Oncogenic Ras differentially regulates metabolism and anoikis in extracellular matrix-detached cells.

In order for cancer cells to survive during metastasis, they must overcome anoikis, a caspase-dependent cell death process triggered by extracellular matrix (ECM) detachment, and rectify detachment-induced metabolic defects that compromise cell survival. However, the precise signals used by cancer cells to facilitate their survival during metastasis remain poorly understood. We have discovered that oncogenic Ras facilitates the survival of ECM-detached cancer cells by using distinct effector pathways to regulate metabolism and block anoikis. Surprisingly, we find that while Ras-mediated phosphatidylinositol (3)-kinase signaling is critical for rectifying ECM-detachment-induced metabolic deficiencies, the critical downstream effector is serum and glucocorticoid-regulated kinase-1 (SGK-1) rather than Akt. Our data also indicate that oncogenic Ras blocks anoikis by diminishing expression of the phosphatase PHLPP1 (PH Domain and Leucine-Rich Repeat Protein Phosphatase 1), which promotes anoikis through the activation of p38 MAPK. Thus, our study represents a novel paradigm whereby oncogene-initiated signal transduction can promote the survival of ECM-detached cells through divergent downstream effectors.

In vitro and in vivo infection of neural cells by a recombinant measles virus expressing enhanced green fluorescent protein.

This study focused on the in vitro infection of mouse and human neuroblastoma cells and the in vivo infection of the murine central nervous system with a recombinant measles virus. An undifferentiated mouse neuroblastoma cell line (TMN) was infected with the vaccine strain of measles virus (MVeGFP), which expresses enhanced green fluorescent protein (EGFP). MVeGFP infected the cells, and cell-to-cell spread was studied by virtue of the resulting EGFP autofluorescence, using real-time confocal microscopy. Cells were differentiated to a neuronal phenotype, and extended processes, which interconnected the cells, were observed. It was also possible to infect the differentiated neuroblastoma cells (dTMN) with MVeGFP. Single autofluorescent EGFP-positive cells were selected at the earliest possible point in the infection, and the spread of EGFP autofluorescence was monitored. In this instance the virus used the interconnecting processes to spread from cell to cell. Human neuroblastoma cells (SH-SY-5Y) were also infected with MVeGFP. The virus infected these cells, and existing processes were used to initiate new foci of infection at distinct regions of the monolayer. Transgenic animals expressing CD46, a measles virus receptor, and lacking interferon type 1 receptor gene were infected intracerebrally with MVeGFP. A productive infection ensued, and the mice exhibited clinical signs of infection, such as ataxia and an awkward gait, identical to those previously observed for the parental virus (Edtag). Mice were sacrificed, and brain sections were examined for EGFP autofluorescence by confocal scanning laser microscopy over a period of 6 h. EGFP was detected in discrete focal regions of the brain and in processes, which extended deep into the parenchyma. Collectively, these results indicate (i) that MVeGFP can be used to monitor virus replication sensitively, in real time, in animal tissues, (ii) that infection of ependymal cells and neuroblasts provides a route by which measles virus can enter the central nervous system in mouse models of encephalitis, and (iii) that upon infection, the virus spreads transneuronally.