The Role of Ykl-40, a Secreted Heparin-Binding Glycoprotein, in Tumor Angiogenesis, Metastasis, and Progression: a Potential Therapeutic Target

A new concept quickly gaining ground in the field of cancer research is that the inflammatory process plays a key role in cancer development and metastasis; however, the molecular mechanisms of such an involvement in cancer progression remain largely unspecified. YKL-40, also known as human cartilage glycoprotein 39, is a secreted heparin-binding protein with ties to both cancers and inflammatory disease. In these diseases, YKL-40 has been suggested to play a role in regulating tissue and extracellular matrix remodeling. It has been found that in certain cancers, including breast, colorectal and brain, that high YKL-40 serum levels correlate with poor outcome, and consequently it may serve as a biomarker. Our recent study has shown that tumor-derived YKL-40 acts as an angiogenic factor due to its ability to up-regulate vessel formation and metastasis during tumor development. However, blockade of the function of YKL-40, which implicates therapeutic value, has not been explored yet. The goal of this project was to better understand the importance of tumor-derived YKL-40 in angiogenesis through both functional and structural studies. By establishing a monoclonal YKL-40 antibody for blocking YKL-40, the function of tumor-derived YKL-40 in inducing endothelial cell angiogenesis and tumor cell survival was uncovered, confirming YKL-40\u27s importance in tumor signaling as well as offering evidence in the benefit of its neutralization. Additionally, a postulated heparin-binding domain on YKL-40 was mutated in hopes of revealing the relevance of this binding ability on YKL-40\u27s function and whether this could serve as a target in inhibiting YKL-40 signaling

A novel function for lysyl oxidase in pluripotent mesenchymal cell proliferation and relevance to inflammation-associated osteopenia.

Lysyl oxidase is a multifunctional enzyme required for collagen biosynthesis. Various growth factors regulate lysyl oxidase during osteoblast differentiation, subject to modulation by cytokines such as TNF-α in inflammatory osteopenic disorders including diabetic bone disease. Canonical Wnt signaling promotes osteoblast development. Here we investigated the effect of Wnt3a and TNF-α on lysyl oxidase expression in pluripotent C3H10T1/2 cells, bone marrow stromal cells, and committed osteoblasts. Lysyl oxidase was up-regulated by a transcriptional mechanism 3-fold in C3H10T1/2 cells, and 2.5-fold in bone marrow stromal cells. A putative functional TCF/LEF element was identified in the lysyl oxidase promoter. Interestingly, lysyl oxidase was not up-regulated in committed primary rat calvarial- or MC3T3-E1 osteoblasts. TNF-α down-regulated lysyl oxidase both in Wnt3a-treated and in non-treated C3H10T1/2 cells by a post-transcriptional mechanism mediated by miR203. Non-differentiated cells do not produce a collagen matrix; thus, a novel biological role for lysyl oxidase in pluripotent cells was investigated. Lysyl oxidase shRNAs effectively silenced lysyl oxidase expression, and suppressed the growth of C3H10T1/2 cells by 50%, and blocked osteoblast differentiation. We propose that interference with lysyl oxidase expression under excess inflammatory conditions such as those that occur in diabetes, osteoporosis, or rheumatoid arthritis can result in a diminished pool of pluripotent cells which ultimately contributes to osteopenia

Lysyl oxidase knockdown prevents osteoblast development of C3H10T1/2 cells.

<p>C3H10T1/2 cells transduced with two different lysyl oxidase shRNAs or empty virus were grown to confluence and then induced to differentiate as indicated in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0100669#s2" target="_blank">Materials and Methods</a>. At intervals, cells were fixed and stained with alizarin red. Fixed and stained cultures were then photographed. This experiment was performed twice with the same outcomes.</p

TNF-α reduces Wnt3a-stimulated lysyl oxidase mRNA stability.

