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, 6 (12), 10146-60

Targeting the Degradation of AXL Receptor Tyrosine Kinase to Overcome Resistance in Gefitinib-Resistant Non-Small Cell Lung Cancer

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Targeting the Degradation of AXL Receptor Tyrosine Kinase to Overcome Resistance in Gefitinib-Resistant Non-Small Cell Lung Cancer

Song Yi Bae et al. Oncotarget.

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.

Keywords: AXL; EGFR-TKI resistance; NSCLC; PS-RIP; yuanhuadine.

Conflict of interest statement

CONFLICT OF INTEREST

The authors declare no conflict of interest.

Figures

Figure 1
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.
Figure 2
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.
Figure 3
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.
Figure 4
Figure 4. Generation of CTF and ICD of AXL by YD
(A) The cells were treated independently with YD, MG132 (proteasomal inhibitor), and chloroquine (lysosomal inhibitor) or co-treated with YD and MG132 or with YD and chloroquine for 3 h. The cell lysates were analyzed by western blot using C-terminal AXL antibody, and β-actin served as a loading control. (B) The cells were co-treated with YD and the indicated concentrations of MG132. The expression of AXL fragments were detected by western blot with an antibody against C-terminal AXL using β-actin as a loading control. (C and D) H292 (C) and H292-Gef (D) cells were treated with YD and/or MG132 for 3 h, and the cells were then harvested and fractionated into the cytosol and membrane fractions. The lysates were subjected to western blot using anti-C-terminal AXL, and GAPDH served as a cytosol marker. T, total cell lysate; C, cytosol; M, membrane. The results are representative of two (A, B) or three (C, D) independent experiments.
Figure 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.
Figure 6
Figure 6. Generation of soluble AXL by YD and the combination effect of gefitinib with YD
(A) The cells were treated or not treated with GM6001, a metalloprotease inhibitor, overnight and then treated with YD for 3 h. (B) H292-Gef cells were treated with YD and/or MG132 for the indicated times. The cell culture media (CM) were harvested, immunoprecipitated with antibody against N-terminal AXL, and immunoblotted using anti-N-terminal AXL. The cultured cells were lysed and analyzed by western blot with anti-C-terminal AXL using β-actin as a loading control. (C) The cells were pre-incubated with GM6001 overnight and then treated with YD and/or MG132 for 3 h. The lysates were subjected to western blot with antibody against C-terminal AXL using β-actin as a loading control. (D) H292-Gef cells were not transfected or transfected with 5 nM scrambled siRNA or AXL siRNA for 24 h, and the gene knockdown was confirmed by western blot with anti-C-terminal AXL using β-actin as a loading control. The transfected cells were seeded for 24 h and then treated with YD for 48 h. The cell proliferation was measured by SRB assay. (E) H292-Gef cells were treated with the indicated concentrations of gefitinib alone or combined with 0.8 nM YD for 48 h. The cell proliferation was determined by SRB assay and the combination effect was measured by calculating CI values. The data are presented as the means ± SD. The results are representative of two (D) or three (A, B, C, E) independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 by t-test.
Figure 7
Figure 7. Alteration of AXL expression by YD in tumor xenograft model
(A) H292 cells were implanted subcutaneously into the flanks of BALB/c-nude mice. Dosing of YD (1 mg/kg body weight) or gefitinib (50 mg/kg body weight) was initiated when the tumor volumes reached approximately 110 mm3. YD and gefitinib were administered orally once a day for 21 days. The tumor volumes were measured on Days 0 and 8 and then every five days during the remaining period (n = 5 mice per group). The error bars represent the means ± SD. (B) BALB/c-nude mice bearing H292-Gef tumors were orally treated with YD (1 mg/kg body weight) or gefitinib (50 mg/kg body weight) once a day for 22 days. The treatment was initiated when the tumor sizes reached approximately 250 mm3. The tumor volumes were measured every 3–4 days (n = 5 mice per group). The error bars represent the means ± SD. (C and D) Immunohistochemical analysis of Ki-67 (C) and AXL (D) was performed using anti-Ki-67 antibody or anti-AXL antibody directed against the C-terminal domain in tumor tissue sections. Scale bar, 50 μm. Histological images shown are representative of three independent experiments. (E) The expression of AXL protein in the frozen xenograft tumor tissue samples was investigated by western blot using β-actin as the loading control. Western blots are representative of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.005 by t-test.

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