Alvaro J. Obaya

Cloning, expression analysis, and structural characterization of seven novel human ADAMTSs, a family of metalloproteinases with disintegrin and thrombospondin-1 domains

ADAMTS (A Disintegrin And Metalloproteinase domain, with ThromboSpondin type-1 modules) is a recently described family of zinc-dependent proteases which play important roles in a variety of normal and pathological conditions, including arthritis and cancer. In this work, we report the identification and cloning of cDNAs encoding seven new human ADAMTSs. These novel enzymes have been called ADAMTS-13, -14, -15, -16, -17, -18, and -19. All of them show a domain organization similar to that of previously characterized family members, consisting of a signal sequence, a propeptide, a metalloproteinase domain, a disintegrin-like domain, a cysteine-rich region, and a variable number of TS-1 repeats. Expression analysis revealed that these ADAMTS genes are mainly expressed in fetal tissues, especially in lung (ADAMTS14, ADAMTS16, ADAMTS17, ADAMTS18, and ADAMTS19), kidney (ADAMTS14, ADAMTS15, and ADAMTS16), and liver (ADAMTS13, ADAMTS15 and ADAMTS18). Reverse transcriptase--polymerase chain reaction analysis also revealed the expression of some of these new ADAMTSs in different human adult tissues, such as prostate (ADAMTS13, ADAMTS17, and ADAMTS18), and brain (ADAMTS13, ADAMTS16, ADAMTS17, and ADAMTS18). High levels of ADAMTSs transcripts were also observed in some tumor biopsies and cells lines, including osteosarcomas (ADAMTS19), melanoma and colon carcinoma cells (ADAMTS13). Chromosomal location analysis indicated that the seven identified ADAMTS genes are dispersed in the human genome mapping to 9q34, 10q21, 11q25, 5p15, 15q24, 16q23, and 5q31, respectively. According to these results, together with a comparative analysis of ADAMTSs in other eukaryotic organisms, we conclude that these enzymes, with at least 18 distinct members encoded within the human genome, represent an example of a widely expanded protease family during metazoan evolution.

Higher sensitivity of Adamts12-deficient mice to tumor growth and angiogenesis.

ADAMTS (a disintegrin and metalloproteinase domain with thrombospondin motifs) constitute a family of endopeptidases related to matrix metalloproteinases. These proteases have been largely implicated in tissue remodeling and angiogenesis associated with physiological and pathological processes. To elucidate the in vivo functions of ADAMTS-12, we have generated a knockout mouse strain (Adamts12−/−) in which Adamts12 gene was deleted. The mutant mice had normal gestations and no apparent defects in growth, life span and fertility. By applying three different in vivo models of angiogenesis (malignant keratinocyte transplantation, Matrigel plug and aortic ring assays) to Adamts12−/− mice, we provide evidence for a protective effect of this host enzyme toward angiogenesis and cancer progression. In the absence of Adamts-12, both the angiogenic response and tumor invasion into host tissue were increased. Complementing results were obtained by using medium conditioned by cells overexpressing human ADAMTS-12, which inhibited vessel outgrowth in the aortic ring assay. This angioinhibitory effect of ADAMTS-12 was independent of its enzymatic activity as a mutated inactive form of the enzyme was similarly efficient in inhibiting endothelial cell sprouting in the aortic ring assay than the wild-type form. Altogether, our results show that ADAMTS-12 displays antiangiogenic properties and protect the host toward tumor progression.

The ADAMTS12 metalloproteinase exhibits anti-tumorigenic properties through modulation of the Ras-dependent ERK signalling pathway

Members of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin motifs) family of proteolytic enzymes are implicated in a variety of physiological processes, such as collagen maturation, organogenesis, angiogenesis, reproduction and inflammation. Moreover, deficiency or overexpression of certain ADAMTS proteins is directly involved in serious human diseases, including cancer. However, the functional roles of other family members, such as ADAMTS12, remain unknown. Here, by using different in vitro and in vivo approaches, we have evaluated the possible role of ADAMTS12 in the development and progression of cancer. First, we show that expression of ADAMTS12 in Madin-Darby canine kidney (MDCK) cells prevents the tumorigenic effects of hepatocyte growth factor (HGF) by blocking the activation of the Ras-MAPK signalling pathway and that this regulation involves the thrombospondin domains of the metalloproteinase. We also show that addition of recombinant human ADAMTS12 to bovine aortic endothelial cells (BAE-1 cells) abolishes their ability to form tubules upon stimulation with vascular endothelial growth factor (VEGF). Additionally, tumours induced in immunodeficient SCID mice injected with A549 cells overexpressing ADAMTS12 show a remarkable growth deficiency in comparison with tumours formed in animals injected with parental A549 cells. Overall, our data suggest that ADAMTS12 confers tumour-protective functions upon cells that produce this proteolytic enzyme.

