-transfected cells, relative to controls. Synergistic effects were seen following co-transfection with both Rap1N17 and Rac1N17, with cell migration significantly lower than that of individual treatments alone (Figure 6A). Cell migration was inhibited by ~25% and 20% in Rap1N17- and Rac1N17-treated cells, respectively, compared to ~50% in Rap1N17 and Rac1N17 co-transfected cells (Figure 6B). Additional inhibitory effects of Rap1N17 or/and Rac1N17 were confirmed using a Boyden chamber under H/R conditions (Figure 6C). In addition, Rap1 and Rac1 activity were inhibited by Rap1N17 and Rac1N17, respectively, under normoxic and H/R conditions. Rap1 activity was increased in Rac1N17-treated cells under normoxic conditions (Figure 6D), which is consistent with the results shown in Figure 4, although the effects of these inhibitors on the individual signaling molecules driving these phenotypes is unknown. Taken together, the data presented here highlight the role of Tβ4-mediated Rap1 activation as an encouraging target for the prevention of cell migration in Rac1-inhibitor-treated cells by preventing compensatory activation of Rap1 (Figure 6E).

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