EML4 promotes the loading of NUDC to the spindle for mitotic progression

<div><p>Echinoderm microtubule-associated protein (EMAP)-like (EML) family proteins are microtubule-associated proteins that have a conserved hydrophobic EMAP-like protein (HELP) domain and multiple WD40 domains. In this study, we examined the role of EML4, which is a member of the EML family, in cell division. Time-lapse microscopy analysis demonstrated that EML4 depletion induced chromosome misalignment during metaphase and delayed anaphase initiation. Further analysis by immunofluorescence showed that EML4 was required for the organization of the mitotic spindle and for the proper attachment of kinetochores to microtubules. We searched for EML4-associating proteins by mass spectrometry analysis and found that the nuclear distribution gene C (NUDC) protein, which is a critical factor for the progression of mitosis, was associated with EML4. This interaction was mediated by the WD40 repeat of EML4 and by the C-terminus of NUDC. In the absence of EML4, NUDC was no longer able to localize to the mitotic spindle, whereas NUDC was dispensable for EML4 localization. Our results show that EML4 is critical for the loading of NUDC onto the mitotic spindle for mitotic progression.</p></div

SHCBP1 is required for midbody organization and cytokinesis completion

<div><p>The centralspindlin complex, which is composed of MKLP1 and MgcRacGAP, is one of the crucial factors involved in cytokinesis initiation. Centralspindlin is localized at the middle of the central spindle during anaphase and then concentrates at the midbody to control abscission. A number of proteins that associate with centralspindlin have been identified. These associating factors regulate furrowing and abscission in coordination with centralspindlin. A recent study identified a novel centralspindlin partner, called Nessun Dorma, which is essential for germ cell cytokinesis in <i>Drosophila melanogaster</i>. SHCBP1 is a human ortholog of Nessun Dorma that associates with human centralspindlin. In this report, we analyzed the interaction of SHCBP1 with centralspindlin in detail and determined the regions that are required for the interaction. In addition, we demonstrate that the central region is necessary for the SHCBP1 dimerization. Both MgcRacGAP and MKLP1 are degraded once cells exit mitosis. Similarly, endogenous and exogenous SHCBP1 were degraded with mitosis progression. Interestingly, SHCBP1 expression was significantly reduced in the absence of centralspindlin, whereas centralspindlin expression was not affected by SHCBP1 knockdown. Finally, we demonstrate that SHCBP1 depletion promotes midbody structure disruption and inhibits abscission, a final stage of cytokinesis. Our study gives novel insight into the role of SHCBP in cytokinesis completion.</p></div

Novel Ways to Target Podoplanin to Combat Cancer

<p>Cancer kills almost 8 million people per year worldwide (about 13 people every minute). Clearly, current cancer treatments are not completely effective. In addition, many chemotherapeutic reagents attack rapidly dividing cells, which renders them toxic to a wide array of normal cells in addition to cancer cells. This lack of specificity can cause collateral damage and significant side effects in patients. More targeted therapies are needed to successfully combat cancer. Tumor cell motility leads to invasion and metastasis that cause the vast majority of cancer deaths. We and others have found that the podoplanin (PDPN) receptor promotes tumor cell motility that contributes to many types of cancer. PDPN has a short intracellular domain of 9 amino acids which include two conserved serine residues. PDPN also has a large extracellular domain that is extensively O-glycosylated with α2,3-sialic acid linked to galactose. We are developing reagents to target the intracellular and extracellular domains of PDPN to inhibit cancer progression. For example, we have found that some activators of protein kinase A can induce phosphorylation of the intracellular serine residues of PDPN to inhibit tumor cell motility. In addition, we have found that Maackia amurensis seed lectin (MASL) can target the extracellular domain of PDPN to inhibit transformed cell growth and motility. The biological activity of this lectin survives gastrointestinal proteolysis and enters the cardiovascular circulatory system to inhibit tumor vascularization and progression. Furthermore, we utilized live cell imaging to find that PDPN expression in cancer associated fibroblasts can promote neighboring tumor cell migration and survival. Thus, reagents can be used to target PDPN from inside of the cell and outside of the cell to inhibit tumor cell migration and combat cancer progression. This work illuminates strategies designed to exploit PDPN as a functionally relevant biomarker and chemotherapeutic target.</p

Human melanoma cells express PDPN and respond to MASL.

<p>(<b>a</b>) PDPN was detected by Western blotting of protein (15 µg per lane) from a variety of human specimens including normal skin, primary melanoma, melanoma in transit prior to lymph node metastasis, nodal metastasis, lung metastasis, and three melanoma cells lines (HT-144, SK-MEL-2, SK-MEL-5). Detection of MAPK and nonspecific bands by Western blotting and India ink staining of membranes is also shown to verify equal loading of these samples from different patients and cell lines. (<b>b</b>) Wound healing migration assays were performed on confluent HT-144 monolayers treated with MASL as indicated. Data are shown as the number of cells that migrated into a 400×500 micron area along the center of the wound in 24 hours (mean + SEM, n = 5). (<b>d</b>) HT-144 cells were treated with MASL, and cell viability was evaluated by Alamar blue assay. Data are shown as percent of nontreated cells (mean + SEM, n = 2). Single, double, and triple asterisks indicate p<0.05, p<0.01, and p<0.001 compared to untreated cells, respectively.</p

MASL associates with PDPN and inhibits migration of Src transformed cells.