Genetic inactivation of ADAMTS15 metalloprotease in human colorectal cancer.

Matrix metalloproteinases have been traditionally linked to cancer dissemination through their ability to degrade most extracellular matrix components, thus facilitating invasion and metastasis of tumor cells. However, recent functional studies have revealed that some metalloproteases, including several members of the ADAMTS family, also exhibit tumor suppressor properties. In particular, ADAMTS1, ADAMTS9, and ADAMTS18 have been found to be epigenetically silenced in malignant tumors of different sources, suggesting that they may function as tumor suppressor genes. Herein, we show that ADAMTS15 is genetically inactivated in colon cancer. We have performed a mutational analysis of the ADAMTS15 gene in human colorectal carcinomas, with the finding of four mutations in 50 primary tumors and 6 colorectal cancer cell lines. Moreover, functional in vitro and in vivo studies using HCT-116 and SW-620 colorectal cancer cells and severe combined immunodeficient mice have revealed that ADAMTS15 restrains tumor growth and invasion. Furthermore, the presence of ADAMTS15 in human colorectal cancer samples showed a negative correlation with the histopathologic differentiation grade of the corresponding tumors. Collectively, these results provide evidence that extracellular proteases, including ADAMTS15, may be targets of inactivating mutations in human cancer and further validate the concept that secreted metalloproteases may show tumor suppressor properties.

The ADAMTS12 metalloprotease gene is epigenetically silenced in tumor cells and transcriptionally activated in the stroma during progression of colon cancer

Proteases have long been associated with tumor progression, given their ability to degrade extracellular matrix components and facilitate invasion and metastasis. However, recent findings indicate that different proteases can also act as tumor-suppressor enzymes. We have recently reported that lung carcinoma cells expressing the ADAMTS-12 metalloprotease show a remarkable impairment of growth in immunodeficient mice as compared with parental cells. Here, we show that ADAMTS12 promoter is hypermethylated in cancer cell lines and tumor tissues. Interestingly, ADAMTS12 expression in the stromal cells surrounding epithelial malignant cells is higher than in the paired normal tissues. Moreover, the expression of this metalloprotease in colon fibroblasts co-cultured with colon cancer cell lines is higher than in those cultured alone. Furthermore, the expression of ADAMTS-12 by these fibroblasts is linked with an anti-proliferative effect on tumor cells. Based on these findings, we hypothesize that ADAMTS-12 is a novel anti-tumor protease that can reduce the proliferative properties of tumor cells. This function is lost by epigenetic silencing in tumor cells, but concurrently induced in stromal cells, probably as part of a response of the normal tissue aimed at controlling the progression of cancer.

Epigenetic repression of ROR2 has a Wnt-mediated, pro-tumourigenic role in colon cancer

BackgroundWnt factors control cell differentiation through semi-independent molecular cascades known as the β-catenin-dependent (canonical) and -independent (non-canonical) Wnt signalling pathways. Genetic and epigenetic alteration of components of the canonical Wnt signalling pathway is one of the primary mechanisms underlying colon cancer. Despite increasing evidence of the role of the non-canonical pathways in tumourigenesis, however, the underlying molecular mechanisms are poorly understood.ResultsHere we report that the receptor tyrosine kinase-like orphan receptor 2 (ROR2), a transmembrane receptor for Wnt factors that activates non-canonical pathways, is frequently repressed by aberrant promoter hypermethylation in human colon cancer cell lines and primary tumours. By restoring ROR2 activity in colon cancer cells harbouring ROR2 promoter hypermethylation, we show that the role of ROR2 in colon cancer cells is mediated, at least in part, by canonical Wnt and that its epigenetic-dependent loss can be pro-tumourigenic.ConclusionsOur data show the importance of epigenetic alterations of ROR2 in colon cancer, highlighting the close interconnection between canonical and non-canonical Wnt signalling pathways in this type of tumour.

Cell-cell adhesion genes CTNNA2 and CTNNA3 are tumour suppressors frequently mutated in laryngeal carcinomas.

