Gallic Acid: A Natural Phenolic Compound Exerting Antitumoral Activities in Colorectal Cancer via Interaction with G-Quadruplexes.

Natural phenolic compounds have gained momentum for the prevention and treatment of cancer, but their antitumoral mechanism of action is not yet well understood. In the present study, we screened the antitumoral potential of several phenolic compounds in a cellular model of colorectal cancer (CRC). We selected gallic acid (GA) as a candidate in terms of potency and selectivity and extensively evaluated its biological activity. We report on the role of GA as a ligand of DNA G-quadruplexes (G4s), explaining several of its antitumoral effects, including the transcriptional inhibition of ribosomal and CMYC genes. In addition, GA shared with other established G4 ligands some effects such as cell cycle arrest, nucleolar stress, and induction of DNA damage. We further confirmed the antitumoral and G4-stabilizing properties of GA using a xenograft model of CRC. Finally, we succinctly demonstrate that GA could be explored as a therapeutic agent in a patient cohort with CRC. Our work reveals that GA, a natural bioactive compound present in the diet, affects gene expression by interaction with G4s both in vitro and in vivo and paves the way towards G4s targeting with phenolic compounds.

Extracellular Protease ADAMTS1 Is Required at Early Stages of Human Uveal Melanoma Development by Inducing Stemness and Endothelial-Like Features on Tumor Cells.

Extracellular matrix remodeling within the tumor microenvironment has been recognized as a relevant dynamic framework during tumor growth. However, research on proteases that trigger this remodeling keeps revealing a wide range of actions including both pro- and anti-tumorigenic. The extracellular protease ADAMTS1 exemplifies this dual role. In this work, we first confirmed a positive correlation of ADAMTS1 with endothelial-like phenotype of human melanoma cells together with the finding of associated signatures, including key genes such as endothelial CDH5. Using a CRISPR-Cas9 approach, we observed that the inhibition of ADAMTS1 in an aggressive uveal melanoma model compromised its endothelial-like properties, and more importantly, caused a robust blockade on the progression of tumor xenografts. Although vasculature emerged affected in ADAMTS1-deficient tumors, the most relevant action implied the downregulation of endothelial CDH5 in tumor cells, in association with stemness markers. Indeed, melanoma sphere assays also revealed a deficient commitment to form spheres in the absence of ADAMTS1, directly correlating with stemness markers and, remarkably, also with CDH5. Finally, taking advantage of advanced bioinformatics tools and available public data of uveal melanomas, we disclosed new prognosis factors, including endothelial elements and ADAMTS proteases. Our findings support the key role of ADAMTS proteases for uveal melanoma development since earlier stages, modulating the complex crosstalk between extracellular matrix and the induction of stemness and endothelial-like features. To our knowledge, this is the first report that supports the development of therapeutic targets on the extracellular matrix to overcome uveal melanoma.

ADAMTS1 Supports Endothelial Plasticity of Glioblastoma Cells with Relevance for Glioma Progression.

Gliomas in general and the more advanced glioblastomas (GBM) in particular are the most usual tumors of the central nervous system with poor prognosis. GBM patients develop resistance to distinct therapies, in part due to the existence of tumor cell subpopulations with stem-like properties that participate in trans-differentiation events. Within the complex tumor microenvironment, the involvement of extracellular proteases remains poorly understood. The extracellular protease ADAMTS1 has already been reported to contribute to the plasticity of cancer cells. Accordingly, this basic knowledge and the current availability of massive sequencing data from human gliomas, reinforced the development of this work. We first performed an in silico study of ADAMTS1 and endothelial markers in human gliomas, providing the basis to further assess these molecules in several primary glioblastoma-initiating cells and established GBM cells with the ability to acquire an endothelial-like phenotype. Using a co-culture approach of endothelial and GBM cells, we noticed a relevant function of ADAMTS1 in GBM cells leading the organization of endothelial-like networks and, even more significantly, we found a blockade of the formation of tumor-spheres and a deficient response to hypoxia in the absence of ADAMTS1. Our data support a chief role of this protease modulating the phenotypic plasticity of GBM.

Inhibition of ADAMTS1 Expression by Lentiviral CRISPR/Cas9 Gene Editing Technology.

The continuous improvement of gene editing tools has allowed a major revolution in biological sciences. Although a variety of gain and loss-of-function approaches have been widely used for the last decades, some limitations arose from non-specific targeting or lack of complete inhibition of the gene of interest. CRISPR/Cas9 editing technology introduced new and significant advantages because it can directly modify the gene of interest and completely blocks its expression.In the context of cancer studies, the heterogeneity of the tumor microenvironment requires comprehensive approaches to unveil the contribution of multiple genes. For example, a deeper understanding of the biology of proteases such as ADAMTS (a disintegrin and metalloproteinase with thrombospondin type 1 motifs) will improve our perspective of complex phenomena affected by extracellular matrix remodeling, including embryonic development, angiogenesis, immune infiltration, metastasis, and tumor plasticity. Here, we present a method using CRISPR/Cas9 technology to inhibit the expression of the representative ADAMTS1 in cancer cells. Following the first steps of gene edition, we pursue further selection of silenced cells and provide a detailed description of sequence analysis and validation assays. This method leads to inactivation of ADAMTS1 in cancer cells, providing a relevant biological tool that will allow subsequent in vivo and in vitro ADAMTS1 functional analysis.

