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Targeting the degradation of AXL receptor tyrosine kinase to overcome resistance in gefitinib-resistant non-small cell lung cancer.

Bae SY, Hong JY, Lee HJ, Park HJ, Lee SK - Oncotarget (2015)

Bottom Line: Here, we first demonstrate that AXL is overexpressed in an acquired gefitinib-resistant cell line (H292-Gef) as a result of slow turnover and that AXL is degraded by presenilin-dependent regulated intramembrane proteolysis (PS-RIP).Treatment with YD effectively suppressed the cancer cell survival in vitro and in vivo.Mechanistically, YD accelerated the turnover of AXL by PS-RIP and resulted in the down-regulation of the full-length AXL.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Seoul National University, Seoul 151-742, Korea.

ABSTRACT
Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, remains a major problem in non-small cell lung cancer (NSCLC) treatment. Increased activation of AXL has been identified as a novel mechanism for acquired resistance to EGFR-TKIs in NSCLC treatment. However, the cause of uncontrolled AXL expression is not fully understood. Here, we first demonstrate that AXL is overexpressed in an acquired gefitinib-resistant cell line (H292-Gef) as a result of slow turnover and that AXL is degraded by presenilin-dependent regulated intramembrane proteolysis (PS-RIP). Based on the findings, we attempted to enhance AXL degradation to overcome acquired gefitinib-resistance by the treatment of gefitinib-resistant NSCLC cells with yuanhuadine (YD), a potent antitumor agent in NSCLC. Treatment with YD effectively suppressed the cancer cell survival in vitro and in vivo. Mechanistically, YD accelerated the turnover of AXL by PS-RIP and resulted in the down-regulation of the full-length AXL. Therefore, the modulation of the proteolytic process through degradation of overexpressed AXL may be an attractive therapeutic strategy for the treatment of NSCLC and EGFR-TKI-resistant NSCLC.

No MeSH data available.


Related in: MedlinePlus

Blockage of ICD generation by γ-secretase inhibitor and fate of ICD(A) After overnight treatment with compound E, a γ-secretase inhibitor, the cells were treated with YD alone or co-treated with MG132 for 3 h. The collected cell lysates were analyzed by western blot with an antibody against C-terminal AXL using β-actin as a loading control. (B) The cells were treated with YD alone or co-treated with MG132 for 3 h. The lysates were immunoprecipitated with anti-C-terminal AXL and immunoblotted using anti-ubiquitin (Ub). AXL western blotting was performed on the total lysates. The β-actin immunoblotting of the total lysates is shown to normalize the input. The results are representative of three independent experiments.
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Figure 5: Blockage of ICD generation by γ-secretase inhibitor and fate of ICD(A) After overnight treatment with compound E, a γ-secretase inhibitor, the cells were treated with YD alone or co-treated with MG132 for 3 h. The collected cell lysates were analyzed by western blot with an antibody against C-terminal AXL using β-actin as a loading control. (B) The cells were treated with YD alone or co-treated with MG132 for 3 h. The lysates were immunoprecipitated with anti-C-terminal AXL and immunoblotted using anti-ubiquitin (Ub). AXL western blotting was performed on the total lysates. The β-actin immunoblotting of the total lysates is shown to normalize the input. The results are representative of three independent experiments.

Mentions: We investigated whether γ-secretase is involved in the generation of AXL-ICD by YD. After pretreatment with compound E, the effect of YD on the release of AXL-ICD was impaired and the CTF was accumulated, whereas the full-length AXL was still decreased in both H292 and H292-Gef cells (Figure 5A). As expected, the AXL-ICD was not detected with anti-AXL N-terminal antibody (Supplementary Figure 5). We then observed the fate of the AXL-ICD produced by YD treatment. The AXL-ICD was ubiquitinated after 3 h of treatment with YD and MG132, the proteasomal inhibitor (Figure 5B). Thus, the loss of AXL induced by YD in H292 and H292-Gef cells results from the γ-secretase-mediated generation of ICD, which is rapidly removed through proteasomal degradation.


Targeting the degradation of AXL receptor tyrosine kinase to overcome resistance in gefitinib-resistant non-small cell lung cancer.