<p>(<b>a</b>) Src transformed cells were exposed to MASL conjugated to HiLyte Fluor TR (red), and PDPN was detected by immunofluorescence microscopy (green). Colocalization of lectin and PDPN (yellow) is apparent in merged images, including orthogonal views of the z-axis which is 14 microns thick (bar = 20 microns). (<b>b</b>) Protein from Src transformed cells (750 μg) was precipitated with agarose beads linked to MASL, or control beads, and examined for PDPN by Western blotting. Cell lysate (15 μg/lane) were also examined as indicated. (<b>c</b>) Wound healing migration assays were performed on confluent monolayers of cells treated with concentrations of MASL as indicated. Data are shown as the number of cells that migrated into a 300x300 micron area along the center of the wound in 24 hours (mean + SEM, n = 7). Double asterisks indicate p<0.01 compared to nontransformed cells.</p

Dietary MASL inhibits melanoma growth <i>in vivo</i>.

<p>(<b>a</b>) Mice were fed MASL to achieve a dosage of 0 or 25 mg/kg once weekly (indicated by <b>asterisks</b>) and inoculated subcutaneously with B16 melanoma cells (100,000 cells per mouse on day indicated by <b>arrow</b>). Tumors were measured daily by caliper, and data is shown as cubic cm (mean + SEM, n = 4). (<b>b</b>) Tumors were evaluated for PDPN expression by IHC as indicated (bar  = 50 microns). “bc” indicates blood filled vascular spaces lined by tumor cells. (<b>c</b>) Tumors were examined by hematoxylin and eosin staining (H&E) to visualize vascularization and morphology. (<b>c</b>) Tumor vascularization was quantified as the percent of each field (0.8 mm2) occupied by blood vessels and shown as mean + SEM (n = 7).</p

Comparison of Pdpn and Vegfr2 (Kdr) mRNA expression in human tissues.

<p>Values from Affymetrix probe sets representing Pdpn and Vegfr2 are shown as presented on BioGPS (<a href="http://biogps.gnf.org" target="_blank">http://biogps.gnf.org</a>).</p

MASL inhibits melanoma cell motility and viability.

<p>(<b>a</b>) PDPN and β-actin were detected by Western blotting of protein (15 µg per lane) from Melan-a melanocytes and B16 melanoma cells. (<b>b</b>) Wound healing migration assays were performed on confluent MelanA and B16 monolayers. Data are shown as the number of cells that migrated into a 400×500 micron area along the center of the wound in 24 hours (mean + SEM, n = 5). (<b>c</b>) Wound healing migration assays were performed on confluent monolayers treated with MASL as indicated. Data are shown as the number of cells that migrated into a 400×500 micron area along the center of the wound in 24 hours (mean + SEM, n = 5). (<b>d</b>) Melan-a and B16 cells were treated with MASL, and cell viability was evaluated by Alamar blue assay. Data are shown as percent of nontreated cells (mean + SEM, n = 2). (<b>e</b>) Melanoma cell viability and Transwell migration assays were performed on 600,000 cells plated on cell culture inserts containing membranes with an 8 micron pore size in 6-well plates. Cell viability was evaluated by Alamar blue assay and shown as percent of nontreated cells, while Transwell migration was measured after 24<b> </b>hours as the percent of cells found on the underside of the membrane (mean + SEM, n = 2). Double and triple asterisks indicate p<0.01 and p<0.001 compared to corresponding treatments of untreated cells or Melan-a cells in panels d and e, respectively.</p

MASL targets PDPN to inhibit melanoma cell growth.

<p>(<b>a</b>) PDPN and β-actin were detected by Western blotting of protein (5 µg per lane) from B16 melanoma cells transfected with control siRNA or siRNA targeted against PDPN, as indicated. (<b>b</b>) Wound healing migration assays were performed on confluent B16 monolayers transfected with control siRNA or siRNA targeted against PDPN, as indicated. Data are shown as the number of cells that migrated into a 500×400 micron area along the center of the wound in 24 hours (mean + SEM, n = 4). (<b>c</b>) MASL toxicity was evaluated by Trypan blue staining of cells, and shown as the percent of live cells from each well (mean + SEM, n = 2). Single, double, and triple asterisks indicate p<0.05, p<0.01, and p<0.001, respectively, compared to nontransformed cells, untreated Src transformed cells, or control transfectants as indicated.</p

Dietary MASL bioactivity survives gastrointestinal proteolysis to enter the cardiovascular circulatory system and inhibit melanoma cell migration.

<p>Wound healing migration assays were performed on confluent monolayers of B16 melanoma cells treated with serum from mice fed MASL to achieve doses of 1, 100 mg/kg, or 200 mg/kg, or without mouse serum (controls) as indicated. Data are shown as the number of cells that migrated into a 200×300 micron area along the center of the wound in 24 hours (mean + SEM, n = 7). Triple asterisks indicate p<0.001 compared to controls.</p