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The proto-oncogene c-Src and its downstream signaling pathways are inhibited by the metastasis suppressor, NDRG1.

Liu W, Yue F, Zheng M, Merlot A, Bae DH, Huang M, Lane D, Jansson P, Lui GY, Richardson V, Sahni S, Kalinowski D, Kovacevic Z, Richardson DR - Oncotarget (2015)

Bottom Line: Moreover, NDRG1 suppressed Rac1 activity by modulating phosphorylation of a c-Src downstream effector, p130Cas, and its association with CrkII, which acts as a "molecular switch" to activate Rac1.Hence, the role of NDRG1 in decreasing cell migration is, in part, due to its inhibition of c-Src activation.In addition, novel pharmacological agents, which induce NDRG1 expression and are currently under development as anti-metastatic agents, markedly increase NDRG1 and decrease c-Src activation.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R.China.

ABSTRACT
N-myc downstream regulated gene-1 (NDRG1) is a potent metastasis suppressor that plays a key role in regulating signaling pathways involved in mediating cancer cell invasion and migration, including those derived from prostate, colon, etc. However, the mechanisms and molecular targets through which NDRG1 reduces cancer cell invasion and migration, leading to inhibition of cancer metastasis, are not fully elucidated. In this investigation, using NDRG1 over-expression models in three tumor cell-types (namely, DU145, PC3MM and HT29) and also NDRG1 silencing in DU145 and HT29 cells, we reveal that NDRG1 decreases phosphorylation of a key proto-oncogene, cellular Src (c-Src), at a well-characterized activating site (Tyr416). NDRG1-mediated down-regulation of EGFR expression and activation were responsible for the decreased phosphorylation of c-Src (Tyr416). Indeed, NDRG1 prevented recruitment of c-Src to EGFR and c-Src activation. Moreover, NDRG1 suppressed Rac1 activity by modulating phosphorylation of a c-Src downstream effector, p130Cas, and its association with CrkII, which acts as a "molecular switch" to activate Rac1. NDRG1 also affected another signaling molecule involved in modulating Rac1 signaling, c-Abl, which then inhibited CrkII phosphorylation. Silencing NDRG1 increased cell migration relative to the control and inhibition of c-Src signaling using siRNA, or a pharmacological inhibitor (SU6656), prevented this increase. Hence, the role of NDRG1 in decreasing cell migration is, in part, due to its inhibition of c-Src activation. In addition, novel pharmacological agents, which induce NDRG1 expression and are currently under development as anti-metastatic agents, markedly increase NDRG1 and decrease c-Src activation. This study leads to important insights into the mechanism involved in inhibiting metastasis by NDRG1 and how to target these pathways with novel therapeutics.

No MeSH data available.


Related in: MedlinePlus

NDRG1 decreases cancer cell migration in a c-Src-dependent manner (A–D) Dp44mT and DpC increase NDRG1 expression and also decrease activation of c-Src (E)(A–D) Migration assays demonstrating that NDRG1 silencing significantly increased DU145 (A, C) and HT29 (B, D) cell migration, as determined by the xCELLigence real-time cell analysis migration assay (see Materials and Methods). In contrast, transiently silencing of c-Src with siRNA, or inhibition of c-Src activity with SU6656, reversed the effect of silencing NDRG1. (E) The levels of NDRG1, p-Src(Tyr416) and c-Src measured by western analysis in DU145 following a 24 h/37°C incubation with control medium, DFO (250 μM), Dp44mT (5 μM), DpC (5 μM), DFO:Fe (1:1; 250 μM), Dp44mT:Fe (2:1; 5 μM), DpC:Fe (2:1; 5 μM), FeCl3 (250 μM), or Dp2mT (5 μM). Results are expressed as mean ± S.D. (3–5 experiments); *p < 0.05; **p < 0.01; ***p < 0.001, relative to sh-control cells, #p < 0.05; ##p < 0.01; ###p < 0.001, relative to sh-control (si-control) or sh-NDRG1 (si-control) cells, as appropriate (A, B); or *p < 0.05; **p < 0.01; ***p < 0.001, relative to sh-control cells, #p < 0.05; ##p < 0.01; ###p < 0.001, relative to sh-control or sh-NDRG1 cells incubated with control medium only, as appropriate (C, D); *p < 0.05; **p < 0.01; ***p < 0.001, relative to cells incubated with control medium only (E).
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Figure 9: NDRG1 decreases cancer cell migration in a c-Src-dependent manner (A–D) Dp44mT and DpC increase NDRG1 expression and also decrease activation of c-Src (E)(A–D) Migration assays demonstrating that NDRG1 silencing significantly increased DU145 (A, C) and HT29 (B, D) cell migration, as determined by the xCELLigence real-time cell analysis migration assay (see Materials and Methods). In contrast, transiently silencing of c-Src with siRNA, or inhibition of c-Src activity with SU6656, reversed the effect of silencing NDRG1. (E) The levels of NDRG1, p-Src(Tyr416) and c-Src measured by western analysis in DU145 following a 24 h/37°C incubation with control medium, DFO (250 μM), Dp44mT (5 μM), DpC (5 μM), DFO:Fe (1:1; 250 μM), Dp44mT:Fe (2:1; 5 μM), DpC:Fe (2:1; 5 μM), FeCl3 (250 μM), or Dp2mT (5 μM). Results are expressed as mean ± S.D. (3–5 experiments); *p < 0.05; **p < 0.01; ***p < 0.001, relative to sh-control cells, #p < 0.05; ##p < 0.01; ###p < 0.001, relative to sh-control (si-control) or sh-NDRG1 (si-control) cells, as appropriate (A, B); or *p < 0.05; **p < 0.01; ***p < 0.001, relative to sh-control cells, #p < 0.05; ##p < 0.01; ###p < 0.001, relative to sh-control or sh-NDRG1 cells incubated with control medium only, as appropriate (C, D); *p < 0.05; **p < 0.01; ***p < 0.001, relative to cells incubated with control medium only (E).

