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.

CD5 is a transmembrane protein that is expressed on the surface of T cells and a subset of B cells. The absence of CD5 rendered thymocytes hyperresponsive to stimulation through the T cell antigen receptor (TCR) in vitro. Selection of T cells expressing three distinct transgenic TCRs was also abnormal in CD5-deficient mice. These observations indicate that CD5 can influence the fate of developing thymocytes by acting as a negative regulator of TCR-mediated signal transduction.

The negative regulation of T-or B-cell antigen receptor signaling by CD5 was proposed based on studies of thymocytes and peritoneal B-1a cells from CD5-deficient mice. Here, we show that CD5 is constitutively associated with phosphotyrosine phosphatase activity in Jurkat T cells. CD5 was found associated with the Src homology 2 (SH2) domain containing hematopoietic phosphotyrosine phosphatase SHP-1 in both Jurkat cells and normal phytohemagglutinin-expanded T lymphoblasts. This interaction was increased upon T-cell receptor (TCR)-CD3 cell stimulation. CD5 co-cross-linking with the TCR-CD3 complex down-regulated the TCR-CD3-increased Ca 2؉ mobilization in Jurkat cells. In addition, stimulation of Jurkat cells or normal phytohemagglutinin-expanded T lymphoblasts through TCR-CD3 induced rapid tyrosine phosphorylation of several protein substrates, which was substantially diminished after CD5 cross-linking. The CD5-regulated substrates included CD3, ZAP-70, Syk, and phospholipase C␥l but not the Src family tyrosine kinase p56 lck . By mutation of all four CD5 intracellular tyrosine residues to phenylalanine, we found the membrane-proximal tyrosine at position 378, which is located in an immunoreceptor tyrosine-based inhibitory (ITIM)-like motif, crucial for SHP-1 association. The F378 point mutation ablated both SHP-1 binding and the down-regulating activity of CD5 during TCR-CD3 stimulation. These results suggest a critical role of the CD5 ITIM-like motif, which by binding to SHP-1 mediates the down-regulatory activity of this receptor.CD5 is a 67-kDa cell surface glycoprotein expressed on thymocytes, mature peripheral T cells, and a subpopulation of peritoneal B cells (B-1a cells) which are increased in some autoimmune diseases and are associated with the production of autoantibodies (7). Molecular cloning of mouse and human CD5 (mCD5 and hCD5) (16, 17) revealed that it belongs to the scavenger receptor cysteine-rich (SRCR) family group B, which comprises a group of leukocyte membrane or soluble proteins with one or more domains homologous to the aminoterminal domain of type I macrophage SRCR domain (21). Thus far, 10 members of this group of proteins have been identified: CD5, CD6, WC1, M130, Sp␣, Pema-SREG, Ebnerin, CPR-ductin, hensin, and gallbladder mucin (2).Biochemical studies suggest that CD5 is associated with CD3 in the T-cell receptor (TCR)-CD3 complex and with the B-cell receptor (BCR) complex (6,24,32). Two different ligands for CD5 have been reported: CD72, a 42-kDa type II constitutively expressed glycoprotein on B cells (28, 51); and CD5L, an activation antigen expressed on splenocytes (3). The physiologic roles of CD5-CD72 and CD5-CD5L interactions are not known but would be consistent with a potential T-cell-B-cell cooperation during antibody-mediated immune responses (8).Early in vitro studies of T lymphocytes and thymocytes demonstrated that monoclonal antibodies (MAbs) to CD5 were costimulatory for T-cell proliferation (9, 18, 42). However, in vivo studies showed that CD5 down-modulation by spe...

One of the P2-integrins found on hematopoietic ceUs is lymphocyte function-associated antigen 1 (LFA-1), a lymphocyte/myeloid cell-specific receptor that binds to members of the intercelular adhesion molecule (ICAM) family on antigen-presenting cels. Stimulation of LFA-1 with antibodies or purified ICAMs induces augmentation of T-cell antigen receptor (TCR)-directed T-cell responsiveness. In the present study, LFA-1 was shown to be linked to the tyrosine kinase signalig pathway that stimulates tyrosine phosphorylation and activation of phospholipase C-y1 (PLC-y1

Six-transmembrane epithelial antigen of the prostate-1 (STEAP-1) is a novel cell surface protein highly expressed in primary prostate cancer, with restricted expression in normal tissues. In this report, we show STEAP-1 expression in prostate metastases to lymph node and bone and in the majority of human lung and bladder carcinomas. We identify STEAP-1 function in mediating the transfer of small molecules between adjacent cells in culture, indicating its potential role in tumor cell intercellular communication. The successful generation of two monoclonal antibodies (mAb) that bind to cell surface STEAP-1 epitopes provided the tools to study STEAP-1 susceptibility to naked antibody therapy. Both mAbs inhibited STEAP-1-induced intercellular communication in a dosedependent manner. Furthermore, both mAbs significantly inhibited tumor growth in mouse models using patientderived LAPC-9 prostate cancer xenografts and established UM-UC-3 bladder tumors. These studies validate STEAP-1 as an attractive target for antibody therapy in multiple solid tumors and provide a putative mechanism for mAb-induced tumor growth inhibition. [Cancer Res 2007;67(12):5798-805]

The localization of the protein tyrosine kinase pp60c-src to the plasma membrane and to the membrane of secretory vesicles in neurally derived bovine chromaffin cells has suggested that tyrosine phosphorylations may be associated with the process of secretion. In the present study we have identified two cytosolic proteins of approximately 42 and 45 kD that become phosphorylated on tyrosine in response to secretagogue treatment. Phosphorylation of these proteins reached a maximum (3 min after stimulation) before maximum catecholamine release was observed (5-10 min after stimulation). Both secretion and tyrosine phosphorylation of p42 and p45 required extracellular Ca2+. Tyrosine-phosphorylated proteins of similar Mr have previously been identified in 3T3-L1 adipocytes stimulated with insulin (MAP kinase; Ray, L. B., and T. W. Sturgill. 1987. Proc. Natl. Acad. Sci. USA. 84:1502-1506) and in avian and rodent fibroblasts stimulated with a variety of mitogenic agents (Cooper, J. A., D. F. Bowen-Pope, E. Raines, R. Ross, and T. Hunter. 1982. Cell. 31:263-273; Nakamura, K. D., R. Martinez, and M. J. Weber. 1983. Mol. Cell. Biol. 3:380-390). Comparisons of the secretion-associated 42-kD protein of chromaffin cells with the 42-kD protein of Swiss 3T3 fibroblasts and 3T3-L1 adipocytes provide evidence that these three proteins are highly related. This evidence includes comigration during one-dimensional SDS-PAGE, cochromatography using ion exchange and hydrophobic matrices, similar isoelectric points, identical cyanogen-bromide peptide maps, and cochromatography of MAP kinase activity with the tyrosine-phosphorylated form of pp42. This protein(s), which appears to be activated in a variety of cell types, may serve a common function, perhaps in signal transduction involving a cascade of kinases.