A Novel EGFR Extracellular Domain Mutant, EGFRΔ768, Possesses Distinct Biological and Biochemical Properties in Neuroblastoma.

UNLABELLED: EGFR is a popular therapeutic target for many cancers. EGFR inhibitors have been tested in children with refractory neuroblastoma. Interestingly, partial response or stable disease was observed in a few neuroblastoma patients. As EGFR mutations are biomarkers for response to anti-EGFR drugs, primary neuroblastoma tumors and cell lines were screened for mutations. A novel EGFR extracellular domain deletion mutant, EGFRΔ768, was discovered and the biologic and biochemical properties of this mutant were characterized and compared with wild-type and EGFRvIII receptors. EGFRΔ768 was found to be constitutively active and localized to the cell surface. Its expression conferred resistance to etoposide and drove proliferation as well as invasion of cancer cells. While EGFRΔ768 had similarity to EGFRvIII, its biologic and biochemical properties were distinctly different from both the EGFRvIII and wild-type receptors. Even though erlotinib inhibited EGFRΔ768, its effect on the mutant was not as strong as that on wild-type EGFR and EGFRvIII. In addition, downstream signaling of EGFRΔ768 was different from that of the wild-type receptor. In conclusion, this is the first study to demonstrate that neuroblastoma express not only EGFRvIII, but also a novel EGFR extracellular domain deletion mutant, EGFRΔ768. The EGFRΔ768 also possesses distinct biologic and biochemical properties which might have therapeutic implications for neuroblastoma as well as other tumors expressing this novel mutant. IMPLICATIONS: Neuroblastoma expressed a novel EGFR mutant which possesses distinct biologic and biochemical properties that might have therapeutic implications. Mol Cancer Res; 14(8); 740-52. ©2016 AACR.

Live attenuated influenza virus vaccines by computer-aided rational design.

Despite existing vaccines and enormous efforts in biomedical research, influenza annually claims 250,000-500,000 lives worldwide, motivating the search for new, more effective vaccines that can be rapidly designed and easily produced. We applied the previously described synthetic attenuated virus engineering (SAVE) approach to influenza virus strain A/PR/8/34 to rationally design live attenuated influenza virus vaccine candidates through genome-scale changes in codon-pair bias. As attenuation is based on many hundreds of nucleotide changes across the viral genome, reversion of the attenuated variant to a virulent form is unlikely. Immunization of mice by a single intranasal exposure to codon pair-deoptimized virus conferred protection against subsequent challenge with wild-type (WT) influenza virus. The method can be applied rapidly to any emerging influenza virus in its entirety, an advantage that is especially relevant when dealing with seasonal epidemics and pandemic threats, such as H5N1- or 2009-H1N1 influenza.

Deregulation of the cell cycle by breast tumor kinase (Brk).

Brk is a cytoplasmic nonreceptor tyrosine kinase that is overexpressed in breast tumors but undetectable in normal or benign mammary tissues. Brk promotes proliferation of human mammary epithelial cells and tumor growth in a mouse model, but the role of Brk in cell cycle regulation is not known. In this study, we describe the mechanism of Brk-induced deregulation of the cell cycle. We provide evidence that Brk antagonizes the transcriptional activity of the transcription factor FoxO family of proteins by inhibiting its nuclear localization. As a result, the cell cycle inhibitor p27, a FoxO target gene, is down-regulated. This event is accompanied by G1/S cell cycle progression of quiescent cells. As p27 is a key regulator of the G1/S cell cycle checkpoint, these data suggest that perturbation of p27 expression induced by Brk causes S phase entrance. Deregulation of the cell cycle is a key event in neoplasia, and thus, the mechanism presented here likely contributes to breast cancer development.

Perturbation of cytoskeleton dynamics by the opposing effects of Rac1 and Rac1b.

Rac1, a ubiquitously expressed member of the Rho GTPase family, plays a pivotal role in the regulation of multiple cellular processes including cytoskeleton reorganization, cell growth, differentiation and motility. Here we show that the tumor-specific splice variant of Rac1, Rac1b, negatively regulates Rac1 activity. The expression of Rac1b in HeLa cells interferes with Rac1 activation by PDGF, leads to a reduction in membrane-bound Rac1 and promotes an increase in Rho activity. The antagonistic relationship between Rac1 and Rac1b perturbs the regulatory circuitry that controls actin cytoskeleton dynamics thereby leading to tumor-linked alterations in cell morphology and motility.

