ABSTRACT
Acquired resistance to EGF receptor (EGFR) tyrosine kinase inhibitors (TKIs) is inevitable in metastatic EGFR-mutant lung cancers. Here, we modeled disease progression using EGFR-mutant human tumor cell lines. Although five of six models displayed alterations already found in humans, one harbored an unexpected secondary NRAS Q61K mutation; resistant cells were sensitive to concurrent EGFR and MEK inhibition but to neither alone. Prompted by this finding and because RAS/RAF/MEK mutations are known mediators of acquired resistance in other solid tumors (colon cancers, gastrointestinal stromal tumors, and melanomas) responsive to targeted therapies, we analyzed the frequency of secondary KRAS/NRAS/BRAF/MEK1 gene mutations in the largest collection to date of lung cancers with acquired resistance to EGFR TKIs. No recurrent NRAS, KRAS, or MEK1 mutations were found in 212, 195, or 146 patient samples, respectively, but 2 of 195 (1%) were found to have mutations in BRAF (G469A and V600E). Ectopic expression of mutant NRAS or BRAF in drug-sensitive EGFR-mutant cells conferred resistance to EGFR TKIs that was overcome by addition of a MEK inhibitor. Collectively, these positive and negative results provide deeper insight into mechanisms of acquired resistance to EGFR TKIs in lung cancer and inform ongoing clinical trials designed to overcome resistance. In the context of emerging knowledge about mechanisms of acquired resistance to targeted therapies in various cancers, our data highlight the notion that, even though solid tumors share common signaling cascades, mediators of acquired resistance must be elucidated for each disease separately in the context of treatment.
Subject(s)
Drug Resistance, Neoplasm , ErbB Receptors/antagonists & inhibitors , ErbB Receptors/metabolism , Lung Neoplasms/enzymology , MAP Kinase Kinase 1/metabolism , Mutation, Missense , Protein Kinase Inhibitors/pharmacology , Proto-Oncogene Proteins B-raf/metabolism , Proto-Oncogene Proteins/metabolism , ras Proteins/metabolism , Amino Acid Substitution , Cell Line, Tumor , Clinical Trials as Topic , ErbB Receptors/genetics , Female , Humans , Lung Neoplasms/drug therapy , MAP Kinase Kinase 1/genetics , Male , Protein Kinase Inhibitors/therapeutic use , Proto-Oncogene Proteins/genetics , Proto-Oncogene Proteins B-raf/genetics , Proto-Oncogene Proteins p21(ras) , ras Proteins/geneticsABSTRACT
The identification of somatically acquired tumor mutations is increasingly important in the clinical management of cancer because the sensitivity of targeted drugs is related to the genetic makeup of individual tumors. Thus, mutational profiles of tumors can help prioritize anticancer therapy. We report herein the development and validation of two multiplexed assays designed to detect in DNA from FFPE tissue more than 40 recurrent mutations in nine genes relevant to existing and emerging targeted therapies in lung cancer. The platform involves two methods: a screen (SNaPshot) based on multiplex PCR, primer extension, and capillary electrophoresis that was designed to assess for 38 somatic mutations in eight genes (AKT1, BRAF, EGFR, KRAS, MEK1, NRAS, PIK3CA, and PTEN) and a PCR-based sizing assay that assesses for EGFR exon 19 deletions, EGFR exon 20 insertions, and HER2 exon 20 insertions. Both the SNaPshot and sizing assays can be performed rapidly, with minimal amounts of genetic material. Compared with direct sequencing, in which mutant DNA needs to compose 25% or more of the total DNA to easily detect a mutation, the SNaPshot and sizing assays can detect mutations in samples in which mutant DNA composes 1.56% to 12.5% and 1.56% to 6.25% of the total DNA, respectively. These robust, reliable, and relatively inexpensive assays should help accelerate adoption of a genotype-driven approach in the treatment of lung cancer.
Subject(s)
Carcinoma, Non-Small-Cell Lung/diagnosis , Carcinoma, Non-Small-Cell Lung/genetics , Genetic Testing/methods , Lung Neoplasms/diagnosis , Lung Neoplasms/genetics , Mutation , Cell Line, Tumor , DNA, Neoplasm/genetics , High-Throughput Nucleotide Sequencing , Humans , Reproducibility of ResultsABSTRACT
We report the first example of a coordinated dual action of epidermal growth factor (EGF) in stimulating the nuclear-cytoplasmic export and translation of a select messenger RNA (mRNA). The effect of EGF is mediated by the RNA-binding protein Grb7 (growth factor receptor-bound protein 7), which serves as an adaptor for a specific mRNA-protein export complex and a translational regulator. Using the kappa-opioid receptor (OR [KOR]) as a model, we demonstrate that EGF activates nuclear SHP-2 (Src homology region 2-containing tyrosine phosphatase), which dephosphorylates Grb7 in the nucleus. Hypophosphorylated Grb7 binds to the KOR mRNA and recruits the Hu antigen R-exportin-1 (CRM1) complex to form a nuclear-cytoplasmic export complex that exports KOR mRNA. EGF also activates focal adhesion kinase in the cytoplasm to rephosphorylate Grb7, releasing KOR mRNA for active translation. In summary, this study uncovers a coordinated, dual activity of EGF in facilitating nuclear export of a specific mRNA-protein complex as well as translational activation of the exported mRNA.
Subject(s)
Epidermal Growth Factor/metabolism , GRB7 Adaptor Protein/metabolism , Protein Biosynthesis/physiology , RNA, Messenger/metabolism , Receptors, Opioid, kappa/biosynthesis , Animals , Cell Line , Cell Nucleus/genetics , Cell Nucleus/metabolism , Cytoplasm/genetics , Cytoplasm/metabolism , Epidermal Growth Factor/pharmacology , Focal Adhesion Kinase 1/genetics , Focal Adhesion Kinase 1/metabolism , GRB7 Adaptor Protein/genetics , Karyopherins/genetics , Karyopherins/metabolism , Phosphorylation/drug effects , Phosphorylation/physiology , Protein Biosynthesis/drug effects , Protein Tyrosine Phosphatase, Non-Receptor Type 11/genetics , Protein Tyrosine Phosphatase, Non-Receptor Type 11/metabolism , RNA, Messenger/genetics , Rats , Receptors, Cytoplasmic and Nuclear/genetics , Receptors, Cytoplasmic and Nuclear/metabolism , Receptors, Opioid, kappa/genetics , Exportin 1 ProteinABSTRACT
RIP140 (receptor-interacting protein 140) is a transcriptional co-repressor that regulates diverse genes such as those responsive to hormones and involved in metabolic processes. The expression of RIP140 is regulated by multiple hormonal activities in adipose tissue and cancer cell lines. However, it is unclear whether and how RIP140 is regulated post-transcriptionally. Using 5'RACE (rapid amplification of 5' cDNA ends), we have identified a novel 5' splice variant of RIP140 mRNA in mouse brain and P19 cells. A target sequence for miRNA (microRNA) mir-346 was found in the 5'UTR (5'-untranslated region) of RIP140 mRNA; this miRNA is also expressed endogenously in mouse brain and P19 cells. Gain- and loss-of-function studies demonstrated that mir-346 elevates RIP140 protein levels by facilitating association of its mRNA with the polysome fraction. Furthermore, the activity of mir346 does not require Ago-2 (Argonaute 2). The expression of mir-346 enhances the gene repressive activity of RIP140. This is the first report demonstrating post-transcriptional regulation of RIP140 mRNA, involving the enhancing effect of a specific miRNA that targets RIP140's 5'UTR.