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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

YD-induced down-regulation of the full-length AXL expression in H292 and H292-Gef Cells(A) Scheme of the AXL degradation by PS-RIP process. (B) The cells were treated with YD for 72 h, and the proliferation of the cells was determined using SRB assay. The IC50 values were calculated using the TableCurve 2D software, and the data are presented as the means ± SD. (C) The cells were treated with 10 nM YD for the indicated times, and the cell lysates were analyzed by western blot using β-actin as a loading control. (D) The cells were treated with 25 μg/ml CHX and 10 nM YD for the indicated times. The cell lysates were analyzed by western blot with antibody against C-terminal AXL using β-actin as a loading control. The AXL expression levels were quantified by densitometry using ImageJ. (E) The cells were treated with YD for 3 h, and the indicated markers were detected by western blot using β-actin as a loading control. (F) Cells treated with YD for 24 h were subjected to immunocytochemistry. The cells were stained with AXL and DAPI. Scale bars, 20 μm. The results are representative of three independent experiments.
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Figure 3: YD-induced down-regulation of the full-length AXL expression in H292 and H292-Gef Cells(A) Scheme of the AXL degradation by PS-RIP process. (B) The cells were treated with YD for 72 h, and the proliferation of the cells was determined using SRB assay. The IC50 values were calculated using the TableCurve 2D software, and the data are presented as the means ± SD. (C) The cells were treated with 10 nM YD for the indicated times, and the cell lysates were analyzed by western blot using β-actin as a loading control. (D) The cells were treated with 25 μg/ml CHX and 10 nM YD for the indicated times. The cell lysates were analyzed by western blot with antibody against C-terminal AXL using β-actin as a loading control. The AXL expression levels were quantified by densitometry using ImageJ. (E) The cells were treated with YD for 3 h, and the indicated markers were detected by western blot using β-actin as a loading control. (F) Cells treated with YD for 24 h were subjected to immunocytochemistry. The cells were stained with AXL and DAPI. Scale bars, 20 μm. The results are representative of three independent experiments.

Mentions: We assumed that the degradation of AXL should be processed as described in Figure 3A. Overexpressed AXL could be removed from the cell membrane by increasing the cleavage of extracellular and intracellular domain of AXL. Moreover, the generated ICD could be removed through proteasomal degradation. Therefore, to confirm our hypothesis and explore the potential to inhibit cell proliferation by regulating the degradation of AXL, we applied YD, a natural product-derived antitumor agent [19, 20], to H292 and H292-Gef cells. YD effectively suppressed the proliferation of these two cell lines with IC50 values of 0.1 nM in H292 cells and 4.3 nM in H292-Gef cells (72 h; Figure 3B). The alterations in AXL expression was then monitored for up to 6 h after treatment with 10 nM YD (Figure 3C). The level of full-length AXL was decreased time-dependently in H292 and H292-Gef cells. The decrease of AXL level was first detected at 2 h in H292 cells and 3 h in H292-Gef cells after exposure to YD. We further determined the effect of YD on the half-life of AXL. After treatment with YD and CHX, the AXL expression was detected in H292 and H292-Gef cells (Figure 3D, upper panel). In both cell lines, the half-life of AXL was observed between 0.5 and 1 h, which was faster than the half-life measured in Figure 2C (Figure 3D, lower panel). Moreover, treatment with YD for 3 h suppressed phosphorylated AKT (Ser473), one of the key molecules of AXL downstream signaling pathway, in both H292 and H292-Gef cells (Figure 3E). Consistent with the findings shown in Figure 3C, we observed the decrease of AXL expression in the cells treated with YD for 24 h by immunocytochemistry (Figure 3F). The action of YD targeted on the expression of AXL rather than the kinase activity of AXL, as an AXL kinase activity assay confirmed that YD has no effect on the kinase activity (Supplementary Figure 3). Collectively, YD effectively decreases the full-length AXL and shortens the half-life of AXL. These events consequently inhibit the growth of cancer cells by down-regulating the AXL downstream signaling pathway.