Bae SY, Hong JY, Lee HJ, Park HJ, Lee SK - Oncotarget (2015)

Blockage of ICD generation by γ-secretase inhibitor and fate of ICD(A) After overnight treatment with compound E, a γ-secretase inhibitor, the cells were treated with YD alone or co-treated with MG132 for 3 h. The collected cell lysates were analyzed by western blot with an antibody against C-terminal AXL using β-actin as a loading control. (B) The cells were treated with YD alone or co-treated with MG132 for 3 h. The lysates were immunoprecipitated with anti-C-terminal AXL and immunoblotted using anti-ubiquitin (Ub). AXL western blotting was performed on the total lysates. The β-actin immunoblotting of the total lysates is shown to normalize the input. The results are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4496346&req=5

Figure 5: Blockage of ICD generation by γ-secretase inhibitor and fate of ICD(A) After overnight treatment with compound E, a γ-secretase inhibitor, the cells were treated with YD alone or co-treated with MG132 for 3 h. The collected cell lysates were analyzed by western blot with an antibody against C-terminal AXL using β-actin as a loading control. (B) The cells were treated with YD alone or co-treated with MG132 for 3 h. The lysates were immunoprecipitated with anti-C-terminal AXL and immunoblotted using anti-ubiquitin (Ub). AXL western blotting was performed on the total lysates. The β-actin immunoblotting of the total lysates is shown to normalize the input. The results are representative of three independent experiments.
Mentions: We investigated whether γ-secretase is involved in the generation of AXL-ICD by YD. After pretreatment with compound E, the effect of YD on the release of AXL-ICD was impaired and the CTF was accumulated, whereas the full-length AXL was still decreased in both H292 and H292-Gef cells (Figure 5A). As expected, the AXL-ICD was not detected with anti-AXL N-terminal antibody (Supplementary Figure 5). We then observed the fate of the AXL-ICD produced by YD treatment. The AXL-ICD was ubiquitinated after 3 h of treatment with YD and MG132, the proteasomal inhibitor (Figure 5B). Thus, the loss of AXL induced by YD in H292 and H292-Gef cells results from the γ-secretase-mediated generation of ICD, which is rapidly removed through proteasomal degradation.

Bottom Line: Here, we first demonstrate that AXL is overexpressed in an acquired gefitinib-resistant cell line (H292-Gef) as a result of slow turnover and that AXL is degraded by presenilin-dependent regulated intramembrane proteolysis (PS-RIP).Treatment with YD effectively suppressed the cancer cell survival in vitro and in vivo.Mechanistically, YD accelerated the turnover of AXL by PS-RIP and resulted in the down-regulation of the full-length AXL.

View Article: PubMed Central - PubMed

Affiliation: College of Pharmacy, Seoul National University, Seoul 151-742, Korea.

ABSTRACT
Acquired resistance to epidermal growth factor receptor tyrosine kinase inhibitors (EGFR-TKIs), such as gefitinib, remains a major problem in non-small cell lung cancer (NSCLC) treatment. Increased activation of AXL has been identified as a novel mechanism for acquired resistance to EGFR-TKIs in NSCLC treatment. However, the cause of uncontrolled AXL expression is not fully understood. Here, we first demonstrate that AXL is overexpressed in an acquired gefitinib-resistant cell line (H292-Gef) as a result of slow turnover and that AXL is degraded by presenilin-dependent regulated intramembrane proteolysis (PS-RIP). Based on the findings, we attempted to enhance AXL degradation to overcome acquired gefitinib-resistance by the treatment of gefitinib-resistant NSCLC cells with yuanhuadine (YD), a potent antitumor agent in NSCLC. Treatment with YD effectively suppressed the cancer cell survival in vitro and in vivo. Mechanistically, YD accelerated the turnover of AXL by PS-RIP and resulted in the down-regulation of the full-length AXL. Therefore, the modulation of the proteolytic process through degradation of overexpressed AXL may be an attractive therapeutic strategy for the treatment of NSCLC and EGFR-TKI-resistant NSCLC.

No MeSH data available.


Related in: MedlinePlus