Stroma-derived but not tumor ADAMTS1 is a main driver of tumor growth and metastasis

The matrix metalloprotease ADAMTS1 (A Disintegrin And Metalloprotease with ThromboSpondin repeats 1) has been involved in tumorigenesis although its contributions appeared ambiguous. To understand the multifaceted actions of this protease, it is still required a deeper knowledge of its implication in heterogeneous tumor-stroma interactions. Using a syngeneic B16F1 melanoma model in wild type and ADAMTS1 knockout mice we found distinct stroma versus tumor functions for this protease. Genetic deletion of ADAMTS1 in the host microenvironment resulted in a drastic decrease of tumor growth and metastasis. However, the downregulation of tumor ADAMTS1 did not uncover relevant effects. Reduced tumors in ADAMTS1 KO mice displayed a paradoxical increase in vascular density and vascular-related genes; a detailed characterization revealed an impaired vasculature, along with a minor infiltration of macrophages. In addition, ex-vivo assays supported a chief role for ADAMTS1 in vascular sprouting, and melanoma xenografts showed a relevant induction of its expression in stroma compartments. These findings provide the first genetic evidence that supports the pro-tumorigenic role of stromal ADAMTS1

ADAMTS1 cleaves aggrecan at multiple sites and is differentially inhibited by metalloproteinase inhibitors

ADAMTS1 is a secreted protein that belongs to the recently described ADAMTS (a disintegrin and metalloprotease with thrombospondin repeats) family of proteases. Evaluation of ADAMTS1 catalytic activity on a panel of extracellular matrix proteins showed a restrictive substrate specificity which includes some proteoglycans. Our results demonstrated that human ADAMTS1 cleaves aggrecan at a previously shown site by its mouse homolog, but we have also identified additional cleavage sites that ultimately confirm the classification of this protease as an 'aggrecanase'. Specificity of ADAMTS1 activity was further verified when a point mutation in the zinc-binding domain abolished its catalytic effects, and latency conferred by the prodomain was also demonstrated using a furin cleavage site mutant. Suppression of ADAMTS1 activity was accomplished with a specific monoclonal antibody and some metalloprotease inhibitors, including tissue inhibitor of metalloproteinases 2 and 3. Finally, we developed an activity assay using an artificial peptide substrate based on the interglobular domain cleavage site (E(373)-A) of rat aggrecan

The Cleavage of Semaphorin 3C Induced by ADAMTS1 Promotes Cell Migration*

Metastasis is a sequential process that allows cells to move from the primary tumor and grow elsewhere. Because of their ability to cleave a variety of extracellular signaling and adhesion molecules, metalloproteases have been long considered key components of the metastatic program. However, the function of certain metalloproteases, such as ADAMTS1, is not clear and seems to depend on the cellular environment and/or the stage of tumor progression. To characterize the function of ADAMTS1, we performed two alternative proteomic approaches, difference gel electrophoresis and stable isotope labeling by amino acids in cell culture, to identify novel substrates of the metalloprotease. Both techniques showed that overexpression of ADAMTS1 leads to the release of semaphorin 3C from the extracellular matrix. Although semaphorins are well known regulators of axon guidance, accumulating evidence shows that they may also participate in tumor progression. Here, we show that the cleavage of semaphorin 3C induced by ADAMTS1 promotes the migration of breast cancer cells, indicating that the co-expression of these molecules in tumors may contribute to the metastatic program

PARP inhibition down-regulates vimentin expression and inhibits endothelial-to-mesenchymal transition in HUVECs.

<p>Cell extracts from HUVEC either treated with vehicle or 40 µM DPQ were subjected to 2D electrophoresis as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003531#s4" target="_blank">Materials and Methods</a>. Image analysis software (DeCyder) indicated that seven proteins exhibited decreased expression in HUVEC treated with DPQ compared to untreated cells. Proteins were identified using MALDI-TOF. Spots labeled with arrows indicate proteins that were identified by mass spectrometry (see <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003531#pgen-1003531-g002" target="_blank">Figure 2</a>). (<b>A</b>) The spot with the arrow is vimentin. (<b>B</b>) PARP inhibition reduced the expression of both vimentin and Snail1 and up-regulated VE-cadherin in human endothelial cells (HUVEC) as determined by immunoblotting, indirect immunofluorescence (<b>C</b>), and mRNA levels (<b>D</b>). PARP inhibition decreased HUVEC cell migration (<b>E</b>). (**<i>P</i><0.01, ***<i>P<</i>0.001 PARP inhibitor groups <i>versus</i> DPQ).</p

PARP inhibition inhibits the acquisition of an EMT phenotype in malignant melanoma cells.

