Hematogenous metastasis involves the adhesion of circulating tumor cells to vascular endothelium of the secondary site. We hypothesized that breast cancer cell adhesion is mediated by interaction of endothelial E-selectin with its glycoprotein counter-receptor(s) expressed on breast cancer cells. At a hematogenous wall shear rate, ZR-75-1 breast cancer cells specifically adhered to E-selectin expressing human umbilical vein endothelial cells when tested in parallel plate flow chamber adhesion assays. Consistent with their E-selectin ligand activity, ZR-75-1 cells expressed flow cytometrically detectable epitopes of HECA-452 mAb, which recognizes high efficiency E-selectin ligands typified by sialofucosylated moieties. Multiple E-selectin reactive proteins expressed by ZR-75-1 cells were revealed by immunoprecipitation with E-selectin chimera (E-Ig chimera) followed by Western blotting. Mass spectrometry analysis of the 72 kDa protein, which exhibited the most prominent E-selectin ligand activity, corresponded to Mac-2 binding protein (Mac-2BP), a heretofore unidentified E-selectin ligand. Immunoprecipitated Mac-2BP expressed sialofucosylated epitopes and possessed E-selectin ligand activity when tested by Western blot analysis using HECA-452 mAb and E-Ig chimera, respectively, demonstrating that Mac-2BP is a novel high efficiency E-selectin ligand. Furthermore, silencing the expression of Mac-2BP from ZR-75-1 cells by shRNA markedly reduced their adhesion to E-selectin expressing cells under physiological flow conditions, confirming the functional E-selectin ligand activity of Mac-2BP on intact cells. In addition to ZR-75-1 cells, several other E-selectin ligand positive breast cancer cell lines expressed Mac-2BP as detected by Western blot and flow cytometry, suggesting that Mac-2BP may be an E-selectin ligand in a variety of breast cancer types. Further, invasive breast carcinoma tissue showed co-localized expression of Mac-2BP and HECA-452 antigens by fluorescence microscopy, underscoring the possible role of Mac-2BP as an E-selectin ligand. In summary, breast cancer cells express Mac-2BP as a novel E-selectin ligand, potentially revealing a new prognostic and therapeutic target for breast cancer.
Introduction-Invasion of other tissues during bloodborne metastasis in part requires adhesion of cancer cells to vascular endothelium by specific fluid shear-dependent receptor-ligand interactions. This study investigates the hypothesis that the adhesion is mediated by ligands shared between endothelial E-selectin and Galectin-1 (Gal-1), both of which are upregulated during inflammation and cancer. Methods-Flow chamber adhesion and dynamic biochemical tissue analysis (DBTA) assays were used to evaluate whether Gal-1 modulates E-selectin adhesive interactions of breast cancer cells and tissues under dynamic flow conditions, while immunocytochemistry, immunohistochemistry, western blotting, and fluorescence anisotropy were used to study molecular interactions under static conditions. Results-Dynamic adhesion assays revealed a shear-dependent binding interaction between Gal-1hFc treated breast cancer cells and tissues and E-selectin-coated beads, caus-ing~300% binding increase of the beads compared to negative controls. Immunocyto-and immunohistochemical analyses showed that Gal-1 and E-selectin fluorescent signals colocalized on cells and tissues at~75% for each assay. Immunoprecipitation and Western blotting of Mac-2BP from breast cancer cell lysates revealed that Gal-1 and Eselectin share Mac-2BP as a ligand, while fluorescence anisotropy and circulating tumor cell model systems exhibited competitive or antagonistic binding between Gal-1 and E-selectin for shared ligands, including Mac-2BP. Furthermore, Mac-2BP functional blockade inhibited the effects of Gal-1 on E-selectin binding. Conclusions-In summary, this investigation reveals a sheardependent interaction between E-selectin and Gal-1 that may be due to intermediation by a similar or shared ligand(s), including Mac-2BP, which may provide a rational basis for development of novel diagnostics or therapeutics for breast cancer.
