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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

Down-regulated Turnover of AXL in Gefitinib Resistant H292 (H292-Gef) Cell Line(A) H292 and H292-Gef cells were treated with gefitinib for 72 h, and the proliferation of the cells was measured using the SRB assay. The IC50 values were calculated using the TableCurve 2D software, and the data are presented as the means ± SD. (B) The basal protein expression of AXL was determined by western blot using β-actin as the loading control. (C) The cells were treated with 25 μg/ml CHX for the indicated times. The lysates were analyzed by western blot analysis with antibody against C-terminal AXL using β-actin as a loading control. The expression levels were quantified by densitometry using ImageJ. (D) The mRNA expression of the indicated markers in cells was determined by real-time PCR, and the β-actin mRNA levels were used for normalization. The data are presented as the mean fold changes ± SD relative to the H292 control. (E) H292-Gef cells were treated with GM6001 and/or compound E overnight and then with MG132 for 3 h before being collected for western blot analysis using β-actin as a loading control. For determination of NTF, the culture medium (CM) was collected, immunoprecipitated with antibody against N-terminal AXL, and immunoblotted using anti-N-terminal AXL. The results are representative of two (C, E) or three (A, B, D) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 by t-test.
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Figure 2: Down-regulated Turnover of AXL in Gefitinib Resistant H292 (H292-Gef) Cell Line(A) H292 and H292-Gef cells were treated with gefitinib for 72 h, and the proliferation of the cells was measured using the SRB assay. The IC50 values were calculated using the TableCurve 2D software, and the data are presented as the means ± SD. (B) The basal protein expression of AXL was determined by western blot using β-actin as the loading control. (C) The cells were treated with 25 μg/ml CHX for the indicated times. The lysates were analyzed by western blot analysis with antibody against C-terminal AXL using β-actin as a loading control. The expression levels were quantified by densitometry using ImageJ. (D) The mRNA expression of the indicated markers in cells was determined by real-time PCR, and the β-actin mRNA levels were used for normalization. The data are presented as the mean fold changes ± SD relative to the H292 control. (E) H292-Gef cells were treated with GM6001 and/or compound E overnight and then with MG132 for 3 h before being collected for western blot analysis using β-actin as a loading control. For determination of NTF, the culture medium (CM) was collected, immunoprecipitated with antibody against N-terminal AXL, and immunoblotted using anti-N-terminal AXL. The results are representative of two (C, E) or three (A, B, D) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 by t-test.

Mentions: To further investigate the status of AXL in acquired gefitinib-resistance, we established a gefitinib-resistant cell line, H292-Gef, through the continuous exposure of the parental-drug-sensitive H292 cells to gefitinib. H292-Gef cells exhibited an approximately 500-fold greater resistance to gefitinib than did the parental cells (IC50 value of gefitinib = 2.3 × 10−2 μM in H292 cells; IC50 value of gefitinib = 11.6 μM in H292-Gef cells, Figure 2A). Consistent with the findings in the gefitinib-resistant NSCLC cell lines, the AXL expression was markedly up-regulated in H292-Gef cells compared with H292 cells (Figure 2B). Based on the finding, we attempted to elucidate the cause of the higher AXL level in H292-Gef cells. We first determined the degradation of AXL over time by measuring AXL expression in H292 and H292-Gef cells after treatment with cycloheximide (CHX), a protein synthesis inhibitor (Figure 2C, left panel). The half-life of AXL was approximately 3 h in H292 cells and 16 h in H292-Gef cells (Figure 2C, right panel). Accordingly, we assumed that the degradation of AXL was suppressed in H292-Gef cells compared with H292 cells, and this event may be highly associated with gefitinib-acquired resistance in NSCLC cells. We then further elucidated the mechanism of AXL degradation in H292-Gef cells.


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)

Down-regulated Turnover of AXL in Gefitinib Resistant H292 (H292-Gef) Cell Line(A) H292 and H292-Gef cells were treated with gefitinib for 72 h, and the proliferation of the cells was measured using the SRB assay. The IC50 values were calculated using the TableCurve 2D software, and the data are presented as the means ± SD. (B) The basal protein expression of AXL was determined by western blot using β-actin as the loading control. (C) The cells were treated with 25 μg/ml CHX for the indicated times. The lysates were analyzed by western blot analysis with antibody against C-terminal AXL using β-actin as a loading control. The expression levels were quantified by densitometry using ImageJ. (D) The mRNA expression of the indicated markers in cells was determined by real-time PCR, and the β-actin mRNA levels were used for normalization. The data are presented as the mean fold changes ± SD relative to the H292 control. (E) H292-Gef cells were treated with GM6001 and/or compound E overnight and then with MG132 for 3 h before being collected for western blot analysis using β-actin as a loading control. For determination of NTF, the culture medium (CM) was collected, immunoprecipitated with antibody against N-terminal AXL, and immunoblotted using anti-N-terminal AXL. The results are representative of two (C, E) or three (A, B, D) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 by t-test.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 2: Down-regulated Turnover of AXL in Gefitinib Resistant H292 (H292-Gef) Cell Line(A) H292 and H292-Gef cells were treated with gefitinib for 72 h, and the proliferation of the cells was measured using the SRB assay. The IC50 values were calculated using the TableCurve 2D software, and the data are presented as the means ± SD. (B) The basal protein expression of AXL was determined by western blot using β-actin as the loading control. (C) The cells were treated with 25 μg/ml CHX for the indicated times. The lysates were analyzed by western blot analysis with antibody against C-terminal AXL using β-actin as a loading control. The expression levels were quantified by densitometry using ImageJ. (D) The mRNA expression of the indicated markers in cells was determined by real-time PCR, and the β-actin mRNA levels were used for normalization. The data are presented as the mean fold changes ± SD relative to the H292 control. (E) H292-Gef cells were treated with GM6001 and/or compound E overnight and then with MG132 for 3 h before being collected for western blot analysis using β-actin as a loading control. For determination of NTF, the culture medium (CM) was collected, immunoprecipitated with antibody against N-terminal AXL, and immunoblotted using anti-N-terminal AXL. The results are representative of two (C, E) or three (A, B, D) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 by t-test.
Mentions: To further investigate the status of AXL in acquired gefitinib-resistance, we established a gefitinib-resistant cell line, H292-Gef, through the continuous exposure of the parental-drug-sensitive H292 cells to gefitinib. H292-Gef cells exhibited an approximately 500-fold greater resistance to gefitinib than did the parental cells (IC50 value of gefitinib = 2.3 × 10−2 μM in H292 cells; IC50 value of gefitinib = 11.6 μM in H292-Gef cells, Figure 2A). Consistent with the findings in the gefitinib-resistant NSCLC cell lines, the AXL expression was markedly up-regulated in H292-Gef cells compared with H292 cells (Figure 2B). Based on the finding, we attempted to elucidate the cause of the higher AXL level in H292-Gef cells. We first determined the degradation of AXL over time by measuring AXL expression in H292 and H292-Gef cells after treatment with cycloheximide (CHX), a protein synthesis inhibitor (Figure 2C, left panel). The half-life of AXL was approximately 3 h in H292 cells and 16 h in H292-Gef cells (Figure 2C, right panel). Accordingly, we assumed that the degradation of AXL was suppressed in H292-Gef cells compared with H292 cells, and this event may be highly associated with gefitinib-acquired resistance in NSCLC cells. We then further elucidated the mechanism of AXL degradation in H292-Gef cells.

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