Purpose EMT has been associated with metastatic spread and EGFR inhibitor resistance. We developed and validated a robust 76-gene EMT signature using gene expression profiles from four platforms using NSCLC cell lines and patients treated in the BATTLE study. Methods We conducted an integrated gene expression, proteomic, and drug response analysis using cell lines and tumors from NSCLC patients. A 76-gene EMT signature was developed and validated using gene expression profiles from four microarray platforms of NSCLC cell lines and patients treated in the BATTLE (Biomarker-integrated Approaches of Targeted Therapy for Lung Cancer Elimination) study, and potential therapeutic targets associated with EMT were identified. Results Compared with epithelial cells, mesenchymal cells demonstrated significantly greater resistance to EGFR and PI3K/Akt pathway inhibitors, independent of EGFR mutation status, but more sensitivity to certain chemotherapies. Mesenchymal cells also expressed increased levels of the receptor tyrosine kinase Axl and showed a trend towards greater sensitivity to the Axl inhibitor SGI-7079, while the combination of SGI-7079 with erlotinib reversed erlotinib resistance in mesenchymal lines expressing Axl and in a xenograft model of mesenchymal NSCLC. In NSCLC patients, the EMT signature predicted 8-week disease control in patients receiving erlotinib, but not other therapies. Conclusion We have developed a robust EMT signature that predicts resistance to EGFR and PI3K/Akt inhibitors, highlights different patterns of drug responsiveness for epithelial and mesenchymal cells, and identifies Axl as a potential therapeutic target for overcoming EGFR inhibitor resistance associated with the mesenchymal phenotype

Transformation of cells by the src oncogene results in elevated tyrosine phosphorylation of two related proteins, p80 and p85 (p80/85). Immunostaining with specific monoclonal antibodies revealed a striking change of subcellular localization of p80/85 in src-transformed cells. p80/85 colocalizes with F-actin in peripheral extensions of normal cells and rosettes (podosomes) of src-transformed cells. Sequence analysis of cDNA clones encoding p80/85 revealed an amino-terminal domain composed of six copies of a direct tandem repeat, each repeat containing 37 amino acids, a carboxyl-terminal SH3 domain, and an interdomain region composed of a highly charged acidic region and a region rich in proline, serine, and threonine. The multidomain structure of p80/85 and its colocalization with F-actin in normal and src-transformed cells suggest that these proteins may associate with components of the cytoskeleton and contribute to organization of cell structure.Transformation of cells by tyrosine kinase oncogenes leads to alterations of cell shape, cellular metabolism, growth control, and gene expression (16,31,54). A substantial body of evidence indicates that many if not all of these changes are a direct result of the tyrosine kinase activity of the oncogene product (reviewed in references 31, 54, and 66). Rous sarcoma virus (RSV) encodes an enzymatically activated, 60-kDa tyrosine protein kinase, pp60v"src (6,15,44). However, the product of the normal cellular homolog of src, pp60c-sc, is enzymatically down regulated and does not induce significant alterations in cellular growth or changes in cell morphology when overexpressed in rodent or avian cells (30,53,67). Oncogenic activation of c-src and concomitant activation of tyrosine kinase activity can be achieved by mutation of the regulatory site of tyrosine phosphorylation, 40,58,63). Thus, expression of pp6Ov-src or activated forms of the c-src protein pp60527F results in efficient cellular transformation and the increased tyrosine phosphorylation of approximately 15 to 30 cellular proteins (27,33,45,61).Genetic studies have shown that structural perturbation of several different domains of pp6Osrc leads to alterations in the pattern of tyrosine phosphorylation of specific cellular proteins and accompanying changes in morphological phenotypes (reviewed in references 31 and 54). For example, mutation of the site of myristylation (e.g., Gly-2 to Ala) of pp60v-src or pp60527F blocks cellular transformation (9, 32, 61) and the tyrosine phosphorylation of a 120-kDa cellular protein (35,45,61). pp60src contains two regions that share amino acid sequence similarity with other nonreceptor tyrosine protein kinases (55) and regulatory proteins such as phospholipase C--y, Crk, and GTPase-activating protein (GAP) (70,73,76). Structural alterations within these regions alter or abolish the transforming activity of src (18,28,51,59,77,79) Whereas recent experiments have shown that tyrosine phosphorylation of some cellular proteins appears to direct stable protein-protein interactio...

Chronic hepatitis B virus (HBV) infection is a major health concern worldwide, frequently leading to liver cirrhosis, liver failure and hepatocellular carcinoma. Evidence exists that high viral antigen load may play a role in chronicity. Production of viral proteins is thought to depend on transcription of viral covalently closed circular DNA (cccDNA). In a human clinical trial with ARC-520, a RNA interference (RNAi)-based therapeutic targeting HBV transcripts, HBV S antigen (HBsAg) was strongly reduced in treatment-naïve patients positive for HBV e antigen (HBeAg) but was reduced significantly less in patients that were HBeAg negative or had received long-term therapy with nucleos(t)ide viral replication inhibitors (NUCs). The molecular basis for this unexpected differential response was investigated in chimpanzees chronically infected with HBV. Several independent lines of evidence demonstrated that HBsAg was expressed not only from the episomal cccDNA minichromosome, but also from transcripts arising from HBV DNA integrated into the host genome. The latter was the dominant source in HBeAg negative chimpanzees. Many of the integrants detected in chimpanzees lacked target sites for the siRNAs in ARC-520, explaining the reduced response in HBeAg negative chimpanzees and by extension in HBeAg negative patients. Our results uncover a heretofore under-recognized source of HBsAg that may represent a strategy adopted by HBV to maintain chronicity in the presence of host immune surveillance and could alter trial design and endpoint expectations of new therapies for chronic HBV.