Regular articleSmall interfering RNAs suppress the expression of endogenous and GFP-fused epidermal growth factor receptor (erbB1) and induce apoptosis in erbB1-overexpressing cells
Introduction
Cancer cells frequently overexpress growth factor receptors, resulting in both enhanced proliferation and dependence of tumor progression on the overexpression phenotype [1]. The erbB family of growth factor receptors are transmembrane receptor tyrosine kinases (RTKs) that are involved in the development and progression of a wide range of human cancers [2]. The four members of the family (erbB1-4) engage in a hierarchical network of interactions resulting in signal diversification [3]. Inhibition of the activity of one family member often induces profound effects by subverting the coordinated interactions within the network [4]. Thus, the erbB molecules constitute ideal targets in cancer therapy [5]. For example, a monoclonal antibody inhibits proliferation of erbB2-overexpressing cancer cells [6], and its humanized form (trastuzumab, Herceptin) is being employed in the successful immunotherapy of breast cancer [7], [8].
The epidermal growth factor receptor (EGFR or erbB1) is likewise overexpressed in a wide range of cancers [9] and thus constitutes another important therapeutic target [2], [10]. A monoclonal anti-erbB1 antibody, cetuximab, is undergoing promising clinical trials in the treatment of lung and of head and neck cancers [11], as are a variety of small molecule ATP binding site inhibitors [5], [12]. Although the detailed understanding of the biology and biochemistry of erbB proteins has led to the introduction of such receptor-targeted drugs in the treatment of human cancers [5], new approaches and combined therapies are required due to therapeutic failure in individual cases and the evolution of drug resistance [13], [14].
In addition to therapeutic approaches targeting the expressed proteins directly, their biosynthesis can also be inhibited, as exemplified by antisense oligonucleotides specific for erbB1 [15]. However, difficulties related to stability or specificity of the oligonucleotides or problems in target sequence selection hamper the widespread use of this approach [16]. More promising is the selective degradation of the corresponding mRNAs by RNA interference (RNAi), a process that avoids the global depression of protein synthesis induced by the double-stranded RNA-inducible interferon system present in mammalian cells [17]. In one implementation of RNAi, selective degradation of target mRNAs in mammalian cells is achieved by transfection with double-stranded, short interfering RNAs (siRNAs), leading to rapid and efficient degradation of the target [17]. Expression of siRNAs under the control of an RNA polymerase III promoter provides the means for achieving long-term suppression of protein synthesis [18], [19].
RNAi of transmembrane proteins, and of RTKs in particular, has not been reported to date. Consideration of the importance of the erbB family in human cancers and the efficiency of RNAi led us to design siRNAs against erbB1 and test their effects on the proliferation and EGF-induced responses of erbB1-(over)expressing cells. We show that siRNA-induced suppression of erbB1 expression results in significant inhibition of EGF-induced tyrosine phosphorylation and in induction of apoptosis in A431 human epidermoid carcinoma cells, a cell line widely used in the study of erbB1 because it expresses this RTK to a level of ∼2×106 [20]. The inhibitory effect of high concentrations of EGF on the proliferation of A431 was suppressed in siRNA-treated cells displaying a pronounced decrease in the expression level of erbB1. In A431 cells transfected to express an erbB1–EGFP (EGFP, enhanced green fluorescent protein) fusion protein in addition to the endogenous erbB1, inhibition of both molecules was achieved with an siRNA directed against the RTK moiety whereas selective suppression of the fusion protein ensued with an siRNA specific for the EGFP segment of the mRNA. The successful application of siRNA for inhibition of proliferation in erbB1-overexpressing cells extends the list of available therapeutic modalities in the treatment of human cancer.
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Cell culture and transfection
Normal and stably transfected CHO, A431, and HeLa cells were cultured in Dulbecco’s modified Eagle’s medium supplemented with 10% FCS and penicillin/streptomycin (Invitrogen, Karlsruhe, Germany) in 5% CO2. Cells were reseeded at a confluency of 30–60% in the absence of antibiotics 1 day before transfection. Transfection with plasmids and siRNAs were carried out with Lipofectamine2000 and Oligofectamine (Invitrogen), respectively, according to the manufacturer’s specifications. Chemically
Inhibition of expression of transfected EGFP and erbB1 in CHO cells
We designed three double-stranded, 21-nucleotide-long siRNAs with TT dinucleotide 3′ overhangs against the coding sequence of erbB1 (GI 12002211) and one siRNA against EGFP (GI 1373318) (Table 1, Fig. 1). Chinese hamster ovary (CHO) cells were transfected with an EGFP plasmid (pAD3) [23] or cotransfected with pAD3 and the GFP-2 siRNA. The ratio of positive cells was determined by fluorescence microscopy. The ratio of EGFP-positive cells decreased from 55% in cells transfected with plasmid
Discussion
ErbB1 has long been considered an important oncoprotein in the development of human malignancies and is one of the emerging targets in tumor therapy [2]. Although considerable progress has been made in the application of oncoprotein-targeted antibodies and small molecule tyrosine kinase inhibitors, none of these agents is curative [5]. In an effort to find new approaches to target erbB1-overexpressing cancer cells, we set out to characterize the RNAi-induced knock-down of endogenous and
Acknowledgements
We are greatly indebted to Thomas Tuschl and Sayda Elbashir for advice and discussions about the RNAi experiments and provision of facilities for synthesizing the siRNAs. This work was supported by EU Grant FP5 QLRT-1999-3126 (“ErbB in Breast Tumor”) and the Max Planck Society. P.N. was the recipient of a fellowship from the EU Grant.
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