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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 inhibition induced co-localization of c-Cbl and EGFR and promoted c-Cbl mRNA expression in lung adenocarcinoma cellsA, A549 cells expressing c-Cbl-GFP (green) and EGFR-RFP (red) were exposed to 1 μM lysotraker (blue) for 30 minutes, and imaged for 120 minutes. Quantification of c-Cbl co-localized with EGFR (left panel). Plotted are the coefficient of co-localization between c-Cbl and EGFR at indicated times after treatment with 5 μM WJ. Co-localization between c-Cbl and EGFR in A549 cells after treatment with 5 μM WJ for 30 minutes. Co-localized signal is pseudo colored in yellow. Scale bars: 20 μm. Confocal microscopy of c-Cbl (green), EGFR (red) and lysosome (blue) in A549 cells after treatment with 5 μM WJ for indicated times (right panel). Scale bars: 20 μm. B, Induction of c-Cbl mRNA expression in A549 cells after WJ treatment (left panel). A549 cells were treated with WJ for indicated doses and time. Induction of c-Cbl RNA expression in A549 cells after MS-275 or apicidin treatment (right panel). The relative mRNA expression levels of c-Cbl were examined by Q-PCR. The levels of c-Cbl mRNA were normalized to level of GAPDH mRNA. *P<0.05, **P<0.01 versus basal. C, A549 cells were transfected with siRNA against HDAC1, HDAC2, HDAC3 or control (scramble) siRNA. *P<0.05, **P<0.01 versus basal. The mRNA expression level of c-Cbl was examined by RT-PCR (upper panel). The relative mRNA expression level of c-Cbl were also examined by Q-PCR (lower panel). The levels of c-Cbl mRNA were normalized to level of GAPDH mRNA. *P<0.05, **P<0.01 versus control. D, Histone acetylation and methylation within the c-Cbl promoter was regulated by WJ. Anti-H3K27-ac or anti-H3K27-me3 antibody was used in ChIP-qPCR to measure the levels of histone markers on c-Cbl promoter. Data were analyzed by the CT method and plotted as percent (%) input DNA. qChIP values were calculated by the following formula: percent (%) input recovery = 100 × 2 (input CT - bound CT). **P <0.01 versus basal. E, Effect of mithromycin A (MTM) on WJ-induced c-Cbl up-regulation. A549 cells were treated with 5 uM WJ, 1 uM MTM, or their combination for 24 hours. Total cell lysates were prepared and western blot was performed using indicated antibiodies.
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Figure 3: HDAC inhibition induced co-localization of c-Cbl and EGFR and promoted c-Cbl mRNA expression in lung adenocarcinoma cellsA, A549 cells expressing c-Cbl-GFP (green) and EGFR-RFP (red) were exposed to 1 μM lysotraker (blue) for 30 minutes, and imaged for 120 minutes. Quantification of c-Cbl co-localized with EGFR (left panel). Plotted are the coefficient of co-localization between c-Cbl and EGFR at indicated times after treatment with 5 μM WJ. Co-localization between c-Cbl and EGFR in A549 cells after treatment with 5 μM WJ for 30 minutes. Co-localized signal is pseudo colored in yellow. Scale bars: 20 μm. Confocal microscopy of c-Cbl (green), EGFR (red) and lysosome (blue) in A549 cells after treatment with 5 μM WJ for indicated times (right panel). Scale bars: 20 μm. B, Induction of c-Cbl mRNA expression in A549 cells after WJ treatment (left panel). A549 cells were treated with WJ for indicated doses and time. Induction of c-Cbl RNA expression in A549 cells after MS-275 or apicidin treatment (right panel). The relative mRNA expression levels of c-Cbl were examined by Q-PCR. The levels of c-Cbl mRNA were normalized to level of GAPDH mRNA. *P<0.05, **P<0.01 versus basal. C, A549 cells were transfected with siRNA against HDAC1, HDAC2, HDAC3 or control (scramble) siRNA. *P<0.05, **P<0.01 versus basal. The mRNA expression level of c-Cbl was examined by RT-PCR (upper panel). The relative mRNA expression level of c-Cbl were also examined by Q-PCR (lower panel). The levels of c-Cbl mRNA were normalized to level of GAPDH mRNA. *P<0.05, **P<0.01 versus control. D, Histone acetylation and methylation within the c-Cbl promoter was regulated by WJ. Anti-H3K27-ac or anti-H3K27-me3 antibody was used in ChIP-qPCR to measure the levels of histone markers on c-Cbl promoter. Data were analyzed by the CT method and plotted as percent (%) input DNA. qChIP values were calculated by the following formula: percent (%) input recovery = 100 × 2 (input CT - bound CT). **P <0.01 versus basal. E, Effect of mithromycin A (MTM) on WJ-induced c-Cbl up-regulation. A549 cells were treated with 5 uM WJ, 1 uM MTM, or their combination for 24 hours. Total cell lysates were prepared and western blot was performed using indicated antibiodies.

Mentions: Since WJ induced c-Cbl to degrade EGFR, the localization of c-Cbl and EGFR was evaluated in live cells by time-lapse confocal microscopy. The co-localization of c-Cbl and EGFR was observed at 30 minutes after WJ treatment (Figure 3A), and EGFR degradation ocurred at 60 minutes (Figure 3A and movie 1 in supplementary material), suggesting that WJ induced co-localization of c-Cbl and EGFR to degrade EGFR.


