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Induction of c-Cbl contributes to anti-cancer effects of HDAC inhibitor in lung cancer.

Wei TT, Lin YC, Lin PH, Shih JY, Chou CW, Huang WJ, Yang YC, Hsiao PW, Chen CC - Oncotarget (2015)

Bottom Line: Here we found loss of c-Cbl, an E3 ligase, expression in non-small cell lung cancer (NSCLC) compared with its adjacent normal tissue in patient specimens.HDAC inhibition by WJ or knockdown of HDAC 1, HDAC2, HDAC3 or HDAC6 all induced c-Cbl.Furthermore, WJ inhibited lung tumor growth through c-Cbl induction in orthotopic and tail vein injected models.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Here we found loss of c-Cbl, an E3 ligase, expression in non-small cell lung cancer (NSCLC) compared with its adjacent normal tissue in patient specimens. HDAC inhibition by WJ or knockdown of HDAC 1, HDAC2, HDAC3 or HDAC6 all induced c-Cbl. Ectopic expression of c-Cbl induced decreased EGFR, inhibited growth in NSCLC cells. Knockdown of EGFR inhibited NSCLC growth. Mutation of EGFR at Y1045 decreased WJ-induced growth inhibition as well as in vivo anti-cancer effect and EGFR degradation mediated by WJ. Time-lapse confocal analysis showed co-localization of c-Cbl and EGFR after WJ treatment. Furthermore, WJ inhibited lung tumor growth through c-Cbl induction in orthotopic and tail vein injected models. C-Cbl up-regulation induced by HDACi is a potential strategy for NSCLC treatment.

No MeSH data available.


Related in: MedlinePlus

HDAC inhibitor reduced EGFR expression through c-Cbl-dependent lysosomal degradation pathwayA, NSCLC cells were treated with 1, 5, 10 μM WJ for 24 hours, or treated with 5 μM WJ for 6, 12, 24 hours. Total cell lysates were prepared and western blot was performed using indicated antibiodies. B, Effect of siRNA-mediated knockdown of c-Cbl on WJ-induced EGFR degradation in A549 cells. C, A549 cells were pre-treated with 20 mM NH4Cl or 10 μM lactacystin (LC) for 30 minutes followed by 5 μM WJ for 24 hours (upper panel). EGFR associates with late endosome/lysosome (lower panel). A549 cells were treated with DMSO, 5 uM WJ, 20 mM NH4Cl or WJ plus NH4Cl for 24 hours. The evaluation of EGFR and LAMP1 expression were detected by laser scan confocal microscopy. Scale bars: 50 μm. D, Effect of phosphorylations of EGFR in A549 cells after WJ treatment. A549 cells were pre-treated with 20 mM NH4Cl for 30 minutes followed by 5 μM WJ for 0.5 to 6 hours. E, Effect of siRNA-mediated knockdown of EGFR on cell viability (left panel). A549 cells were transfected with EGFR siRNA or control (scramble) siRNA. **P<0.01 versus control. Effect of NH4Cl on WJ-induced cytotoxicity (right panel). A549 cells were pre-treated with 20 mM NH4Cl for 30 minutes followed by WJ for 24 hours. The cell viability was measured by MTT assay. *P<0.05, **P<0.01 versus basal. ##P<0.01.
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Figure 2: HDAC inhibitor reduced EGFR expression through c-Cbl-dependent lysosomal degradation pathwayA, NSCLC cells were treated with 1, 5, 10 μM WJ for 24 hours, or treated with 5 μM WJ for 6, 12, 24 hours. Total cell lysates were prepared and western blot was performed using indicated antibiodies. B, Effect of siRNA-mediated knockdown of c-Cbl on WJ-induced EGFR degradation in A549 cells. C, A549 cells were pre-treated with 20 mM NH4Cl or 10 μM lactacystin (LC) for 30 minutes followed by 5 μM WJ for 24 hours (upper panel). EGFR associates with late endosome/lysosome (lower panel). A549 cells were treated with DMSO, 5 uM WJ, 20 mM NH4Cl or WJ plus NH4Cl for 24 hours. The evaluation of EGFR and LAMP1 expression were detected by laser scan confocal microscopy. Scale bars: 50 μm. D, Effect of phosphorylations of EGFR in A549 cells after WJ treatment. A549 cells were pre-treated with 20 mM NH4Cl for 30 minutes followed by 5 μM WJ for 0.5 to 6 hours. E, Effect of siRNA-mediated knockdown of EGFR on cell viability (left panel). A549 cells were transfected with EGFR siRNA or control (scramble) siRNA. **P<0.01 versus control. Effect of NH4Cl on WJ-induced cytotoxicity (right panel). A549 cells were pre-treated with 20 mM NH4Cl for 30 minutes followed by WJ for 24 hours. The cell viability was measured by MTT assay. *P<0.05, **P<0.01 versus basal. ##P<0.01.

Mentions: Since WJ could induce c-Cbl expression, its effect on EGFR was evaluated. WJ induced EGFR down-regulation in A549 cells, as well as SAHA and TSA did (Figure 2A and Supplementary Figure S1C). Its effect on wild-type or mutant EGFR lung adenocarcinoma cells was further evaluated. WJ down-regulated EGFR expression in all NSCLC cells and inhibited its downstream AKT and ERK in a dose- and time-dependent manner (Figure 2A). Knockdown of c-Cbl reversed WJ-mediated EGFR inhibition (Figure 2B), suggesting that WJ mediated EGFR inhibition through c-Cbl expression.


