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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 promotes tumour development in T2D mice.(a) Blood glucose/insulin/IGF-1 was measured in C57 BL/6 and KK-Ay mice. Dashed lines indicate the normal range of blood glucose (3.5–9.8 mM) or insulin (0.2–0.6 ng ml−1). Data are mean±s.e.m. of three assays (n=8 per group). Inserts show TRB3 expression in the liver and lungs. (b) KK-Ay or C57 BL/6 mice were s.c. injected with B16-F10 cells (1.5 × 105). Data are the mean volumes±s.e.m. at indicated times and representative mice (n=7 per group). Insert shows TRB3 expression in xenograft tumour. (c) Photographs of representative tumour and quantified tumour weight. Data are mean weight±s.e.m. (n=7 per group). Scale bar, 1 cm. (d) KK-Ay or C57 BL/6 mice were i.v. injected with B16-F10 cells (3 × 105). Data are representative of macroscopy and bioluminescence images with total tumour volumes at multiple metastatic sites (mean±s.e.m.; n=12 per group). (e) Male KK-Ay (n=11) and C57 BL/6 (n=9) mice were p.o. gavaged with 0.1 ml (total 1 mg) DMBA once a week for 5 weeks. Data are timeline (upper) and macroscopic, and histopathological analysis of tumours (lower) with numbers of tumour nodules (mean±s.e.m.) and expression of TRB3 and γH2AX in the liver and lung. Scale bar, 10 μm. (f) Kaplan–Meier survival curve for mice fed with DMBA (n=20 per group). Statistical significance was determined by Kaplan–Meier log-rank test; *P<0.05. (g) Overexpression of TRB3 promotes DEN-induced tumorigenesis. Data are timeline and TRB3 expression in the liver and lungs (upper), and haematoxylin and eosin-stained sections plus incidence and number of tumour nodules (lower). Data are mean±s.e.m. (n=10 per group). Scale bar, 10 μm. (h) Blood glucose/insulin/IGF-1 was measured in mice infected with control-shRNA or TRB3-shRNA lentiviral particles (2 × 105). Data are mean±s.e.m. of three assays with triplicates (n=8 per group). The inserts show TRB3 expression in the liver and lung. (i,j) TRB3 knockdown (n=9) or control KK-Ay (n=8) were s.c. inoculated with B16-F10 cells (1.5 × 105). Data are the mean volumes±s.e.m. at indicated times and representative mice (i) and tumours along with tumour weight (j). Scale bars, 1 cm. (k) TRB3 knockdown (n=9) or control KK-Ay (n=8) were i.v. injected with B16-F10 cells (3 × 105). Data are representatives of macroscopy and bioluminescence images with total tumour volumes (mean±s.e.m) at multiple metastatic sites. Except for f, statistical significance was determined with Student's t-test; *P<0.05; **P<0.01; ***P<0.001.
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f2: TRB3 promotes tumour development in T2D mice.(a) Blood glucose/insulin/IGF-1 was measured in C57 BL/6 and KK-Ay mice. Dashed lines indicate the normal range of blood glucose (3.5–9.8 mM) or insulin (0.2–0.6 ng ml−1). Data are mean±s.e.m. of three assays (n=8 per group). Inserts show TRB3 expression in the liver and lungs. (b) KK-Ay or C57 BL/6 mice were s.c. injected with B16-F10 cells (1.5 × 105). Data are the mean volumes±s.e.m. at indicated times and representative mice (n=7 per group). Insert shows TRB3 expression in xenograft tumour. (c) Photographs of representative tumour and quantified tumour weight. Data are mean weight±s.e.m. (n=7 per group). Scale bar, 1 cm. (d) KK-Ay or C57 BL/6 mice were i.v. injected with B16-F10 cells (3 × 105). Data are representative of macroscopy and bioluminescence images with total tumour volumes at multiple metastatic sites (mean±s.e.m.; n=12 per group). (e) Male KK-Ay (n=11) and C57 BL/6 (n=9) mice were p.o. gavaged with 0.1 ml (total 1 mg) DMBA once a week for 5 weeks. Data are timeline (upper) and macroscopic, and histopathological analysis of tumours (lower) with numbers of tumour nodules (mean±s.e.m.) and expression of TRB3 and γH2AX in the liver and lung. Scale bar, 10 μm. (f) Kaplan–Meier survival curve for mice fed with DMBA (n=20 