Redox-dependent downregulation of Rho by Rac.

Rac and Rho GTPases function as critical regulators of actin cytoskeleton remodelling during cell spreading and migration. Here we demonstrate that Rac-mediated reactive oxygen species (ROS) production results in the downregulation of Rho activity. The redox-dependent decrease in Rho activity is required for Rac-induced formation of membrane ruffles and integrin-mediated cell spreading. The pathway linking generation of ROS to downregulation of Rho involves inhibition of the low-molecular-weight protein tyrosine phosphatase (LMW-PTP) and then an increase in the tyrosine phosphorylation and activation of its target, p190Rho-GAP. Our findings define a novel mechanism for the coupling of changes in cellular redox state to the control of actin cytoskeleton rearrangements by Rho GTPases.

The pleckstrin homology (PH) domain-interacting protein couples the insulin receptor substrate 1 PH domain to insulin signaling pathways leading to mitogenesis and GLUT4 translocation.

Receptor-mediated tyrosine phosphorylation of the insulin receptor substrate 1 (IRS-1) is required for the propagation of many of insulin's biological effects. The amino-terminal pleckstrin homology (PH) domain of IRS-1 plays a pivotal role in promoting insulin receptor (IR)-IRS-1 protein interactions. We have recently reported the isolation of a PH domain-interacting protein, PHIP, which selectively binds to the IRS-1 PH domain and is stably associated with IRS-1 in mammalian cells. Here we demonstrate that overexpression of PHIP in fibroblasts enhances insulin-induced transcriptional responses in a mitogen-activated protein kinase-dependent manner. In contrast, a dominant-negative mutant of PHIP (DN-PHIP) was shown to specifically block transcriptional and mitogenic signals elicited by insulin and not serum. In order to examine whether PHIP/IRS-1 complexes participate in the signal transduction pathway linking the IR to GLUT4 traffic in muscle cells, L6 myoblasts stably expressing a myc-tagged GLUT4 construct (L6GLUT4myc) were transfected with either wild-type or dominant-interfering forms of PHIP. Whereas insulin-dependent GLUT4myc membrane translocation was not affected by overexpression of PHIP, DN-PHIP caused a nearly complete inhibition of GLUT4 translocation, in a manner identical to that observed with a dominant-negative mutant of the p85 subunit of phosphatidylinositol 3-kinase (Deltap85). Furthermore, DN-PHIP markedly inhibited insulin-stimulated actin cytoskeletal reorganization, a process required for the productive incorporation of GLUT4 vesicles at the cell surface in L6 cells. Our results are consistent with the hypothesis that PHIP represents a physiological protein ligand of the IRS-1 PH domain, which plays an important role in insulin receptor-mediated mitogenic and metabolic signal transduction.

The two hats of SOS.

Son of sevenless (SOS) is a guanine nucleotide exchange factor that activates Ras in response to growth factor stimulation. SOS also appears to serve as a guanine nucleotide exchanger for Rac and, thus, may be involved in cytoskeleton reorganization. Nimnual and Bar-Sagi discuss how these two activities of SOS can be regulated and how SOS may be recruited to different cellular locations through interactions with the adaptor proteins Grb2 and E3b1.

The Bax subfamily of Bcl2-related proteins is essential for apoptotic signal transduction by c-Jun NH(2)-terminal kinase.

Targeted gene disruption studies have established that the c-Jun NH(2)-terminal kinase (JNK) signaling pathway is required for stress-induced release of mitochondrial cytochrome c and apoptosis. Here we demonstrate that activated JNK is sufficient to induce rapid cytochrome c release and apoptosis. However, activated JNK fails to cause death in cells deficient of members of the Bax subfamily of proapoptotic Bcl2-related proteins. Furthermore, exposure to stress fails to activate Bax, cause cytochrome c release, and induce death in JNK-deficient cells. These data demonstrate that proapoptotic members of the Bax protein subfamily are essential for JNK-dependent apoptosis.