Autophagosome-mediated EGFR down-regulation induced by the CK2 inhibitor enhances the efficacy of EGFR-TKI on EGFR-mutant lung cancer cells with resistance by T790M

Abstract

Protein kinase CK2 has diverse functions promoting and maintaining cancer phenotypes. We investigated the effect of CK2 inhibition in lung cancer cells with T790M-mediated resistance to the EGFR-TK inhibitor. Resistant sublines of PC-9 to gefitinib (PC-9/GR) and erlotinib (PC-9/ER) were established by previous study, and T790M secondary mutation was found in both resistant sublines. A decrease of EGFR by siRNA treatment effectively controlled the growth of resistant cells, thus suggesting that they still have EGFR-dependency. CX-4945, a potent and selective CK2 inhibitor, induced autophagy in PC-9/GR and PC-9/ER, and which was supported by the induction of autophagic vacuoles and microtubule-associated protein 1 light chain 3 (LC3) expression, and the increase of punctate fluorescent signals in resistant cells pre-transfected with green fluorescent protein (GFP)-tagged LC3. However, the withdrawal of CX-4945 led to the recovery of cancer cells with autophagy. We found that the induction of autophagy by CX-4945 in both resistant cells was CK2 dependent by using small interfering RNA against CK2. The treatment with CX-4945 alone induced a minimal growth inhibition in resistant cells. However, combined treatment of CX-4945 and EGFR-TKI effectively inhibited cancer-cell proliferation and induced apoptosis. CX-4945 increased the translocation of EGFR from the cell surface into the autophagosome, subsequently leading to the decrease of EGFR while inhibition of autophagy by 3MA or Atg7-targeted siRNA pretreatment reduced the decrease of EGFR by CX-4945. Accordingly, apoptosis by a combination of CX-4945 and EGFR-TKI was suppressed by 3MA or Atg7-targeted siRNA pretreatment, thus suggesting that autophagosome-mediated EGFR down-regulation would have an important role regarding apoptotic cell death by EGFR-TKI. Combined treatment of the CK2 inhibitor and EGFR-TKI may be a promising strategy for overcoming T790M-mediated resistance.

Figure 1. CX-4945 induced the autophagy in EGFR-TKI-resistant PC-9 cells.

A, Cells were treated with different concentrations of CX-4945 for 72 h, and the rate of inhibition was determined by MTT assay. B, Cells were treated with or without CX-4945 (5 µM) for 48 h and were then incubated for 24 h with a drug-free medium containing 10% FBS. Pictures showing the autophagic vacuole formation (AVOs) were taken at ×20 magnification. C and D, Cells were treated with CX-4945 for 48 h. Cell lysates were subjected to Western blot analysis. Quantitative detection of acidic vesicular organelles by acridine orange staining of cells was determined by FACS analysis. *p<0.01 and **p<0.001 compared with the control. E, Cells were transfected with a plasmid to express LC3-GFP. After 24 h transfection, cells were treated with CX-4945 (5 µM) for 24 h. Punctate pattern of LC3 localization analyzed by immunofluorescence microscopy. F and G, Cells were incubated with CX-4945 (5 µM), 17-DMAG (100 nM) or rapamycin (20 µM) for 48 h. Pictures were taken at ×20 magnification. The induction of LC3-I/II was shown by Western blot analysis.

Figure 2. Down-regulation of CK2α by siRNA treatment induced the autophagy in EGFR-TKI-resistant PC-9 cells.

A and B, Cells were transfected with control or CK2α siRNA (100 nM) for 48 h. Cell numbers were determined with an ADAM-MC automatic cell counter. Pictures showing the autophagic vacuole formation (AVOs) were taken at ×20 magnification. *p<0.01 compared with the control. C, After 48 h transfection, CK2α and LC3-I/II was shown by Western blot analysis.

Figure 3. CX-4945 enhanced the efficacy of EGFR-TKIs to overcome drug resistance caused by T790M.

Cells were treated with CX-4945 (5 µM), gefitinib (1 µM), and erlotinib (1 µM) or a combination of CX-4945 and gefitinib or CX-4945 and erlotinib for 48 h. A, Cell numbers were determined using a cell counter (upper panel). Quantitative detection of acidic vesicular organelles by acridine orange staining of cells was determined by FACS analysis (lower panel). †p<0.01 compared with CX-4945 alone. B, Apoptosis was assessed by Annexin V-FITC/Propidium iodide staining and flow cytometry. Diagrams of Annexin V-FITC/Propidium iodide flow cytometry in a representative experiment are presented at the left panel. The results are representative of at least 3 independent experiments, and the error bars signify standard deviations (±SDs). C, Cleavage of PARP-1 and caspase-3 was shown by Western blot analysis. *p<0.01 and **p<0.001 compared with the control.

Figure 4. Addition of CX-4945 to EGFR-TKIs substantially suppressed the EGFR signaling pathway in gefitinib/erlotinib-resistant, PC-9 cells.

A and B, Control and EGFR siRNAs (100 nM) were introduced into parental or resistant cells, and EGFR suppression was confirmed by Western blot analysis. Cell viability was measured using a cell counter 72 h later. *p<0.01 and **p<0.001 compared with the control. C, Cells were treated with drugs as in Fig. 2. Cells were harvested, and the modulation of EGFR signaling in the indicated cell lines was detected by Western blot analysis.

Figure 5. The inhibition of CX-4945-induced autophagy led to decreased apoptosis.

A, PC-9/ER cells were treated with CX-4945 (5 µM) for 48 h and then were fixed with methanol, immunostained with anti-LC3 (red), anti-EGFR (green), and DAPI (blue), and analyzed by confocal microscopy to determine the intracellular localization of EGFR. B, The suppression of Atg7 by siRNA treatment was detected by Western blot analysis. PC-9/ER cells were treated with CX-4945 (5 µM) for 48 h in the presence or absence of 3MA (2 mM) and Atg7 siRNA (100 nM). The modulation of EGFR was detected by Western blot analysis. C and D, Cells were treated with drugs as in Fig. 2 under the presence or absence of 3MA and Atg7 siRNA. Cleavage of PARP-1 and caspase-3 was shown by Western blot analysis. Apoptosis was assessed by Annexin V-FITC/Propidium iodide staining and flow cytometry. The results are representative of at least 3 independent experiments, and the error bars signify standard deviations (±SDs). *p<0.01 and **p<0.001 compared with the combination of CX-4945 and gefitinib or erlotinib.

Figure 6. Illustration of EGFR degradation by CX-4945-induced authophagy.

EGFR is delivered from the plasma membrane to early endosomes in endocytic vesicles. These vesicles are back to the plasma membrane through the recycling pathway. However, vesicles can also fuse with authophagosomes, and then directly with lysosomes leading to degradation of EGFR.