per group). Statistical significance was determined by Kaplan–Meier log-rank test; *P<0.05. (g) Overexpression of TRB3 promotes DEN-induced tumorigenesis. Data are timeline and TRB3 expression in the liver and lungs (upper), and haematoxylin and eosin-stained sections plus incidence and number of tumour nodules (lower). Data are mean±s.e.m. (n=10 per group). Scale bar, 10 μm. (h) Blood glucose/insulin/IGF-1 was measured in mice infected with control-shRNA or TRB3-shRNA lentiviral particles (2 × 105). Data are mean±s.e.m. of three assays with triplicates (n=8 per group). The inserts show TRB3 expression in the liver and lung. (i,j) TRB3 knockdown (n=9) or control KK-Ay (n=8) were s.c. inoculated with B16-F10 cells (1.5 × 105). Data are the mean volumes±s.e.m. at indicated times and representative mice (i) and tumours along with tumour weight (j). Scale bars, 1 cm. (k) TRB3 knockdown (n=9) or control KK-Ay (n=8) were i.v. injected with B16-F10 cells (3 × 105). Data are representatives of macroscopy and bioluminescence images with total tumour volumes (mean±s.e.m) at multiple metastatic sites. Except for f, statistical significance was determined with Student's t-test; *P<0.05; **P<0.01; ***P<0.001.

Mentions: Genetically diabetic KK-Ay mouse has been widely used as a relevant model of human T2D (ref. 20). Adult KK-Ay mice not only exhibited hyperglycaemia, hyperinsulinaemia as well high plasma IGF-1, but also showed higher TRB3 expression in the liver and lungs compared with C57 BL/6 mice (Fig. 2a). We found that B16-F10 melanoma cells grew faster in diabetic KK-Ay mice than in C57 BL/6 mice, especially at the later stage after tumour-cell inoculation; the xenograft tumours from KK-Ay mice showed a higher TRB3 expression in comparison with those from C57BL/6 mice (Fig. 2b,c). Metastatic nodules with huge gross volume were found in multiple organs, including lungs, mesentery, omentum, mediastinum and axillary lymph nodes, in KK-Ay mice, whereas lungs were primary metastatic organ with smaller volume and few metastases were found in the mediastinum of C57 BL/6 mice (Fig. 2d and Supplementary Table 2). We compared tumorigenesis in C57 BL/6 and KK-Ay mice fed with 7,12-dimethylbenz(a) anthracene (DMBA), a chemical carcinogen causing Ras mutation21. More HCC and lung tumours with a higher mortality rate developed in KK-Ay mice than in C57 BL/6 mice (Fig. 2e,f). Consistently, higher expression of TRB3 and γH2AX was found in the liver and lungs of KK-Ay mice (Fig. 2e). We then examined whether ectopically expressed TRB3 promoted carcinogen-evoked tumorigenesis. HCC or lung tumours developed in 80% of TRB3-Ad but in less than 50% of green fluorescent protein (GFP)-Ad-infected C57 BL/6 mice in response to diethylnitrosamine (DEN; Fig. 2g). In addition, more tumour nodules developed in the TRB3-Ad than in GFP-Ad-infected mice (Fig. 2g). To validate the role of TRB3 in diabetes-accelerated tumour development, we examined tumour growth and metastasis in TRB3 knockdown KK-Ay mice. We found that TRB3-short hairpin RNA (shRNA) lentiviral infection reduced TRB3 expression but did not change hyperglycaemia and plasma level of insulin or IGF-1 (Fig. 2h). However, TRB3 knockdown repressed tumour growth and metastasis in KK-Ay mice (Fig. 2i–k). As shown in Fig. 2i, TRB3 expression in tumour nodules from TRB3 knockdown KK-Ay mice was much lower than that from control KK-Ay mice, suggesting that the higher insulin/IGF-1 in TRB3 knockdown KK-Ay mice cannot enhance TRB3 expression in the inoculated tumour cells as it does in the control KK-Ay mice. To further confirm that whether it was the reduced TRB3 in tumour cells responsible for the tumour growth inhibition, B16-F10 cells stably expressing control-shRNA or TRB3-shRNA sequences were subcutaneously (s.c.) or intravenously (i.v.) injected into KK-Ay and C57 BL/6 mice. We found that control B16-F10 cells grew at least three times faster than TRB3 depletion B16-F10 