Invasion of other tissues during bloodborne metastasis requires the adhesion of cancer cells to the endothelium by specific fluid shear-dependent receptor-ligand interactions. It is hypothesized that ligands are shared between the endothelial adhesion molecule known as E-selectin, a transmembrane glycoprotein, and Galectin-1 (Gal-1), a secretory dimer protein, both of which are upregulated during inflammation and cancer. Our previous data strongly indicate that this shear-dependent interaction involves the same or closely related cancer cell-expressed ligands, such as the glycoprotein Mac-2BP. Initial investigation using immuno-staining methods to locate E-selectin and Gal-1 ligands and determine relative proximity to one another revealed that E-selectin and Gal-1 ligands on ZR-75-1 breast cancer cells and breast cancer tissue microarrays had a high percentage of colocalization, as high as 75% in some cases. These results allude to the possibility that these reactive molecules are in close proximity or are in fact the same molecule. Subsequently, E-selectin microspheres were perfused over Gal-1-treated ZR-75-1 cell monolayers and breast cancer tissue microarrays to determine the functional relationship, if any, between Gal-1 and E-selectin ligands during adhesion from flow. Flow assay data show that Gal-1 treatment enhances E-selectin microsphere interactions to a breast cancer monolayer in a shear-dependent manner, with a shear stress of 0.5 dynes/cm2 having an almost 300% increase in binding compared to the untreated monolayer. This amount of binding at a physiological shear stress gives credence to the hypothesis that Gal-1 influences E-selectin receptor-ligand interactions via the same or similar ligands during breast cancer cell adhesion. Dynamic biochemical tissue analysis (DBTA), a novel flow-based tissue interrogation technique developed in our lab, was then implemented to test human invasive ductal breast carcinoma tissue sections. Similar to ZR-75-1 cell line flow assay data, a 142% increase in shear-dependent binding interactions between the E-selectin microspheres and the Gal-1 treated breast cancer tissue sections at 0.5 dynes/cm2 shear stress was observed. Colocalization of reactive signals and increased shear-dependent flow interactions indicate that metastatic binding in breast cancer is aided through E-selectin to Gal-1 interaction via a shared ligand intermediate. Ascertaining the mechanism behind this interaction would lead to a better understanding of the circulating breast cancer cells’ adhesion cascade during metastasis. Citation Format: Nathan M. Reynolds, Claire R. Hall, Sean E. Thomas, Monica M. Burdick. Breast cancer adhesion cascade is influenced by Galectin-1 and E-selectin receptor-ligand interactions. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 342. doi:10.1158/1538-7445.AM2015-342
Cutaneous T-cell lymphomas (CTCL) are the most common primary skin lymphomas and characterized by accumulation of malignant T-cells in the skin. The early lesion resembles both clinical and histological benign inflammatory disorders with hyperproliferative epidermis with infiltration of T-cells. Despite considerable progress in the understanding of molecular mechanisms involved in the malignant transformation of T-cells, it is largely unknown what causes the morphological and histopathological features of the disease. Here we take advantage of a novel, organotypic 3D-model of CTCL to show that malignant T-cells profoundly changes the behavior and growth of keratinocytes. Thus, organotypic 3D-cultures with human keratinocytes, fibroblasts and malignant T-cells display a disorganized stratification and hypercromatic cells resembling the early hyperproliferative stage of CTCL. Furthermore, hyperproliferation was followed by a down regulation of differentiation markers such as keratin 10 and involucrin. Interestingly, we observed a loss of attachment between the epithelial-and mesenchymal compartment, and immunohistochemistry revealed a greatly reduced expression of E-cadherin compared to controls. In conclusion, we provide the first evidence that malignant T-cells orchestra the histopathological skin changes seen in CTCL. The human Dermokine gene (DMKN) is located at 19q13.1 within the « Stratified epithelium Secreted peptides Complex » (SSC) locus, surrounded by 2 other genes encoding proteins secreted in the stratified epithelia, namely Suprabasin (SBSN) and Keratinocyte-differentiation-associated protein (KRTDAP). DMKN expression leads to production of 4 isoform families, each of them displaying a different histological and subcellular location. We recently showed that the ubiquitous and intracellular DMKN δ isoforms are involved in endovesicular trafficking via activation of the small GTPase Rab5. Contrary to δ isoforms, DMKN β and γ are specifically expressed and secreted by the granular keratinocytes. Thus they could play a specific role in the late steps of epidermal differentiation. We studied the Dmkn gene expression in the mouse. SSC locus is located in the syntenic region of murine chromosome 7. RT-PCR and RACE-PCR experiments as well as in silico analysis of the murine genomic sequence show mRNA expression for the α, β and γ families but not for the δ one. Splicing of the Dmkn mRNA is thus less complex than its human counterpart. Quantitative RT-PCR analysis revealed that Dmkn, Sbsn and Krtdap expression is upregulated in the skin of adult and old mice compared to that of newborns. During skin wound healing, Dmkn β and γ are highly expressed in the late stages of re-epithelialization, like many other epidermal differentiation markers. Finally, we developed Dmkn βγ knockout mice. The animals are viable and show no visible differences compared to wild type mice. However, quantitative RT-PCR and immunoblot experiments revealed a deregulated expression of late epidermis differentiation genes. In particular, ...
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