ADAMTS1 protease is required for a balanced immune cell repertoire and tumour inflammatory response.

Recent advances have emphasized the relevance of studying the extracellular microenvironment given its main contribution to tissue homeostasis and disease. Within this complex scenario, we have studied the extracellular protease ADAMTS1 (a disintegrin and metalloprotease with thrombospondin motif 1), implicated in vascularization and development, with reported anti- and pro-tumorigenic activities. In this work we performed a detailed study of the vasculature and substrates in adult organs of wild type and Adamts1-deficient mice. In addition to the expected alterations of organs like kidney, heart and aorta, we found that the lack of ADAMTS1 differently affects lymphocyte and myeloid populations in the spleen and bone marrow. The study of the substrate versican also revealed its alteration in the absence of the protease. With such premises, we challenged our mice with subcutaneous B16F1 syngeneic tumours and closely evaluated the immune repertoire in the tumours but also in the distant spleen and bone marrow. Our results confirmed a pro-inflammatory landscape in the absence of ADAMTS1, correlating with tumour blockade, supporting its novel role as a modulator of the immune cell response.

Evaluation of Tumor Vasculature Using a Syngeneic Tumor Model in Wild-Type and Genetically Modified Mice.

The relevance of tumor vasculature has been extensively recognized, and it is still the focus of numerous lines of research for basic, translational, and clinical scientists. Indeed, the knowledge of some of its regulatory mechanisms has provoked the generation of ongoing cancer therapies. Within the context of the tumor microenvironment, the information that the analysis of the vasculature provides is very valuable, and it might reveal not just its quality and the response against a specific therapy but also its close relationship with neighboring stromal and tumor players.Studies during last decades already supported the contribution of extracellular proteases in neovascularization events, including ADAMTS. However, deeper analyses are still required to better understand the modulation of their proteolytic activity in the tumor microenvironment. Future studies will clearly benefit from existing and ongoing genetically modified mouse models.Here we emphasize the use of syngeneic models to study the vasculature during tumor progression, supported by their intact immunocompetent capacities and also by the range of possibilities to play with engineered mice and with modified tumor cells. Although various high-tech and sophisticated approaches have already been reported to evaluate tumor neovascularization, here we describe a simple and easily reproduced methodology based in the immunofluorescence detection of vascular-specific molecules. A final in silico analysis guarantees an unbiased quantification of tumor vasculature under different conditions.

Relevance of IGFBP2 proteolysis in glioma and contribution of the extracellular protease ADAMTS1.

Expression of IGFBP2 (Insulin-like Growth Factor Binding Protein 2) has been positively correlated with glioma progression. Although the proteolysis of IGFBP2 has been widely recognized, with consequences as a major modulator of IGFII signaling, the relevance of this post-translational modification has not been well studied in tumors. Using an in vivo proteomic approach by Isotope-Coded Protein Label (ICPL), we identified IGFBP2 as a target of the extracellular protease ADAMTS1 (A Disintegrin And Metalloproteinase with ThromboSpondin motifs 1). Notably, the proteolytic pattern of IGFBP2 was also detected in human glioma culture cells and, more importantly, in all glioma samples evaluated. In addition, high expression of ADAMTS1 correlates with higher levels of cleaved IGFBP2 in glioblastoma multiforme cases. Using gene expression public databases, we confirmed that IGFBP2 is a poor prognosis marker for gliomas, and we also observed an important contribution of ADAMTS1.Finally, we showed the impact of ADAMTS1 on IGFII-mediated IGF1R phosphorylation and cellular migration. Our results support a functional interaction between IGFBP2 and ADAMTS1 and suggest the need to evaluate post-translational modifications of IGFBP2 in glioma, in order to approach new therapies.

Contribution of ADAMTS1 as a tumor suppressor gene in human breast carcinoma. Linking its tumor inhibitory properties to its proteolytic activity on nidogen-1 and nidogen-2.

The extracellular protease ADAMTS1 (A disintegrin and metalloprotease with thrombospondin repeats 1) has been described as an anti-angiogenic molecule and its role as a putative tumor protective molecule has also been suggested. Here, we have used a tumor xenograft model to determine the role of ADAMTS1 in tumor growth and angiogenesis. Increasing levels of the protease led to the complete inhibition of tumor growth. In an attempt to elucidate the mechanism of action of this protease, we focused our attention on its proteolytic activity on nidogens, one of the main components of the vascular basement membrane. The increased expression of ADAMTS1 was accompanied by increased proteolysis of nidogen-1 and -2 and their almost complete removal from vascular structures, together with major morphological alterations of tumor blood vessels and a decreased vessel density. The clinical relevance of this work is supported by our observations that ADAMTS1 expression is decreased in breast tumor specimens when compared with healthy tissue. Our studies also reveal that the cleavage of nidogen-1 and -2 is partially inhibited in human tumor samples. Moreover, the deposition of both nidogens surrounding vascular structures is drastically altered, implying a possible reduction in the maintenance of vessel integrity. Our studies reflect the requirement to explore the functional interactions between proteases and specific substrates in cancer biology.

