ANGELONI, DEBORA

BRAGA E

DEAN M

DUH FM

LERMAN MI

MOODY M

SENTCHENKO V

ZABAROVSKY ER

Archivio della ricerca della Scuola Superiore Sant'Anna

C to A single nucleotide polymorphism in intron 18 of the humanMST1R (RON) gene that maps at 3p21.3Debora Angelonia,*, Fuh-Mei Duhb, Michele Moodya, Michael Deanc, Eugene R. Zabarovskyd,Vera Sentchenkod, Eleonora Bragad, Michael I. LermanaaLaboratory of Immunobiology, National Cancer Institute at Frederick, Frederick, MD 21702-1201, USAbIntramural Research Support Program, SAIC, National Cancer Institute at Frederick, Frederick, MD 21702-1201, USAcLaboratory of Genomic Diversity, National Cancer Institute at Frederick, Frederick, MD 21702-1201, USAdMTC and CGR, Karolinska Institute, Stockholm 17177, SwedenReceived 6 May 2002; accepted for publication 4 November 2002AbstractThe MST1R (RON) gene, that maps at 3p21.3, encodes a protein tyrosine kinase receptor comprised of an extra-cellular domain thatcontains the ligand binding pocket and an intracellular region where the kinase domain is located. It controls cell survival and motilityprograms related to invasive growth. With the single strand conformation polymorphism (SSCP) method, a C to A nucleotide polymorphism(SNP) was found in intron 18 of the gene. The SNP has a frequency of 0.28 among African–American, 0.25 among Caucasian CEPH and0.09 among Asian healthy individuals. During these studies, an alternatively spliced cDNA of MST1R, lacking exon 19, was also found thatmay result from this change.q 2003 Elsevier Science Ltd. All rights reserved.Keywords: Alternative splicing; Breast cancer; MST1R (RON); Center d’Etude du Polymorphisme Humain (CEPH); Single nucleotide polymorphism (SNP);Single strand conformation polymorphism (SSCP)1. IntroductionThe macrophage stimulating 1 receptor, MST1R (RON,OMIM; 600168) gene maps at 3p21.3, a region critical forbreast and lung cancer [1]. MST1R protein signaling can beabnormally activated through several mechanisms.The experimental introduction of tumorigenic pointmutations found in MET and KIT activates the oncogenicpotential of RON [2]. The mouse and avian orthologs ofRON (stk and sea, respectively) are involved in oncogenesisthrough the participation of viral component [3,4].Rare splicing variants found in gastric carcinomas andcell lines lead to aberrant dimerization and constitutiveactivation of RON [5].However, so far RON has been found to be involved innaturally occurring human tumors through over-expression.This is the case of breast and colon cancer. In normal breastepithelium and benign lesions, the protein is expressed ata very low level but the expression level increasessignificantly in infiltrating carcinomas [6]. Similarly, coloncarcinoma cell lines show a high level of expression andconstitutive activation of RON whereas normal epithelialcolon cells do not [7,8]. Moreover, Ron was found highlyexpressed also in a number of non-small cell lung cancer [9]and activated in bronchioalveolar carcinomas (Danilkovitchet al., personal communication).Using the single strand conformation polymorphism(SSCP) method we performed mutation analysis of severalbreast, renal and lung cancer samples in which the RONgene and the surrounding genomic region were foundamplified (Zabarowsky et al., manuscript in preparation).During these studies a new single nucleotide polymorphism(SNP) was found: a C to A transversion that occurs 10 bpupstream of the acceptor splice site of intron 18 (GenBankentry: AF164652). The SNP frequency was evaluated inthree different ethnic groups. An alternatively spliced cDNA0890-8508/03/$ - see front matter q 2003 Elsevier Science Ltd. All rights reserved.doi:10.1016/S0890-8508(02)00115-9Molecular and Cellular Probes 17 (2003) 