(A) TMA from sunitinib treated or untreated tumor from RCC patients were stained for AXL. The AXL levels were quantified and compared between the two groups of patients (*** indicates p
(A) 786-O cells were acutely treated with sunitinib (+Suni, 1μM, 24h) or chronically treated with sunitinib (Chronic Suni, 1μM, >2weeks) as indicated. The cells in the last lane were treated in sunitinib free media for 4 days (−Suni) before lysis. Cell lysates were examined by western blot with specific antibodies as indicated. Data represent three independent experiments. (B) Chronic sunitinib pre-treated cells were treated in sunitinib free media for 3 days. The cells and 786-O parental cells were then serum starved for 24 hours followed by HGF (100ng/ml, 30minutes) or GAS6 (200ng/ml, 30 minutes) stimulation. The cells were then lysed and examined by western blot with specific antibodies as indicated. To better visualize the phosphorylated MET and AXl signal with HGF and GAS6 stimulation, shorter exposure time was used compared to the blots in (A). Data represent three independent experiments. (C). 786-O cells that were chronically pretreated with sunitinib were deprived of sunitinib (S786-O), the cells and 786-O parental cells were then seeded for wound-healing experiment as described in Methods. The cells were treated with DMSO or sunitinib (1μM, 24 hours) as indicated. The migration distances from four independent experiments were measured and analyzed by comparing to the migration distance of 786-O cells with DMSO treatment (* indicates p
Figure 3. Chronic sunitinib treatment induced EMT…
Figure 3. Chronic sunitinib treatment induced EMT is AXL and MET dependent
Figure 3. Chronic sunitinib treatment induced EMT is AXL and MET dependent (A) 786-O cells, 786-O cells that stably expressed AXL or MET shRNA constructs were chronically treated with sunitinib (1 μM) and then deprived of sunitinib for 48 hours. The cells were examined by western blot with specific antibodies as indicated. Data represent three independent experiments. (B) Chronic sunitinib pre-treated cells (786-O, AXL shRNA, and MET shRNA) were deprived of sunitinib for 3 days. The cells and 786-O parental cells were then serum starved for 24 hours followed by HGF (100ng/ml, 30minutes) or GAS6 (200ng/ml, 30minutes) stimulation as indicated. The cells were then lysed and examined by western blot with specific antibodies as indicated. Data represent three independent experiments. (C). 786-O cells, 786-O cells that were stably infected with AXL or MET shRNA constructs were chronically treated with sunitinib (1 μM) and then deprived of sunitinib, and 786-O parental cells were seeded for wound-healing experiment as described in Methods. The gap distances in four independent experiments were measured and analyzed. (D). The transwells were coated with Matrigel as described in Methods. 786-O cells, 786-O cells that were stably infected with AXL or MET shRNA constructs were chronically treated with sunitinib (1 μM) and then deprived of sunitinib, and 786-O parental cells were seeded (10, 000 per well) in the transwells in serum free medium. Complete Growth Medium was added to the bottom wells. After 24 hours, the invading cells were stained and counted as described in Methods. The data in this figure is representative for 3 shRNA constructs designed to target different sequences in MET and AXL.
Figure 4. Cabozantinib suppressed MET/AXL signaling and…
Figure 4. Cabozantinib suppressed MET/AXL signaling and chronic sunitinib treatment induced EMT
Figure 4. Cabozantinib suppressed MET/AXL signaling and chronic sunitinib treatment induced EMT (A) Chronic sunitinib treated 786-O cells and 786-O parental cells were cultured in medium with or without sunitinib (1μM) for 3 days, and then treated with or without cabozantinib (5μM, 24h) as indicated. The cells were lysed and examined by western blot with specific antibodies as indicated. Data represent three independent experiments. (B) Chronic sunitinib treated 786-O cells were cultured in medium without sunitinib for 3 days and then treated with cabozantinib (5μM, 24h) as indicated in serum free medium. The cells were then stimulated by HGF (100ng/ml, 30minutes) or GAS6 (200ng/ml, 30 minutes). The cells were then lysed and examined by western blot with specific antibodies as indicated. Data represent three independent experiments. (C). Chronic sunitinib treated 786-O cells and 786-O parental cells were cultured in medium without sunitinib before experiment. The cells were then seeded for wound-healing experiment as described in Methods. The cells were treated with DMSO or cabozantinib (5μM, 24h) as indicated. The gap distances in four independent experiments were measured and analyzed. (D). The transwells were coated with Matrigel as described in Methods. Chronic sunitinib pre-treated 786-O cells that were deprived of sunitinib (S786-O) or 786-O parental cells (10, 000 per well) were seeded in transwells in serum free medium supplied with cabozantinib (Cab, 5μM) or DMSO as indicated, Complete Growth Medium with cabozantinib (5μM) or DMSO was added to the bottom wells. After 24 hours, the invaded cells were stained and counted as described in Methods. Data represent four independent experiments.