Induction of c-Cbl contributes to anti-cancer effects of HDAC inhibitor in lung cancer.

Wei TT, Lin YC, Lin PH, Shih JY, Chou CW, Huang WJ, Yang YC, Hsiao PW, Chen CC - Oncotarget (2015)

HDAC inhibitor reduced EGFR expression through c-Cbl-dependent lysosomal degradation pathwayA, NSCLC cells were treated with 1, 5, 10 μM WJ for 24 hours, or treated with 5 μM WJ for 6, 12, 24 hours. Total cell lysates were prepared and western blot was performed using indicated antibiodies. B, Effect of siRNA-mediated knockdown of c-Cbl on WJ-induced EGFR degradation in A549 cells. C, A549 cells were pre-treated with 20 mM NH4Cl or 10 μM lactacystin (LC) for 30 minutes followed by 5 μM WJ for 24 hours (upper panel). EGFR associates with late endosome/lysosome (lower panel). A549 cells were treated with DMSO, 5 uM WJ, 20 mM NH4Cl or WJ plus NH4Cl for 24 hours. The evaluation of EGFR and LAMP1 expression were detected by laser scan confocal microscopy. Scale bars: 50 μm. D, Effect of phosphorylations of EGFR in A549 cells after WJ treatment. A549 cells were pre-treated with 20 mM NH4Cl for 30 minutes followed by 5 μM WJ for 0.5 to 6 hours. E, Effect of siRNA-mediated knockdown of EGFR on cell viability (left panel). A549 cells were transfected with EGFR siRNA or control (scramble) siRNA. **P<0.01 versus control. Effect of NH4Cl on WJ-induced cytotoxicity (right panel). A549 cells were pre-treated with 20 mM NH4Cl for 30 minutes followed by WJ for 24 hours. The cell viability was measured by MTT assay. *P<0.05, **P<0.01 versus basal. ##P<0.01.
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Figure 2: HDAC inhibitor reduced EGFR expression through c-Cbl-dependent lysosomal degradation pathwayA, NSCLC cells were treated with 1, 5, 10 μM WJ for 24 hours, or treated with 5 μM WJ for 6, 12, 24 hours. Total cell lysates were prepared and western blot was performed using indicated antibiodies. B, Effect of siRNA-mediated knockdown of c-Cbl on WJ-induced EGFR degradation in A549 cells. C, A549 cells were pre-treated with 20 mM NH4Cl or 10 μM lactacystin (LC) for 30 minutes followed by 5 μM WJ for 24 hours (upper panel). EGFR associates with late endosome/lysosome (lower panel). A549 cells were treated with DMSO, 5 uM WJ, 20 mM NH4Cl or WJ plus NH4Cl for 24 hours. The evaluation of EGFR and LAMP1 expression were detected by laser scan confocal microscopy. Scale bars: 50 μm. D, Effect of phosphorylations of EGFR in A549 cells after WJ treatment. A549 cells were pre-treated with 20 mM NH4Cl for 30 minutes followed by 5 μM WJ for 0.5 to 6 hours. E, Effect of siRNA-mediated knockdown of EGFR on cell viability (left panel). A549 cells were transfected with EGFR siRNA or control (scramble) siRNA. **P<0.01 versus control. Effect of NH4Cl on WJ-induced cytotoxicity (right panel). A549 cells were pre-treated with 20 mM NH4Cl for 30 minutes followed by WJ for 24 hours. The cell viability was measured by MTT assay. *P<0.05, **P<0.01 versus basal. ##P<0.01.
Mentions: Since WJ could induce c-Cbl expression, its effect on EGFR was evaluated. WJ induced EGFR down-regulation in A549 cells, as well as SAHA and TSA did (Figure 2A and Supplementary Figure S1C). Its effect on wild-type or mutant EGFR lung adenocarcinoma cells was further evaluated. WJ down-regulated EGFR expression in all NSCLC cells and inhibited its downstream AKT and ERK in a dose- and time-dependent manner (Figure 2A). Knockdown of c-Cbl reversed WJ-mediated EGFR inhibition (Figure 2B), suggesting that WJ mediated EGFR inhibition through c-Cbl expression.

Bottom Line: Here we found loss of c-Cbl, an E3 ligase, expression in non-small cell lung cancer (NSCLC) compared with its adjacent normal tissue in patient specimens.HDAC inhibition by WJ or knockdown of HDAC 1, HDAC2, HDAC3 or HDAC6 all induced c-Cbl.Furthermore, WJ inhibited lung tumor growth through c-Cbl induction in orthotopic and tail vein injected models.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology, College of Medicine, National Taiwan University, Taipei, Taiwan.

ABSTRACT
Here we found loss of c-Cbl, an E3 ligase, expression in non-small cell lung cancer (NSCLC) compared with its adjacent normal tissue in patient specimens. HDAC inhibition by WJ or knockdown of HDAC 1, HDAC2, HDAC3 or HDAC6 all induced c-Cbl. Ectopic expression of c-Cbl induced decreased EGFR, inhibited growth in NSCLC cells. Knockdown of EGFR inhibited NSCLC growth. Mutation of EGFR at Y1045 decreased WJ-induced growth inhibition as well as in vivo anti-cancer effect and EGFR degradation mediated by WJ. Time-lapse confocal analysis showed co-localization of c-Cbl and EGFR after WJ treatment. Furthermore, WJ inhibited lung tumor growth through c-Cbl induction in orthotopic and tail vein injected models. C-Cbl up-regulation induced by HDACi is a potential strategy for NSCLC treatment.

No MeSH data available.


Related in: MedlinePlus