Abstract:Vasohibins (VASH1 and VASH2) are recently identified regulators of angiogenesis and cancer cell functions. They are secreted proteins without any classical secretion signal sequences, and are thought to be secreted instead via an unconventional protein secretion (UPS) pathway in a small vasohibin-binding protein (SVBP)-dependent manner. However, the precise mechanism of SVBP-dependent UPS is poorly understood. In this study, we identified a novel UPS regulatory system in which essential domain architecture (VA… Show more
“…When bound to SVBP, VASH2 is efficiently secreted, and this may be responsible for the paracrine effect of VASH2 in the tumor microenvironment. We have previously reported that when VASH2 was knocked down in cancer cells, it prevented tumor angiogenesis in ovarian cancer cells and hepatocellular carcinoma .…”
Vasohibin‐2 (VASH2) is expressed in various cancers and promotes their progression. We recently reported that pancreatic cancer patients with higher VASH2 expression show poorer prognosis. Herein, we sought to characterize the role of VASH2 in pancreatic cancer. We used LSL‐KrasG12D; LSL‐Trp53R172H; Pdx‐1‐Cre (KPC) mice, a mouse model of pancreatic ductal adenocarcinoma (PDAC), and cells isolated from them (KPC cells). Knockdown of Vash2 from PDAC cells did not affect their proliferation, but decreased their migration. When Vash2‐knockdown PDAC cells were orthotopically inoculated, liver metastasis and peritoneal dissemination were reduced, and the survival period was significantly prolonged. When KPC mice were crossed with Vash2‐deficient mice, metastasis was significantly decreased in Vash2‐deficient KPC mice. VASH2 was recently identified to have tubulin carboxypeptidase activity. VASH2 knockdown decreased, whereas VASH2 overexpression increased tubulin detyrosination of PDAC cells, and tubulin carboxypeptidase (TCP) inhibitor parthenolide inhibited VASH2‐induced cell migration. We next clarified its role in the tumor microenvironment. Tumor angiogenesis was significantly abrogated in vivo when VASH2 was knocked down or deleted. We further examined genes downregulated by Vash2 knockdown in KPC cells, and found chemokines and cytokines that were responsible for the recruitment of myeloid derived suppressor cells (MDSC). Indeed, MDSC were accumulated in PDAC of KPC mice, and they were significantly decreased in Vash2‐deficient KPC mice. These findings suggest that VASH2 plays an essential role in the metastasis of PDAC with multiple effects on both cancer cells and the tumor microenvironment, including tubulin detyrosination, tumor angiogenesis and evasion of tumor immunity.
“…When bound to SVBP, VASH2 is efficiently secreted, and this may be responsible for the paracrine effect of VASH2 in the tumor microenvironment. We have previously reported that when VASH2 was knocked down in cancer cells, it prevented tumor angiogenesis in ovarian cancer cells and hepatocellular carcinoma .…”
Vasohibin‐2 (VASH2) is expressed in various cancers and promotes their progression. We recently reported that pancreatic cancer patients with higher VASH2 expression show poorer prognosis. Herein, we sought to characterize the role of VASH2 in pancreatic cancer. We used LSL‐KrasG12D; LSL‐Trp53R172H; Pdx‐1‐Cre (KPC) mice, a mouse model of pancreatic ductal adenocarcinoma (PDAC), and cells isolated from them (KPC cells). Knockdown of Vash2 from PDAC cells did not affect their proliferation, but decreased their migration. When Vash2‐knockdown PDAC cells were orthotopically inoculated, liver metastasis and peritoneal dissemination were reduced, and the survival period was significantly prolonged. When KPC mice were crossed with Vash2‐deficient mice, metastasis was significantly decreased in Vash2‐deficient KPC mice. VASH2 was recently identified to have tubulin carboxypeptidase activity. VASH2 knockdown decreased, whereas VASH2 overexpression increased tubulin detyrosination of PDAC cells, and tubulin carboxypeptidase (TCP) inhibitor parthenolide inhibited VASH2‐induced cell migration. We next clarified its role in the tumor microenvironment. Tumor angiogenesis was significantly abrogated in vivo when VASH2 was knocked down or deleted. We further examined genes downregulated by Vash2 knockdown in KPC cells, and found chemokines and cytokines that were responsible for the recruitment of myeloid derived suppressor cells (MDSC). Indeed, MDSC were accumulated in PDAC of KPC mice, and they were significantly decreased in Vash2‐deficient KPC mice. These findings suggest that VASH2 plays an essential role in the metastasis of PDAC with multiple effects on both cancer cells and the tumor microenvironment, including tubulin detyrosination, tumor angiogenesis and evasion of tumor immunity.
