1、APC-activated long noncoding RNA inhibitscolorectal carcinoma pathogenesis throughreduction of exosome productionFeng-Wei Wang,Rui-Hua Xu,Dan XieJ Clin Invest.2019;129(2):727-743.https:/doi.org/10.1172/JCI122478.The adenomatous polyposis coli(APC)gene plays a pivotal role in the pathogenesis ofcolor
2、ectal carcinoma(CRC)but remains a challenge for drug development.Long noncodingRNAs(lncRNAs)are invaluable in identifying cancer pathologies and providing therapeuticoptions for patients with cancer.Here,we identified a lncRNA(lncRNA-APC1)activated byAPC through lncRNA microarray screening and exami
3、ned its expression in a large cohort ofCRC tissues.A decrease in lncRNA-APC1 expression was positively associated withlymph node and/or distant metastasis,a more advanced clinical stage,as well as a poorprognosis for patients with CRC.Additionally,APC could enhance lncRNA-APC1expression by suppressi
4、ng the enrichment of PPARa on the lncRNA-APC1 promoter.Furthermore,enforced lncRNA-APC1 expression was sufficient to inhibit CRC cell growth,metastasis,and tumor angiogenesis by suppressing exosome production through the directbinding of Rab5b mRNA and a reduction of its stability.Importantly,exosom
5、es derived fromlncRNA-APC1silenced CRC cells promoted angiogenesis by activating the MAPKpathway in endothelial cells,and,moreover,exosomal Wnt1 largely enhanced CRC cellproliferation and migration through noncanonicial Wnt signaling.Collectively,lncRNA-APC1 is a critical lncRNA regulated by APC in
6、the pathogenesis of CRC.Our findingssuggest that an APC-regulated lncRNA-APC1 program is an exploitable therapeuticapproach for the treatment of patients with CRC.Research ArticleGastroenterologyOncologyFind the latest version:http:/jci.me/122478/pdfThe Journal of Clinical Investigation R ES E ARC H
7、 ARTICLE7 2 7jci.org Volume 129 Number 2 February 2019IntroductionAs one of the most common human malignancies,colorectal car-cinoma(CRC)is a leading cause of cancer-related deaths world-wide.It is well established that the pathogenesis of CRC follows the adenoma-carcinoma sequence and involves mult
8、istep tumor-igenesis through the progressive accumulation of abnormalities in both tumor suppressor genes and oncogenes(1,2).Mutations in the gene adenomatous polyposis coli(APC)play a pivotal role in tumorigenesis and progression of CRC(3).To date,targeting certain oncogenes and their related pathw
9、ays represents the best option for cancer treatment and improving the survival of patients at advanced stages of the disease.However,because it is a large scaffold protein with multiple functions,APC remains a challenge to target for translation into drug development.Long noncoding RNAs(lncRNAs)are
10、a large class of tran-scripts longer than 200 bases,with no protein-coding potential(4).Current research indicates that lncRNAs are exquisitely reg-ulated and that they can control gene expression to regulate var-ious aspects of biological and/or pathological processes(5,6).It has been shown that ln
11、cRNAs modulate several important cancer phenotypes,including cellular proliferation,apoptosis,immor-tality,motility,as well as angiogenesis(7,8).As a result,it is now widely understood that lncRNAs are invaluable in their ability to identify cancer pathologies as well as to provide other prognos-tic
12、 value,or even inform therapeutic options for cancer patients.Despite this knowledge,the functions of certain lncRNAs involved in mediating the anticancer role of APC in CRC and any abnor-malities they might possess have yet to be elucidated.Here,we used lncRNA microarray screening to identify a lnc
13、RNA(TCONS_00027227)activated by APC through PPAR,which we named lncRNA-APC1.Examination and function anal-ysis of CRC tissues collected from a large patient cohort showed that lncRNA-APC1 plays a crucial tumor-suppressive role in the pathogenesis of CRC.Further mechanistic studies revealed that lncR