cells in KK-Ay mice (Supplementary Fig. 2a). However, control B16-F10 cells grew only two times faster than TRB3 depletion B16-F10 cells in C57 BL/6 mice (Supplementary Fig. 2c). Furthermore, control B16-F10 cells formed metastatic nodules eight times more than TRB3 depletion B16-F10 cells did in KK-Ay mice (Supplementary Fig. 2d); however, control B16-F10 cells form metastatic nodules only three times more than TRB3 depletion B16-F10 cells inC57 BL/6 mice (Supplementary Fig. 2b). These data suggest that it is the insulin/IGF-induced TRB3 in tumour cells that is responsible for the tumour promotion actions. We further evaluated the metastatic and proliferative effects of cancer cells with TRB3 depletion in BALB/c nude mice. Mice i.v. inoculated with HepG2 cells expressing control-shRNA developed more metastatic nodules than mice with cells expressing TRB3-shRNA1/2 (Supplementary Fig. 3a,b). Thus, mice with TRB3-silenced cells survived much longer than did mice with TRB3-control cells (Supplementary Fig. 3c). Moreover, mice s.c. inoculated with control-shRNA cells developed larger tumours than mice with TRB3-shRNA1/2 cells (Supplementary Fig. 3d–f). Similarly, TRB3 depletion inhibited metastasis and growth of HCT-8 cells (Supplementary Fig. 3g,h). These results suggest that TRB3 plays a crucial role in tumorigenesis, tumour growth and metastasis in mice, particularly in mice with T2D.


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 promotes tumour development in T2D mice.(a) Blood glucose/insulin/IGF-1 was measured in C57 BL/6 and KK-Ay mice. Dashed lines indicate the normal range of blood glucose (3.5–9.8 mM) or insulin (0.2–0.6 ng ml−1). Data are mean±s.e.m. of three assays (n=8 per group). Inserts show TRB3 expression in the liver and lungs. (b) KK-Ay or C57 BL/6 mice were s.c. injected with B16-F10 cells (1.5 × 105). Data are the mean volumes±s.e.m. at indicated times and representative mice (n=7 per group). Insert shows TRB3 expression in xenograft tumour. (c) Photographs of representative tumour and quantified tumour weight. Data are mean weight±s.e.m. (n=7 per group). Scale bar, 1 cm. (d) KK-Ay or C57 BL/6 mice were i.v. injected with B16-F10 cells (3 × 105). Data are representative of macroscopy and bioluminescence images with total tumour volumes at multiple metastatic sites (mean±s.e.m.; n=12 per group). (e) Male KK-Ay (n=11) and C57 BL/6 (n=9) mice were p.o. gavaged with 0.1 ml (total 1 mg) DMBA once a week for 5 weeks. Data are timeline (upper) and macroscopic, and histopathological analysis of tumours (lower) with numbers of tumour nodules (mean±s.e.m.) and expression of TRB3 and γH2AX in the liver and lung. Scale bar, 10 μm. (f) Kaplan–Meier survival curve for mice fed with DMBA (n=20 per group). Statistical significance was determined by Kaplan–Meier log-rank test; *P<0.05. (g) Overexpression of TRB3 promotes DEN-induced tumorigenesis. Data are timeline and TRB3 expression in the liver and lungs (upper), and haematoxylin and eosin-stained sections plus incidence and number of tumour nodules (lower). Data are mean±s.e.m. (n=10 per group). Scale bar, 10 μm. (h) Blood glucose/insulin/IGF-1 was measured in mice infected with control-shRNA or TRB3-shRNA lentiviral particles (2 × 105). Data are mean±s.e.m. of three assays with triplicates (n=8 per group). The inserts show TRB3 expression in the liver and lung. (i,j) TRB3 knockdown (n=9) or control KK-Ay (n=8) were s.c. inoculated with B16-F10 cells (1.5 × 105). Data are the mean volumes±s.e.m. at indicated times and representative mice (i) and tumours along with tumour weight (j). Scale bars, 1 cm. (k) TRB3 knockdown (n=9) or control KK-Ay (n=8) were i.v. injected with B16-F10 cells (3 × 105). Data are representatives of macroscopy and bioluminescence images with total tumour volumes (mean±s.e.m) at multiple metastatic sites. Except for f, statistical significance was determined with Student's t-test; *P<0.05; **P<0.01; ***P<0.001.
