KIT Mutations Induce Intracellular Retention and Activation of an Immature Form of the KIT Protein in Gastrointestinal Stromal Tumors

Abstract

Purpose: Gastrointestinal stromal tumors (GIST) are frequently associated with gain-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. Experimental Design: Subcellular localization, activation, and trafficking of the mature and the immature forms of KIT were investigated in GIST samples and in NIH3T3 cells infected with two different GIST-type exon 11^mutated human KIT cDNA. Results: Paranuclear dot expression of KIT was more frequent in GISTs with homozygous KIT mutations than in those with heterozygous (P = 0.01) or no mutations (P < 0.01). Activation of the immature 125 kDa form of KITwas detected in most GISTs with KIT mutations but not in GISTs without KIT mutations. In NIH3T3 cells, mutant KIT was mainly retained within endoplasmic reticulum and Golgi compartments in an immature constitutively phosphorylated form, whereas the wild-type KIT was expressed at the plasma membrane, in a mature nonphosphorylated form. Imatinib-induced inhibition of the phosphorylation of immature and mature mutant KIT proteins resulted in the restoration of KITexpression at the cell surface. Conclusions: These results show that GIST-type KIT mutations induce an activation-dependent alteration of normal maturation and trafficking, resulting in the intracellular retention of the activated kinase within the cell. These observations likely account for the absence of correlation between response to imatinib and KIT expression using immunohistochemistry and may deserve to be investigated in other tyrosine kinase^activated tumors.

Distinct cellular properties of oncogenic KIT receptor tyrosine kinase mutants enable alternative courses of cancer cell inhibition

“…2 A and B, the presence of WT KIT and the N505I mutant on the cell surface is higher than that of T417IΔ418-419 and Dup A502Y503 mutants. The presence of the cytoplasmic domain mutants V560D, D816V, and V560DY823D on the cell surface was very low, in accordance with previous reports (18,19). We also used fluorescence microscopy to visualize the cellular distribution of a green fluorescent protein (GFP) fused to WT or each oncogenic KIT mutant expressed in COS7 cells.…”

“…We and others (18,19) have shown that glycosylated WT KIT migrates on SDS/PAGE as two distinct bands with apparent molecular masses of 145 kDa and 125 kDa ( Fig. 2 C and D).…”

“…Analysis of cellular localization using fluorescence microscopy showed that, unlike WT KIT, which is mostly confined to the cell membrane, oncogenic KIT with mutations in the TKD or JM domains is primarily localized inside the cell (18,19). Because anti-RTK antibodies that have been successfully applied in targeted therapy of cancers (20) exert their inhibitory activity by binding receptor molecules that are located on the cell surface, we compared the cellular localization and dynamic properties of the most common recurrent oncogenic KIT mutations in human cancers.…”

Les tumeurs stromales gastro-intestinales (GIST)

“…Ainsi, nous avons observé que des formes mutées de KIT ont un devenir biochimique très différent. En effet, elles existent majoritairement à l'état immature (non ou peu glycosylées), sont principalement localisées dans le réticulum endoplasmique ou l'appareil de Golgi, mais sont peu exprimées à la surface cellulaire [30]. De plus, les formes immatures de KIT peuvent être phosphorylées et transmettre un signal d'activation cellulaire ( Figure 5).…”

The role of high‐dose imatinib in the management of patients with gastrointestinal stromal tumor

“…12 KIT mutations have been detected principally (66% to 71%) in gene portions encoding for the juxtamembrane region, most often exon 11, followed by the fifth extracellular immunoglobulin-like region encoded by exon 9 (13%), and less frequently in the tyrosine kinase domain 1 encoded by exon 13 (1% to 4%) and the phosphotransferase domain encoded by exon 17 (>1% to 4%).…”