Identification Of Key Functional LncRNAS And Genes In The EGFR-TKIs Resistance Susceptibility Chromosome 4q12 Loci In Non-small Cell Lung Cancer

Part ?:Screening of potential functional lncRNAs and genes in the chromosome 4q12 lociBackground:Lung cancer is the malignancy with the highest morbidity and mortality worldwide.There are two histopathological subtypes,non-small cell lung cancer(NSCLC)and small cell lung cancer(SCLC).Due to most NSCLC patients are diagnosed at the advanced disease stage,they cannot be treated with surgery.The discovery and clinical applications of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitors(EGFR-TKIs)significantly improved the prognosis of NSCLC patients with advanced diseases.Most NSCLC patients have mutations in the EGFR tyrosine kinase domain,including point mutations in exon 18,20 and 21 as well as the exon 19 deletion mutations,in cancerous tissues.By competitively binding to the EGFR tyrosine kinase domain with ATP,EGFR-TKIs block the activation of the EGFR downstream signaling pathway and inhibit the proliferation,invasion and metastasis of NSCLC cells.There are three generations of EGFR-TKIs currently used in clinic.The first-generation EGFR-TKI mainly includes Gefitinib and Erlotinib,which are the most classic EGFR-TKIs targeting NSCLC cells carrying the EGFR-TKIs sensitive mutations.The second-generation EGFR-TKIs mainly include Afatinib,which is a multi-target TKI.The third-generation EGFR-TKIs mainly include Osimertinib which is mainly used for NSCLC patients with the EGFR T790M resistance mutation.It has been reported that EGFR-TKIs show good respond to NSCLC patients with sensitive EGFR mutations.However,a portion of NSCLC patients carrying sensitive EGFR mutations show primary resistance to EGFR-TKIs and have poor prognosis.In addition,almost all NSCLC patients develop acquired resistance after a certain period of EGFR-TKIs treatment.Therefore,it is an important scientific issue to revealing the molecular mechanism how EGFR-TKIs resistance develops,which currently restricts the clinical applications of EGFR-TKIs.According to 1000 Genomes Project,more than 99%of the human genome DNA sequences in different individuals are same and less than 1%human genome DNA sequences are different.Single nucleotide polymorphisms(SNPs)were the main type of genetic variations leading to the differences in human genome DNA sequences.Genome-wide Association Studies(GWAS)is a powerful tool for genome-wide identification of genetic variants that are significantly associated with specific complex diseases,including cancers,or drug response to certain treatments for diseases.A GWAS cohort study with relatively large sample size in Taiwan region of China found that the chromosome 4q12 loci is a susceptible region for EGFR-TKIs resistance of NSCLC patients in Chinese populations.However,it is still largely unclear how geves in the chrolosome 4t12 loci are involved in development of EGFR-TKIs resistance.Therefore,we detected the differential expression of candidate lncRNAs and protein-coding genes in chromosome 4q12 loci in two NSCLC cohorts in this part,and preliminarily identified two candidate IncRNAs and one candidate gene for the following studies of this paper.Methods and Results:1)The increased expression levels of lncRNA LCETRL3 and lncRNA LCETRL4 in NSCLC tissues:There were 5 lncRNAs in the chromosome 4q12 loci,which were named as LCETRL1(Lung Cancer EGFR-TKIs resistance lncRNA1),LCETRL2,LCETRL3,LCETRL4 and LCETRL5.Through real-time quantitative PCR assays,we examined expression levels of these lncRNAs in 64 paired cancer tissues and normal samples in the discovery cohort and validation cohort.We found that the expression levels of lncRNA LCETRL3 and lncRNA LCETRL4 were significantly increased in NSCLC tissues compared to those in normal tissues.2)The significantly up-regulated expression levels of CLOCK in NSCLC tissues:We analyzed the normal lung tissue RNA-seq data of the GTEx database by expression Quantitative Trait Loci(eQTL)analyses.We found that the expression levels of several coding genes in chromosome 4q12 loci in normal lung tissues were significantly correlated with the EGFR-TKIs resistance susceptibility SNPs.The expression level of PDCL2 was significantly correlated with SNP rs576732.The expression level of CLOCK was significantly correlated with SNPs rs476184,rs1801260,rs1021307 and rs17725115.The expression level of NMU was significantly correlated with SNP rs3805383.The expression level of SRD5A3 was significantly correlated with SNP rs41324951.The expression level of TMEM165 was significantly correlated with SNP rs693367.In the discovery cohort(20 NSCLC tumor tissues and normal tissues),we detected expression of candidate genes including KDR,SRD5A3,TMEM165,CLOCK,PDCL2,NMU,EXOC1 and CEP 135 in the chromosome 4q12 loci.We found that expression levels CLOCK and NMU were significantly up-regulated in NSCLC tumor tissues compared to those in normal tissues.Similarly,we found the evidently elevated