Acquired EGFR-TKIs Resistance In Non-small Lung Cancer By Lysosomal Deficiency-induced SQSTM1 Accumulation

Background:The small molecular inhibitors of Epidermal Growth Factor Receptor coupled Tyrosine Kinase Inhibitors(EGFR-TKIs),occupy an important position in the treatment of non-small cell lung cancer(NSCLC)nowdays.However,primary and acquired drug-resistance limits patients benefit from EGFR-TKIs treatment.Thus,it is of great significance to seek more safe and effective strategies to sensitivate or reverse EGFR-TKIs resistance.As an autophagic adaptor and signaling hub,SQSTM1 plays a crucial role in tumorigenesis and drug resistance.However,there is no eveidence for whether SQSTM1 also plays a role in EGFR-TKIs resistant against NSCLC.Objective:We aim to investigate how EGFR-TKIs regulate SQSTM1 expression,and the role of SQSTM1 on promoting acquired EGFR-TKIs resistence in NSCLC.By this study,we aim to providing effective strategies for reversing the acquired EGFR-TKIs resistance in NSCLC.Method:Western blot was used to detect the protein level of SQSTM1 as well as the makers of apoptosis and autophagy after indicated treatment;MTS assay and flow cytometry apoptosis analysis were performed to evaluate the synergistic effects of SQSTM1 knock down or overexpression with EGFR-TKIs;We knocked down or overexpressed SQSTM1 by transfection of siRNA or plasmid;RT-PCR was applied to assess theexpression level of SQSTM1 mRNA after EGFR-TKIs treatment;Measuring the change of autophagy and lysosomal function by immunofluorescence.Results:EGFR-TKIs(including Gefitinib and AZD9291)could upregulate the protein level of SQSTM1 in concentration and time dependency,and SQSTM1 knock down significantly enhanced the effect of EGFR-TKIs(Gefitinib and AZD9291)against NSCLC by inducing apoptosis.Additionally,overexpression of SQSTM1 could promote EGFR-TKIs resistance in NSCLC.Further study demonstrated that EGFR-TKIs inhibited the degradation of SQSTM1 but not impacted its transcription.In addition,we found that EGFR-TKIs could repress autophagy flux and thus resulted in SQSTM 1 accumulation.Furthermore,we found EGFR-TKIs(Gefitinib and AZD9291)inhibit lysosomal function rather than autophagosome formation and its fusion with lysosome,which resulted in autophagy repression.Conclusion:EGFR-TKIs(Gefitinib and AZD9291)inhibited autophagy by repressing lysosomal function and thus restrained autophagic degradation of SQSTM1.The overexpression of SQSTM 1 promoted acquired EGFR-TKIs resistence in NSCLC and SQSTM 1 knock down enhanced the effect of EGFR-TKIs(Gefitinib and AZD9291)against NSCLC.