14、NA-APC1 exerts its effects through the direct binding of Rab5b mRNA,thereby reducing its stability and ultimately leading to decreased exosome production.This action inhibits the over-activation of the MAPK pathway in endothelial cells and the sub-sequent suppression of angiogenesis.Importantly,we r
15、eveal for the first time to our knowledge an oncogenic role of CRC-derived exosomal Wnt1,which acts in an autocrine manner through non-The adenomatous polyposis coli(APC)gene plays a pivotal role in the pathogenesis of colorectal carcinoma(CRC)but remains a challenge for drug development.Long noncod
16、ing RNAs(lncRNAs)are invaluable in identifying cancer pathologies and providing therapeutic options for patients with cancer.Here,we identified a lncRNA(lncRNA-APC1)activated by APC through lncRNA microarray screening and examined its expression in a large cohort of CRC tissues.A decrease in lncRNA-
17、APC1 expression was positively associated with lymph node and/or distant metastasis,a more advanced clinical stage,as well as a poor prognosis for patients with CRC.Additionally,APC could enhance lncRNA-APC1 expression by suppressing the enrichment of PPAR on the lncRNA-APC1 promoter.Furthermore,enf
18、orced lncRNA-APC1 expression was sufficient to inhibit CRC cell growth,metastasis,and tumor angiogenesis by suppressing exosome production through the direct binding of Rab5b mRNA and a reduction of its stability.Importantly,exosomes derived from lncRNA-APC1silenced CRC cells promoted angiogenesis b
19、y activating the MAPK pathway in endothelial cells,and,moreover,exosomal Wnt1 largely enhanced CRC cell proliferation and migration through noncanonicial Wnt signaling.Collectively,lncRNA-APC1 is a critical lncRNA regulated by APC in the pathogenesis of CRC.Our findings suggest that an APC-regulated
20、 lncRNA-APC1 program is an exploitable therapeutic approach for the treatment of patients with CRC.APC-activated long noncoding RNA inhibits colorectal carcinoma pathogenesis through reduction of exosome productionFeng-Wei Wang,1 Chen-Hui Cao,1 Kai Han,1 Yong-Xiang Zhao,2 Mu-Yan Cai,1 Zhi-Cheng Xian
21、g,1 Jia-Xing Zhang,3 Jie-Wei Chen,1,4 Li-Ping Zhong,2 Yong Huang,2 Su-Fang Zhou,2 Xiao-Han Jin,1 Xin-Yuan Guan,5 Rui-Hua Xu,1 and Dan Xie1,2,41Sun Yat-sen University Cancer Center,State Key Laboratory of Oncology in South China,Collaborative Innovation Center for Cancer Medicine,Guangzhou,China.2Nat
22、ional Center for International Research of Biological Targeting Diagnosis and Therapy,Guangxi Key Laboratory of Biological Targeting Diagnosis and Therapy Research,Collaborative Innovation Center for Targeting Tumor Diagnosis and Therapy,Guangxi Medical University,Nanning,Guangxi,China.3Department o
23、f Oncology,The First Affiliated Hospital,Sun Yat-sen University,Guangzhou,China.4Department of Pathology,Sun Yat-sen University Cancer Center,Guangzhou,China.5Department of Clinical Oncology,The University of Hong Kong,Hong Kong,China.Related Commentary:p.503Authorship note:FWW,CHC,KH,YXZ,and MYC co
24、ntributed equally to this work.Conflict of interest:The authors have declared that no conflict of interest exists.License:Copyright 2019,American Society for Clinical Investigation.Submitted:May 25,2018;Accepted:November 27,2018.Reference information:J Clin Invest.2019;129(2):727743.https:/doi.org/1
25、0.1172/JCI122478.The Journal of Clinical Investigation R ESE A RCH A RTI CL E72 8jci.org Volume 129 Number 2 February 2019Using the 5 and 3 rapid amplification of cDNA ends(RACE)assay,we discovered that lncRNA-APC1 was a 1580-nt inter-gene transcript and poly(A)positive.The sequence of full-length l