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f2: TRB3 promotes tumour development in T2D mice.(a) Blood glucose/insulin/IGF-1 was measured in C57 BL/6 and KK-Ay mice. Dashed lines indicate the normal range of blood glucose (3.5–9.8 mM) or insulin (0.2–0.6 ng ml−1). Data are mean±s.e.m. of three assays (n=8 per group). Inserts show TRB3 expression in the liver and lungs. (b) KK-Ay or C57 BL/6 mice were s.c. injected with B16-F10 cells (1.5 × 105). Data are the mean volumes±s.e.m. at indicated times and representative mice (n=7 per group). Insert shows TRB3 expression in xenograft tumour. (c) Photographs of representative tumour and quantified tumour weight. Data are mean weight±s.e.m. (n=7 per group). Scale bar, 1 cm. (d) KK-Ay or C57 BL/6 mice were i.v. injected with B16-F10 cells (3 × 105). Data are representative of macroscopy and bioluminescence images with total tumour volumes at multiple metastatic sites (mean±s.e.m.; n=12 per group). (e) Male KK-Ay (n=11) and C57 BL/6 (n=9) mice were p.o. gavaged with 0.1 ml (total 1 mg) DMBA once a week for 5 weeks. Data are timeline (upper) and macroscopic, and histopathological analysis of tumours (lower) with numbers of tumour nodules (mean±s.e.m.) and expression of TRB3 and γH2AX in the liver and lung. Scale bar, 10 μm. (f) Kaplan–Meier survival curve for mice fed with DMBA (n=20 per group). Statistical significance was determined by Kaplan–Meier log-rank test; *P<0.05. (g) Overexpression of TRB3 promotes DEN-induced tumorigenesis. Data are timeline and TRB3 expression in the liver and lungs (upper), and haematoxylin and eosin-stained sections plus incidence and number of tumour nodules (lower). Data are mean±s.e.m. (n=10 per group). Scale bar, 10 μm. (h) Blood glucose/insulin/IGF-1 was measured in mice infected with control-shRNA or TRB3-shRNA lentiviral particles (2 × 105). Data are mean±s.e.m. of three assays with triplicates (n=8 per group). The inserts show TRB3 expression in the liver and lung. (i,j) TRB3 knockdown (n=9) or control KK-Ay (n=8) were s.c. inoculated with B16-F10 cells (1.5 × 105). Data are the mean volumes±s.e.m. at indicated times and representative mice (i) and tumours along with tumour weight (j). Scale bars, 1 cm. (k) TRB3 knockdown (n=9) or control KK-Ay (n=8) were i.v. injected with B16-F10 cells (3 × 105). Data are representatives of macroscopy and bioluminescence images with total tumour volumes (mean±s.e.m) at multiple metastatic sites. Except for f, statistical significance was determined with Student's t-test; *P<0.05; **P<0.01; ***P<0.001.
