Limits...
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 suppressed Rac1 activity and its downstream effector PAK1(A, B) A Rac1 activation assay was performed (see Materials and Methods) to detect the active form of Rac1 (GTP-Rac1) in (A) DU145 and (B) HT29 cells. These studies demonstrated that NDRG1 expression inhibited Rac1 activity. (C, D) Immunoblotting showed that NDRG1 suppressed PAK1 phosphorylation (Thr423) in (C) DU145 and (D) HT29 cells. Immunoblotting results are representative of three independent experiments. Densitometry data are mean ± S.D. (3–5 experiments); *p < 0.05; **p < 0.01; ***p < 0.001, relative to vector control or sh-control cells, as appropriate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4496188&req=5

Figure 4: NDRG1 suppressed Rac1 activity and its downstream effector PAK1(A, B) A Rac1 activation assay was performed (see Materials and Methods) to detect the active form of Rac1 (GTP-Rac1) in (A) DU145 and (B) HT29 cells. These studies demonstrated that NDRG1 expression inhibited Rac1 activity. (C, D) Immunoblotting showed that NDRG1 suppressed PAK1 phosphorylation (Thr423) in (C) DU145 and (D) HT29 cells. Immunoblotting results are representative of three independent experiments. Densitometry data are mean ± S.D. (3–5 experiments); *p < 0.05; **p < 0.01; ***p < 0.001, relative to vector control or sh-control cells, as appropriate.

Mentions: To further investigate the finding that NDRG1 plays a negative regulatory role in c-Src activation and its downstream effectors (namely p130Cas; Figures 1, 3), a Rac1 activation assay was performed to assess the effect of NDRG1 on Rac1 activity. This was examined as there is a close association between c-Src and Rac1 activity linked by the p130Cas-CrkII-DOCK180 complex [46]. For both DU145 and HT29 cells, NDRG1 over-expression significantly (p < 0.01–0.05) inhibited Rac1 activation, which was determined by the levels of GTP-bound Rac1 (GTP-Rac1), relative to vector control cells (Figure 4A, 4B). In contrast, silencing NDRG1 significantly (p < 0.01) increased GTP-Rac1 levels relative to the sh-control DU145 and HT29 cells (Figure 4A, 4B). Although alteration in NDRG1 expression markedly influenced GTP-Rac1 levels, it did not significantly (p > 0.05) affect total Rac1 levels in both cell-types (Figure 4A, 4B).


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 suppressed Rac1 activity and its downstream effector PAK1(A, B) A Rac1 activation assay was performed (see Materials and Methods) to detect the active form of Rac1 (GTP-Rac1) in (A) DU145 and (B) HT29 cells. These studies demonstrated that NDRG1 expression inhibited Rac1 activity. (C, D) Immunoblotting showed that NDRG1 suppressed PAK1 phosphorylation (Thr423) in (C) DU145 and (D) HT29 cells. Immunoblotting results are representative of three independent experiments. Densitometry data are mean ± S.D. (3–5 experiments); *p < 0.05; **p < 0.01; ***p < 0.001, relative to vector control or sh-control cells, as appropriate.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496188&req=5

Figure 4: NDRG1 suppressed Rac1 activity and its downstream effector PAK1(A, B) A Rac1 activation assay was performed (see Materials and Methods) to detect the active form of Rac1 (GTP-Rac1) in (A) DU145 and (B) HT29 cells. These studies demonstrated that NDRG1 expression inhibited Rac1 activity. (C, D) Immunoblotting showed that NDRG1 suppressed PAK1 phosphorylation (Thr423) in (C) DU145 and (D) HT29 cells. Immunoblotting results are representative of three independent experiments. Densitometry data are mean ± S.D. (3–5 experiments); *p < 0.05; **p < 0.01; ***p < 0.001, relative to vector control or sh-control cells, as appropriate.
Mentions: To further investigate the finding that NDRG1 plays a negative regulatory role in c-Src activation and its downstream effectors (namely p130Cas; Figures 1, 3), a Rac1 activation assay was performed to assess the effect of NDRG1 on Rac1 activity. This was examined as there is a close association between c-Src and Rac1 activity linked by the p130Cas-CrkII-DOCK180 complex [46]. For both DU145 and HT29 cells, NDRG1 over-expression significantly (p < 0.01–0.05) inhibited Rac1 activation, which was determined by the levels of GTP-bound Rac1 (GTP-Rac1), relative to vector control cells (Figure 4A, 4B). In contrast, silencing NDRG1 significantly (p < 0.01) increased GTP-Rac1 levels relative to the sh-control DU145 and HT29 cells (Figure 4A, 4B). Although alteration in NDRG1 expression markedly influenced GTP-Rac1 levels, it did not significantly (p > 0.05) affect total Rac1 levels in both cell-types (Figure 4A, 4B).

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