Non-small cell lung cancer (NSCLC) comprises 75% to 85% of newly diagnosed lung cancers. Over 70% of NSCLC patients present with advanced disease, and the 5-year survival rate for NSCLC is only 16%. For early-stage or locally-advanced lung cancer, surgery is the most effective treatment, and combined chemotherapy is the standard adjuvant approach. For stage III/IV NSCLC, platinum-based combined chemotherapy is the current standard of care, but with much room for improvement .
In a minority of patients, a mutant epidermal growth factor receptor (EGFR) has become a validated therapeutic target and EGFR tyrosine kinase inhibitors (TKIs) gefitinib and erlotinib are currently the first-line treatment options for these patients [2, 3]. These drugs lead to impressive improvements in progression-free survival (PFS) compared to chemotherapy. However, ultimately these tumors develop resistance to these TKIs through various mechanisms. A frequent mechanism is the emergence of a malignant clone with a second mutation in the EGFR kinase domain, a threonine-to-methionine substitution at amino acid position 790 (T790M) .
The ErbB family includes four related receptor proteins (EGFR/ErbB1/HER1, ErbB2/Neu/HER2, ErbB3/HER3, and ErbB4/HER4). The ErbB family of membrane receptors is a group of transmembrane glycoproteins that consists of an extracellular ligand-binding domain, a transmembrane domain, and an intracellular tyrosine kinase domain mediating signal transduction. The complex EGFR signal transduction pathway involves the RAS/MAPK cascade, phosphatidyl inositol 3-kinase (PI3K), signal transducer and activator of transcription (STAT), and downstream protein kinase C (PKC). Following ligand binding, EGFR can homodimerize or heterodimerize with another member of the ErbB family, causing activation of the intracellular tyrosine kinase domain and receptor transphosphorylation. The newly formed phosphotyrosine residues act as docking sites for various adaptor molecules that consequently activate a number of intracellular signaling cascades, that, in case of constitutive activation of the pathway, leads to cell proliferation, inhibition of apoptosis, angiogenesis, and invasion/metastasis, resulting in tumor growth and progression .
Currently two main anti-EGFR strategies are in clinical use: low-molecular-weight TKIs that compete with ATP for binding to the tyrosine kinase portion of the receptor, and monoclonal antibodies that are directed at the ligand-binding extracellular domain thereby preventing ligand binding, receptor dimerization, and receptor signaling. These two classes of agents have shown solid preclinical and clinical activity in a variety of tumor types .
Among the receptor TKIs, single-agent erlotinib (Tarceva, Genentech, Inc, South San Francisco, CA, USA and OSI Pharmaceuticals Inc., Melville, NY, USA) improves survival in advanced NSCLC patients who progressed after chemotherapy [7–10] and is superior to chemotherapy in the first-line treatment of lung adenocarcinoma with an EGFR mutation in exon 19/21 . The aggregated clinical experience today indicates that only patients whose tumors contain a sensitizing mutation in the EGFR tyrosine kinase domain derive an important and meaningful clinical benefit from these agents. Some randomized studies indicate that in patients not selected for such mutations these drugs might even have an adverse effect on outcome . In an unselected patient population, gefitinib maintenance therapy also failed to show a survival advantage .
Not all patients with tyrosine kinase domain mutations respond to these inhibitors and even patients that respond usually only achieve a partial remission. In addition, some base-line mutations, for example those located in exon 20 of the kinase domain, are resistant or only weakly sensitive to current anti-EGFR TKIs. The efficacy of the inhibitors is also limited in time due to, in nearly half of the cases, the appearance of cells with a second "resistance" mutation, usually T790M located in the receptor tyrosine kinase domain . An additional mechanism is the activation, either at baseline or acquired, of c-Met over-expression. Afatinib (BIBW 2992, Boehringer Ingelheim GmbH), an irreversible dual inhibitor of EGFR and HER2 kinases, retains some activity in tumors with T790M mutations although at doses that are a log higher than what is needed for cancers with only a sensitizing mutation . Afatinib is currently being evaluated in phase III trials [13–17].
The chimerical IgG1 mAb cetuximab (ERBITUX, ImClone Systems Incorporated, New York, NY, USA and Bristol-Myers Squibb Company, Princeton, NJ, USA) is the most comprehensively studied anti-EGFR antibody. By blocking the ligand-receptor interaction, cetuximab down-regulates EGFR signaling, thereby inhibiting cell proliferation, apoptosis, and angiogenesis . Cetuximab in combination with chemotherapy has been approved by the FDA for the treatment of metastatic colorectal cancer (CRC) and in combination with radiotherapy or a platinum derivative for the treatment of locally advanced head and neck cancer (HNC) [18, 19]. Cetuximab has modest activity as a single agent as well as in combination with docetaxel in patients with advanced, chemotherapy-refractory NSCLC . A multinational, multicentre, open-label, phase-III trial has shown that addition of cetuximab to platinum-based chemotherapy improved outcome for patients with advanced NSCLC . However, the effect is small and no clear predictive biomarker has been identified.
The limitations of the clinical results obtained with single agent EGFR TKIs or cetuximab justify the investigation of additional therapeutic strategies, including enhanced targeting of the EGFR. RNA interference (RNAi), has been extensively explored in recent years in many targets. The ability of small interference RNA (siRNA) sequences to modulate gene expression has provided a powerful tool with which to study gene function and is being explored in clinical trials [22, 23]. However, the combined use of RNAi and other types of EGFR targeting has not been explored.
In the current study we investigated whether the combination of EGFR inhibitory agents with EGFR-specific siRNA increases the therapeutic efficacy. To this end, we have examined the effects of either treatment alone versus the combination, in a set of lung cancer cell lines differing in their genomic status.