<p>Human melanoma G361 cells and murine B16- F10 melanoma cells (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1003531#pgen.1003531.s003" target="_blank">Figure S3</a>) were used for these experiments. Cells were treated with either DPQ (40 µM), PJ-34 (10 µM) or KU0058948 (100 nM) for 22 hours. IF, western blot or qPCR assays were performed to evaluate the effects of PARP inhibition on EMT markers. PARP inhibition reduced the expression of vimentin and Snail1 and up-regulated E-cadherin in human melanoma cells as determined by immunoblotting (<b>A</b>), indirect immunofluorescence (<b>B</b>), and mRNA levels (<b>C</b>). (*<i>P<</i>0.05, ***<i>P<</i>0.001, PARP Inhibitor groups <i>versus</i> the control). β-actin was used as an internal control for protein loading. (<b>D</b>) Snail1 and E-cadherin promoter activity are regulated by PARP inhibitors. Luciferase activity was determined after transfecting the constructions into G361 cells. Firefly Luciferase was standarized to the levels of Renilla Luciferase. Cells were cotransfected with 0.5 µg renilla as a transfection control and 0.5 µg of Snail1 or E-cadherin using jetPEI cationic polymer transfection reagent according to the manufacturer's instructions. Cells were compared in the presence or absence of serum (***<i>P<</i>0.001 control <i>versus</i> PJ-34). The expression of both Firefly and Renilla luciferase was analyzed 48 h after transfection. Cloning of the human Snail1 promoter (−869/+59) into pGL3 basic (Promega) was described previously (41). The E-Cadherin promoter was cloned into pGL3-basic (Promega) to generate pGL3-E-cadherin (−178/+92). (<b>E</b>) Inhibitory effect of PARP on B16F10 motility. Treatment with the PARP inhibitor PJ-34 (10 µM) decreased cell migration in vitro. Migration was quantified as distance between Wound Healing limits (*** <i>P<</i>0.001 control <i>versus</i> DPQ).</p

Decreased melanoma-induced lung metastasis following PARP inhibition.

<p>(<b>A</b>) Mice were inoculated with the murine melanoma cell line B16-F10-luc. Localization and the intensity of luciferase expression were monitored by <i>in vivo</i> bioluminescence imaging (dpi, days post cells injection). At the bottom of Figure A two lungs from vehicle (left) or DPQ (right) treated mice are shown. Lungs were extracted to analyze the number of melanoma foci. Quantification of luciferase activity over time shows the average light (photons) emission in photons/s (<b>B</b>) (**P<0.01; ***P<0.001 <i>versus</i> DPQ). (<b>C</b>) The number of metastatic foci/lung were counted macroscopically (***<i>P<</i>0.001). (<b>D</b>) Angiogenesis was measured using a specific endothelial cell marker (tomato lectin) and measured as blood vessels per mm² in tumor sections of lung metastasis (Columns, mean ± SE. *<i>P<</i>0.05, with respect to control and DPQ–treated mice. (<b>E</b>) Immunohistochemistry staining of Snail1 and E-Cadherin in lung metastasis and quantitation using ImageJ , colour deconvolution plugin (<b>F and G</b>) Kaplan-Meyer survival curve shows the survival advantage of DPQ-treated mice following intravenous tail injection of melanoma cells as previously described in mice treated with DPQ (<b>F</b>) or injected with B16F10 stably silenced for PARP-1 (<b>G</b>) (** <i>P<</i>0. 01).</p

Proteins differentially expressed and identified by mass spectrometry analysis in HUVEC.

<p>The level of expression of various proteins in HUVEC was altered following PARP inhibition as determined by 2D-DIGE, and the proteins were positively identified using mass spectrometry analysis. Of particular interest for this study was vimentin, the major structural protein of intermediary filaments (spot 1). Expression of this protein was decreased in HUVEC following PARP inhibition. The proteins were identified by MALDI-TOF. Sequence coverage (%) and number of peptides were identified with  = 1% FDR (false discovery rate cut-off against decoy-concatenated randomized database). Coverage and score was determined using the MASCOT algorithm. The average ratio of protein expression between the control and cells treated with the PARP inhibitor DPQ was determined in HUVEC.</p

Interaction between vimentin over-expression and the activation of EMT signaling pathway.

<p>(<b>A</b>) Over-expression of vimentin in G361 cells. (<b>B</b>) Forced expression of vimentin drives human breast tumor epithelial cells (MCF7) to a mesenchymal phenotype through the integrin-linked-kinase/GSK-3β axis. 5 mM LiCl was used to inhibit GSK-3β, as detected by the accumulation of beta-catenin. (<b>C</b>) ILK was knocked down to analyze the significance of the interaction between vimentin and ILK in promoting the transition to a mesenchymal phenotype.</p

PARP-1 or vimentin is sufficient to reverse EMT and confer increased cell motility.

<p>(<b>A</b>) Melanoma (G361) and endothelial (HUVEC) (<b>B</b>) cells were silenced for PARP-1 or vimentin and the expression levels of Axl, E-/VE-cadherin, Snail1, ILK, β-catenin, GSK-3β, PARP-1, and vimentin were determined by immunoblot. (<b>C</b>) HUVEC were silenced for vimentin and wound healing was measured. After over-expression of vimentin wound healing closure was measured in HUVEC cells (<b>D</b>) or B16-F10 (<b>E</b>). (<b>F</b>) Cell migration was analyzed in epithelial cell line Madin Darby canine kidney (MDCK) cells transfected with either GFP or GFP-vimentin using video-microscopy and MetaMorph Image Analysis software. While vimentin was able to increase the length of the trajectories in the absence or presence of hepatocyte growth factor (HGF), treatment with PARP inhibitor resulted in a sustained reduction in cell motility (*<i>P<</i>0.05 PJ-34 or olaparib <i>versus</i> control).</p