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TRB3 links insulin/IGF to tumour promotion by interacting with p62 and impeding autophagic/proteasomal degradations.

Hua F, Li K, Yu JJ, Lv XX, Yan J, Zhang XW, Sun W, Lin H, Shang S, Wang F, Cui B, Mu R, Huang B, Jiang JD, Hu ZW - Nat Commun (2015)

Bottom Line: Here we report a previously unrecognized tumour-promoting mechanism for stress protein TRB3, which mediates a reciprocal antagonism between autophagic and proteasomal degradation systems and connects insulin/IGF to malignant promotion.TRB3 interacts with autophagic receptor p62 and hinders p62 binding to LC3 and ubiquitinated substrates, which causes p62 deposition and suppresses autophagic/proteasomal degradation.Interrupting TRB3/p62 interaction produces potent antitumour efficacies against tumour growth and metastasis.

View Article: PubMed Central - PubMed

Affiliation: Immunology and Cancer Pharmacology Group, State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100050, China.

ABSTRACT
High insulin/IGF is a biologic link between diabetes and cancers, but the underlying molecular mechanism remains unclear. Here we report a previously unrecognized tumour-promoting mechanism for stress protein TRB3, which mediates a reciprocal antagonism between autophagic and proteasomal degradation systems and connects insulin/IGF to malignant promotion. We find that several human cancers express higher TRB3 and phosphorylated insulin receptor substrate 1, which correlates negatively with patient's prognosis. TRB3 depletion protects against tumour-promoting actions of insulin/IGF and attenuates tumour initiation, growth and metastasis in mice. TRB3 interacts with autophagic receptor p62 and hinders p62 binding to LC3 and ubiquitinated substrates, which causes p62 deposition and suppresses autophagic/proteasomal degradation. Several tumour-promoting factors accumulate in cancer cells to support tumour metabolism, proliferation, invasion and metastasis. Interrupting TRB3/p62 interaction produces potent antitumour efficacies against tumour growth and metastasis. Our study opens possibility of targeting this interaction as a potential novel strategy against cancers with diabetes.

No MeSH data available.