“…The amino acid sequences of VASH-SIa and VASH-PS, which contain SIb and SIc, are highly conserved among the VASH family members in vertebrates. 8 These findings suggested that SVBP-dependent UPS may be common within the VASH family. Figure 1.…”
Section: Splicing Products Of the Vasohibinsmentioning
Vasohibin-1 is an intrinsic angiogenesis inhibitor, and is expressed in endothelial cells via induction by pro-angiogenesis factors. It is known to inhibit several processes of angiogenesis, with different mechanisms from extrinsic angiogenesis inhibitors. Vasohibin-2 is mainly expressed by mononuclear cells which have been mobilized from bone marrow. It not only promotes angiogenesis, but also modulates the releases of FGF-2 and VEGF, which are the two major inducers for vasohibin1. Hypoxic environment induces the expression of hypoxia-inducible Factor 1α with a result of VEGF release nearly in all tumor cell lines and tissues. However, it has been observed that hypoxia reduces the inducible effects of VEGF on vasohibin, which indicates that a complicated mechanism exists in the angiogenesis. Vasohibin and its family members play important roles in both the physiological and pathological procedures, in contrary but complementary patterns. Furthermore, human aortic smooth muscle cells and fibroblast have also been detected to express vasohibin on a moderate to weak scale range. Recently, the results of an increasing number of studies in vivo have shown that vasohibin can also be detected in several cancers, and is associated with micro-vessel densities, histology grades, invasions, poor clinical features, metastasis, and dissemination in abdominal cavities, as well as EMT. In more recent reports, it has been confirmed that, along with being angiogenesis regulators, a variety of other roles have been associated with this family. The focus of this study was the upstream regulatory mechanisms of vasohibin expressions, and their role in regard to the downstream target proteins of vasohibin, especially in carcinoma. Vasohibin is considered to be an original angiogenesis inhibitor, and has a much broader significance in pathological processes. It can be taken as an independent prognostic factor, as well as a potential strategy for cancer therapy programs.
“…The FLAPs with high binding affinity for HER2 were experimentally identified by screening with a fast and easy method using bioluminescence, which is a highly sensitive method to quantitatively analyse target-binding proteins even without protein purification 17 . The FLAP candidates fused with glutathione-S-transferase (GST) and Renilla luciferase 8.6-535 (RLuc) 18 were expressed as a dimeric form in Escherichia coli, and the resulting crude extract was used to examine the binding activities of FLAP candidates to HER2 using a bioluminescence imaging (BLI)-ELISA ( Fig.…”
Section: Rapid Identification Of Antigen-binding Flapsmentioning
300 s. The dissociation of the interaction was subsequently measured for 300 s. Systematic baseline drift correction was done by subtracting the shift recorded for sensors loaded with ligand but incubated without analyte. Data analysis and curve fitting were done using Octet software version 11.0. Experimental data were fitted with the binding equations available for a 1:1 interaction with local fitting and the mean ± standard error of the mean (SEM) values of k on and k off were calculated from the data of five different concentrations of an analyte. The K D was calculated as the ratio of k off /k on. cell immunostaining. The human cervical cancer cell line HeLa and human gastric cancer cell line N87 or human breast cancer cells expressing firefly luciferase SK-BR-3/Luc were obtained from the RIKEN Bio-Resource Center (Tsukuba, Japan) or JCRB Cell Bank (Osaka, Japan), respectively, and maintained at 37 °C in 5% FBS-DMEM (Nacalai Tesque, Kyoto, Japan) supplemented with penicillin (100 U/ml) and streptomycin (100 µg/ml) (Nacalai Tesque). The cells (1.0 × 10 4 cells/well) were seeded on a slide chamber plate, cultured for 16 h and fixed by treatment with a 4% paraformaldehyde solution (Nacalai Tesque) for 10 min at 25 °C. The cells were blocked with 1% BSA/PBS for 1 h at 25 °C. After the addition of 50 nM purified His-tagged FLAPs or 5 nM trastuzumab in 1% BSA/PBS to the chamber, the cells were incubated for 16 h at 4 °C. Thousand-fold diluted Alexa Fluor 488-conjugated mouse anti-His tag secondary antibody (Medical & Biological Laboratories, Aichi, Japan) or Alexa Fluor 488-conjugated mouse anti-human IgG Fc secondary antibody in 1% BSA/PBS were used to fluorescently label the His-tagged FLAPs or trastuzumab, respectively. After washing three times with PBS, the stained cells were mounted with Fluoromount (Diagnostic ByoSystems, CA, USA) containing 1/1000 Hoechst 33342 (Nacalai Tesque). All photos were taken using a BZ-X700 microscope (Keyence, Osaka, Japan). Statistical analysis. Data are presented as means ± SEM and were statistically analysed with a two-sided Student's t-test; p values of < 0.05 were considered statistically significant.
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