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 inhibition induced co-localization of c-Cbl and EGFR and promoted c-Cbl mRNA expression in lung adenocarcinoma cellsA, A549 cells expressing c-Cbl-GFP (green) and EGFR-RFP (red) were exposed to 1 μM lysotraker (blue) for 30 minutes, and imaged for 120 minutes. Quantification of c-Cbl co-localized with EGFR (left panel). Plotted are the coefficient of co-localization between c-Cbl and EGFR at indicated times after treatment with 5 μM WJ. Co-localization between c-Cbl and EGFR in A549 cells after treatment with 5 μM WJ for 30 minutes. Co-localized signal is pseudo colored in yellow. Scale bars: 20 μm. Confocal microscopy of c-Cbl (green), EGFR (red) and lysosome (blue) in A549 cells after treatment with 5 μM WJ for indicated times (right panel). Scale bars: 20 μm. B, Induction of c-Cbl mRNA expression in A549 cells after WJ treatment (left panel). A549 cells were treated with WJ for indicated doses and time. Induction of c-Cbl RNA expression in A549 cells after MS-275 or apicidin treatment (right panel). The relative mRNA expression levels of c-Cbl were examined by Q-PCR. The levels of c-Cbl mRNA were normalized to level of GAPDH mRNA. *P<0.05, **P<0.01 versus basal. C, A549 cells were transfected with siRNA against HDAC1, HDAC2, HDAC3 or control (scramble) siRNA. *P<0.05, **P<0.01 versus basal. The mRNA expression level of c-Cbl was examined by RT-PCR (upper panel). The relative mRNA expression level of c-Cbl were also examined by Q-PCR (lower panel). The levels of c-Cbl mRNA were normalized to level of GAPDH mRNA. *P<0.05, **P<0.01 versus control. D, Histone acetylation and methylation within the c-Cbl promoter was regulated by WJ. Anti-H3K27-ac or anti-H3K27-me3 antibody was used in ChIP-qPCR to measure the levels of histone markers on c-Cbl promoter. Data were analyzed by the CT method and plotted as percent (%) input DNA. qChIP values were calculated by the following formula: percent (%) input recovery = 100 × 2 (input CT - bound CT). **P <0.01 versus basal. E, Effect of mithromycin A (MTM) on WJ-induced c-Cbl up-regulation. A549 cells were treated with 5 uM WJ, 1 uM MTM, or their combination for 24 hours. Total cell lysates were prepared and western blot was performed using indicated antibiodies.
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Figure 3: HDAC inhibition induced co-localization of c-Cbl and EGFR and promoted c-Cbl mRNA expression in lung adenocarcinoma cellsA, A549 cells expressing c-Cbl-GFP (green) and EGFR-RFP (red) were exposed to 1 μM lysotraker (blue) for 30 minutes, and imaged for 120 minutes. Quantification of c-Cbl co-localized with EGFR (left panel). Plotted are the coefficient of co-localization between c-Cbl and EGFR at indicated times after treatment with 5 μM WJ. Co-localization between c-Cbl and EGFR in A549 cells after treatment with 5 μM WJ for 30 minutes. Co-localized signal is pseudo colored in yellow. Scale bars: 20 μm. Confocal microscopy of c-Cbl (green), EGFR (red) and lysosome (blue) in A549 cells after treatment with 5 μM WJ for indicated times (right panel). Scale bars: 20 μm. B, Induction of c-Cbl mRNA expression in A549 cells after WJ treatment (left panel). A549 cells were treated with WJ for indicated doses and time. Induction of c-Cbl RNA expression in A549 cells after MS-275 or apicidin treatment (right panel). The relative mRNA expression levels of c-Cbl were examined by Q-PCR. The levels of c-Cbl mRNA were normalized to level of GAPDH mRNA. *P<0.05, **P<0.01 versus basal. C, A549 cells were transfected with siRNA against HDAC1, HDAC2, HDAC3 or control (scramble) siRNA. *P<0.05, **P<0.01 versus basal. The mRNA expression level of c-Cbl was examined by RT-PCR (upper panel). The relative mRNA expression level of c-Cbl were also examined by Q-PCR (lower panel). The levels of c-Cbl mRNA were normalized to level of GAPDH mRNA. *P<0.05, **P<0.01 versus control. D, Histone acetylation and methylation within the c-Cbl promoter was regulated by WJ. Anti-H3K27-ac or anti-H3K27-me3 antibody was used in ChIP-qPCR to measure the levels of histone markers on c-Cbl promoter. Data were analyzed by the CT method and plotted as percent (%) input DNA. qChIP values were calculated by the following formula: percent (%) input recovery = 100 × 2 (input CT - bound CT). **P <0.01 versus basal. E, Effect of mithromycin A (MTM) on WJ-induced c-Cbl up-regulation. A549 cells were treated with 5 uM WJ, 1 uM MTM, or their combination for 24 hours. Total cell lysates were prepared and western blot was performed using indicated antibiodies.
Mentions: Since WJ induced c-Cbl to degrade EGFR, the localization of c-Cbl and EGFR was evaluated in live cells by time-lapse confocal microscopy. The co-localization of c-Cbl and EGFR was observed at 30 minutes after WJ treatment (Figure 3A), and EGFR degradation ocurred at 60 minutes (Figure 3A and movie 1 in supplementary material), suggesting that WJ induced co-localization of c-Cbl and EGFR to degrade EGFR.

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