The Treatment Effects Of Arsenic On Non-small Cell Lung Cancer And Its Mechanism And A Functional Study Of A Point Mutation Of Zebrafish C-cbl Gene In The Ring Finger Domain

Lung cancer is one of the malignancies of highest morbidity and mortality worldwide,and non-small cell lung cancer(NSCLC)accounts for about 85% of all cases.The lifetime of NSCLC patients in several sub-groups has been prolonged since the advent of molecularly targeted therapy,particularly the drugs targeting epidermal growth factor receptor(EGFR)such as Gefitinib.However,resistance to therapy ultimately develops in the vast majority of patients,and the overall survival of NSCLC patients still needs to be improved.Hence,it is urgent to find new treatment for NSCLC.As a drug originated from the traditional Chinese medicine,arsenic has been shown to be able to cure the great majority of patients with acute promyelocytic leukemia.Arsenic can also exert therapeutic effects on chronic myeloid leukemia.Nevertheless,so far few studies were reported about arsenic treatment in solid tumors.In the present study,we explored the effects of arsenic on NSCLC at the levels of cell lines,animal models and fresh samples from patients.The molecular and cellular mechanisms underlying the therapeutic effects of arsenic have been addressed by using methods such as immunofluorescence,transmission electron microscopy,western blot and co-immunoprecipitation.Our results showed that arsenic could inhibit the proliferation of NSCLC cell lines,especially the cell line with resistance to Gefitinib.Arsenic was shown to induce autophagy of NSCLC cells,enhance the interaction between the autophagy-related protein p62 and EGFR by directly binding to p62,and eventually trigger the degradation of EGFR through autophagy-lysosome pathway.Of note,in NSCLC mouse models,arsenic suppressed the tumor growth in subcutaneous nude mouse xenografts and prolonged the survival of in situ NOD-SCID models.Clinically the injection of arsenic trioxide into the pleural cavity of the late stage NSCLC patients reduced the number of cancer cells in pleural effusions.Based on these data,we believe that arsenic may have potential therapeutic value for NSCLC either as a single drug or in combination with other agents.Further studies are thus warranted to evaluate the clinical benefits of arsenic such as the improvement of the prognosis or of the quality of life in NSCLS patients.Controlled self-renewal and differentiation of hematopoietic stem/progenitor cells(HSPCs)are critical for vertebrate development and survival.These processes are tightly regulated by the transcription factors,signaling molecules,and epigenetic factors.Impaired regulations of their function could result in hematological diseases,including malignancies.Using a large-scale zebrafish N-ethyl-N-nitrosourea(ENU)mutagenesis screening,we identified a line named LDD731,which presented significantly increased HSPCs in hematopoietic organs without other apparently abnormal phenotypes.Further analysis revealed that the erythoid/myeloid lineages in definitive hematopoiesis were increased while the primitive hematopoiesis was not affected.The causal mutation was located by positional cloning on chromosome 15 in the c-cbl gene,of which the human ortholog,c-CBL,is found frequently mutated in myeloproliferative neoplasms(MPN)or acute leukemia.Sequence analysis showed the mutation in LDD731 caused a histidine-to-tyrosine substitution of the amino acid(aa)codon 382 within the RING finger domain of c-Cbl,which could result in the loss of function for c-Cbl as an E3 ubiquitin ligase.The homozygous mutation was lethal with a median survival time around 14–15 days post-fertilization.Moreover,the myeloproliferative phenotype in zebrafish seemed dependent on the Flt3(fms-like tyrosine kinase 3)signaling,consistent with that observed in both mice and humans.Our study may shed new lights on the pathogenesis of MPN and provide a useful in vivo vertebrate model for drug screenings for this syndrome.