The Role And Mechanism Of HSP70 In The Acquired Resistance Of EGFR-TKI-sensitive Non-small Cell Lung Cancer

Lung carcinoma is the leading cause of cancer-related deaths worldwide.Although the measures against primary lung cancer such as operation,radiotheraphy and chemotherapy had been developed,the prognosis of lung cancer is still worst with 15%of 5-year-viability.Non-small cell lung cancer(NSCLC)accounts for approximately 80-85%of lung cancer.Recently,considerable progress has been achieved in the treatment of NSCLC due to the development of molecular targeted therapies against epidermal growth factor receptor(EGFR).Gefitinib and erlotinib,two EGFR tyrosine kinase inhibitors(EGFR-TKIs),have been FDA approved for the treatment of advanced or metastatic NSCLC.Growing evidence has been demonstrated that most advanced NSCLCs with activating somatic EGFR mutations occurring in the tyrosine kinase domain(mostly exon 19 deletions and L858R exon 21 mutations)initially respond to gefitinib or erlotinib with hypersensitivity.However,over time(9-12 months of treatment),almost all patients eventually develop acquired resistance to EGFR-TKI.The most common mechanism that confers resistance involves a second-site point mutation that substitutes methionine for threonine at position 790(T790M),which is believed to render the receptor refractory to inhibition by these EGFR-TKIs.Although numerous new TKIs have been designed to directly target EGFR T790M mutant NSCLC cells,most of them are still far from being used clinically because of their relatively weak binding activity to the EGFR or severe toxicity.Consequently,an unmet need exists for the development of novel strategies in preventing the onset of resistance in order to enhance the clinical effectiveness of Gefitinib and Erlotinib.All cells are confronted with tens of thousands of DNA lesions each day by endogenous and exogenous DNA-damaging agents.The base excision repair(BER)pathway is considered as a highly conserved and important DNA repair system.Although BER pathway removes damaged bases and protects genome from mutagenesis,it is one of the sources of mutation because of misincorporation during repair.Given that EGFR T790M mutation is a C-T transversion at nucleotide position 2,369 of EGFR gene,we speculate that the production of EGFR secondary mutation is related to the capacity of BER pathway after the EGFR-TKI treatment.Heat shock peotein 70(HSP70)is one of the most important HSPs during normal cellular growth as well as pathophysiologycal conditions.HSP70 functions as an ATP-dependent molecular chaperone that assists in folding of newly synthesized polypeptides,the assembly of multiprotein complexes,transport of proteins across cellular membranes,and targeting of proteins for lysosomal degradation.It has recently been reported that HSP70 is associated with DNA protection.HSP70 is postulated to stimulate the activities of repair enzymes of BER pathway to reduce DNA damage.In the present study,the role of HSP70 in the BER has been further studied and the possible causative relationship between the HSP70-mediated BER and the EGFR secondary mutation-mediated drug resistance has been investigated.Our results are listed as follows:1.Long-term exposure of erlotinib induced an emergence of T790M mutation in HCC827 cells detected by PCR and DNA sequencing assay.2.EGFR-TKI treatment suppressed the capacity of BER pathway and induced a protein level reduction of HSP70 in EGFR-TKI-sensitive HCC827 cells.3.Transient overexpression of HSP70 ameliorated the EGFR-TKI-elicited DNA damage.We also established an HSP70-stably overexpressing HCC827 cell line in order to evaluate a possibility that HSP70 might suppress gene mutation.By HPRT gene mutation assay,we found that stable overexpression of HSP70 significantly inhibited the increase of gene mutation rate induced by the treatment of drug or MMS.Importantly,HSP70 overexpression delays the emergence of erlotinib-induced EGFR T790M mutation.4.HSP70 directly binds to FEN 1 and Pol ?.Recombinant HSP70 promotes FEN1 activity and enhances the Pol P fidelity,thereby promoting the capacity of BER pathway.5.EGFR-TKI induced the reduction of HSP70 through the inhibition of EGFR signaling and the inhibition of phosphorylation of HSP70 at tysosine 41,probably resulting in the enhanced ubiquitination of HSP70 and subsequently,in the degradation of HSP70 through proteasome pathway.Based on above results,we provide a potential explanation for the clinical observation that tumors bearing EGFR-activating mutation may undergo dramatic shrinkage after the administration of EGFR-TKI,however,EGFR blockade may paradoxically enhance the gene mutation rate via reduction of HSP70-mediated capacity of BER pathway,thus promoting the emergence of secondary mutations upon the drug selection.Our observations reveal the relationship between HSP70 protein stability and the gene mutation,and the mechanism by which HSP70 promotes BER capacity to inhibit gene mutation.We believe our study have instructive significance not only in the elucidation of HSP70 multi-function in cancer,but also in providing new strategies to overcome drug resistance in targeted therepy of lung cancer.