Drug Resistance of Human Glioblastoma Cells Conferred by A
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Proc. Natl. Acad. Sci. USA Vol. 95, pp. 5724–5729, May 1998 Medical Sciences Drug resistance of human glioblastoma cells conferred by a tumor-specific mutant epidermal growth factor receptor through modulation of Bcl-XL and caspase-3-like proteases \ MOTOO NAGANE*, ALEXANDER LEVITZKI†,AVIV GAZIT†,WEBSTER K. CAVENEE*‡§¶, AND H.-J. SU HUANG*‡ *Ludwig Institute for Cancer Research, ‡Department of Medicine, §Center for Molecular Genetics, and ¶Cancer Center, University of California at San Diego, La Jolla, CA 92093-0660; and †Department of Biological Chemistry, The Institute of Life Sciences, The Hebrew University of Jerusalem, Jerusalem 91904, Israel Contributed by Webster K. Cavenee, March 5, 1998 ABSTRACT Alterations of the epidermal growth factor The malignant progression of gliomas involves accumulation receptor (EGFR) gene occur frequently in human malignant of genetic alterations that inactivate tumor suppressor genes gliomas. The most common of these is deletion of exons 2–7, such as p53, p16, RB, and PTEN, or activate oncogenes resulting in truncation of the extracellular domain (DEGFR including the epidermal growth factor receptor (EGFR), or EGFRvIII), which occurs in a large fraction of de novo CDK4, CDK6, and MDM2 genes (6, 7). EGFR gene amplifi- malignant gliomas (but not in progressive tumors or those cation occurs frequently in gliomas, is restricted to high-grade lacking p53 function) and enhances tumorigenicity, in part by tumors that are usually of the de novo type and express decreasing apoptosis through up-regulation of Bcl-XL. Here, wild-type p53 (8), and occurs at a frequency of 40–50% of all we demonstrate that the DEGFR concomitantly confers re- grade IV gliomas (9, 10). Several clinical and histopathological sistance to the chemotherapeutic drug cisplatin (CDDP) by studies have shown that the presence of EGFR amplification suppression of CDDP-induced apoptosis. Expression of correlates with a shorter interval to disease relapse and lower Bcl-XL was elevated in U87MG.DEGFR cells prior to and rates of survival in patients receiving adjuvant therapies, during CDDP treatment, whereas it decreased considerably in suggesting that it may affect responsiveness of malignant CDDP-treated parental cells. CDDP-induced activation of gliomas to treatment (10). The majority of such EGFR gene caspase-3-like proteases was suppressed significantly in amplifications also include rearrangements (9, 11), the most U87MG.DEGFR cells. These responses were highly specific to common being a genomic deletion of exons 2–7, resulting in a D constitutively kinase-active DEGFR, because overexpression mutant receptor truncated in its extracellular domain ( EGFR of kinase-deficient DEGFR (DK) or wild-type EGFR had no or EGFRvIII) (11). This specific genetic alteration has also such effects. Correspondingly, DEGFR specific tyrosine ki- been found frequently in lung and breast cancers (12, 13). Introduction of DEGFR into the U87MG human glioma cell nase inhibitors reduced Bcl-XL expression and potentiated CDDP-induced apoptosis in U87MG.DEGFR cells. Ectopic line resulted in cell surface expression of a truncated receptor having a ligand-independent, weak but constitutively active, overexpression of Bcl-XL in parental U87MG cells also re- sulted in suppression of both caspase activation and apoptosis and unattenuated kinase and enhanced tumorigenicity in nude induced by CDDP. These results may have important clinical mice (14), which was mediated by both an increase in prolif- eration and a decrease in apoptosis of tumor cells. In contrast, implications for the use of CDDP in the treatment of those overexpression of wild-type (wt) EGFR did not confer a malignant gliomas expressing DEGFR. similar growth advantage (15, 16). Bcl-XL, an inhibitor of the Bcl-2 family of apoptotic proteins, was up-regulated in Persistent invasion of malignant glioma tumor cells into the U87MG.DEGFR tumors, which was inversely correlated with adjacent normal brain parenchyma renders surgical resection their reduced apoptotic rate (16). Overexpression of Bcl-XL incomplete and necessitates adjuvant treatments such as ra- has been shown to confer drug resistance in some tumor cells diation and chemotherapy (1). However, most gliomas even- (17) and also to suppress activation of caspases, the cysteine tually become drug-resistant, limiting the effectiveness of proteases that play a key role in the execution phase of chemotherapy. A number of mechanisms may contribute to apoptosis (18). cellular drug resistance, including reduced intracellular drug Here we report that DEGFR expression in glioma cells concentrations, rapid inactivation of the drug, and increased confers resistance to some commonly