Mentions: As demonstrated in Figure 9A–9D, relative to DU145 or HT29 sh-control cells (si-control), there was a significant (p < 0.05) increase in the migratory capacity of sh-NDRG1 cells (si-control) after only 4–8 h of incubation and remained significantly (p < 0.001–0.05) increased for up to 24 h. This is consistent with our recent reports demonstrating that inhibition of NDRG1 expression increases cellular migration [29, 31]. However, upon silencing c-Src, a significant (p < 0.001–0.05) decrease in migration was observed in both sh-control and sh-NDRG1 cells in comparison to si-control transfected cells for both cell-types (Figure 9A, 9B). Importantly, these observations were confirmed by incubating cells with the c-Src inhibitor, SU6656, upon which the migration of sh-control and sh-NDRG1 cells were significantly (p < 0.001–0.05) reduced in both cell-types relative to the control (Figure 9C, 9D). In summary, these observations above indicate that silencing NDRG1 increases cell migration relative to the control and that inhibition of c-Src can prevent this increase. Hence, the role of NDRG1 in decreasing cell migration is, at least in part, due to its effects on inhibiting c-Src activation.


The proto-oncogene c-Src and its downstream signaling pathways are inhibited by the metastasis suppressor, NDRG1.

Liu W, Yue F, Zheng M, Merlot A, Bae DH, Huang M, Lane D, Jansson P, Lui GY, Richardson V, Sahni S, Kalinowski D, Kovacevic Z, Richardson DR - Oncotarget (2015)

