Study Of NY-ESO-1 As A Therapeutic Target For Multiple Myeloma

Background:Multiple myeloma(MM)is a malignant disease characterised by proliferation of clonal plasma cells,which can lead to hematopoietic inhibition and osteolytic lesions.With the development of high dose chemotherapy and hematopoietic stem cell transplantation therapy,as well as the use of novel drugs such as proteasome inhibitors and immunomodulatory drugs has increased response rates and survival substantially of MM.Nevertheless,MM is still regarded as an incurable disease for most patients.Minimal residual myeloma cells are considered to be an important factor in the progression and recurrence of myeloma after MM has escaped from conventional chemotherapy.Therefore,the selection of highly specific target molecules for the reduction of minimal residual myeloma cells and consolidation therapy is one of the current research priorities of MM.NY-ESO-1(New York esophageal squamous cell carcinoma 1)is an important member of the cancer-testis antigen family.It is one of the most immunogenic tumor antigens and can induce spontaneous humoral and cellular immune responses.NY-ESO-1 is widely expressed in many types of tumors such as MM,neuroblastoma,melanoma and ovarian cancer,etc.In addition,the expression of NY-ESO-1 in tumor has a certain correlation with tumor type,malignancy degree and prognosis,and its expression can be regulated by epigenetic drugs.It was found that NY-ESO-1 is highly expressed in MM patients with cytogenetic abnormality or poor prognosis.During the progression or recurrence of the disease,the titer of specific NY-ESO-1 antibody was significantly increased,and these antibody could mediate killing of the primary MM cells.NY-ESO-1 is an ideal target for immunotherapy of MM.However,the role of NY-ESO-1 in the occurrence and development of MM remains unclear.It is important to explore the biological function and mechanism of NY-ESO-1.Objective:To investigate the effect of NY-ESO-1 on biology properties of MM cells,and to provide theoretical and experimental evidences for targeting NY-ESO-1 of tumor immunotherapy.Methods and contents:1.Expression of NY-ESO-1 in U266 and RPMI8226 was detected by qPCR and Western Blot.Two shRNAs targeting humen NY-ESO-1 mRNA and a scrambled shRNA as negative control were designed and synthesized.The annealed oligonucleotide fragments were subcloned into pHBLV vector.Virus particles were collected after shRNA vector was cotransfected with the psPAX2 packaging plasmid and the envelope plasmid pMD2.G into293T cells,then U266 cells were infected with virus condensed by high speed centrifugation.Limit dilution method was used to separate GFP~+cells,then stable cell lines were established.QPCR and Western Blot were used to detect the expression of NY-ESO-1 after lentivirus infection.2.To initially investigate the effects and mechanisms of down-regulated NY-ESO-1expression on biology characteristics of U266 cells.The CCK-8 assay was used to evaluate the effect on cell proliferation and chemotherapy resistance.Soft agar assay was used to evaluate the effect on clonal formation.Transwell Assays were used to evaluate the effect on cell migration and invasion.The cell cycle status and apoptosis of cells were detected by flow cytometry.QPCR was used to detect the mRNA level of cycle-related molecules such as Cyclin D1 and p21.Western Blot was applied to detect the expression of proteins,including p21 and indicator molecules for EMT(E-cadherin,N-cadherin,Vimentin).All the assays were carried out in U266,LV-shNC-U266 and LV-shNY-ESO-1-U266 cells.Effects of down-regulating NY-ESO-1 expression on the oncogenicity,bone mineral density,tumor formation times and tumor sizes were respectively observed by subcutaneous injection of U266,LV-shNC-U266 and LV-shNY-ESO-1-U266 cells in nonobese diabetic/serious combined immunodeficiency disease(NOD/SCID)mice.In addition,bone mineral density was analyzed by Micro-CT scan assay.The expression of NY-ESO-1,p53 and E-cadherin in tumor tissue was also detected by qPCR,Western Blot or immunohistochemistry.Results:1.The results of qPCR and Western Blot showed the high expression of NY-ESO-1 in U266cells,and low expression or no expression in RPMI8226 cells.The nucleotide sequencing showed that recombinant lentivirus vectors were constructed successfully.The mRNA and protein levels of NY-ESO-1 were reduced significantly in cells after lentivirus infection and the inhibitory effect of LV-shNY-ESO-1#2 group was better than that of LV-shNY-ESO-1#1 group,suggesting that stable infected U266 cell lines were established.2.The activities of proliferation,clonal formation,chemotherapy resistance,migration and invasion in the LV-shNY-ESO-1-U266 cells were all significantly lower than that U266 cells and LV-shNC-U266 cells in vitro.Both of the proportion of G1-phase cells and the expression of p21 were increased in the LV-shNY-ESO-1-U266 cells.The differences were statistically significant(P<0.05).3.The tumorigenicity was significantly decreased and the bone density was markedly increased in LV-shNY-ESO-1 group compared with the control group.The expression of NY-ESO-1 was lower,while the expression of p53 and E-cadherin was higher in tumor tissue in NOD/SCID mice injected with LV-shNY-ESO-1-U266 cells compared with the mice injected with LV-shNC-U266 and U266.The differences were statistically significant(P<0.05 or P<0.01).Conclusions:The down-regulation of NY-ESO-1 reduced the ability of cell proliferation,clonal formation,chemotherapy resistance,migration and invasion as well as the tumorigenecity of MM U266 cells.This findings suggested that NY-ESO-1 can be used as a potential target for multiple myeloma immunotherapy.