Identification of mRNA splicing factors as the endothelial receptor for carbohydrate-dependent lung colonization of cancer cells

Abstract

Cell surfaces of epithelial cancer are covered by complex carbohydrates, whose structures function in malignancy and metastasis. However, the mechanism underlying carbohydrate-dependent cancer metastasis has not been defined. Previously, we identified a carbohydrate-mimicry peptide designated I-peptide, which inhibits carbohydrate-dependent lung colonization of sialyl Lewis X-expressing B16-FTIII-M cells in E/P-selectin doubly-deficient mice. We hypothesized that lung endothelial cells express an unknown carbohydrate receptor, designated as I-peptide receptor (IPR), responsible for lung colonization of B16-FTIII-M cells. Here, we visualized IPR by in vivo biotinylation, which revealed that the major IPR is a group of 35-kDa proteins. IPR proteins isolated by I-peptide affinity chromatography were identified by proteomics as Ser/Arg-rich alternative pre-mRNA splicing factors or Sfrs1, Sfrs2, Sfrs5, and Sfrs7 gene products. annexin ͉ galectin ͉ metastasis ͉ selectin ͉ vasculature T he apical cell surface of epithelia is covered by numerous carbohydrates attached to membrane proteins and lipids. When epithelial cells are transformed, the structure of these carbohydrates changes (1). Many investigators have suggested a correlation between cancer-associated carbohydrate antigens and poor patient prognosis, including metastasis (2-8). Despite extensive structural analysis and clinical observations using monoclonal antibodies against cancer-associated carbohydrate antigens, mechanisms underlying carbohydrate-dependent cancer metastasis through the circulation remain elusive. Because E-selectin is expressed on the inflammatory endothelial cell surface and binds to cancer-associated carbohydrate antigens such as sialyl Lewis A (sLeA) and sialyl Lewis X (sLeX), E-selectin provides a mechanism for sLeA and/or sLeX antigenexpressing cancer cells to be metastasized through the hematogenous route (4-6). Involvement of P-selectin and L-selectin in cancer metastasis has been also described (7,8). However, substantial clinical data indicate the existence of selectin-independent but carbohydrate-dependent cancer metastasis (6, 10, 11).In our previous studies, we screened a peptide-displaying phage library using a monoclonal anti-Lewis A antibody (clone 7LE) and identified a short peptide IELLQAR, designated I-peptide (12-14). When I-peptide was injected intravenously into a mouse, it bound to a specific subset of lung capillary endothelial cells but not to blood vessels of other organs including the liver (12). I-peptide administration inhibited carbohydrate-dependent lung colonization of fucosyltransferase-3-transfected sialyl Lewis X antigenpositive B16 (B16-FTIII-M) cells (15) in wild-type mice (12) and in E-and P-selectin doubly-deficient mutant mice (13), suggesting that the lung endothelial surface expresses unknown carbohydratebinding receptor(s) enabling colonization of B16-FTIII-M cells. We designated this presumptive receptor I-peptide receptor (IPR) (13).In this study, we isolated IPR candidate proteins by I-peptideaff...

Targeted drug delivery to tumor vasculature by a carbohydrate mimetic peptide

“…Because I-peptide targets normal lung through pre-mRNA splicing factor and it is likely targets the tumor vasculature through Anxa1 (12) (Fig. S1A), we looked for a peptide sequence specifically targeting tumor but not normal lung vasculature.…”

“…However, sialyl Lewis X-dependent B16FT-IIIM colonization occurred in E-selectin/P-selectin doubly-deficient mutant mice, and I-peptide inhibited that colonization, suggesting the presence of a yet unknown I-peptide receptor distinct from E-or P-selectins (11). We then isolated the putative receptor by I-peptide affinity chromatography and subjected it to proteomic analysis, which identified the I-peptide receptor as a pre-mRNA splicing factor (12). During these experiments, we found unexpectedly that I-peptide binds to a 15 kDa fragment of annexin 1 (Anxa1) (12).…”

“…We then isolated the putative receptor by I-peptide affinity chromatography and subjected it to proteomic analysis, which identified the I-peptide receptor as a pre-mRNA splicing factor (12). During these experiments, we found unexpectedly that I-peptide binds to a 15 kDa fragment of annexin 1 (Anxa1) (12).…”

Localization and Dynamics of Nuclear Speckles in Plants

“…In animals, SRs are known to perform many other diverse functions in the nucleus as well as in the cytoplasm, including mRNA transport, localization, translation, and decay, as well as in genome stability and microRNA biogenesis (Twyffels et al, 2011). Interestingly, some SRs in animals are present at the cell surface and exhibit carbohydratebinding activity (Hatakeyama et al, 2009). The SR proteins are major regulators of CS and AS Long and Caceres, 2009;.…”

COP35, a Cholangiocarcinoma-Binding Oligopeptide, Interacts with the Clathrin Heavy Chain Accompanied by GRP78

“…The biopanning technique has been shown to be a powerful tool for identifying specific ligands on target organs and tumors, as it enables forced evolution of very high-affinity targeting peptides through rounds of selection of living libraries of peptides presented on the surface of the bacteriophage particle (9)(10)(11)(12). This technique has classically been used to iteratively produce peptides binding purified cell surface markers (13,14), cultured cell lines (15,16), or tumor-bearing animals (17)(18)(19). We utilized a peptide-presenting bacteriophage library on the basis of a combinatorial library of random peptide 12mers fused to a minor coat protein pIII of the M13 bacteriophage containing approximately 2.7 Â 10 9 different sequences, and different peptides selective for the target cells were identified with successive rounds of biopanning.…”