NDRG1 decreases cancer cell migration in a c-Src-dependent manner (A–D) Dp44mT and DpC increase NDRG1 expression and also decrease activation of c-Src (E)(A–D) Migration assays demonstrating that NDRG1 silencing significantly increased DU145 (A, C) and HT29 (B, D) cell migration, as determined by the xCELLigence real-time cell analysis migration assay (see Materials and Methods). In contrast, transiently silencing of c-Src with siRNA, or inhibition of c-Src activity with SU6656, reversed the effect of silencing NDRG1. (E) The levels of NDRG1, p-Src(Tyr416) and c-Src measured by western analysis in DU145 following a 24 h/37°C incubation with control medium, DFO (250 μM), Dp44mT (5 μM), DpC (5 μM), DFO:Fe (1:1; 250 μM), Dp44mT:Fe (2:1; 5 μM), DpC:Fe (2:1; 5 μM), FeCl3 (250 μM), or Dp2mT (5 μM). Results are expressed as mean ± S.D. (3–5 experiments); *p < 0.05; **p < 0.01; ***p < 0.001, relative to sh-control cells, #p < 0.05; ##p < 0.01; ###p < 0.001, relative to sh-control (si-control) or sh-NDRG1 (si-control) cells, as appropriate (A, B); or *p < 0.05; **p < 0.01; ***p < 0.001, relative to sh-control cells, #p < 0.05; ##p < 0.01; ###p < 0.001, relative to sh-control or sh-NDRG1 cells incubated with control medium only, as appropriate (C, D); *p < 0.05; **p < 0.01; ***p < 0.001, relative to cells incubated with control medium only (E).
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Figure 9: NDRG1 decreases cancer cell migration in a c-Src-dependent manner (A–D) Dp44mT and DpC increase NDRG1 expression and also decrease activation of c-Src (E)(A–D) Migration assays demonstrating that NDRG1 silencing significantly increased DU145 (A, C) and HT29 (B, D) cell migration, as determined by the xCELLigence real-time cell analysis migration assay (see Materials and Methods). In contrast, transiently silencing of c-Src with siRNA, or inhibition of c-Src activity with SU6656, reversed the effect of silencing NDRG1. (E) The levels of NDRG1, p-Src(Tyr416) and c-Src measured by western analysis in DU145 following a 24 h/37°C incubation with control medium, DFO (250 μM), Dp44mT (5 μM), DpC (5 μM), DFO:Fe (1:1; 250 μM), Dp44mT:Fe (2:1; 5 μM), DpC:Fe (2:1; 5 μM), FeCl3 (250 μM), or Dp2mT (5 μM). Results are expressed as mean ± S.D. (3–5 experiments); *p < 0.05; **p < 0.01; ***p < 0.001, relative to sh-control cells, #p < 0.05; ##p < 0.01; ###p < 0.001, relative to sh-control (si-control) or sh-NDRG1 (si-control) cells, as appropriate (A, B); or *p < 0.05; **p < 0.01; ***p < 0.001, relative to sh-control cells, #p < 0.05; ##p < 0.01; ###p < 0.001, relative to sh-control or sh-NDRG1 cells incubated with control medium only, as appropriate (C, D); *p < 0.05; **p < 0.01; ***p < 0.001, relative to cells incubated with control medium only (E).
Mentions: As demonstrated in Figure 9A–9D, relative to DU145 or HT29 sh-control cells (si-control), there was a significant (p < 0.05) increase in the migratory capacity of sh-NDRG1 cells (si-control) after only 4–8 h of incubation and remained significantly (p < 0.001–0.05) increased for up to 24 h. This is consistent with our recent reports demonstrating that inhibition of NDRG1 expression increases cellular migration [29, 31]. However, upon silencing c-Src, a significant (p < 0.001–0.05) decrease in migration was observed in both sh-control and sh-NDRG1 cells in comparison to si-control transfected cells for both cell-types (Figure 9A, 9B). Importantly, these observations were confirmed by incubating cells with the c-Src inhibitor, SU6656, upon which the migration of sh-control and sh-NDRG1 cells were significantly (p < 0.001–0.05) reduced in both cell-types relative to the control (Figure 9C, 9D). In summary, these observations above indicate that silencing NDRG1 increases cell migration relative to the control and that inhibition of c-Src can prevent this increase. Hence, the role of NDRG1 in decreasing cell migration is, at least in part, due to its effects on inhibiting c-Src activation.

Bottom Line: Moreover, NDRG1 suppressed Rac1 activity by modulating phosphorylation of a c-Src downstream effector, p130Cas, and its association with CrkII, which acts as a "molecular switch" to activate Rac1.Hence, the role of NDRG1 in decreasing cell migration is, in part, due to its inhibition of c-Src activation.In addition, novel pharmacological agents, which induce NDRG1 expression and are currently under development as anti-metastatic agents, markedly increase NDRG1 and decrease c-Src activation.

View Article: PubMed Central - PubMed

Affiliation: Department of General Surgery, Ruijin Hospital, Shanghai Jiao Tong University School of Medicine, Shanghai 200025, P.R.China.

ABSTRACT
N-myc downstream regulated gene-1 (NDRG1) is a potent metastasis suppressor that plays a key role in regulating signaling pathways involved in mediating cancer cell invasion and migration, including those derived from prostate, colon, etc. However, the mechanisms and molecular targets through which NDRG1 reduces cancer cell invasion and migration, leading to inhibition of cancer metastasis, are not fully elucidated. In this investigation, using NDRG1 over-expression models in three tumor cell-types (namely, DU145, PC3MM and HT29) and also NDRG1 silencing in DU145 and HT29 cells, we reveal that NDRG1 decreases phosphorylation of a key proto-oncogene, cellular Src (c-Src), at a well-characterized activating site (Tyr416). NDRG1-mediated down-regulation of EGFR expression and activation were responsible for the decreased phosphorylation of c-Src (Tyr416). Indeed, NDRG1 prevented recruitment of c-Src to EGFR and c-Src activation. Moreover, NDRG1 suppressed Rac1 activity by modulating phosphorylation of a c-Src downstream effector, p130Cas, and its association with CrkII, which acts as a "molecular switch" to activate Rac1. NDRG1 also affected another signaling molecule involved in modulating Rac1 signaling, c-Abl, which then inhibited CrkII phosphorylation. Silencing NDRG1 increased cell migration relative to the control and inhibition of c-Src signaling using siRNA, or a pharmacological inhibitor (SU6656), prevented this increase. Hence, the role of NDRG1 in decreasing cell migration is, in part, due to its inhibition of c-Src activation. In addition, novel pharmacological agents, which induce NDRG1 expression and are currently under development as anti-metastatic agents, markedly increase NDRG1 and decrease c-Src activation. This study leads to important insights into the mechanism involved in inhibiting metastasis by NDRG1 and how to target these pathways with novel therapeutics.

No MeSH data available.


Related in: MedlinePlus