A human ESC model for MLL-AF4 leukemic fusion gene reveals an impaired early hematopoietic-endothelial specification.

The MLL-AF4 fusion gene is a hallmark genomic aberration in high-risk acute lymphoblastic leukemia in infants. Although it is well established that MLL-AF4 arises prenatally during human development, its effects on hematopoietic development in utero remain unexplored. We have created a human-specific cellular system to study early hemato-endothelial development in MLL-AF4-expressing human embryonic stem cells (hESCs). Functional studies, clonal analysis and gene expression profiling reveal that expression of MLL-AF4 in hESCs has a phenotypic, functional and gene expression impact. MLL-AF4 acts as a global transcriptional activator and a positive regulator of homeobox gene expression in hESCs. Functionally, MLL-AF4 enhances the specification of hemogenic precursors from hESCs but strongly impairs further hematopoietic commitment in favor of an endothelial cell fate. MLL-AF4 hESCs are transcriptionally primed to differentiate towards hemogenic precursors prone to endothelial maturation, as reflected by the marked upregulation of master genes associated to vascular-endothelial functions and early hematopoiesis. Furthermore, we report that MLL-AF4 expression is not sufficient to transform hESC-derived hematopoietic cells. This work illustrates how hESCs may provide unique insights into human development and further our understanding of how leukemic fusion genes, known to arise prenatally, regulate human embryonic hematopoietic specification.

ADAMTS1 contributes to the acquisition of an endothelial-like phenotype in plastic tumor cells.

Cancer stem cells have been hypothesized to explain tumor plasticity, including the capability to adopt distinct differentiation commitments. Among the mechanisms of tumor neovascularization, the ability of some malignant cells to mimic an endothelial phenotype has been recognized by a capacity to form matrix-enriched pseudovascular structures. In addition to the expression of genes associated with an endothelial nature, the molecular dynamism of specific microenvironments may also be critical. Here, we report the identification of the extracellular protease ADAMTS1 as a critical molecule for tumor cells to acquire endothelial-like properties. In a fibrosarcoma model, ADAMTS1 increased tumor growth rate in an angiogenesis-independent manner, influencing the tumor cells to display an exclusive endothelial-like gene signature. We documented the relevant expression of ADAMTS1 in aggressive and highly plastic melanoma and Ewing sarcoma cells. Notably, inhibiting ADAMTS1 action compromised the endothelial mimetic attributes observed in this setting. Our findings provide insights into how the tumor microenvironment can elicit endothelial mimicry by tumor cells.

Cleavage of syndecan-4 by ADAMTS1 provokes defects in adhesion.

Syndecan-4 is a membrane-bound heparan sulfate proteoglycan that participates in cell-cell and cell-matrix interactions and modulates adhesion and migration of many cell types. Through its extracellular domain, syndecan-4 cooperates with adhesion molecules and binds matrix components relevant for cell migration. Importantly, syndecan-4 is a substrate of extracellular proteases, however the biological significance of this cleavage has not been elucidated. Here, we show that the secreted metalloprotease ADAMTS1, involved in angiogenesis and inflammatory processes, cleaves the ectodomain of syndecan-4. We further showed that this cleavage results in altered distribution of cytoskeleton components, functional loss of adhesion, and gain of migratory capacities. Using syndecan-4 null cells, we observed that ADAMTS1 proteolytic action mimics the outcome of genetic deletion of this proteoglycan with regards to focal adhesion. Our findings suggest that the shedding of syndecan-4 by ADAMTS1 disrupts cell adhesion and promotes cell migration.

Identification of substrates of the extracellular protease ADAMTS1 by DIGE proteomic analysis.

Proteolytic modification of components of the extracellular milieu by metalloproteinases plays important roles in the regulation of multiple cellular and physiological processes and pathological conditions. ADAMTS1 is a secreted enzyme of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteases, which is related to angiogenesis and inflammation processes. Here, we describe a proteomic screening for putative ADAMTS1 substrates by analyzing the protein profiles obtained from cultures of transfected cells overexpressing the protease as compared to parental cells. Conditioned medium proteins of cultures of the two cell lines have been quantitatively compared by DIGE. Proteins showing differential levels have been identified by MS techniques leading to the finding of five potential new substrates of ADAMTS1: the basement membrane proteins nidogen-1 and -2, the desmosomal protein desmocollin-3, and the extracellular glycoproteins dystroglycan 1 and Mac-2-binding protein. Nidogen-1 and -2 have been further validated as substrates by immunochemical analysis. Our results demonstrate the utility of the DIGE proteomic technique for the discovery of specific substrates of matrix proteases.