Related in: MedlinePlus

TRB3 mediates insulin/IGF-induced p62 accumulation.(a) HepG2 cells were treated with indicated stimulators for 12 h. The mRNAs were determined with RT–PCR using specific primer sets. Data are means±s.e.m of three independent assays. (b) HepG2 cells were incubated with cycloheximide (CHX) (10 μg ml−1) for indicated time points after insulin/IGF-1 stimulation. Indicated proteins were detected with immunoblotting. Data are means±s.e.m of three independent assays. (c) Control or TRB3-silenced cells were incubated with CHX (10 μg ml−1) for indicated time points after IGF-1 stimulation. Cell lysates were isolated for immunoblotting. Data are means±s.e.m of three independent assays. (d) HepG2 cells expressing p62- or control-siRNA were co-transfected with UbG76V-GFP. The cells were treated with IGF-1 for 12 h. The cell lysates were isolated for immunoblotting. Data are representative of immunoblots of three independent assays. (e) Cells expressing control-shRNA or TRB3-shRNAs were transfected with UbG76V-GFP plasmid plus or minus with p62-DDK-expressing plasmid. The lysates were isolated for immunoblotting. Data are representative immunoblots of three independent assays. (f) HepG2 cells expressing p62- or control-siRNA were infected with adenovirus containing mRFP-GFP-LC3-PE. After 24 h, the cells were treated with IGF-1 for 12 h, and the flux rate of autophagy was detected with Live Cell Imaging Microscopy. Data are representative images of three independent assays. Red colour shows autophagolysosomes and double-colour red/green shows autophagosomes. Scale bar, 10 μm. (g) Control or p62-silencing cells were treated with or without IGF-1 for 12 h. Indicated proteins were evaluated with immunoblotting. Data are representative immunoblots of three independent assays. (h) BALB/c nude mice were i.v. injected with HepG2 cells expressing control-shRNA or p62-shRNA (3 × 106). Data are representative of number of metastases in lungs and lungs. Metastatic nodules were indicated by arrows (n=8 per group). (i) BALB/c nude mice were s.c. inoculated with HepG2 cells expressing control- or p62-shRNA (1.5 × 106). Data are the mean volumes±s.e.m. at indicated times and representative tumours along with tumour weight (n=8 per group). Scale bar, 1.5 cm. Statistical significance was determined with Student's t-test; *P<0.05.
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f4: TRB3 mediates insulin/IGF-induced p62 accumulation.(a) HepG2 cells were treated with indicated stimulators for 12 h. The mRNAs were determined with RT–PCR using specific primer sets. Data are means±s.e.m of three independent assays. (b) HepG2 cells were incubated with cycloheximide (CHX) (10 μg ml−1) for indicated time points after insulin/IGF-1 stimulation. Indicated proteins were detected with immunoblotting. Data are means±s.e.m of three independent assays. (c) Control or TRB3-silenced cells were incubated with CHX (10 μg ml−1) for indicated time points after IGF-1 stimulation. Cell lysates were isolated for immunoblotting. Data are means±s.e.m of three independent assays. (d) HepG2 cells expressing p62- or control-siRNA were co-transfected with UbG76V-GFP. The cells were treated with IGF-1 for 12 h. The cell lysates were isolated for immunoblotting. Data are representative of immunoblots of three independent assays. (e) Cells expressing control-shRNA or TRB3-shRNAs were transfected with UbG76V-GFP plasmid plus or minus with p62-DDK-expressing plasmid. The lysates were isolated for immunoblotting. Data are representative immunoblots of three independent assays. (f) HepG2 cells expressing p62- or control-siRNA were infected with adenovirus containing mRFP-GFP-LC3-PE. After 24 h, the cells were treated with IGF-1 for 12 h, and the flux rate of autophagy was detected with Live Cell Imaging Microscopy. Data are representative images of three independent assays. Red colour shows autophagolysosomes and double-colour red/green shows autophagosomes. Scale bar, 10 μm. (g) Control or p62-silencing cells were treated with or without IGF-1 for 12 h. Indicated proteins were evaluated with immunoblotting. Data are representative immunoblots of three independent assays. (h) BALB/c nude mice were i.v. injected with HepG2 cells expressing control-shRNA or p62-shRNA (3 × 106). Data are representative of number of metastases in lungs and lungs. Metastatic nodules were indicated by arrows (n=8 per group). (i) BALB/c nude mice were s.c. inoculated with HepG2 cells expressing control- or p62-shRNA (1.5 × 106). Data are the mean volumes±s.e.m. at indicated times and representative tumours along with tumour weight (n=8 per group). Scale bar, 1.5 cm. Statistical significance was determined with Student's t-test; *P<0.05.

Mentions: We examined how TRB3 mediated the insulin/IGF-induced p62 accumulation. Insulin/IGF-1 did not change p62 transcription (Fig. 4a) but rather markedly enhanced the half-life of p62 degradation from 8.5 to 24 h (Fig. 4b). Silencing TRB3 promoted p62 degradation in the presence or absence of IGF-1 (Fig. 4c), indicating that TRB3 mediates insulin/IGF-induced p62 accumulation. Interestingly, p62 depletion not only reduced basal but also blocked IGF-1-induced expression of UbG76V-GFP (Fig. 4d), coinciding with a report that p62 accumulation by autophagy inhibition slows down the UPS-specific substrate clearance29. To verify whether inhibitory effect of TRB3 on UPS was mediated by p62 accumulation, the p62 level was recovered by ectopic expression of p62 in TRB3-silenced cells. Overexpressing p62 rescued UbG76V-GFP activity in TRB3-silenced cells, indicating that TRB3 blocks UPS clearance through inducing p62 accumulation (Fig. 4e). In addition, silencing p62 moderately enhanced autophagic flux in quiescent cells and partially restored the IGF-1-suppressed autophagic flux (Fig. 4f). However, silencing p62 had a mixed effect on expression of the tumour-promoting factors in quiescent cells, which was quite different from the effects induced by TRB3 depletion; it could not protect them from the IGF-1 induction as did by TRB3 depletion (Fig. 4g). Thus, p62 depletion produced only a moderate antitumour role in tumour metastasis and growth (Fig. 4h,i). These results indicate that TRB3 mediates the IGF-induced p62 accumulation, which compromises UPS as well as (partially) autophagic degradation in cancer cells.


