Research On The Role And Mechanism Of LINE-1 In The Occurrence And Development Of Breast Cancer

Breast cancer is one of the most prevalent cancers in women.Triple negative breast cancer(TNBC)is a subtype of breast cancer that lacks expression of estrogen receptor,progesterone receptor and HER2.It is characterized by high-grade malignancy,high heterogeneity,lack of specific therapeutic targets,and poor prognosis.Therefore,there is an urgent need to further explore the molecular characteristics and tumor biology of TNBCGenomic instability is one of the main features of tumor,which is an important driving force for the occurrence and development of tumor by providing raw materials for evolution,endowing cells with varied genetic backgrounds that allow for the selection of cells with growth advantages under various selection pressure.Our previous studies found that p53-mutant TNBC cells showed more increased genomic instability than ERa-positive breast cancer cells under chemotherapy stress.However,the mechanisms involved are unclear.Long interspersed element-1(LINE-1)is an abundant class of retrotransposon broadly distributed throughout the human genome,which encodes two protein products(ORF1 and ORF2).The ORF2 possesses endonuclease and reverse transcriptase activity.LINE-1 is always silenced in normal cells,but could be reactivated due to decreased methylation and other epgenetical modification changes in tumor cells.LINE-1 retrotransposition could insert itself or other transposons into other locations in the genome,modulating gene expressions and reprogramming cell genome.In addition,DNA damage induced by the endonuclease activity of ORF2 also pose a huge threat to genomic stability.Previous studies found that higher expression of LINE-1 could contribute to the progression of multiple tumors.And stress stimuli are important inducement of LINE-1 activation.However,the role of chemotherapy stress in LINE-1 activation and the subsequent biological effects of genomic instability induced by LINE-1 remains unknown and needs to be explored.Genomic instability could impact the established genome structure and gene expression pattern,which would be detrimental to host cells.Consequently,the tolerance of genomic instability caused by aberrant expression of LINE-1 would be an essential precondition for tumor cells evolving.Thus,to explore the mechanisms by which TNBC cells can tolerate the genomic instability and accumulate genetic variations will be of great significance for a better understanding of TNBC and its treatment.As an important guarantee of cell genome,p53 helps in maintaining genomic stability by undergoing cellular arrest for DNA repair or triggering apoptosis of cells with severe damage.However,p53 gene was found to be mutated in approximately 80%of TNBC.Interestingly,the expression level of LINE-1 seems to be affected by the status of p53,since tumors with mutant p53 express higher LINE-1.These phenomena indicate that loss of p53 function is an important precondition of the tolerance of LINE-1-induced genomic insability in tumor cells.We investigated the functional relationship between chemotherapy stress,LINE-1 induced genomic stability and p53 status in TNBC cells and their biological effects using Comet,immunofluorescence assay,flow cytometry and other techniques.The results showed that genomic instability was significantly elevated by chemotherapy drug accompanied with remarkably increased LINE-1 expression.The aberrant expression of LINE-1 could contribute to genomic instability in TNBC cells.Loss of p53 function is an important precondition of the tolerance of LINE-1-induced genomic insability,while restoration of wild type p53 inhibited the encrichment of cells with genomic instability.Finally,we found that LINE-1 high expression cells showed higher migration,invasion and chemotherapy resistance.And the treatment of LINE-1 inhibitor EFV could reverse the malignant phenotype described above.Our work will help to understand the highly heterogeneous formation mechanism and provide new potential therapeutic strategies for TNBC.Breast cancer is one of the most common malignant tumors in women with the ascending tendency in recent years.The pathogenesis of breast cancer is not yet fully understood,but the inactivation of tumor suppressor genes is one of the most important mechanisms.ATM is a well known tumor suppressor of breast cancer.Loss of function caused by ATM downregualtion is an important reason for the occurrence of breast cancer,however,the molecular mechanisms involved remains to be resolved.LINE-1 is an abundant class of retrotransposon widely distributed in the genome,which can be located within genes,intragenic,or between genes,intergenic.The 5'UTR contains a bidirectional promoter:the sense promoter transcribes the two open reading frames,and the antisense promoter(ASP)can direct transcription of the adjacent genomic DNA in an opposite direction to generate an antisense RNA.Antisense transcripts are reported to regulate the corresponding sense gene expression in a variety of ways,including acting as chromatin modification regulators.Human ATM gene is located in chromosome 11q22-q23,with a length of about 150kb,and consisted of 63 exons.By bioinformatic analysis,we found that ATM contains an entire LINE-1 in its 61st intron.The purpose of this research was to explore whether Ll-ASP located in the intron of ATM gene could drive an antisense RNA,and whether and how the transcript could regulate the expression of ATM.We demonstrated that LI-ASP could drive transcription of an antisense RNA(named as L1-ATM-AS)using PCR assay.And the expression of L1-ATM-AS was downregulated in breast cancer cells compared to the normal ones,showing a positive correlation with the expression of ATM.A similar result was observed in 65 pairs of human clinical samples,that both L1-ATM-AS and ATM were significantly decreased in breast cancer tissues compared with matched para-carcinoma tissues.Reporter gene assay found that L1-ATM-AS could enhance the transcriptional activity of ATM promoter.L1-ATM-AS may exert its regulatory function through modifying chromatin configuration based on the phenomenon that inhibition of Ll-ATM-AS was associated with decreased active chromatin modification and increased repressive chromatin modification at ATM promoter.Furthermore,downregulation of ATM in normal cells inhibited the function of ATM,including decreased DNA repair capacity and malignant transformation.Our results suggest that L1-ATM-AS transcribed by L1-ASP located in the intron of ATM gene is a positive regulator of ATM expression,and is an important guarantee for the antitumor activity of ATM.The aberrant low expression of L1-ATM-AS contributed to decreased ATM expression and subsequent loss of function in breast cancer cells.Our findings revealed a novel mechanism of ATM regulation,further confirmed a retrotransposition-independent role for LINE-1 elements in the occurrence of breast cancer,and provided novel ideas for clinical understanding of pathogenesis and exploring effective treatment strategies of breast cancer.