The Role Of FAK Splicing Variants (FAK^6/7) In Small Cell Lung Cancer And Pancreatic Tumors

Background:Small cell lung cancer(SCLC)is a highly malignant neuroendocrine cancer,characterized by short doubling time,rapid tumor cell growth,extensive metastasis in the early stage,and poor prognosis,accounting for 15%of all lung cancer.In recent years,multi-omics studies have greatly deepened the understanding of the pathogenesis of SCLC and proposed potential therapeutic targets.However,the prognosis of SCLC is still very poor,and the five-year survival rate is less than 7%.It is urgent to find new effective therapeutic targets.Alternative splicing(AS)is a process in which mRNA precursors produce different mRNA splicing isomers through different splicing methods(choosing different splicing site combinations).AS is involved in a variety of physiological processes and is associated with a variety of diseases,including cancer.Therefore,it is of great significance to study SCLC from alternative splicing to find new therapeutic targets.Methods:Transcriptome sequencing was employed to investigate differential alternative splicing expression profiles in tumor and normal tissues from SCLC paired samples.Alternative splicing variants of focal adhesion kinase(FAK)were identified by polymerase chain reaction(PCR),agarose gel electrophoresis and Sanger sequencing.In situ expression of FAK6/7 splicing variants was detected by Basescope assay.Available survival information and clinical characteristics were utilized to analyze the correlation between FAK splicing variants and clinical characteristics and survival.CCLE database was used to study the splicing expression of FAK in SCLC cell lines.Immunohistochemistry(IHC)and western blot(WB)were used to detect the expression level of pFAK and other proteins.Kinase activity assay was used to detect the tyrosine kinase activity of FAK splicing isoforms.Protein structure of FAK splicing isoforms was predicted by AlphaFold.CCK8,IncuCyte,Transwell,and colony formation assay were employed to investigate cell proliferation,migration and clone formation ability.Cell cycle was detected by flow cytometry(FC).Mouse tumor-bearing experiment and patient-derived tumor xenograft(PDX)model were used to evaluate the effects of FAK inhibitors on tumors.Results:A total of 1196 differential alternative splicing events(ASEs)were detected in 832 genes in SCLC,among which 25 genes,including FAK,had differential ASEs in 27 patients.Among 136 SCLC patients,103(75.7%)expressed FAK splicing variants FAK6/7(FAK6,FAK7,FAK6,7).FAK6/7 was negatively correlated with paients’ survival(P=0.05),but had no association with gender,age,smoking status,histological type and TNM stage.FAK6/7 was highly expressed in SCLC cell lines,accounting for 80%(40/50).Compared with wild-type FAK,FAK7 and FAK6,7 showed significantly enhanced autophosphorylation and kinase activity in structural prediction and functional assays.FAK inhibitors PF562271 and PF573228 can inhibit cell proliferation and migration,and induce cell cycle arrestted at G2/M phases,and FAK6/7 is more sensitive to FAK inhibitors.The tumor-bearing experiments in mice showed that the inhibitory effects of FAK inhibitors on H82(expressing FAK6,7)tumors was stronger than that of DMS114(expressing wild-type FAK).PDX model showed that PF562271 could significantly inhibit the growth of 2 SCLC patients derived tumors.Conclusions:Alternative splicing variants FAK6/7 is highly expressed in SCLC,in which the autophosphorylation level and kinase activity of FAK7 and FAK6,7 are significantly elevated.FAK7 and FAK6,7 are more sensitive to FAK inhibitors than wild-type FAK.FAK inhibitor has better inhibitory effect on the tumor expressing FAK6/7.FAK6/7 is an effective antitumor therapeutic target in SCLC.Background:Pancreatic cancer is a recalcitrant cancer with a poor five-year overall survival and is a common cause of cancer-related death worldwide.Alternative splicing(AS)plays an important role in cancer.FAK splicing variants have been investigated in non-small cell lung cancer,colorectal cancer and papillary thyroid carcinoma,while it remains unclear in pancreatic tumors.Methods:Polymerase chain reaction(PCR)and Sanger sequencing were employed to identify FAK splicing variants,in 157 pancreatic tumor samples and pancreas cell lines.The association of FAK6/7 status and clinical factors were analyzed in pancreatic adenocarcinoma(PAAD)patients from The Cancer Genome Atlas(TCGA)database and pancreatic tumors in our cohort,including pancreatic ductal adenocarcinoma(PDAC),pancreatic neuroendocrine neoplasm(pNEN),pancreatic solid pseudopapillary neoplasm(SPN).TCGA and Cancer Cell Line Encyclopedia(CCLE)database were used to investigate FAK6/7(including BOX6 and/or BOX7)in PAAD patients and pancreas cell lines.Gene set enrichment analysis(GSEA)was conducted to investigate the differences between wt FAK and FAK6/7 group in TCGA-PDAC,a subgroup of TCGA-PAAD patients.Results:In 157 pancreatic cancer samples,36.3%(57/157)cases were found existing FAK6/7 variants,including 75.6%(34/45)in pNENs,47.5%(19/40)in SPNs and 2.9%(2/69)in PDACs.Significant differences of FAK6/7 ratio were found among 3 subtypes and pNEN and SPN had a higher FAK6/7 expression than that in PD AC(P<0.001).The overall survival time was longer in pNEN and SPN patients than in PD AC patients.No association was found between FAK6/7 status and other clinical factors.In TCGA-PAADs,27 of 178 patients were identified BOX6 and/or BOX7(BOX6/7)positive.The expression ratio of BOX6/7 was higher in neuroendocrine carcinoma(NEC)(87.5%)than in PD AC(11.9%,P<0.001).The expression ratio of BOX6/7 was higher in non-smokers than in smokers(P=0.028),while no association was found between BOX6/7 status and other clinical factors(all P>0.05).It is revealed that genes in regulation of synapse and neurotransmitter receptor are enriched in BOX6/7-containing group by GSEA.Among the detected pancreas cell lines,only QGP-1 harboured FAK6/7 splicing variants.Conclusions:This work for the first time reported the FAK6/7 expression pattern in pancreatic cancers with different histological subtypes,and found that the ratio of FAK6/7 is high in pNEN and SPN,low in PDAC,suggesting that FAK6/7 is a potential differential diagnosis and prognosis marker for pancreatic tumors.