expression levels of CLOCK in NSCLC tissue samples in the validation cohort(44 pairs of NSCLC tumor tissues and normal lung tissues).Conclusions:In this part,we detected the expression of 5 lncRNAs and 8 protein-coding genes in chromosome 4q12 loci in NSCLC tumor tissues and normal lung tissues in samples from discovery cohort and validation cohort.We found significantly increased expression of lncRNA LCETRL3,LCETRL4 and CLOCK in NSCLC tissues.As a result,we systematically explore how lncRNA LCETRL3,LCETRL4 and CLOCK promote development of NSCLC cells and lead to EGFR-TKIs treatment resistance of NSCLC cells in the 2nd and 3rd parts of the paper.Part ?:Identification of oncogenic lncRNAs LCETRL3 and LCETRL4 and their role in gefitinib resistance in NSCLCBackground:Long non-coding RNAs(lncRNA)are a class of more than 200 nucleotides ncRNAs,which regulate gene expression at the epigenetic,transcriptional and post-transcriptional levels.It has been shown that lncRNAs can promote the proliferation,invasion and metastasis of NSCLC and development of chemotherapy resistance of NSCLC cells.However,it is still unclear how lncRNAs are involved in development of EGFR-TKIs resistance of NSCLC cells.TDP43(Transactive response DNA-binding protein 43)is an RNA-binding protein,which is involved in the regulation of DNA replication,gene transcription,nRNA splicing and translation.TDP43 plays an important role in cancer development.EIF2S1(Eukaryotic Translation Initiation Factor 2 Subunit 1)is directly involved in the regulation of several key cancer-related intracellular signaling pathways,including transcriptional initiation complex recruitment and integrated emergency response.The potential role of TDP43 and EIF2S1 in development of EGFR-TKIs resistance of NSCLC cells remains largely unclear.Methods and Results:1)lncRNAs LC-ETRL3 and LCETRL4 significantly promoted the malignant phenotype of NSCLC cells:Functional assays showed that,silencing of LCERL3 or LCETRL4 significantly reduced cell proliferation,clone formation,as well as invasion and metastasis ability of NSCLC cells,overexpression of LCERL3 or LCETRL4 significantly promoted these malignant phenotypes of NSCLC cells.LCETRL3 or LCETRL4 significantly promoted in vivo proliferation of NSCLC xenografts in nude mice.2)lncRNAs LCETRL3 and LCETRL4 inhibit the sensitivity of Gefitinib in NSCLC:It has been shown that silencing of LCETRL3 or LCETRL4 significantly inhibited proliferation of PC9 cells treated with 5 ?M to 10 ?M Gefitinib compared to cells treated with Gefitinib alone.On the contrary,Overexpression of LCERL3 or LCETRL4 significantly promoted proliferation of PC9 cells compared to cells treated with Gefitinib alone.Similar results were observed in H1299 cells treated with 60-80 ?M Gefitinib.In line with these results,LCETRL3 or LCETRL4 can significantly reduce the sensitivity of NSCLC xenografts to gefitinib in vivo.3)lncRNA LCETRL3 enhances the stability of TDP43 and activates the AKT signaling:RNA Pulldown assay,Protein mass spectrometry and RNA-binding Protein Immunoprecipitation(RIP)assay showed that oncogenic LCETRL3 could bind TDP43 and up-regulate its protein expression levels in NSCLC cells.It has been reported that TDP43 promotes NOTCH1 transcription and reduces PTEN expression.We found that overexpression of LCETRL3 increased the levels of NOTCH1 in NSCLC cells,which led to the decreased expression levels of PTEN.PTEN is an important inhibitor of the phosphorylation of AKT.LCETRL3 inhibited PTEN expression and led to increased phosphorylation levels of AKT.The activation of AKT signaling could decreased sensitivity of NSCLC cells to Gefitinib.4)lncRNA LCETRL4 inhibits the ubiquitin-proteasome degradation of EIF2S1 and activates the AKT signaling:We found that LCETRL4 can interact with EIF2S1 by RNA Pulldown and protein profiling analyses.The RIP assays verified that EIF2S1 protein could bind to LCETRL4 in NSCLC cells.Overexpression of LCETRL4 can significantly up-regulate the expression levels of EIF2S1 in NSCLC cells.It has been reported that EIF2S1 can promote the phosphorylation of PDK1 and the phosphorylation of AKT.We found that LCETRL4 can increase the phosphorylation levels of AKT and then activate the AKT signaling in NSCLC cells,which leading to significantly decreased drug sensitivity of NSCLC cells to Gefitinib.Conclusions:In this part,we found that lncRNA LCETRL3 and LCETRL4 in the chromosome 4q12 loci can regulate the stability and expression levels of TDP43 and EIF2S1,respectively.Both LCETRL3 and LCETRL4 can activate the AKT signaling in NSCLC cells.Overexpression of lncRNA LCETRL3 or LCETRL4 could promote the malignant phenotypes of NSCLC cells and significantly reduce the sensitivity of NSCLC cells to Gefitinib.Part ?:CLOCK activates the EGFR signaling pathway to promote EGFR-TKIs resistance of NSCLC cellsBackground:Due to revolution and rotation of Earth,many organisms have formed a 24-hour functional cycle,which is known as the circadian clock.Organisms can carry out behaviors