TRB3 links insulin/IGF to tumour promotion by interacting with p62 and impeding autophagic/proteasomal degradations.

Hua F, Li K, Yu JJ, Lv XX, Yan J, Zhang XW, Sun W, Lin H, Shang S, Wang F, Cui B, Mu R, Huang B, Jiang JD, Hu ZW - Nat Commun (2015)

TRB3 mediates insulin/IGF-induced p62 accumulation.(a) HepG2 cells were treated with indicated stimulators for 12 h. The mRNAs were determined with RT–PCR using specific primer sets. Data are means±s.e.m of three independent assays. (b) HepG2 cells were incubated with cycloheximide (CHX) (10 μg ml−1) for indicated time points after insulin/IGF-1 stimulation. Indicated proteins were detected with immunoblotting. Data are means±s.e.m of three independent assays. (c) Control or TRB3-silenced cells were incubated with CHX (10 μg ml−1) for indicated time points after IGF-1 stimulation. Cell lysates were isolated for immunoblotting. Data are means±s.e.m of three independent assays. (d) HepG2 cells expressing p62- or control-siRNA were co-transfected with UbG76V-GFP. The cells were treated with IGF-1 for 12 h. The cell lysates were isolated for immunoblotting. Data are representative of immunoblots of three independent assays. (e) Cells expressing control-shRNA or TRB3-shRNAs were transfected with UbG76V-GFP plasmid plus or minus with p62-DDK-expressing plasmid. The lysates were isolated for immunoblotting. Data are representative immunoblots of three independent assays. (f) HepG2 cells expressing p62- or control-siRNA were infected with adenovirus containing mRFP-GFP-LC3-PE. After 24 h, the cells were treated with IGF-1 for 12 h, and the flux rate of autophagy was detected with Live Cell Imaging Microscopy. Data are representative images of three independent assays. Red colour shows autophagolysosomes and double-colour red/green shows autophagosomes. Scale bar, 10 μm. (g) Control or p62-silencing cells were treated with or without IGF-1 for 12 h. Indicated proteins were evaluated with immunoblotting. Data are representative immunoblots of three independent assays. (h) BALB/c nude mice were i.v. injected with HepG2 cells expressing control-shRNA or p62-shRNA (3 × 106). Data are representative of number of metastases in lungs and lungs. Metastatic nodules were indicated by arrows (n=8 per group). (i) BALB/c nude mice were s.c. inoculated with HepG2 cells expressing control- or p62-shRNA (1.5 × 106). Data are the mean volumes±s.e.m. at indicated times and representative tumours along with tumour weight (n=8 per group). Scale bar, 1.5 cm. Statistical significance was determined with Student's t-test; *P<0.05.
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f4: TRB3 mediates insulin/IGF-induced p62 accumulation.(a) HepG2 cells were treated with indicated stimulators for 12 h. The mRNAs were determined with RT–PCR using specific primer sets. Data are means±s.e.m of three independent assays. (b) HepG2 cells were incubated with cycloheximide (CHX) (10 μg ml−1) for indicated time points after insulin/IGF-1 stimulation. Indicated proteins were detected with immunoblotting. Data are means±s.e.m of three independent assays. (c) Control or TRB3-silenced cells were incubated with CHX (10 μg ml−1) for indicated time points after IGF-1 stimulation. Cell lysates were isolated for immunoblotting. Data are means±s.e.m of three independent assays. (d) HepG2 cells expressing p62- or control-siRNA were co-transfected with UbG76V-GFP. The cells were treated with IGF-1 for 12 h. The cell lysates were isolated for immunoblotting. Data are representative of immunoblots of three independent assays. (e) Cells expressing control-shRNA or TRB3-shRNAs were transfected with UbG76V-GFP plasmid plus or minus with p62-DDK-expressing plasmid. The lysates were isolated for immunoblotting. Data are representative immunoblots of three independent assays. (f) HepG2 cells expressing p62- or control-siRNA were infected with adenovirus containing mRFP-GFP-LC3-PE. After 24 h, the cells were treated with IGF-1 for 12 h, and the flux rate of autophagy was detected with Live Cell Imaging Microscopy. Data are representative images of three independent assays. Red colour shows autophagolysosomes and double-colour red/green shows autophagosomes. Scale bar, 10 μm. (g) Control or p62-silencing cells were treated with or without IGF-1 for 12 h. Indicated proteins were evaluated with immunoblotting. Data are representative immunoblots of three independent assays. (h) BALB/c nude mice were i.v. injected with HepG2 cells expressing control-shRNA or p62-shRNA (3 × 106). Data are representative of number of metastases in lungs and lungs. Metastatic nodules were indicated by arrows (n=8 per group). (i) BALB/c nude mice were s.c. inoculated with HepG2 cells expressing control- or p62-shRNA (1.5 × 106). Data are the mean volumes±s.e.m. at indicated times and representative tumours along with tumour weight (n=8 per group). Scale bar, 1.5 cm. Statistical significance was determined with Student's t-test; *P<0.05.
Mentions: We examined how TRB3 mediated the insulin/IGF-induced p62 accumulation. Insulin/IGF-1 did not change p62 transcription (Fig. 4a) but rather markedly enhanced the half-life of p62 degradation from 8.5 to 24 h (Fig. 4b). Silencing TRB3 promoted p62 degradation in the presence or absence of IGF-1 (Fig. 4c), indicating that TRB3 mediates insulin/IGF-induced p62 accumulation. Interestingly, p62 depletion not only reduced basal but also blocked IGF-1-induced expression of UbG76V-GFP (Fig. 4d), coinciding with a report that p62 accumulation by autophagy inhibition slows down the UPS-specific substrate clearance29. To verify whether inhibitory effect of TRB3 on UPS was mediated by p62 accumulation, the p62 level was recovered by ectopic expression of p62 in TRB3-silenced cells. Overexpressing p62 rescued UbG76V-GFP activity in TRB3-silenced cells, indicating that TRB3 blocks UPS clearance through inducing p62 accumulation (Fig. 4e). In addition, silencing p62 moderately enhanced autophagic flux in quiescent cells and partially restored the IGF-1-suppressed autophagic flux (Fig. 4f). However, silencing p62 had a mixed effect on expression of the tumour-promoting factors in quiescent cells, which was quite different from the effects induced by TRB3 depletion; it could not protect them from the IGF-1 induction as did by TRB3 depletion (Fig. 4g). Thus, p62 depletion produced only a moderate antitumour role in tumour metastasis and growth (Fig. 4h,i). These results indicate that TRB3 mediates the IGF-induced p62 accumulation, which compromises UPS as well as (partially) autophagic degradation in cancer cells.

