Definition of molecular determinants of prostate cancer cell bone extravasation.

Advanced prostate cancer commonly metastasizes to bone, but transit of malignant cells across the bone marrow endothelium (BMEC) remains a poorly understood step in metastasis. Prostate cancer cells roll on E-selectin(+) BMEC through E-selectin ligand-binding interactions under shear flow, and prostate cancer cells exhibit firm adhesion to BMEC via ß1, ß4, and αVß3 integrins in static assays. However, whether these discrete prostate cancer cell-BMEC adhesive contacts culminate in cooperative, step-wise transendothelial migration into bone is not known. Here, we describe how metastatic prostate cancer cells breach BMEC monolayers in a step-wise fashion under physiologic hemodynamic flow. Prostate cancer cells tethered and rolled on BMEC and then firmly adhered to and traversed BMEC via sequential dependence on E-selectin ligands and ß1 and αVß3 integrins. Expression analysis in human metastatic prostate cancer tissue revealed that ß1 was markedly upregulated compared with expression of other ß subunits. Prostate cancer cell breaching was regulated by Rac1 and Rap1 GTPases and, notably, did not require exogenous chemokines as ß1, αVß3, Rac1, and Rap1 were constitutively active. In homing studies, prostate cancer cell trafficking to murine femurs was dependent on E-selectin ligand, ß1 integrin, and Rac1. Moreover, eliminating E-selectin ligand-synthesizing α1,3 fucosyltransferases in transgenic adenoma of mouse prostate mice dramatically reduced prostate cancer incidence. These results unify the requirement for E-selectin ligands, α1,3 fucosyltransferases, ß1 and αVß3 integrins, and Rac/Rap1 GTPases in mediating prostate cancer cell homing and entry into bone and offer new insight into the role of α1,3 fucosylation in prostate cancer development.

Metabolic inhibition of galectin-1-binding carbohydrates accentuates antitumor immunity.

Galectin-1 (Gal-1) has been shown to play a major role in tumor immune escape by inducing apoptosis of effector leukocytes and correlating with tumor aggressiveness and disease progression. Thus, targeting the Gal-1/Gal-1 ligand axis represents a promising cancer therapeutic approach. Here, to test the Gal-1-mediated tumor immune evasion hypothesis and demonstrate the importance of Gal-1-binding N-acetyllactosamines in controlling the fate and function of antitumor immune cells, we treated melanoma- or lymphoma-bearing mice with peracetylated 4-fluoro-glucosamine (4-F-GlcNAc), a metabolic inhibitor of N-acetyllactosamine biosynthesis, and analyzed tumor growth and immune profiles. We found that 4-F-GlcNAc spared Gal-1-mediated apoptosis of T cells and natural killer (NK) cells by decreasing their expression of Gal-1-binding determinants. 4-F-GlcNAc enhanced tumor lymphocytic infiltration and promoted elevations in tumor-specific cytotoxic T cells and IFN-γ levels, while lowering IL-10 production. Collectively, our data suggest that metabolic lowering of Gal-1-binding N-acetyllactosamines may attenuate tumor growth by boosting antitumor immune cell levels, representing a promising approach for cancer immunotherapy.

Alpha 1,3 fucosyltransferases are master regulators of prostate cancer cell trafficking.

How cancer cells bind to vascular surfaces and extravasate into target organs is an underappreciated, yet essential step in metastasis. We postulate that the metastatic process involves discrete adhesive interactions between circulating cancer cells and microvascular endothelial cells. Sialyl Lewis X (sLe(X)) on prostate cancer (PCa) cells is thought to promote metastasis by mediating PCa cell binding to microvascular endothelial (E)-selectin. Yet, regulation of sLe(X) and related E-selectin ligand expression in PCa cells is a poorly understood factor in PCa metastasis. Here, we describe a glycobiological mechanism regulating E-selectin-mediated adhesion and metastatic potential of PCa cells. We demonstrate that alpha1,3 fucosyltransferases (FT) 3, 6, and 7 are markedly elevated in bone- and liver-metastatic PCa and dictate synthesis of sLe(X) and E-selectin ligands on metastatic PCa cells. Upregulated FT3, FT6, or FT7 expression induced robust PCa PC-3 cell adhesion to bone marrow (BM) endothelium and to inflamed postcapillary venules in an E-selectin-dependent manner. Membrane proteins, CD44, carcinoembryonic antigen (CEA), podocalyxin-like protein (PCLP), and melanoma cell adhesion molecule (MCAM) were major scaffolds presenting E-selectin-binding determinants on FT-upregulated PC-3 cells. Furthermore, elevated FT7 expression promoted PC-3 cell trafficking to and retention in BM through an E-selectin dependent event. These results indicate that alpha1,3 FTs could enhance metastatic efficiency of PCa by triggering an E-selectin-dependent trafficking mechanism.

Spatial and temporal expression of dADAR mRNA and protein isoforms during embryogenesis in Drosophila melanogaster.

Adenosine Deaminases Acting on RNA (ADARs) function to co-transcriptionally deaminate specific (or non-specific) adenosines to inosines within pre-mRNAs, using double-stranded RNAs as substrate. In both Drosophila and mammals, the best-studied ADAR functions are to catalyze specific nucleotide conversions within mRNAs encoding various ligand- or voltage-gated ion channel proteins within the adult brain. In contrast, ADARs within developing fly embryos have scarcely been studied, in part because they contain little or no editase activity, raising interesting questions as to their functional significance. Quantitative RT-PCR shows that two major developmentally regulated mRNA isoform classes are produced (full-length and truncated), which arise by alternative splicing and also alternative 3'-end formation. In situ localization of specific dADAR mRNA isoforms during embryogenesis reveals that the full-length class is found primarily within the developing germ band and central nervous system, whereas the truncated isoform is mostly located in gut endothelium. Developmental Western immunoblots show that both isoform classes are expressed into protein during embryogenesis. Both the rnp-4f 5'-UTR unspliced isoform and the full-length dADAR mRNA primarily localize in the embryonic germ band and subsequently throughout the developing central nervous system. Previous studies have shown that some rnp-4f pre-mRNAs are extensively edited by dADAR in the adult brain. Computer predictions suggest that intron-exon pairing promotes formation of an evolutionarily conserved secondary structure in the rnp-4f 5'-UTR, forming a 177-nt RNA duplex resembling an editing site complementary sequence, which is shown to be associated with splicing failure and to generate a long isoform. Taken together, these observations led us to explore the possibility that interaction between rnp-4f pre-mRNA and nuclear full-length dADAR protein may occur during embryogenesis. In dADAR null mutants, rnp-4f 5'-UTR alternative splicing is significantly diminished, suggesting a non-catalytic role for dADAR in splicing regulation. A working model is proposed which provides a possible molecular mechanism.