or perform biological functions at the appropriate time depended on their circadian clock.CLOCK is an important component of circadian clock and is involved in the transcriptional regulation of multiple target genes.CLOCK can form heterodimer with BMAL1 to perform biological functions.The dimer is an important positive regulator of mammalian circadian rhythm.It can bind to the E-box motif of the transcriptional initiation region of target genes and participate in the transcriptional regulation of several cancer-related genes.As an important member of laminin family,LAMC2 is an crucial component of cell membrane receptor proteins and extracellular matrix.It has been shown that LAMC2 is involved in the invasion and metastasis of multiple malignancies.It has been reported that LAMC2 could activate the PI3K-AKT signaling.Methods and Results:1)The allelic regulation of CLOCK expression by SNP rs 1021307:We analyzed the genetic variation data and RNA-seq data of lung tissues in the GTEx database using eQTL analyses.We found that EGFR-TKIs resistance susceptibility SNPs rs476184,rs 1801260,rs 1021307 and rs 17725110 were significantly correlated with CLOCK expression levels in lung tissues.Via the Haploreg V4.1 software,we found that rs 1021307 locating in the transcription regulation region of CLOCK was in the consensus binding motif of the transcription factor TCF7L2,and could potentially affect TCF7L2 binding capability.Using double luciferase reporter gene assays and EMSA assays,we found that the G allele of rs 1021307 could bind to TCF7L2 to regulate CLOCK transcription and expression levels,while the rs 1021307 A allele could not bind to the transcription factor TCF7L2 in NSCLC cells.2)CLOCK significantly promoted the malignant phenotypes of NSCLC cells:The cell counting,clone formation,scratch healing and Transwell assays showed that silencing of CLOCK significantly inhibited the proliferation,clone formation,cell migration,invasion and metastasis capabilities of PC9 and H1299 cells.Overexpression of CLOCK significantly promoted proliferation,clonal formation,migration,invasion and metastasis capabilities of NSCLC cells.Silencing of CLOCK evidently blocked PC9 and H1299 cells in G0/G1 phase.Overexpression of CLOCK significantly promoted in vivo proliferation of NSCLC xenografts in nude mice.3)CLOCK significantly promoted resistance of NSCLC cells to EGFR-TKIs:Gefitinib(one of the first-generation EGFR-TKIs)and Osimertinib(the third-generation EGFR-TKI)are widely used in clinic to treat NSCLC.Therefore,we examined how CLOCK impacts the treatment resistance of NSCLC cells to Gefitinib and Osimertinib respectively.We found that silencing of CLOCK significantly improved the inhibitory abilities of Gefitinib and Osimertinib.NSCLC cells developed drug resistance to Gefitinib and Osimertinib after overexpression of CLOCK.CLOCK could significantly reduce in vivo drug sensitivity of NSCLC xenografts to Gefitinib and Osimertinib in nude mice.4)CLOCK can promote LAMC2 transcription:We analyzed the RNA-seq data and found that CLOCK can regulate several important signaling pathways,including the cell cycle signaling pathway,the MAPK signaling pathway and the P53 signaling pathway.These data further explain the effects of CLOCK on the proliferation,clone formation and cell cycle of NSCLC cells.Combined with ChIP-seq data,we found that the signal of H3K4me3 in the transcription initiation region of LAMC2 was significantly up-regulated after the overexpression of CLOCK,suggesting that LAMC2 transcription ability was significantly enhanced.The ChIP-qPCR assays indicated that CLOCK can bind to the transcription initiation region of LAMC2 and promote transcription and expression of LAMC2,which plays an important role in promoting proliferation of NSCLC cells and EGFR-TKIs resistance of NSCLC cells.5)LAMC2 promotes the EGFR signaling pathway:It has been reported that LAMC2 can promote the phosphorylation and activation of PI3K and ERK1/2.Therefore,we examined the protein expression levels and protein phosphorylation levels of PI3K,AKT,KRAS and ERK1/2 in NSCLC cells.,We found that overexpression of CLOCK can significantly increase PI3K phosphorylation levels and KRAS expression levels;which can enhance phosphorylation of AKT and ERK1/2.The activation of these signaling pathways may lead to the resistance of NSCLC cells to EGFR-TKIs.Conclusions:In summary,we found that the G allele of EGFR-TKIs drug-resistant susceptibility SNP rs 1021307 can promote the transcription of CLOCK by binding to the transcription factor TCF7L2.There are significantly increased the expression levels of CLOCK in NSCLC tissues compared to normal tissues.CLOCK acts as an oncogene in NSCLC and promotes proliferation,invasion and metastasis of NSCLC cells.As a transcription factor,CLOCK can directly promote the transcription of LAMC2 and up-regulate the expression levels of LAMC2,which thus activating the EGFR signaling pathway and promoting the resistance of NSCLC cells to Gefitinib and Osimertinib.