Study On The Mechanism Of CD47 Protein Promoting Resistance Of Targeted Drugs In Non-small Lung Cancer And Melanoma

Malignant tumor is one of the main threat to human life and health.At the early stage of the tumor,some patients can be cured by surgery combined with chemotherapy and radiotherapy.But some patients still relapse within several years.However,once the tumor is metastatic,there is generally no specific treatment regimen.With the deepening understanding of the human genome in recent decades,it has been found that there are some specific gene mutations in different cancers.Targeted therapy for mutant genes has become a standard treatment for advanced malignant tumors.However,the efficacy time of drug is generally short,the patient will relapse in a few weeks to several months,and our study aims to investigate resistant mechanism of targeted drugs for two kinds of tumors,non-small cell lung cancer(NSCLC)as well as melanoma.Lung cancer is one of the leading causes of death in men and women,about 80%-85% of patients were diagnosed with non-small cell lung cancer(NSCLC),more than 80% of NSCLC cases were diagnosed at late stage,among which,30% of patients has epidermal growth factor Receptor(EGFR)mutations.The first-line epidermal growth factor receptor tyrosine kinase inhibitor(EGFR TKI)is a standard treatment for patients with advanced EGFR mutations in non-small cell lung cancer(NSCLC),but the drug response very shortly.Current studies have shown a variety of resistance mechanisms,including primitive or induced T790 M mutations,human EGFR2 amplification and activation of secondary conduction signals,such as MET amplification or phosphatidylinositol 3-kinase mutations.In addition,epithelial stromal transformation of lung cancer cells has also been identified as a mechanism for acquired anti-EGFR-TKIs.There is growing evidence that noncoding RNAs include long non-coding RNA and micro RNAs that also promote drug resistance.Recently,it has been closely related to the function of the immune system.Studies have shown that PD-L1 is involved in the production of drug resistance in patients with EGFR mutant lung cancer.PD-L1 is a transmembrane protein with a molecular weight of 41 k Da.Although the current understanding of EGFR resistance mechanism has promoted the treatment of lung cancer,providing a variety of combined therapy strategy,its resistance has not yet been fundamentally resolved.The rate of BRAF mutation in melanoma is about 50%,Targeting BRAF,MEK,and co-targeting BRAF and MEK using specific inhibitors have become the standard of care for patients with late-stage mutant BRAF melanomas.However,the benefits are often of limited duration due to rapid development of resistance.Many mechanisms have been shown to contribute to the resistance.However,the potential effect of MAPK inhibitors on the interaction between melanoma cells and the immune system seems paradoxical.On one hand,there is a large body of evidence showing that MAPK activation in melanoma cells contributes to immunosuppression and MAPK inhibitors trigger melanoma-specific immune responses,which are typically manifested by a rapid increase in tumour infiltrating lymphocytes(TILs)consisting mainly of cytotoxic T lymphocytes(CTLs).On the other,TIL numbers decline progressively after the initial increase,suggesting that the balance between immunostimulatory and immunosuppressive mechanisms in the melanoma environment skews from the former to the latter.While MAPK inhibitor-triggered anti-melanoma immune responses are associated with the increased expression of melanoma antigens and reduction in immunosuppressive cytokines such as interleukin(IL)-6 and IL-8,the immunosuppressive effect of MAPK inhibitors has been linked to upregulation of programmed death ligand 1(PD-L1)on the melanoma cell surface and stimulation of tumour-associated macrophages(TAMs)that in turn promote melanoma growth.We investigated the potential effect of EGFR and BRAF/MEK inhibitors on CD47 expression in NSCLC cells and melanoma cells and its functional consequences.We detected the expression of CD47 in drug-resistant and non-resistant strains of lung cancer and melanoma.Comparison of macrophages between drug-resistant and non-resistant cell strains phagocytosis differences.It was found that the expression levels of CD47 were up-regulated in gefitinib-resistant lung cancer cell lines and vemurafenib-resistant melanoma cell lines relative to their respective non-resistant strains.Similarly,melanoma cells selected for resistance to vemurafenib in vitro expressed higher levels of CD47.Moreover,CD47 was expressed at increased levels in 3 of 5 primary melanoma cell cultures from biopsies post-treatment with vemurafenib compared with those from paired pre-treatment tissues.The increase in CD47 in gefitinib-resistant lung cancer cell lines and vemurafenib-resistant melanoma cell lines was mediated by the transcription factor NRF-1,which was also responsible for the constitutive CD47 expression.In particular,CD47 upregulation is an important mechanism that dumps the anti-immune response initially activated by EGFR and BRAF/MEK inhibitors.The combination of CD47 blockade and EGFR or BRAF/MEK inhibitors may be a promising approach to improve their therapeutic efficacy.Cluster of Differentiation 47(CD47)is a transmembrane protein of the immunoglobulin superfamily that is ubiquitously expressed in mammalian tissues as a “self-marker”.It transmits “don't eat me” signalling through interacting with signal-regulatory protein(SIRP? on the surface of phagocytic cells.Binding of SIRP? with CD47 results in phosphorylation of the immunoreceptor tyrosine-based inhibition motif(ITIM)on the cytoplasmic tail of SIRP?.This results in inhibition of phagocytosis through preventing myosin-IIA accumulation at the phagocytic synapse.In fact,CD47-SIRP? is the only known ligand-receptor pair that negatively regulates phagocytosis.In contrast to CD47,calreticulin(CRT),a protein normally located to the lumen of the endoplasmic reticulum(ER),transmits “eat-me” signalling once it is exposed to the outer leaflet of the cell membrane through binding to low density lipo-protein receptor–related protein 1(LRP1)on the surface of phagocytic cells[16,19].Noticeably,CRT is often absent on the normal cell surface but is expressed on the surface of cancer cells and can further induced when cells undergo immunogenic cell death.The expression of CD47 is frequently increased in cancer cells.Although the mechanism responsible for this remains largely unknown,blockade of the interaction between CD47 and SIRP? is emerging as a promising immunotherapeutic approach in the treatment of cancer.Indeed,targeting CD47 has demonstrated potent preclinical activity against various cancers including melanoma.Although this is directly related to phagocytosis of cancer cells by macrophages,CD47 blockade also triggers anti-cancer T cell responses through macrophages as well as dendritic cells(DCs).Humanized anti-CD47 Abs have entered early clinical trials in the treatment of various types of cancers(clinicaltrials.gov).In conclusion,in the two epithelial original malignant tumors,different targeting drugs have a common mechanism of generated resistance-increasing the CD47 protein expression to inhibit the function of immune system.This provides guidelines for the combined use of CD47 blockers in lung cancer and melanoma.Do other targeted drugs in both tumors as well as targeted drugs in other tumors have this common drug resistance mechanism? This is worthy of our thought,for this part,we will conduct a deeper study in the future,to provide more theoretical basis for the treatment of malignant tumors.