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Protein Kinase C Is Highly Expressed in Gastrointestinal Stromal Tumors

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Protein Kinase C Is Highly Expressed in Gastrointestinal Stromal Tumors Vol. 10, 4089–4095, June 15, 2004 Clinical Cancer Research 4089 Protein Kinase C ␪ Is Highly Expressed in Gastrointestinal Stromal Tumors But Not in Other Mesenchymal Neoplasias Pilar Blay,1 Aurora Astudillo,2 Jose´M. Buesa,1 enchymal or epithelial tumors, including non-GIST KIT- Elı´as Campo,4 Mar Abad,5 Juan Garcı´a-Garcı´a,2 positive tumors. PKC-␪ immunoreactivity was also observed 4 6 1 in interstitial cells of Cajal. Rosa Miquel, Vicente Marco, Marta Sierra, ␪ 1 1 3 Conclusions: Our results show that PKC- is easily Raquel Losa, Angel Lacave, Alejandro Bran˜a, detected by immunohistochemistry in GIST specimens and 7 7 Milagros Balbı´n, and Jose´M. P. Freije that it could be a sensitive and specific marker for the 1Servicios de Oncologı´a Me´dica, 2Anatomı´a Patolo´gica, and diagnosis of this malignancy. 3Traumatologı´a, Instituto Universitario de Oncologı´a, Hospital Central de Asturias, Oviedo; 4Laboratorio de Patologı´a, Hospital Clinic, Institut d’Investigacions Biomediques August Pi i Sunyer, University of Barcelona, Barcelona; 5Departamento de Biologı´a Celular y INTRODUCTION Patologı´a, Universidad de Salamanca, Salamanca; 6Servicio de The term gastrointestinal stromal tumor (GIST) refers to a Anatomı´a Patolo´gica, Hospital General de Catalunya, Barcelona; and newly defined entity that is composed of a heterogeneous group 7 Departamento de Bioquı´mica y Biologı´a Molecular, Instituto of tumors of mesenchymal origin, most of them arising in the Universitario de Oncologı´a, Universidad de Oviedo, Oviedo, Spain gastrointestinal tract. GISTs were designated previously as leiomyoblastoma, plexosarcoma, gastrointestinal autonomic tu- ABSTRACT mor, or gastrointestinal pacemaker cell tumor, depending on its Purpose: Gastrointestinal stromal tumors (GIST) are a appearance. A general characteristic of these tumors is their distinctive group of mesenchymal neoplasms of the gastro- resistance to treatment with conventional chemotherapy. GISTs intestinal tract. The oncogene KIT has a central role in the are currently considered to be originated from interstitial cells of pathogenesis of GIST, with c-kit receptor tyrosine kinase Cajal (ICC) or from a common undifferentiated cell and are (KIT) protein expression being the gold standard in its immunohistochemically defined as c-kit receptor tyrosine ki- diagnosis. The identification of GIST patients has become nase (KIT)-positive tumors. Thus, KIT positivity serves to dif- crucial, because the tyrosine kinase inhibitor Imatinib is ferentiate GISTs from other gastrointestinal spindle cell tumors effective in the treatment of this malignancy. However, a (1, 2). small set of GISTs remain unrecognized, because KIT pro- A new drug, the protein kinase inhibitor Imatinib (STI571, tein expression is not always evident. The aim of this study Glivec), has been shown to be active against GISTs and is used was the identification of new markers for the differential currently in the clinic (3). This drug selectively inhibits the diagnosis of GIST. tyrosine signaling of a group of closely related tyrosine kinase Experimental Design: By analyzing publicly available receptors, including KIT, platelet-derived growth factor receptor data from transcriptional profiling of sarcomas, we found (PDGFR), and ABL (4). Most GISTs carry specific KIT muta- that protein kinase C ␪ (PKC-␪), a novel PKC isotype tions leading to ligand-independent activation of the KIT tyro- involved in T-cell activation, is highly and specifically ex- sine kinase activity, and it is now accepted that these mutations pressed in GIST. PKC-␪ expression in GIST was confirmed play a causal role in the development of this malignancy (5). by reverse transcription-PCR and Western blot. PKC-␪ was However, there are GISTs in which KIT protein expression is analyzed by immunohistochemistry in a panel of 26 GIST, not detectable, despite having activating KIT mutations (6). 12 non-GIST soft-tissue sarcomas, and 35 tumors from other Moreover, alternative mutations in the tyrosine kinase receptor ␣ histologies. PDGFR have been described recently (7). Thus, the existence Results: We found that all of the GISTs expressed of KIT-negative GISTs highlights the need of additional mark- PKC-␪, whereas this protein was undetectable in other mes- ers for the diagnosis of this neoplasia. Gene expression studies using DNA microarrays have re- vealed that GISTs show a distinct and uniform gene expression profile, which allows distinguishing of these tumors from other Received 3/31/04; accepted 4/6/04. malignancies of mesenchymal origin (8, 9). Among the tran- Grant support: Comisio´n Interministerial de Ciencia y Tecnologı´a- scripts identified