<p>Serum starved C3H10T1/2 were pre-treated with Wnt3a- or conditioned media for 16 hours and then treated with or without TNF-α (20 ng/ml) in the presence of dichlorobenzimidazole riboside (an inhibitor of mRNA transcription) for various intervals. Total RNA was isolated from cell lysates and subjected to real time PCR. Data were plotted as percent log remaining lysyl oxidase mRNA levels (normalized to 18 s rRNA mRNA levels) vs time. The TNF-α induced loss of lysyl oxidase mRNA stability was calculated from the relative slopes of the lines of best fit. Student's t-test statistical analyses were performed by comparing the slopes and intercepts of these two lines and showed a P value of 0.006 (GraphPad Prism 5). Data are means ± SD (n = 3 for each time point) and are from one of two representative experiments with the same outcome.</p

Wild-type and Mutant Sequences of TCF/LEF Elements in the Lysyl Oxidase Promoter.

<p><b>TCF/LEF consensus sequence: CTTTG(A/T)(A/T).</b></p

A potential cis-element for Wnt3a regulation of lysyl oxidase is located at −1321 to −1328 bp upstream of lysyl oxidase translation start site.

<p>Three putative TCF/LEF cis-elements within the first 1.5 kbp of the murine lysyl oxidase promoter (pLOXFL) were mutated by site-directed mutagenesis individually, in pairs, and all three together. C3H10T1/2 cells were transfected with a Renilla luciferase thymidine kinase (pRL-TK) and either wild type pLOXFFL or mutant pLOXFFL reporter constructs. After 24 hours, transfected cells were serum starved and treated with Wnt3a- or control-conditioned media for 24 hours. Luciferase activity was assessed as explained in Experimental Procedures. The fold change of lysyl oxidase transcriptional activity of wild-type and mutated reporter constructs in response to Wn3a are presented as means ± SD. Data are pooled from three independent experiments (n = 9; *, p<0.05; Student's t-test).</p

TNF-α post-transcriptionally attenuates Wnt3a-stimulated lysyl oxidase mRNA levels.

<p><b>A)</b> The effect of TNF-α on Wnt3a-stimulated lysyl oxidase mRNA levels in C3H10T1/2 cells was examined by treating cells with Wnt3a- or control-conditioned media in the presence or absence of increasing concentrations of TNF-α. Lysyl oxidase mRNA analysis of total RNA extracted from these cells was performed by real time PCR. Lysyl oxidase mRNA levels were normalized to the levels of GAPDH mRNA. Data are means ± SD (n = 3; *, p<0.05; Student's t-test). <b>B)</b> Luciferase reporter was used to functionally assess lysyl oxidase transcriptional activity in response to TNF-α. Data are means ± SD (n = 6, N.S, not significant; Student's t-test). Data shown were pooled from two independent experiments.</p

Analysis of miR203 functionality to down-regulate lysyl oxidase mRNA levels.

<p><b>A)</b> miR203 down-regulates lysyl oxidase mRNA levels: C3H10T1/2 cells were transfected with miR203 mimic or non-specific control micro RNA. Cell lysates were collected after five days post-transfection and total extracted RNA subjected to real time PCR analysis for lysyl oxidase and 18S rRNA mRNA for normalization. Data are presented as means ± SD (n = 3; *, p<0.05; Student's t-test). Data are from one of two independent experiments with the same outcome. <b>B</b>) To functionally evaluate TNF-α up-regulation of functional miR203 in C3H10T1/2 cells, cells were transfected with the miR203 reporter plasmid miR203-RenSPL and R01-RenSPL (control) constructs. 24 Hours post-transfection, cells were serum starved and pre-treated with (i) Wnt3a- or (ii) control conditioned media for 24 hours. Cells were then treated with or without TNF-α (20 ng/ml) for 24 hours. Cell lysates were collected and subjected to a luciferase activity assay. Panel (iii) is from cells treated only with or without Wnt3a showing that this reporter for miR203 activity is not affected by Wmt3a alone, as expected. Data are presented as means ± SD (n = 6; *, p<0.05; N.S, not significant; Student's t-test). Data shown are from one of two independent experiments with the same outcomes.</p