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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

Expression of AXL in Lung Cancer Cell Lines(A) The cells were treated with gefitinib for 72 h, and the cell growth was then determined by SRB assay. The IC50 values were calculated using the TableCurve 2D software, and are shown in parentheses. (B) The cells were lysed, and the levels of AXL were analyzed by western blot analysis with antibody against C-terminal AXL using β-actin as a loading control. (C) The mRNA levels of AXL were examined using 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 A549 control. The results are representative of two (A, B) or three (C) independent experiments.
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Figure 1: Expression of AXL in Lung Cancer Cell Lines(A) The cells were treated with gefitinib for 72 h, and the cell growth was then determined by SRB assay. The IC50 values were calculated using the TableCurve 2D software, and are shown in parentheses. (B) The cells were lysed, and the levels of AXL were analyzed by western blot analysis with antibody against C-terminal AXL using β-actin as a loading control. (C) The mRNA levels of AXL were examined using 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 A549 control. The results are representative of two (A, B) or three (C) independent experiments.

Mentions: The identification of the causes of drug resistance in anti-cancer chemotherapies is important for the restoration of drug sensitivity. Recent reports suggest that the activation of AXL is in part associated with EGFR-TKI-resistant NSCLC [6, 7]. Therefore, we assessed the correlation between AXL expression and gefitinib, an EGFR-TKI, sensitivity. We first evaluated the IC50 values of gefitinib in six NSCLC cell lines (Figure 1A and Supplementary Figure 1). Six cell lines were treated with various concentrations of gefitinib ranging from 1.3 nM to 200 μM for 72 h. The growth inhibitory activity was determined by measuring the protein contents of cells using the sulforhodamine B (SRB) assay. Calu-1 and H1299 cells were resistant to gefitinib (IC50 > 10 μM), and A549 cells were regarded as intermediate-sensitive to gefitinib (IC50 = 7.8 μM). H292, H358 and H1993 cells were sensitive to gefitinib with the IC50 values of less than 1 μM. To examine whether the difference in the gefitinib sensitivity of the three groups of cell lines is related to the expression of AXL, we evaluated the AXL expression levels in these six cell lines. The AXL protein levels were considerably high in Calu-1 and H1299 cells, but the AXL protein was barely detected in the remaining four cell lines (Figure 1B). The AXL gene levels in the two gefitinib-resistant cells were 7- to 10-fold higher than those observed in A549 cells (Figure 1C). Although H292 cells exhibited a higher expression level of the AXL gene compared with A549 cells, the AXL gene levels in the gefitinib-sensitive cell lines were fairly low relative to those observed in the gefitinib-resistant cell lines. Therefore, it implies that there is a correlation between high AXL expression and gefitinib-resistance in NSCLC cells, whereas no correlation was found between AXL expression and gefitinib sensitivity in the gefitinib-sensitive 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)

Expression of AXL in Lung Cancer Cell Lines(A) The cells were treated with gefitinib for 72 h, and the cell growth was then determined by SRB assay. The IC50 values were calculated using the TableCurve 2D software, and are shown in parentheses. (B) The cells were lysed, and the levels of AXL were analyzed by western blot analysis with antibody against C-terminal AXL using β-actin as a loading control. (C) The mRNA levels of AXL were examined using 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 A549 control. The results are representative of two (A, B) or three (C) independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 1: Expression of AXL in Lung Cancer Cell Lines(A) The cells were treated with gefitinib for 72 h, and the cell growth was then determined by SRB assay. The IC50 values were calculated using the TableCurve 2D software, and are shown in parentheses. (B) The cells were lysed, and the levels of AXL were analyzed by western blot analysis with antibody against C-terminal AXL using β-actin as a loading control. (C) The mRNA levels of AXL were examined using 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 A549 control. The results are representative of two (A, B) or three (C) independent experiments.
Mentions: The identification of the causes of drug resistance in anti-cancer chemotherapies is important for the restoration of drug sensitivity. Recent reports suggest that the activation of AXL is in part associated with EGFR-TKI-resistant NSCLC [6, 7]. Therefore, we assessed the correlation between AXL expression and gefitinib, an EGFR-TKI, sensitivity. We first evaluated the IC50 values of gefitinib in six NSCLC cell lines (Figure 1A and Supplementary Figure 1). Six cell lines were treated with various concentrations of gefitinib ranging from 1.3 nM to 200 μM for 72 h. The growth inhibitory activity was determined by measuring the protein contents of cells using the sulforhodamine B (SRB) assay. Calu-1 and H1299 cells were resistant to gefitinib (IC50 > 10 μM), and A549 cells were regarded as intermediate-sensitive to gefitinib (IC50 = 7.8 μM). H292, H358 and H1993 cells were sensitive to gefitinib with the IC50 values of less than 1 μM. To examine whether the difference in the gefitinib sensitivity of the three groups of cell lines is related to the expression of AXL, we evaluated the AXL expression levels in these six cell lines. The AXL protein levels were considerably high in Calu-1 and H1299 cells, but the AXL protein was barely detected in the remaining four cell lines (Figure 1B). The AXL gene levels in the two gefitinib-resistant cells were 7- to 10-fold higher than those observed in A549 cells (Figure 1C). Although H292 cells exhibited a higher expression level of the AXL gene compared with A549 cells, the AXL gene levels in the gefitinib-sensitive cell lines were fairly low relative to those observed in the gefitinib-resistant cell lines. Therefore, it implies that there is a correlation between high AXL expression and gefitinib-resistance in NSCLC cells, whereas no correlation was found between AXL expression and gefitinib sensitivity in the gefitinib-sensitive 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