Mentions: Genetically diabetic KK-Ay mouse has been widely used as a relevant model of human T2D (ref. 20). Adult KK-Ay mice not only exhibited hyperglycaemia, hyperinsulinaemia as well high plasma IGF-1, but also showed higher TRB3 expression in the liver and lungs compared with C57 BL/6 mice (Fig. 2a). We found that B16-F10 melanoma cells grew faster in diabetic KK-Ay mice than in C57 BL/6 mice, especially at the later stage after tumour-cell inoculation; the xenograft tumours from KK-Ay mice showed a higher TRB3 expression in comparison with those from C57BL/6 mice (Fig. 2b,c). Metastatic nodules with huge gross volume were found in multiple organs, including lungs, mesentery, omentum, mediastinum and axillary lymph nodes, in KK-Ay mice, whereas lungs were primary metastatic organ with smaller volume and few metastases were found in the mediastinum of C57 BL/6 mice (Fig. 2d and Supplementary Table 2). We compared tumorigenesis in C57 BL/6 and KK-Ay mice fed with 7,12-dimethylbenz(a) anthracene (DMBA), a chemical carcinogen causing Ras mutation21. More HCC and lung tumours with a higher mortality rate developed in KK-Ay mice than in C57 BL/6 mice (Fig. 2e,f). Consistently, higher expression of TRB3 and γH2AX was found in the liver and lungs of KK-Ay mice (Fig. 2e). We then examined whether ectopically expressed TRB3 promoted carcinogen-evoked tumorigenesis. HCC or lung tumours developed in 80% of TRB3-Ad but in less than 50% of green fluorescent protein (GFP)-Ad-infected C57 BL/6 mice in response to diethylnitrosamine (DEN; Fig. 2g). In addition, more tumour nodules developed in the TRB3-Ad than in GFP-Ad-infected mice (Fig. 2g). To validate the role of TRB3 in diabetes-accelerated tumour development, we examined tumour growth and metastasis in TRB3 knockdown KK-Ay mice. We found that TRB3-short hairpin RNA (shRNA) lentiviral infection reduced TRB3 expression but did not change hyperglycaemia and plasma level of insulin or IGF-1 (Fig. 2h). However, TRB3 knockdown repressed tumour growth and metastasis in KK-Ay mice (Fig. 2i–k). As shown in Fig. 2i, TRB3 expression in tumour nodules from TRB3 knockdown KK-Ay mice was much lower than that from control KK-Ay mice, suggesting that the higher insulin/IGF-1 in TRB3 knockdown KK-Ay mice cannot enhance TRB3 expression in the inoculated tumour cells as it does in the control KK-Ay mice. To further confirm that whether it was the reduced TRB3 in tumour cells responsible for the tumour growth inhibition, B16-F10 cells stably expressing control-shRNA or TRB3-shRNA sequences were subcutaneously (s.c.) or intravenously (i.v.) injected into KK-Ay and C57 BL/6 mice. We found that control B16-F10 cells grew at least three times faster than TRB3 depletion B16-F10 cells in KK-Ay mice (Supplementary Fig. 2a). However, control B16-F10 cells grew only two times faster than TRB3 depletion B16-F10 cells in C57 BL/6 mice (Supplementary Fig. 2c). Furthermore, control B16-F10 cells formed metastatic nodules eight times more than TRB3 depletion B16-F10 cells did in KK-Ay mice (Supplementary Fig. 2d); however, control B16-F10 cells form metastatic nodules only three times more than TRB3 depletion B16-F10 cells inC57 BL/6 mice (Supplementary Fig. 2b). These data suggest that it is the insulin/IGF-induced TRB3 in tumour cells that is responsible for the tumour promotion actions. We further evaluated the metastatic and proliferative effects of cancer cells with TRB3 depletion in BALB/c nude mice. Mice i.v. inoculated with HepG2 cells expressing control-shRNA developed more metastatic nodules than mice with cells expressing TRB3-shRNA1/2 (Supplementary Fig. 3a,b). Thus, mice with TRB3-silenced cells survived much longer than did mice with TRB3-control cells (Supplementary Fig. 3c). Moreover, mice s.c. inoculated with control-shRNA cells developed larger tumours than mice with TRB3-shRNA1/2 cells (Supplementary Fig. 3d–f). Similarly, TRB3 depletion inhibited metastasis and growth of HCT-8 cells (Supplementary Fig. 3g,h). These results suggest that TRB3 plays a crucial role in tumorigenesis, tumour growth and metastasis in mice, particularly in mice with T2D.

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