Bottom Line: Here we report a previously unrecognized tumour-promoting mechanism for stress protein TRB3, which mediates a reciprocal antagonism between autophagic and proteasomal degradation systems and connects insulin/IGF to malignant promotion.TRB3 interacts with autophagic receptor p62 and hinders p62 binding to LC3 and ubiquitinated substrates, which causes p62 deposition and suppresses autophagic/proteasomal degradation.Interrupting TRB3/p62 interaction produces potent antitumour efficacies against tumour growth and metastasis.

View Article: PubMed Central - PubMed

Affiliation: Immunology and Cancer Pharmacology Group, State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia Medica, Chinese Academy of Medical Sciences, Peking Union Medical College, Beijing 100050, China.

ABSTRACT
High insulin/IGF is a biologic link between diabetes and cancers, but the underlying molecular mechanism remains unclear. Here we report a previously unrecognized tumour-promoting mechanism for stress protein TRB3, which mediates a reciprocal antagonism between autophagic and proteasomal degradation systems and connects insulin/IGF to malignant promotion. We find that several human cancers express higher TRB3 and phosphorylated insulin receptor substrate 1, which correlates negatively with patient's prognosis. TRB3 depletion protects against tumour-promoting actions of insulin/IGF and attenuates tumour initiation, growth and metastasis in mice. TRB3 interacts with autophagic receptor p62 and hinders p62 binding to LC3 and ubiquitinated substrates, which causes p62 deposition and suppresses autophagic/proteasomal degradation. Several tumour-promoting factors accumulate in cancer cells to support tumour metabolism, proliferation, invasion and metastasis. Interrupting TRB3/p62 interaction produces potent antitumour efficacies against tumour growth and metastasis. Our study opens possibility of targeting this interaction as a potential novel strategy against cancers with diabetes.

No MeSH data available.


Related in: MedlinePlus