utilized chemotherapeu- rate of DNA repair (2). Inhibition of apoptosis, a genetically tic agents. The resistance was associated with suppression of controlled form of cell death, may also be important for drug drug-induced apoptosis, which was largely mediated by in- resistance because the primary mechanism by which most creased expression of Bcl-XL and subsequent inhibition of chemotherapeutic agents having disparate modes of action and caspase-3-like protease activation. These effects required con- cellular targets induce cell death appears to be apoptosis (3). stitutive signaling by DEGFR, because overexpression of ki- The observations that tumors which were either deficient in nase-deficient DEGFR (DK) or wt EGFR had no such effects. the p53 tumor suppressor gene or those in which expression of Moreover, suppression of DEGFR enzymatic function by the antiapoptotic protein Bcl-2 was elevated, were resistant to specific inhibitors sensitized the cells to drug treatment. These apoptosis and showed poor response to radiotherapy and results suggest a new treatment strategy for glioma in which chemotherapy (4, 5) suggest that tumor-specific genetic lesions DEGFR inhibition could be effectively combined with che- may bestow this property to tumor cells, resulting in a survival motherapy. advantage. Abbreviations: EGFR, epidermal growth factor receptor; wt, wild The publication costs of this article were defrayed in part by page charge type; CDDP, cis-diamminedichloroplatinum(II); TUNEL, terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling; payment. This article must therefore be hereby marked ‘‘advertisement’’ in PARP, poly(ADP-ribose) polymerase. accordance with 18 U.S.C. §1734 solely to indicate this fact. \To whom reprint requests should be addressed at: Ludwig Institute for © 1998 by The National Academy of Sciences 0027-8424y98y955724-6$2.00y0 Cancer Research, San Diego Branch 3080 CMM-East, 9500 Gilman PNAS is available online at http:yywww.pnas.org. Drive, La Jolla, CA 92093-0660. e-mail: [email protected]. 5724 Downloaded by guest on October 3, 2021 Medical Sciences: Nagane et al. Proc. Natl. Acad. Sci. USA 95 (1998) 5725 MATERIALS AND METHODS Caspase Activity Assay. Protease activity was assayed as described previously (20) with minor modifications. One unit Cells. The human glioma cell line U87MG, which expresses D of protease activity was defined as the amount of enzyme a low amount of wt EGFR, and its sublines, U87MG. EGFR, required to release 1 pmol p-nitroanilide (pNA)ymin at 37°C. U87MG.DK, and U87MG.wtEGFR, which overexpress Western Blotting. Analysis was as described previously (16) D D EGFR, a kinase-deficient mutant of EGFR (DK), and and utilized antibodies to Bcl-XL (Transduction Laboratories, exogenous wt EGFR, respectively, were described previously Lexington, KY), poly(ADP-ribose) polymerase (PARP) (C2– (15). U87MG cells were transfected with either pSFFVneo- 10, Enzyme Systems Products, Livermore, CA), EGFR (C13) bcl-XL or its control vector pSFFV-neo plasmids (gifts of S. J. (15), or phosphotyrosine (4G10, Upstate Biotechnology, Lake Korsmeyer, Washington University, St. Louis) by using the Placid, NY). calcium phosphate precipitation method and selected in the m y y presence of 400 g ml of G418 (GIBCO BRL). Clones ex- RESULTS pressing high levels of Bcl-XL were used for experiments. All cells were cultured as described (16). To determine the level DEGFR Confers CDDP Resistance in U87MG Human of resistance of the cells to the chemotherapeutic agents Glioma Cells. Because DEGFR expression in glioma cells has cisplatin [cis-diamminedichloroplatinum(II), CDDP], pacli- been shown to decrease the rate of apoptosis both under taxel (Taxol), and vincristine (Sigma), in the presence or serum-starved culture conditions and in implanted tumors absence of tyrosine kinase inhibitors, tyrphostins AG1478 derived from such cells (16), we tested the sensitivity of D (Calbiochem), AG1479, AG1517, and AG1536 (chemically EGFR-expressing glioma cells to the chemotherapeutic drug, synthesized at The Hebrew University of Jerusalem), colony- CDDP, a DNA-damaging agent known to induce apoptosis in forming efficiency assays were performed as described (19). tumor cells and that often has been used in glioma therapy (21, In Situ Terminal Deoxynucleotidyltransferase-Mediated 22). Endogenous and exogenous wt EGFR were moderately dUTP Nick End Labeling (TUNEL) of Apoptotic DNA Frag- autophosphorylated under the culture condition used (10% mentation. Apoptotic cells were detected by using TUNEL of serum), suggesting that wt EGFR was constantly stimulated by ligand in the serum (Fig. 1A). Consistent with previous studies, apoptotic DNA strand breaks as described (16). DEGFR was also constitutively autophosphorylated while a kinase-deficient mutant of DEGFR (DK)