Laryngeal squamous cell carcinoma is a frequent and significant cause of morbidity and mortality. Here we explore the biological basis of this aggressive tumour, and identify two cell-cell adhesion genes as recurrently mutated in this malignancy. We first perform exome sequencing of four laryngeal carcinomas and their matched normal tissues. Among the 569 genes found to present somatic mutations, and based on their recurrence or functional relevance in cancer, we select 40 for further validation in 86 additional laryngeal carcinomas. We detect frequent mutations (14 of 90, 15%) in CTNNA2 and CTNNA3-encoding α-catenins. Functional studies reveal an increase in the migration and invasive ability of head and neck squamous cell carcinoma cells producing mutated forms of CTNNA2 and CTNNA3 or in cells where both α-catenins are silenced. Analysis of the clinical relevance of these mutations demonstrates that they are associated with poor prognosis. We conclude that CTNNA2 and CTNNA3 are tumour suppressor genes frequently mutated in laryngeal carcinomas.

Accelerated ageing : from mechanism to therapy through animal models

Ageing research benefits from the study of accelerated ageing syndromes such as Hutchinson-Gilford progeria syndrome (HGPS), characterized by the early appearance of symptoms normally associated with advanced age. Most HGPS cases are caused by a mutation in the gene LMNA, which leads to the synthesis of a truncated precursor of lamin A known as progerin that lacks the target sequence for the metallopotease FACE-1/ZMPSTE24 and remains constitutively farnesylated. The use of Face-1/Zmpste24-deficient mice allowed us to demonstrate that accumulation of farnesylated prelamin A causes severe abnormalities of the nuclear envelope, hyper-activation of p53 signalling, cellular senescence, stem cell dysfunction and the development of a progeroid phenotype. The reduction of prenylated prelamin A levels in genetically modified mice leads to a complete reversal of the progeroid phenotype, suggesting that inhibition of protein farnesylation could represent a therapeutic option for the treatment of progeria. However, we found that both prelamin A and its truncated form progerin can undergo either farnesylation or geranylgeranylation, revealing the need of targeting both activities for an efficient treatment of HGPS. Using Face-1/Zmpste24-deficient mice as model, we found that a combination of statins and aminobisphosphonates inhibits both types of modifications of prelamin A and progerin, improves the ageing-like symptoms of these mice and extends substantially their longevity, opening a new therapeutic possibility for human progeroid syndromes associated with nuclear-envelope defects. We discuss here the use of this and other animal models to investigate the molecular mechanisms underlying accelerated ageing and to test strategies for its treatment.

Immune-Dependent and Independent Antitumor Activity of GM-CSF Aberrantly Expressed by Mouse and Human Colorectal Tumors

Granulocyte-macrophage colony-stimulating factor (GM-CSF/CSF2) is a cytokine produced in the hematologic compartment that may enhance antitumor immune responses, mainly by activation of dendritic cells. Here, we show that more than one-third of human colorectal tumors exhibit aberrant DNA demethylation of the GM-CSF promoter and overexpress the cytokine. Mouse engraftment experiments with autologous and homologous colon tumors engineered to repress the ectopic secretion of GM-CSF revealed the tumor-secreted GM-CSF to have an immune-associated antitumor effect. Unexpectedly, an immune-independent antitumor effect was observed that depended on the ectopic expression of GM-CSF receptor subunits by tumors. Cancer cells expressing GM-CSF and its receptor did not develop into tumors when autografted into immunocompetent mice. Similarly, 100% of the patients with human colon tumors that overexpressed GM-CSF and its receptor subunits survived at least 5 years after diagnosis. These data suggest that expression of GM-CSF and its receptor subunits by colon tumors may be a useful marker for prognosis as well as for patient stratification in cancer immunotherapy.

The dual role of fibulins in tumorigenesis

The human fibulin family consists of seven complex extracellular glycoproteins originally characterized as components of elastic fibers in connective tissue. However, beyond its structural role, fibulins are involved in complex biological processes such as cell adhesion, migration or proliferation. Indeed, they have proved to be essential elements in normal physiology, as shown by mouse models lacking these proteins, that evidence several developmental abnormalities and pathological features. Their relevance is also apparent in tumorigenesis, an aspect that has started to be intensely studied. Distinct fibulins are expressed in both tumor and stromal cells and are subjected to multiple expression regulations with either anti or pro-tumor effects. The mechanistic insights that underlie these observations are now commencing to emerge, portraying these proteins as very versatile and active constituents of connective tissue. The aim of this review is to highlight the most relevant connections between fibulins and cancer.