Summary Cell surface growth factor receptors couple environmental cues to the regulation of cytoplasmic homeostatic process including autophagy, and aberrant activation of such receptors is a common feature of human malignancies. Here, we defined the molecular basis by which the epidermal growth factor receptor (EGFR) tyrosine kinase regulates autophagy. Active EGFR binds to the autophagy protein Beclin 1, leading to its multisite tyrosine phosphorylation, enhanced binding to inhibitors, and decreased Beclin 1-associated Class III phosphatidylinositol-3 kinase activity. EGFR tyrosine kinase inhibitor (TKI) therapy disrupts Beclin 1 tyrosine phosphorylation and binding to its inhibitors, and restores autophagy in non-small cell lung carcinoma (NSCLC) cells with a TKI-sensitive EGFR mutation. In NSCLC tumor xenografts, the expression of a tyrosine phosphomimetic Beclin 1 mutant leads to reduced autophagy, enhanced tumor growth, tumor dedifferentiation, and resistance to TKI therapy. Thus, oncogenic receptor tyrosine kinases directly regulate the core autophagy machinery, which may contribute to tumor progression and chemoresistance.

[ Autophagy 4:8, 989-997; 16 November 2008]; ©2008 Landes Bioscience γ-Herpesviruses (γHVs), including important human pathogens such as Epstein Barr virus, Kaposi's sarcoma-associated HV, and the murine γHV68, encode homologs of the antiapoptotic, cellular Bcl-2 (cBcl-2) to promote viral replication and pathogenesis. The precise molecular details by which these proteins function in viral infection are poorly understood. Autophagy, a lysosomal degradation pathway, is inhibited by the interaction of cBcl-2s with a key autophagy effector, Beclin 1, and can also be inhibited by γHV Bcl-2s. Here we investigate the γHV68 M11-Beclin 1 interaction in atomic detail, using biochemical and structural approaches. We show that the Beclin 1 BH3 domain is the primary determinant of binding to M11 and other Bcl-2s, and this domain binds in a hydrophobic groove on M11, reminiscent of the binding of different BH3 domains to other Bcl-2s. Unexpectedly, regions outside of, but contiguous with, the Beclin 1 BH3 domain also contribute to this interaction. We find that M11 binds to Beclin 1 more strongly than do KSHV Bcl-2 or cBcl-2. Further, the differential affinity of M11 for different BH3 domains is caused by subtle, yet significant, variations in the atomic details of each interaction. Consistent with our structural analysis, we find that Beclin 1 residues L116 and F123, and M11 residue pairs G86 + R87 and Y60 + L74, are required for M11 to bind to Beclin 1 and downregulate autophagy. Thus, our results suggest that M11 inhibits autophagy through a mechanism that involves the binding of the Beclin 1 BH3 domain in the M11 hydrophobic surface groove.

After internalization of triglyceride-rich lipoproteins (TRL) in hepatoma cells, TRL particles are immediately disintegrated in the early endosomal compartment. This involves the targeting of lipids and apoprotein B along the degradative pathway and the recycling of TRL-derived apoE through recycling endosomes. Re-secretion of apoE is accompanied by the concomitant association of apoE and cellular cholesterol with high-density lipoproteins (HDL). Since epidemiological data showed that apoE3 and apoE4 have differential effects on HDL metabolism, we investigated whether the intracellular processing of TRL-derived apoE4 differs from apoE3-TRL. In this study, we demonstrated by radioactive and immunofluorescence uptake experiments that cell-surface binding and internalization of TRL-derived apoE4 are increased compared with apoE3 in hepatoma cells. Pulse-chase experiments revealed that HDL-induced recycling, but not disintegration and degradation, of apoE4-enriched TRL is strongly reduced in these cells. Furthermore, impaired HDL-induced apoE4 recycling is associated with reduced cholesterol efflux. Studies performed in Tangier fibroblasts showed that apoE recycling does not depend on ATP-binding cassette transporter A1 activity. These studies provide initial evidence that impaired recycling of apoE4 could interfere with intracellular cholesterol transport and contribute to the pathophysiological lipoprotein profile observed in apoE4 homozygotes.Intestinal chylomicrons and liver-derived very low-density lipoproteins (VLDL) 1 represent triglyceride-rich lipoproteins (TRL) that deliver lipids and lipophilic vitamins to other cells of the body. Lipoprotein lipase-mediated hydrolysis of TRL at the luminal side of endothelial cells resulted in the formation of TRL remnants, which are rapidly cleared by the liver (for review see Refs. 1 and 2). Lipoprotein lipase remained associated with these remnants, which simultaneously became enriched with high-density lipoprotein (HDL)-derived apoprotein E (apoE). Both lipoprotein lipase and apoE then facilitated the internalization of TRL remnants via the LDL receptor-related protein 1 and the low-density lipoprotein receptor (3-7). After receptor-mediated endocytosis, the intracellular processing of TRL is very complex. We and others (8 -12) demonstrated that TRL is disintegrated in peripheral endosomes, which is followed by a differential sorting of TRL components. In human hepatoma cells and fibroblasts, the majority of TRL lipids is targeted to the lysosomal compartment, whereas TRLderived apoE is found in peripheral recycling endosomes (9, 10). Subsequently, substantial amounts of TRL-derived apoE are recycled back to the cell-surface, re-secreted (9), and found associated with newly synthesized or exogenous lipoproteins (10,11,13,14). We recently discovered that HDL stimulates and serves as an acceptor for recycled apoE in hepatocytes in vitro and in vivo (10,13). This new link between TRL-derived apoE and HDL metabolism is associated with cholesterol efflux and involves the ...

Although next-generation sequencing (NGS) has been the domain of large genome centers, it is quickly becoming more accessible to general pathology laboratories. In addition to finding single-base changes, NGS allows for the detection of larger structural variants, including insertions/deletions, translocations, and viral insertions. We describe the use of targeted NGS on DNA extracted from formalin-fixed, paraffin-embedded (FFPE) tissue, and show that the short read lengths of NGS are ideally suited to fragmented DNA obtained from FFPE tissue. Further, we describe a novel method for performing hybrid-capture target enrichment using PCR-generated capture probes. As a model, we captured the 5.3-kb Merkel cell polyomavirus (MCPyV) genome in FFPE cases of Merkel cell carcinoma using inexpensive, PCR-derived capture probes, and achieved up to 37,000-fold coverage of the MCPyV genome without prior virus-specific PCR amplification. This depth of coverage made it possible to reproducibly detect viral genome deletions and insertion sites anywhere within the human genome. Out of four cases sequenced, we identified the 5' insertion sites in four of four cases and the 3' sites in three of four cases. These findings demonstrate the potential for an inexpensive gene targeting and NGS method that can be easily adapted for use with FFPE tissue to identify large structural rearrangements, opening up the possibility for further discovery from archival tissue.

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