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)

YD-induced down-regulation of the full-length AXL expression in H292 and H292-Gef Cells(A) Scheme of the AXL degradation by PS-RIP process. (B) The cells were treated with YD for 72 h, and the proliferation of the cells was determined using SRB assay. The IC50 values were calculated using the TableCurve 2D software, and the data are presented as the means ± SD. (C) The cells were treated with 10 nM YD for the indicated times, and the cell lysates were analyzed by western blot using β-actin as a loading control. (D) The cells were treated with 25 μg/ml CHX and 10 nM YD for the indicated times. The cell lysates were analyzed by western blot with antibody against C-terminal AXL using β-actin as a loading control. The AXL expression levels were quantified by densitometry using ImageJ. (E) The cells were treated with YD for 3 h, and the indicated markers were detected by western blot using β-actin as a loading control. (F) Cells treated with YD for 24 h were subjected to immunocytochemistry. The cells were stained with AXL and DAPI. Scale bars, 20 μm. The results are representative of three independent experiments.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Figure 3: YD-induced down-regulation of the full-length AXL expression in H292 and H292-Gef Cells(A) Scheme of the AXL degradation by PS-RIP process. (B) The cells were treated with YD for 72 h, and the proliferation of the cells was determined using SRB assay. The IC50 values were calculated using the TableCurve 2D software, and the data are presented as the means ± SD. (C) The cells were treated with 10 nM YD for the indicated times, and the cell lysates were analyzed by western blot using β-actin as a loading control. (D) The cells were treated with 25 μg/ml CHX and 10 nM YD for the indicated times. The cell lysates were analyzed by western blot with antibody against C-terminal AXL using β-actin as a loading control. The AXL expression levels were quantified by densitometry using ImageJ. (E) The cells were treated with YD for 3 h, and the indicated markers were detected by western blot using β-actin as a loading control. (F) Cells treated with YD for 24 h were subjected to immunocytochemistry. The cells were stained with AXL and DAPI. Scale bars, 20 μm. The results are representative of three independent experiments.
Mentions: We assumed that the degradation of AXL should be processed as described in Figure 3A. Overexpressed AXL could be removed from the cell membrane by increasing the cleavage of extracellular and intracellular domain of AXL. Moreover, the generated ICD could be removed through proteasomal degradation. Therefore, to confirm our hypothesis and explore the potential to inhibit cell proliferation by regulating the degradation of AXL, we applied YD, a natural product-derived antitumor agent [19, 20], to H292 and H292-Gef cells. YD effectively suppressed the proliferation of these two cell lines with IC50 values of 0.1 nM in H292 cells and 4.3 nM in H292-Gef cells (72 h; Figure 3B). The alterations in AXL expression was then monitored for up to 6 h after treatment with 10 nM YD (Figure 3C). The level of full-length AXL was decreased time-dependently in H292 and H292-Gef cells. The decrease of AXL level was first detected at 2 h in H292 cells and 3 h in H292-Gef cells after exposure to YD. We further determined the effect of YD on the half-life of AXL. After treatment with YD and CHX, the AXL expression was detected in H292 and H292-Gef cells (Figure 3D, upper panel). In both cell lines, the half-life of AXL was observed between 0.5 and 1 h, which was faster than the half-life measured in Figure 2C (Figure 3D, lower panel). Moreover, treatment with YD for 3 h suppressed phosphorylated AKT (Ser473), one of the key molecules of AXL downstream signaling pathway, in both H292 and H292-Gef cells (Figure 3E). Consistent with the findings shown in Figure 3C, we observed the decrease of AXL expression in the cells treated with YD for 24 h by immunocytochemistry (Figure 3F). The action of YD targeted on the expression of AXL rather than the kinase activity of AXL, as an AXL kinase activity assay confirmed that YD has no effect on the kinase activity (Supplementary Figure 3). Collectively, YD effectively decreases the full-length AXL and shortens the half-life of AXL. These events consequently inhibit the growth of cancer cells by down-regulating the AXL downstream signaling pathway.

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