Figure 5. Chronic Sunitinib treatment induced AXL…
Figure 5. Chronic Sunitinib treatment induced AXL and MET dependent expression of VEGF, and promoted…
Figure 5. Chronic Sunitinib treatment induced AXL and MET dependent expression of VEGF, and promoted HUVEC angiogenesis (A) 786-O cells, 786-O cells that stably expressed AXL or MET shRNA constructs were chronically treated with sunitinib (1 μM) and then deprived of sunitinib, and 786-O parental cells were seeded (10, 000 per well) in Matrigel (1:2 diluted) in 96 well plates, HUVEC cells were seeded on top of Matrigel (5, 000 per well). After 24 hours, HUVEC cell tube formation was recorded with a bright field microscope. Data represent three independent experiments. The tube lengths were measured and quantified with Image J software. (B) Chronic sunitinib pre-treated 786-O that were deprived of sunitinib (S786-O) or 786-O parental cells (10, 000 per well) were seeded in Matrigel (1:2 diluted) in 96 well plates, HUVEC cells were seeded on top of Matrigel (5, 000 per well). The cell co-culture was treated with DMSO, sunitinib (+Suni, 1 μM) or cabozantinib (+Cab, 5μM) for 24 hours, HUVEC tube formation was recorded with a bright field microscope. Data represent three independent experiments. The tube lengths were measured and quantified with Image J software. (C) 786-O cells, 786-O cells that were stably expressed with AXL or MET shRNA constructs were chronically treated with sunitinib (1 μM) and then deprived of sunitinib, and 786-O parental cells were cultured as described in Methods. The conditioned media were collected and analyzed for VEGF levels. The results from four independent experiments were assessed for statistical significance (** indicates p<0.01). (D) Chronic sunitinib pre-treated 786-O that were deprived of sunitinib (S786-O) or 786-O parental cells were cultured as described in Methods. The conditioned media were collected and analyzed for VEGF concentration. The resulted from four independent experiments were assessed for statistical significance (** indicates p<0.01). The data in this figure is representative for 3 shRNA constructs designed to target different sequences in MET and AXL.
Figure 6. Chronic sunitinib treatment induced AXL…
Figure 6. Chronic sunitinib treatment induced AXL and MET signaling and angiogenesis in xenograft mouse…
Figure 6. Chronic sunitinib treatment induced AXL and MET signaling and angiogenesis in xenograft mouse models (A) 1x107 786-O cells (786-O) or chronic sunitinib pre-treated 786-O (S786-O) were injected into the flank of each NCr-nu/nu mouse (10 mice per group). The xenograft tumor sizes were measured after 4 weeks’ stabilization. The size difference between two groups at same time point was analyzed using the Student t-test. (* indicates p<0.05, ** indicates p<0.01). (B) The mice were sacrificed after 9 weeks, the tumor tissue was obtained and equal amounts of protein lysate were analyzed by western blot with specific antibodies as indicated. (C) Representative immunohistochemistry staining of CD31 (red) and cell nucleus (DAPI, blue) in xenograft tumor sections from the experiment performed in (B). (D) Diagram describes the experimental design of in vivo sunitinib-resistant (20mg/kg, daily) tumor generation followed with cabozantinib (40mg/kg, daily) treatment (10 mice per group). (E) The measurement of tumor size during the experiment of (D). The size difference between sunitinib and saline treated groups at same time point was analyzed using Student t-test. (* indicates p<0.05, ** indicates p<0.01). (F) The mice were sacrificed after saline/sunitinib or cabozantinib treatment, the tumor tissue were obtained and equal amount of protein lysate were analyzed by western blot with specific antibodies as indicated. (G) Representative immunohistochemistry staining of CD31 (red) and cell nucleus (DAPI, blue) in xenograft tumor sections from the experiment performed in (F). (H) Proposed model for chronic sunitinib treatment-induced drug resistance.
Figure 6. Chronic sunitinib treatment induced AXL…
Figure 6. Chronic sunitinib treatment induced AXL and MET signaling and angiogenesis in xenograft mouse…
Figure 6. Chronic sunitinib treatment induced AXL and MET signaling and angiogenesis in xenograft mouse models (A) 1x107 786-O cells (786-O) or chronic sunitinib pre-treated 786-O (S786-O) were injected into the flank of each NCr-nu/nu mouse (10 mice per group). The xenograft tumor sizes were measured after 4 weeks’ stabilization. The size difference between two groups at same time point was analyzed using the Student t-test. (* indicates p<0.05, ** indicates p<0.01). (B) The mice were sacrificed after 9 weeks, the tumor tissue was obtained and equal amounts of protein lysate were analyzed by western blot with specific antibodies as indicated. (C) Representative immunohistochemistry staining of CD31 (red) and cell nucleus (DAPI, blue) in xenograft tumor sections from the experiment performed in (B). (D) Diagram describes the experimental design of in vivo sunitinib-resistant (20mg/kg, daily) tumor generation followed with cabozantinib (40mg/kg, daily) treatment (10 mice per group). (E) The measurement of tumor size during the experiment of (D). The size difference between sunitinib and saline treated groups at same time point was analyzed using Student t-test. (* indicates p<0.05, ** indicates p<0.01). (F) The mice were sacrificed after saline/sunitinib or cabozantinib treatment, the tumor tissue were obtained and equal amount of protein lysate were analyzed by western blot with specific antibodies as indicated. (G) Representative immunohistochemistry staining of CD31 (red) and cell nucleus (DAPI, blue) in xenograft tumor sections from the experiment performed in (F). (H) Proposed model for chronic sunitinib treatment-induced drug resistance.