The severe acute respiratory syndrome coronavirus (SARS-CoV) genome contains open reading frames (ORFs) that encode for several genes that are homologous to proteins found in all known coronaviruses. These are the replicase gene 1a/1b and the four structural proteins, nucleocapsid (N), spike (S), membrane (M), and envelope (E), and these proteins are expected to be essential for the replication of the virus. In addition, this genome also contains nine other potential ORFs varying in length from 39 to 274 amino acids. The largest among these is the first ORF of the second longest subgenomic RNA, and this protein (termed U274 in the present study) consists of 274 amino acids and contains three putative transmembrane domains. Using antibody specific for the C terminus of U274, we show U274 to be expressed in SARS-CoV-infected Vero E6 cells and, in addition to the full-length protein, two other processed forms were also detected. By indirect immunofluorescence, U274 was localized to the perinuclear region, as well as to the plasma membrane, in both transfected and infected cells. Using an N terminus myc-tagged U274, the topology of U274 and its expression on the cell surface were confirmed. Deletion of a cytoplasmic domain of U274, which contains Yxx and diacidic motifs, abolished its transport to the cell surface. In addition, U274 expressed on the cell surface can internalize antibodies from the culture medium into the cells. Coimmunoprecipitation experiments also showed that U274 could interact specifically with the M, E, and S structural proteins, as well as with U122, another protein that is unique to SARS-CoV.

The surface marker CD44 has been identified as one of several markers associated with cancer stem cells (CSC) in solid tumors, but its ubiquitous expression in many cell types, including hematopoietic cells, has hindered its use in targeting CSCs. In this study, 28 paired primary tumor and adjacent nontumor gastric tissue samples were analyzed for cell surface protein expression. Cells that expressed pan-CD44 were found to occur at significantly higher frequency in gastric tumor tissues. We identified CD44v8-10 as the predominant CD44 variant expressed in gastric cancer cells and verified its role as a gastric CSC marker by limiting dilution and serial transplantation assays. Parallel experiments using CD133 failed to enrich for gastric CSCs. Analyses of another 26 primary samples showed significant CD44v8-10 upregulation in gastric tumor sites. Exogenous expression of CD44v8-10 but not CD44 standard (CD44s) increased the frequency of tumor initiation in immunocompromised mice. Reciprocal silencing of total CD44 resulted in reduced tumor-initiating potential of gastric cancer cells that could be rescued by CD44v8-10 but not CD44s expression. Our findings provide important functional evidence that CD44v8-10

Preclinical models of diffuse-type gastric cancer (DGC) that reliably predict clinical activity of novel compounds are lacking. To overcome the problem of poor tumor cellularity in DGC, we used cells from malignant ascites to establish DGC patientderived xenograft (PDX) models that recapitulate the primary cancer. Cells in PDX model GAGA6 with FGFR2 amplification were sensitive to AZD4547, a potent FGFR inhibitor that is being clinically evaluated for FGFR-aberrant cancer types. Intermittent in vivo treatment of GAGA6 tumors with AZD4547 gave rise to PDX tumors with acquired resistance to AZD4547, GAGA6-R. Surprisingly, there were no mutations in the FGFR2 gene in GAGA6-R, negating gatekeeper mutations as a mechanism of drug resistance. Phosphorylation of FGFR2 and downstream signaling molecules AKT/PKB and MAPK/ERK remained inhibited by AZD4547. Further analysis of signaling pathways identified AKT-independent phosphorylation and inhibition of GSK3b as a mechanism of drug resistance in GAGA6-R cells. Treatment of GAGA6-R cells with protein kinase C (PKC) inhibitor H7 in combination with AZD4547 led to dephosphorylation and activation of GSK3b with concomitant downregulation of MCL-1 and BCL-XL. Combined treatment with AZD4547 and H7 in vitro synergistically enhanced cell death in GAGA6-R but not GAGA6 cells. Furthermore, midostaurin, a multikinase inhibitor with PKC-inhibiting activity, in part reversed resistance of GAGA6-R tumor to AZD4547 in vivo. Our results suggest that upon challenge with FGFR inhibitors, FGFR2-amplified tumors that are highly dependent on FGFR2 signaling for survival rapidly develop resistance by switching to a PKC-mediated inhibition of GSK3b to gain a survival advantage.