55–57www.elsevier.com/locate/ymcpr* Corresponding author. Scuola Superlore Sant’Anna, Piazza Martiridella Liberta, 33-56100 Pisa, Italy. Tel.: þ1-301-846-7328; fax: þ1-301-846-6145.E-mail address: angeloni@sssup.it (D. Angeloni).lacking exon 19 was also found, along with the wild typecDNA, in a lung cancer cell line with the polymorphism.2. Materials and methods2.1. Human DNASigned, informed consent was obtained from allparticipants in the study, according to the NCI institutionalreview board-approved protocol.2.2. Cell linesLung cancer cell lines NCI-H358, NCI-H1650,NCI-H1666 and NCI-H1781 were obtained from ATCC(Manassas, VA).2.3. PCR primersPrimers were synthesized by ABI Applied Biosystems(Frederick, MD).in 18 Fw: 50 TTC CAG CTG AAG GAC TCT GGin 19 Rv: 50 ACT ACC ACC TCC ACA TAC TCex 18 Fw: 50 ATC CTG GAC AGG GAG TACex5 Rv: 50 TCC CCA AGC AGT GCA GAC ACT ATCTG2.4. PCR–SSCP analysisThe radioactive labeling reaction was performed in atotal reaction volume of 12.5 ml, containing 100 ng ofgenomic DNA, 12.5 pmol of each primer (in 18-Fw, in19-Rv), 200 mM dNTPs, 1.5 mM MgCl2, 1.25 nCia35S-dATP. Primers amplified a single band (255 bp)under the following cycling conditions: 3 min at 95 8C;(30 s at 95 8C, 30 s at 60 8C, 30 s at 72 8C) for 35 cycles;7 min extension at 72 8C. After heat denaturation (8 min at90 8C) in formamide buffer (Stop Solution, Amersham,Arlington Heights, IL), PCR products were run overnight ina 0.5 £ MDE gel (FMC Bioproducts, Rockland, ME),0.6 £ TBE, at room temperature, 8 W constant power;transferred on 3 mm paper, dried and exposed to auto-radiography film (X-OMAT AR, Kodak, Rochester, NY).2.5. RT-PCRFirst strand cDNAs were reverse transcribed fromrandom primed total RNA isolated from cell lines above(cDNA Cycle Kit, Invitrogen, Carslbad, CA). MST1RcDNA was amplified with the primers ex18-Fw, ex5-Rv(PCR product: 356 bp) under the following conditions:Table 1Frequency of the SNP in three different ethnic groupsEthnic group Genotype Frequency of allele ACC CA AAAfrican–American 15 5 1 0.28Asian 18 2 0 0.09Caucasian (CEPH) 19 8 0 0.25Fig. 1. SSCP analysis of the CEPH Family 1362 [10] shows Mendelianinheritance of the C to A SNP located in intron 18 (GenBank entry:AF164652) of the MST1R gene. (A) pedigree of Family 1362. (B) SSCPprofile of allele ‘C’ and allele ‘A’ and corresponding sequence ofsubcloning products.Fig. 2. Alternative splicing of exon 19 is present along with wildtypesplicing in the SNP-carrier cell line H1650. (A) SSCP analysis of gDNA.(1) H1650 (‘AA’), (2) NCI-H358, (3) NCI-H1781, (4) NCI-H1666 (all‘CC’). (B) Sequence of the alternatively spliced cDNA. The arrow points tothe splice junction between exons 18 and 20.D. Angeloni et al. / Molecular and Cellular Probes 17 (2003) 55–57563 min at 95 8C; (30 s at 95 8C, 30 s at 62 8C, 30 s at 72 8C)for 35 cycles; 7 min extension at 72 8C.2.6. SequencingPCR products were cloned into the pCR2.1-TOPO vector(TA Cloning Kit, Invitrogen, Carslbad, CA) and severalsingle colonies were isolated and sequenced.Sequencing reactions were performed on an ABI 373Stretch Automated DNA Sequencer (Applied Biosystems,Foster City, CA).3. Results and discussionA C to A transversion was found 10 bp upstream theacceptor splice site of the MST1R gene intron 18(nucleotide 414 GenBank entry AF164652), it introducesa new XcmI site at base 92 of the 255 bp PCR product(Section 2). Three different ethnic groups were analyzed toestablish the frequency of allele A in the general healthypopulation (Table 1). One carrier family (CEPH Family1362 [10]) was analyzed to prove Mendelian inheritance ofthe genotypic trait (Fig. 1).To investigate a possible effect of the SNP on the splicingprocess, we analyzed MST1R cDNA from cell lineshomozygous for the ‘A’ (NCI-H1650), ‘C’ (NCI-H358,NCI-H1781 and NCI-H1666) alleles (Fig. 2A). Amplifica-tion of NCI-H1650 cDNA with PCR primers designedacross exon 19 (ex18Fw, ex5Rv) yielded two bands (data notshown): the expected 356 bp product with wildtypesequence and a less abundant, alternatively spliced 219 bpproduct, lacking exon 19 (Fig. 2B). Under the conditionsused the alternatively spliced cDNA was not amplified fromcell lines with the C allele.Because this polymorphism affects exon 19, a part of theMST1R kinase domain, we plan to test the hypothesis that itmay lead to a predisposition for cancer.AcknowledgementsThis project has been funded in toto with funds from theNational Cancer Institute, National Institutes of Health,under Contract No. NO1-CO-56000 and NO1-CO-12400.The content of the publication does not necessarily reflectthe views or policies of the Department of Health andHuman Services, nor does mention of trade names,commercial products, or organizations imply endorsementby the US Government. The Swedish group was supportedby research grants from the Swedish Cancer Society, theSwedish Research Council, STINT, Pharmacia Corporationand the Karolinska Institute.References[1] Lerman MI, Minna JD. The 630-kb lung cancer homozygous deletionregion on human chromosome 3p21.3: identification and evaluation ofthe resident candidate tumor suppressor genes. The International LungCancer Chromosome 3p21.3 Tumor Suppressor Gene Consortium.Cancer Res 2000;60:6116–33.[2] Santoro MM, Penengo L, Minetto M, Orecchia S, Cilli M, Gaudino G.Point mutations in the tyrosine kinase domain release the oncogenicand metastatic potential of the RON receptor. Oncogene 1998;17:741–9.[3] Persons DA, Paulson RF, Loyd MR, Herley MT, Bodner SM,Bernstein A, Correll PH, Ney PA. Fv2 encodes a truncated form of theStk receptor tyrosine kinase. Nat Genet 1999;23:159–65.[4] Smith DR, Vogt PK, Hayman MJ. The v-sea oncogene of avianerythroblastosis retrovirus S13: another member of the protein-tyrosine kinase gene family. Proc Natl Acad Sci USA 1989;86:5291–5.[5] Collesi C, Santoro MM, Gaudino G, Comoglio PM. A Splicing variantof the RON transcript induces constitutive tyrosine kinase activity andan invasive phenotype. Mol Cell Biol 1996;16:5518–26.[6] Maggiora P, Marchio S, Stella MC, Giai M, Belfiore A, De Bortoli M,Di Renzo MF, Costantino A, Sismondi P, Comoglio PM. Over-expression of the RON gene in human breast carcinoma. Oncogene1998;16:2927–33.[7] Okino T, Egami H, Ohmachi H, Takai E, Tamori Y, Nakagawa K,Nakano S, Akagi J, Sakamoto O, Suda T, Ogawa M. Presence of RONreceptor tyrosine kinase and its splicing variant in malignant andnon-malignant human colonic mucosa. Int J Oncol 1999;15:709–14.[8] Chen YQ, Zhou YQ, Angeloni D, Kurtz AL, Qiang XZ, Wang MH.Overexpression and activation of the RON receptor tyrosine kinase ina panel of human colorectal carcinoma cell lines. Exp Cell Res 2000;261:229–38.[9] Willett CG, Wang MH, Emanuel RL, Graham SA, Smith DI,Shridhar V, Sugarbaker DJ, Sunday ME. Macrophage-stimulatingprotein and its receptor in non-small-cell lung tumors: induction ofreceptor tyrosine phosphorylation and cell migration. Am J RespirCell Mol Biol 1998;18:489–96.[10] Dausset J, Cann H, Cohen D, Lathrop M, Lalouel JM, White R.Centre d’etude du polymorphisme humain (CEPH): collaborativegenetic mapping of the human genome. Genomics 1990;6:575–7.D. Angeloni et al. / Molecular and Cellular Probes 17 (2003) 55–57 57

C to A Single Nucleotide Polymorphism in Intron 18 of the Human MST1R (RON) Gene that Maps at 3p21.3.

C to A Single Nucleotide Polymorphism in Intron 18 of the Human MST1R (RON) Gene that Maps at 3p21.3.

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Archivio della ricerca della Scuola Superiore Sant'Anna