26、ncRNA-APC1 is presented in Supplemental Figure 1,A and C(sup-plemental material available online with this article;https:/doi.org/10.1172/JCI122478DS1).Northern blot analysis confirmed the size of lncRNA-APC1 in the CRC cell lines(Supplemental Fig-ure 1B).Further analysis of the sequences using the
27、NCBIs Nation-al Center for Biotechnology Information ORFfinder(https:/www.ncbi.nlm.nih.gov/orffinder/)failed to predict a protein of more than 55 aa.Additionally,we calculated its coding potential using the Coding Potential Calculator(CPC)(http:/ Wnt signaling.Collectively,our data uncovered an APC
28、signaling mechanism,APC/PPAR/lncRNA-APC1/Rab5b,in the pathogenic process of CRC and revealed the potential for several prognostic and/or therapeutic targets for human CRC.ResultslncRNA-APC1 is upregulated by APC in CRCs.Inactivated mutations in the APC gene are the initiating mutation driving CRC tu
29、morigen-esis and/or progression(3).In this study,we sought to investigate the abnormal dynamics and underlying roles of certain lncRNAs that are involved in this process and applied a lncRNA microarray technique to select and identify which lncRNAs were regulated by APC in CRC cells.We first reinduc
30、ed WT APC full-length coding sequence(CDS)into the SW480 and DLD-1 human CRC cell lines(Figure 1,A and B),both of which express an endogenous truncated APC protein(mutated at aa 1338 and 1427,respectively)that con-stitutively activates-catenin/T cell factor 4mediated(-catenin/TCF4mediated)transcript
31、ion.The 2 cell lines were examined in 2 independently repeated microarray tests.We found that 3 lncRNAs were upregulated and 2 lncRNAs were downregulated by more than 2-fold and that these events were induced after ectopic overexpres-sion of WT APC in both lines(Figure 1C and Table 1).Among these,TC
32、ONS_00027227,which we named lncRNA-APC1,is encoded by a gene at chromosome 19p12 and was consistently upregulated by more than 17-fold,as confirmed by quantitative reverse transcrip-tion PCR(qRT-PCR)(Figure 1D).Table 1.lncRNAs regulated by ectopic APC expression in both SW480 and DLD-1 cell linesFol
33、d ChangeTranscript IDGene name2.3660802DownENST00000538380AC091878.13.5207261DownTCONS_00013163XLOC_0064323.5765904UpENST00000465880RP11-80H8.42.5030724UpENST00000581029RP11-838N2.46.7291048UpTCONS_00027227XLOC_013265Transcript IDs and gene names can be found in the LNCipedia database(https:/lnciped
34、ia.org/).Figure 1.Upregulation of lncRNA-APC1 by APC.Expression of APC in the indicated cell lines transfected with control or WT APC vector,as measured by qRT-PCR(A)and Western blotting(B).(C)Number of altered lncRNAs in the indicated cells examined in 2 inde-pendently repeated lncRNA microarray te
35、sts.(D)qRT-PCR verification of lncRNAs potentially regulated by APC.(E)Expression of lncRNA-APC1 was detected by FISH.Scale bars:20 m.(F)Relative expression of lncRNA-APC1 in paired CRC primary tumor tissues and nontumor colonic tissues(n=30).(G)Kaplan-Meier survival analysis of patients with CRC(n=
36、110)according to lncRNA-APC1 expression(cutoff value is the median).Experiments in F and G were repeated twice with similar results.Data in A,E,and F represent the mean SD of 3 separate experiments.*P 0.01,*P 0.001,and*P 0.0001,by independent Students t test(A and F)or log-rank test(G).NC,negative c
37、ontrol.The Journal of Clinical Investigation R ES E ARC H ARTICLE7 29jci.org Volume 129 Number 2 February 2019els of lncRNA-APC1(64.3 months,95%CI:60.168.7;P 0.001,log-rank test)(Figure 1G and Table 3).These results indicated that a decrease in lncRNA-APC1,which is downstream of APC,could play an im
38、portant oncogenic role in regulating CRC progression.lncRNA-APC1 is regulated by APC through PPAR in CRC cells.Previous studies have found that truncated APC in CRC cells contributes to tumor cell migration via interaction with the Rac-specific guanine nucleo-tide exchange factor Asef(9,10).Hence,we