as discriminatory in these studies, the gene Spain, Fundacio´n para el Desarrollo de la Oncologı´a, and European encoding protein kinase C ␪ (PKC-␪) was overexpressed in Union. The Instituto Universitario de Oncologı´a del Principado de GISTs samples. PKC-␪ is a novel isotype of PKC involved in Asturias is funded by Obra Social CajAstur, Asturias. The costs of publication of this article were defrayed in part by the T-cell activation (10, 11), in skeletal muscle signal transduction payment of page charges. This article must therefore be hereby marked (12), and in neuronal differentiation (13). This protein kinase advertisement in accordance with 18 U.S.C. Section 1734 solely to has raised interest as a potential drug target due to its role in indicate this fact. leukemia T-cell proliferation and survival (10, 11). Here we Requests for reprints: Jose´M. P. Freije, Departamento de Bioquı´mica ␪ y Biologı´a Molecular, 4.9, Universidad de Oviedo, Campus del Cristo, report that PKC- is expressed at the RNA and protein level in 33006 Oviedo, Spain. Phone: 34-985-106281; Fax: 34-985-103564; GISTs but not in other malignancies of epithelial or mesenchy- E-mail: [email protected]. mal origin, including KIT-positive tumors. PKC-␪ was easily Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2004 American Association for Cancer Research. 4090 Protein Kinase C ␪ in GIST Fig. 1 Microarray data on KIT and protein kinase C ␪ (PKC␪) expression levels in human sarcomas. Arbitrary expression units (Y axis) for KIT and PKC-␪ are shown for different sarcoma samples (X axis). A, cDNA microarray data from Allander et al. (8);8 the panel labeled as KIT corresponds to IMAGE clone 269806, whereas the two panels labeled as PKC-␪ correspond to IMAGE clones 205239 and 2164126. B, cDNA microarray data from Nielsen et al. (9).9 The panel labeled as KIT corresponds to the expression data for IMAGE clone 265060 and the panel labeled as PKC-␪ to IMAGE clone 205239. C, expression analysis with Affymetrix GeneChips, published by Segal et al. (14).10 The panel labeled as KIT represents the signals for the probe 1888_s_at, whereas the panels labeled as PKC-␪ represent the signals for probes 38949_at and 2081_s_at. All of the samples identified as gastrointestinal stromal tumor (GIST)inA and B express PKC-␪, but 1 GIST samples in B (sample 19) does not express KIT. All of the samples in C that express KIT also express PKC-␪. detected by immunohistochemistry in all of the samples from a were imported in Microsoft Excel spreadsheets to compose the panel of GISTs, suggesting its potential as a molecular marker graphs shown in Fig. 1. for the diagnosis of this tumor type. Immunoreactivity was also Tumor Samples. Formalin-fixed, paraffin-embedded tis- observed in ICCs, supporting the common lineage of these cell sue blocks and freshly frozen tumor samples were collected types. from the Hospital Central de Asturias, the Hospital Clı´nic de Barcelona, and the Hospital Universitario de Salamanca. Tu- MATERIALS AND METHODS mors included 26 GIST from different locations, 12 non-GIST Microarray Data. Microarray data on gene expression soft-tissue sarcomas, and 35 benign and malignant tumors from profiles of human sarcomas have been reported previously and different histologies (Table 1). GISTs were histologically clas- are publicly available (8, 9, 14).8,9,10 sified as predominantly spindled, epithelioid, or mixed, as well The relative hybridization values from the cDNA micro- as benign-appearing, borderline, or malignant based on cellu- arrays or the signal intensities from the oligonucleotide chip larity, pleomorphism, mitotic activity, necrosis, and growth pat- tern. Reverse Transcription-PCR. Total RNA from tumor samples and cell lines was isolated as described (15). One ␮g 8 each of total RNA was reverse-transcribed using the Termo- Internet address: http://research.nhgri.nih.gov/microarray/gist_data.txt. script Reverse Transcription-PCR kit (Life Technologies, Inc., 9 Internet address: http://genome-www.stanford.edu/sarcoma/data/ 74252filtered(SAM_GIST)final.xls/. Rockville, MD). PCR primers for PKC-␪ were 5Ј-TGGA- 10 Internet address: http://www.mskcc.org/genomic/ccsmsp. CAATCCCTTTTACCCACG (sense) and 5Ј-GTCTCTG- Downloaded from clincancerres.aacrjournals.org on September 24, 2021. © 2004 American Association for Cancer Research. Clinical Cancer Research 4091 Table 1 Histological and immunohistochemical features of the fetal bovine serum. The filters were washed three times for 5 tumors included in this study min each with PBS (pH 7.4) and 0.1% Tween 20 and then Tumor KITa PKC-␪ incubated for 1 h with a horseradish peroxidase-conjugated goat GIST (n ϭ 26) antimouse antibody at a 1:20,000 dilution (Pierce Biotechnol- Esophagus ϩϩ ogy, Rockford, IL). The secondary antibody was detected using Stomach (n ϭ 14) ϩϩ the PicoSignal chemiluminescent
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