Cell surface protease activation during RAS transformation: Critical role of the plasminogen receptor, S100A10.

The link between oncogenic RAS expression and the acquisition of the invasive phenotype has been attributed to alterations in cellular activities that control degradation of the extracellular matrix. Oncogenic RAS-mediated upregulation of matrix metalloproteinase 2 (MMP-2), MMP-9 and urokinase-type plasminogen activator (uPA) is critical for invasion through the basement membrane and extracellular matrix. The uPA converts cell surface-bound plasminogen to plasmin, a process that is regulated by the binding of plasminogen to specific receptors on the cell surface, however, the identity of the plasminogen receptors that function in this capacity is unclear. We have observed that transformation of cancer cells with oncogenic forms of RAS increases plasmin proteolytic activity by 2- to 4-fold concomitant with a 3-fold increase in cell invasion. Plasminogen receptor profiling revealed RAS-dependent increases in both S100A10 and cytokeratin 8. Oncogenic RAS expression increased S100A10 gene expression which resulted in an increase in S100A10 protein levels. Analysis with the RAS effector-loop mutants that interact specifically with Raf, Ral GDS pathways highlighted the importance of the RalGDS pathways in the regulation of S100A10 gene expression. Depletion of S100A10 from RAS-transformed cells resulted in a loss of both cellular plasmin generation and invasiveness. These results strongly suggest that increases in cell surface levels of S100A10, by oncogenic RAS, plays a critical role in RAS-stimulated plasmin generation, and subsequently, in the invasiveness of oncogenic RAS expressing cancer cells.

Oncolytic virus-initiated protective immunity against prostate cancer.

Recently reovirus-based oncotherapy has been successfully implemented for the treatment of prostate cancer. In this report, we show that apart from its primary direct cancer-killing activity, reovirus oncotherapy overrides tumor-associated immune evasion strategies and confers protective antiprostate cancer immunity. Prostate cancer represents an ideal target for immunotherapies. However, currently available immune interventions fail to induce clinically significant antiprostate cancer immune responses, owing to the immunosuppressive microenvironment associated with this disease. We show here that during the process of oncolysis, reovirus acts upon prostate cancer cells and initiates proinflammatory cytokines and major histocompatibility complex (MHC) class I molecule expression. In an immunocompetent transgenic adenocarcinoma of mouse prostate (TRAMP) model, reovirus oncotherapy induces the homing of CD8(+) T and NK cells in tumors and the display of tumor-associated antigens (TAAs) on antigen-presenting cells (APCs), and endows dendritic cells (DCs) with a capacity to successfully present TAAs to tumor-specific CD8(+) T cells. These newly generated immunological events lead to the development of strong antiprostate cancer T cell responses, which restrict the growth of subsequently, implanted syngeneic tumor in an antigen-specific, but reovirus-independent manner. Such reovirus-initiated antiprostate cancer immunity represents a clinically valuable entity that can promote long-term cancer-free health even after discontinuation of the primary oncotherapy.

Oncogenic Ras promotes reovirus spread by suppressing IFN-beta production through negative regulation of RIG-I signaling.

Reovirus is the first naturally occurring human virus reported to exploit activated Ras signaling in the host cell for infection, and is currently undergoing clinical trials as a cancer therapeutic. Recent evidence suggests that Ras transformation promotes three reoviral replication steps during the first round of infection: uncoating of the incoming virion, generation of progeny viruses with enhanced infectivity, and virus release through enhanced apoptosis. Whether oncogenic Ras also enhances reovirus spread in subsequent rounds of infection through other mechanisms has not been examined. Here, we show that compared with nontransformed cells, Ras-transformed cells are severely compromised not only in their response to IFN-beta, but also in the induction of IFN-beta mRNA following reovirus infection. Defects in both IFN-beta production and response allow for efficient virus spread in Ras-transformed cells. We show that the MEK/ERK pathway downstream of Ras is responsible for inhibiting IFN-beta expression by blocking signaling from the retinoic acid-inducible gene I (RIG-I) which recognizes viral RNAs. Overexpression of wild-type RIG-I restores INF-beta expression in reovirus-infected Ras-transformed cells. In vitro-synthesized viral mRNAs also invoke robust RIG-I-mediated IFN-beta production in transfected nontransformed cells, but not in Ras-transformed cells. Collectively, our data suggest that oncogenic Ras promotes virus spread by suppressing viral RNA-induced IFN-beta production through negative regulation of RIG-I signaling.

Cloning and sequencing of tubulin cDNAs from Artemia franciscana: evidence for differential expression of alpha- and beta-tubulin genes.

Tubulin is a heterodimeric protein composed of alpha- and beta-tubulin. In most organisms, they are encoded by multiple gene families whose members are subject to differential regulation. The objective of the work described herein was to better understand tubulin gene expression in the extremophile Artemia franciscana To this end tubulin cDNAs were cloned and sequenced. alphaAT2, an alpha-tubulin cDNA, differed by one nucleotide from alphaAT1, a previously cloned Artemia cDNA. This change, possibly generated by allelic variation, caused an M313V substitution in alpha-tubulin. The amino acid sequence of beta-tubulin encoded by betaAT1, one of only a very limited number of cloned crustacean beta-tubulin cDNA sequences yet available, and the first from Artemia, was similar to other beta-tubulins. However, betaAT1 possessed four degenerate TATA boxes in the 5' untranslated region, although authentic TATA and CCAAT boxes occurred in the 3' non-coding sequence. Analyses by quantitative PCR demonstrated that the amount of tubulin mRNA declined relative to total mRNA in progressive life history stages of Artemia and also that the organism contained more alphaAT2- than betaAT1-tubulin mRNA at all developmental phases examined.

Functional characterization of artemin, a ferritin homolog synthesized in Artemia embryos during encystment and diapause.

Oviparously developing embryos of the crustacean Artemia franciscana encyst and enter diapause, exhibiting a level of stress tolerance seldom seen in metazoans. The extraordinary stress resistance of encysted Artemia embryos is thought to depend in part on the regulated synthesis of artemin, a ferritin superfamily member. The objective of this study was to better understand artemin function, and to this end the protein was synthesized in Escherichia coli and purified to apparent homogeneity. Purified artemin consisted of oligomers approximately 700 kDa in molecular mass that dissociated into monomers and a small number of dimers upon SDS/PAGE. Artemin inhibited heat-induced aggregation of citrate synthase in vitro, an activity characteristic of molecular chaperones and shown here to be shared by apoferritin and ferritin. This is the first report that apoferritin/ferritin may protect cells from stress other than by iron sequestration. Stably transfected mammalian cells synthesizing artemin were more resistant to heat and H(2)O(2) than were cells transfected with vector only, actions also shared by molecular chaperones such as the small heat shock proteins. The data indicate that artemin is a structurally modified ferritin arising either from a common ancestor gene or by duplication of the ferritin gene. Divergence, including acquisition of a C-terminal peptide extension and ferroxidase center modification, eliminated iron sequestration, but chaperone activity was retained. Therefore, because artemin accumulates abundantly during development, it has the potential to protect embryos from stress during encystment and diapause without adversely affecting iron metabolism.