39、 further examined whether mutant APC affects lncRNA-APC1 expression.Two common mutant APC plasmids(gifts of Bert Vogelstein,Johns Hopkins University,Baltimore,Maryland,USA),shown in Supplemental Figure 2A,were transfected into the CRC cell line HCT116(WT APC)(11).qRT-PCR and immunoblot analysis show
40、ed that the 2 APC plasmids produced comparable amounts of APC mRNA and protein(Supplemental Figure 2,B and C).Surprisingly,we found that mutant APC331 had no effect on lncRNA-APC1 expression and that APC1309-mutant overexpression slightly suppressed lncRNA-APC1 expression(Supplemental Figure 2D).In
41、addition,we transfected siRNAs specific for APC into SW480 and DLD-1 cells and observed similar results(Supplemental Figure 2,E and F).These results suggest that WT but not truncated APC is mainly responsible for regulating lncRNA-APC1 expression.It is well accepted that regulating-catenin sta-biliz
42、ation is the most prominent function of APC(12).Therefore,we tested whether-catenin could regulate lncRNA-APC1 expression in CRC.Our results showed that inhibition of-catenin by a specific siRNA resulted in only slightly elevated expression of lncRNA-APC1 in the CRC cell lines HCT116(WT APC,-catenin
43、 mutant)and DLD-1(Sup-plemental Figure 3,A and B).We consistently found that neither and the Coding Potential Assessment Tool(CPAT)(http:/ CPC(using ORF_ FRAME FINDER)predicted a lncRNA-APC1 score of 36.13,and the CPAT predicted a coding probability of 0.008,further supporting the notion that lncRNA
44、-APC1 has no protein-cod-ing potential.Moreover,FISH analysis showed that lncRNA-APC1 was primarily located in the cytoplasm(Figure 1E).Subsequent qRT-PCR analysis in our study revealed that expression of lncRNA-APC1 was significantly lower in CRC tissues than that in the 30 corresponding samples of
45、 nontumor colorectal tissues(Figure 1F).Fur-thermore,we measured the expression lev-els of lncRNA-APC1 in CRC tissues from 110 patients,and our correlation analysis revealed that low expression levels of lncRNA-APC1 were positively correlated with lymph node and/or distant metastasis of CRC as well
46、as with a more advanced clinical stage(P 60A512625 605928310.712Sex Female532726 Male5727300.708Tumor location Colon663234 Rectum4422220.876Histological grade(WHO)G12874542 G3239140.283pT status T1T229218 T3T48133480.003Clinical stage I+II 421527 III+IV6839290.027Lymph node status No metastasis42152
47、7 Metastasis6839290.027CEA level 5(g/l)4121200.731AMean age;B2 test.CEA,carcinoembryonic antigen;pN,pathological staging of lymph node;pT,pathological staging of tumor.Table 3.Univariate and multivariate Cox regression analysis of different prognostic variables for patients with CRCVariableSubsetHR
48、for DSS(95%CI)P valueUnivariate analysis(n=110)Age(yr)60A vs.601.470(0.7173.014)0.293 SexMale vs.female0.712(0.3431.479)0.363 Tumor locationColon vs.rectum0.598(0.2741.307)0.198 Histological grade(WHO)G12 vs.G32.907(1.3816.116)0.005 Clinical stageI+II vs.III+IV2.648(1.2775.490)0.009 pT statusT1+2 vs
49、.T3+44.174(1.25813.845)0.020 pN statusN0 vs.N12.648(1.2775.490)0.009 CEA level5(g/l)1.615(0.7883.311)0.190 lncRNA-APC1 expression levelLow vs.high expression0.198(0.0810.487)0.001Multivariate analysis(n=110)Histological grade(WHO)G12 vs.G32.319(1.0844.959)0.030 pT statusT1+2 vs.T3+42.046(0.5817.208)
50、0.265 pN statusN0 vs.N11.764(0.8333.733)0.138 lncRNA-APC1 expression levelLow vs.high expression0.253(0.1010.632)0.003AMean age.DSS,disease specific survival.The Journal of Clinical Investigation R ESE A RCH A RTI CL E730jci.org Volume 129 Number 2 February 2019,which encode proteins sharing a highl