KIT MutationsInduceIntracellularRetentionandActivationofan ImmatureFormoftheKITProteininGastrointestinalStromalTumors Se¤verineTabone-Eglinger, 1 Fre¤ de¤ ricSubra, 5 Hiba El Sayadi, 1 LaurentAlberti, 1 Eric Tabone, 2 Jean-Philippe Michot, 2 NathalieThe¤ou-Anton, 4 Antoinette Lemoine, 4 Jean-Yves Blay, 1,3 andJean-Franc ¸ ois Emile 4,6,7 Abstract Purpose: Gastrointestinalstromaltumors(GIST)arefrequentlyassociatedwithgain-of-function mutations of KIT, which can be inhibited by imatinib both in vitro and in vivo. The survival of patients with GIST, following imatinib therapy, has been correlated with the nature of mutations but not with KITexpression. ExperimentalDesign: Subcellular localization, activation, and trafficking of the mature and the immature forms of KITwere investigated in GISTsamples and in NIH3T3 cells infected with two differentGIST-typeexon11^mutatedhuman KIT cDNA. Results: Paranuclear dot expression of KITwas more frequent in GISTs with homozygous KIT mutations than in those with heterozygous ( P = 0.01) or no mutations (P < 0.01). Activationof theimmature125kDaformofKITwasdetectedinmostGISTswith KIT mutationsbutnotinGISTs without KIT mutations. In NIH3T3 cells, mutant KITwas mainly retained within endoplasmic reticulum and Golgi compartments in an immature constitutively phosphorylated form, whereas the wild-type KITwas expressed at the plasma membrane, in a mature nonphosphorylated form. Imatinib-induced inhibition of the phosphorylation of immature and mature mutant KIT proteins resultedinthe restorationof KITexpressionat the cell surface. Conclusions: Theseresults show thatGIST-type KIT mutations induce anactivation-dependent alterationofnormalmaturationandtrafficking,resultingintheintracellularretentionoftheactivated kinase within the cell.These observations likely account for the absence of correlation between response to imatinib and KITexpression using immunohistochemistry and may deserve to be investigatedinother tyrosine kinase^activated tumors. Gastrointestinal stromal tumors (GIST) are the most frequent sarcomas of the digestive tract and express KIT protein in >95% of cases (1). Gain-of-function mutations in the KIT gene, and more recently, in the PDGFRA gene, have been shown to play a critical oncogenic role in GISTs (2–4). Imatinib mesylate, a KIT and PDGFR tyrosine kinase inhibitor, blocks the activation of both receptors in vitro and induces tumor control in >90% of patients with GIST (5–8). More than 80% of KIT mutations occur in exon 11 with a hotspot in the 550 to 561 codon region, whereas mutations in exon 9 and in exons 13 to 17 are observed in 10% to 15% and <1% of metastatic tumors, respectively (5, 9–13). Correlations between the nature of mutation (exon 11 versus others and sites of mutations within exon 11) and disease outcome prior to imatinib therapy has been reported in some but not all studies (9–12, 14–20). Conversely, response to imatinib therapy has consistently been influenced by the nature of KIT mutations: patients with exon 11 mutations were reported to have a better survival with imatinib than exon 9 or wild-type (WT) forms (5, 6). Of note, deletions involving the distal part of KIT exon 11 may be less favorable for response to imatinib (21). An intriguing observation is that immunohistochemical expression of the KIT protein in KIT-positive GISTs is not correlated with imatinib activity in vivo (22), in contrast with observations in other models such as HER2 amplification in breast cancer (23). In the present work, we observed that KIT mutations result in the modification of the protein subcellular localization. In GISTs, as well as in NIH3T3-infected cells, mutated forms of KIT were mainly expressed in an immature phosphorylated- activated form within the intracellular compartments and with limited expression at the cell surface in this model. Activation Human Cancer Biology Authors’Affiliations: 1 Equipe Cytokines et Cancers, INSERMU590 and 2 Service d’Anatomie Pathologique, Centre Le¤on Be¤rard, 3 Universite¤ Claude Bernard Lyon1 and Ho“ pital Edouard Herriot, Lyon, France, 4 INSERMU602, Ho“ pital Paul Brousse, Villejuif, France, 5 ENS Cachan, LBPA, UMR 8113, Cachan, France, 6 Ho“pital Ambroise Pare¤ APHP, Boulogne, France, and 7 PRES Universud and UVSQ, Versailles,France Received9/5/07;revised11/5/07;accepted11/5/07. Grantsupport: S.Tabone-EglingerisafellowofANRT,withfinancialsupportfrom Centre Le¤on Be¤rard Cancer and Novartis SA. J-Y. Blay andJ-F. Emile received research grants from Novartis. Novartis also kindly provided imatinib mesylate. Association pour la Recherche sur le Cancer, CONTICANET, Networkof Excellence FP6-018806, Ligue Contre le Cancer (Comite¤ de l’Ain et Comite¤ du Rho“ ne), the Breast Cancer Research Foundation, Institut National du Cancer, Cance¤ropo“le LyonAuvergneRho“ ne Alpes,andBQR2007Universite¤ VersaillesSQY. Thecostsofpublicationofthisarticleweredefrayedinpartbythepaymentofpage charges.This article must therefore be hereby marked advertisement in accordance with18U.S.C.Section1734solely toindicatethisfact. Note: J-Y. Blay andJ-F. Emile contributedequally to this work. Requestsforreprints: Jean-Yves Blay, Centre Le¤ on Be¤ rard, Equipe Cytokines et Cancers, 28 rue Lae« nnec, 69373 Lyon cedex 08, France. Phone: 33-47878-7138; Fax: 33-47878-2720;E-mail:blay@lyon.fnclcc.fr. F 2008AmericanAssociationforCancerResearch. doi:10.1158/1078-0432.CCR-07-4102 www.aacrjournals.org ClinCancerRes2008;14(8)April15,2008 2285 Downloaded from http://aacrjournals.org/clincancerres/article-pdf/14/8/2285/1981052/2285.pdf by guest on 21 June 2022