Cyclin D1 promotes nuclear DNA synthesis through phosphorylation and inactivation of the pRb tumor suppressor. Herein, cyclin D1 deficiency increased mitochondrial size and activity that was rescued by cyclin D1 in a Cdk-dependent manner. Nuclear respiratory factor 1 (NRF-1), which induces nuclear-encoded mitochondrial genes, was repressed in expression and activity by cyclin D1. Cyclin D1-dependent kinase phosphorylates NRF-1 at S47. Cyclin D1 abundance thus coordinates nuclear DNA synthesis and mitochondrial function.mitochondria ͉ phosphorylation

Despite clinical approval of erlotinib, most advanced lung cancer patients are primary non-responders. Initial responders invariably develop secondary resistance, which can be accounted for by T790M-EGFR mutation in half of the relapses. We show that MET is highly expressed in lung cancer, often concomitantly with epidermal growth factor receptor (EGFR), including H1975 cell line. The erlotinib-resistant lung cancer cell line H1975, which expresses L858R/T790M-EGFR in-cis, was used to test for the effect of MET inhibition using the small molecule inhibitor SU11274. H1975 cells express wild-type MET, without genomic amplification (CNV ¼ 1.1). At 2 mM, SU11274 had significant in vitro pro-apoptotic effect in H1975 cells, 3.9-fold (P ¼ 0.0015) higher than erlotinib, but had no effect on the MET and EGFR-negative H520 cells. In vivo, SU11274 also induced significant tumour cytoreduction in H1975 murine xenografts in our bioluminescence molecular imaging assay. Using small-animal microPET/MRI, SU11274 treatment was found to induce an early tumour metabolic response in H1975 tumour xenografts. MET and EGFR pathways were found to exhibit collaborative signalling with receptor cross-activation, which had different patterns between wild type (A549) and L858R/ T790M-EGFR (H1975). SU11274 plus erlotinib/CL-387,785 potentiated MET inhibition of downstream cell proliferative survival signalling. Knockdown studies in H1975 cells using siRNA against MET alone, EGFR alone, or both, confirmed the enhanced downstream inhibition with dual MET -EGFR signal path inhibition. Finally, in our time-lapse video-microscopy and in vivo multimodal molecular imaging studies, dual SU11274-erlotinib concurrent treatment effectively inhibited H1975 cells with enhanced abrogation of cytoskeletal functions and complete regression of the xenograft growth. Together, our results suggest that MET-based targeted inhibition using small-molecule MET inhibitor can be a potential treatment strategy for T790M-EGFR-mediated erlotinib-resistant non-small-cell lung cancer. Furthermore, optimised inhibition may be further achieved with MET inhibition in combination with erlotinib or an irreversible EGFR-TKI.

Targeted therapy against epidermal growth factor receptor (EGFR) represents a major therapeutic advance in lung cancer treatment. Somatic mutations of the EGFR gene, most commonly L858R (exon 21) and short in-frame exon 19 deletions, have been found to confer enhanced sensitivity towards the inhibitors gefitinib and erlotinib. We have recently identified an EGFR mutation E884K, in combination with L858R, in a patient with advanced lung cancer who progressed on erlotinib maintenance therapy, and subsequently had leptomeningeal metastases that responded to gefitinib. The somatic E884K substitution appears to be relatively infrequent, and resulted in a mutant lysine residue that disrupts an ion pair with residue R958 in the EGFR kinase domain C-lobe, an interaction that is highly conserved within the human kinome as demonstrated by our sequence analysis and structure analysis. Our studies here, using COS-7 transfection model system, show that E884K works in concert with L858R in-cis, in a dominant fashion, to change downstream signaling, differentially induce MAPK-ERK1/2 signaling and associated cell proliferation, and differentially alter sensitivity of EGFR phosphorylation inhibition by ERBB family inhibitors in an inhibitor-specific fashion. Mutations of the conserved ion pair E884-R958 may result in conformational changes that alter kinase substrate recognition. The analogous E1271K-MET mutation conferred differential sensitivity towards preclinical MET inhibitors SU11274 (unchanged), and PHA665752 (more sensitive). Systematic bioinformatics analysis of the mutation catalog in the human kinome (COSMIC) revealed the presence of cancer-associated mutations involving the conserved E884 homologous residue, and adjacent residues at the ion pair, in known proto-oncogenes (KIT, RET, MET, FAK) and tumor suppressor gene (LKB1). Targeted therapy using small molecule inhibitors should take into account potential cooperative effects of multiple kinase mutations, and their specific effects on downstream signaling and inhibitor sensitivity. Improved efficacy of targeted kinase inhibitors may be achieved by targeting the dominant activating mutations present.