Ana Gutiérrez-Fernández

Whole-genome sequencing identifies recurrent mutations in chronic lymphocytic leukaemia

Chronic lymphocytic leukaemia (CLL), the most frequent leukaemia in adults in Western countries, is a heterogeneous disease with variable clinical presentation and evolution. Two major molecular subtypes can be distinguished, characterized respectively by a high or low number of somatic hypermutations in the variable region of immunoglobulin genes. The molecular changes leading to the pathogenesis of the disease are still poorly understood. Here we performed whole-genome sequencing of four cases of CLL and identified 46 somatic mutations that potentially affect gene function. Further analysis of these mutations in 363 patients with CLL identified four genes that are recurrently mutated: notch 1 (NOTCH1), exportin 1 (XPO1), myeloid differentiation primary response gene 88 (MYD88) and kelch-like 6 (KLHL6). Mutations in MYD88 and KLHL6 are predominant in cases of CLL with mutated immunoglobulin genes, whereas NOTCH1 and XPO1 mutations are mainly detected in patients with unmutated immunoglobulins. The patterns of somatic mutation, supported by functional and clinical analyses, strongly indicate that the recurrent NOTCH1, MYD88 and XPO1 mutations are oncogenic changes that contribute to the clinical evolution of the disease. To our knowledge, this is the first comprehensive analysis of CLL combining whole-genome sequencing with clinical characteristics and clinical outcomes. It highlights the usefulness of this approach for the identification of clinically relevant mutations in cancer.

Accelerated ageing in mice deficient in Zmpste24 protease is linked to p53 signalling activation.

Zmpste24 (also called FACE-1) is a metalloproteinase involved in the maturation of lamin A (Lmna), an essential component of the nuclear envelope. Both Zmpste24- and Lmna-deficient mice exhibit profound nuclear architecture abnormalities and multiple histopathological defects that phenocopy an accelerated ageing process. Similarly, diverse human progeroid syndromes are caused by mutations in ZMPSTE24 or LMNA genes. To elucidate the molecular mechanisms underlying these devastating diseases, we have analysed the transcriptional alterations occurring in tissues from Zmpste24-deficient mice. We demonstrate that Zmpste24 deficiency elicits a stress signalling pathway that is evidenced by a marked upregulation of p53 target genes, and accompanied by a senescence phenotype at the cellular level and accelerated ageing at the organismal level. These phenotypes are largely rescued in Zmpste24-/-Lmna+/- mice and partially reversed in Zmpste24-/-p53-/- mice. These findings provide evidence for the existence of a checkpoint response activated by the nuclear abnormalities caused by prelamin A accumulation, and support the concept that hyperactivation of the tumour suppressor p53 may cause accelerated ageing.

Increased inflammation delays wound healing in mice deficient in collagenase-2 (MMP-8)

Matrix metalloproteinases (MMPs) have been implicated in numerous tissue‐remodeling processes. The finding that mice deficient in collagenase‐2 (MMP‐8) are more susceptible to develop skin cancer, prompted us to investigate the role of this protease in cutaneous wound healing. We have observed a significant delay in wound closure in MMP8−/− mice and an altered inflammatory response in their wounds, with a delay of neutrophil infiltration during the first days and a persistent inflammation at later time points. These changes were accompanied by alterations in the TGF‐β1 signaling pathway and by an apoptosis defect in MMP8−/− mice. The delay in wound healing observed in MMP8−/− mice was rescued by bone marrow transplantation from wild‐type mice. Analysis of other MMPs showed that MMP8−/−mice had a significant increase in the expression of MMP‐9, suggesting that both proteases might act coordi‐nately in this process. This possibility was further supported by the novel finding that MMP‐8 and MMP‐9 form specific complexes in vivo. Taken together, these data indicate that MMP‐8 participates in wound repair by contributing to the resolution of inflammation and open the possibility to develop new strategies for treating wound healing defects.—Gutierrez‐Fernandez, A., Inada, M., Balbín, M., Fueyo, A., Pitiot, A. S., Astudillo, A., Hirose, K., Hirata, M., Shapiro, S. D., Noel, A., Werb, Z., Krane, S. M. Lopez‐Otín, C., Puente, X. S. FASEB J. 21, 2580–2591 (2007)

Matrix Metalloproteinase-8 Functions as a Metastasis Suppressor through Modulation of Tumor Cell Adhesion and Invasion

Collagenase-2 (matrix metalloproteinase-8, MMP-8) is an MMP mainly produced by neutrophils and associated with many inflammatory conditions. We have previously described that MMP-8 plays a protective role in cancer through its ability to regulate the inflammatory response induced by carcinogens. Moreover, it has been reported that experimental manipulation of the expression levels of this enzyme alters the metastatic behavior of human breast cancer cells. In this work, we have used mutant mice deficient in MMP-8 and syngenic melanoma and lung carcinoma tumor cells lines overexpressing this enzyme to further explore the putative antimetastatic potential of MMP-8. We report herein that MMP-8 prevents metastasis formation through the modulation of tumor cell adhesion and invasion. Thus, tumor cells overexpressing MMP-8 have an increased adhesion to extracellular matrix proteins, whereas their invasive ability through Matrigel is substantially reduced when compared with control cells. Analysis of MMP-8 in breast cancer patients revealed that the expression of this metalloproteinase by breast tumors correlates with a lower incidence of lymph node metastasis and confers good prognosis to these patients. On this basis, we propose that MMP-8 is a tumor protective factor, which also has the ability to reduce the metastatic potential of malignant cells in both mice and human.

Exome sequencing and functional analysis identifies BANF1 mutation as the cause of a hereditary progeroid syndrome.

Accelerated aging syndromes represent a valuable source of information about the molecular mechanisms involved in normal aging. Here, we describe a progeroid syndrome that partially phenocopies Hutchinson-Gilford progeria syndrome (HGPS) but also exhibits distinctive features, including the absence of cardiovascular deficiencies characteristic of HGPS, the lack of mutations in LMNA and ZMPSTE24, and a relatively long lifespan of affected individuals. Exome sequencing and molecular analysis in two unrelated families allowed us to identify a homozygous mutation in BANF1 (c.34G>A [p.Ala12Thr]), encoding barrier-to-autointegration factor 1 (BAF), as the molecular abnormality responsible for this Mendelian disorder. Functional analysis showed that fibroblasts from both patients have a dramatic reduction in BAF protein levels, indicating that the p.Ala12Thr mutation impairs protein stability. Furthermore, progeroid fibroblasts display profound abnormalities in the nuclear lamina, including blebs and abnormal distribution of emerin, an interaction partner of BAF. These nuclear abnormalities are rescued by ectopic expression of wild-type BANF1, providing evidence for the causal role of this mutation. These data demonstrate the utility of exome sequencing for identifying the cause of rare Mendelian disorders and underscore the importance of nuclear envelope alterations in human aging.

A genomic view of the complexity of mammalian proteolytic systems.

Proteolytic enzymes play an essential role in different physiological processes, including development, reproduction and host defence, as well as in numerous pathologies, like inflammatory diseases, neurological disorders or cancer. The completion of the human genome sequence allowed us to determine that more than 2% of all human genes are proteases or protease inhibitors, reflecting the importance of proteolysis in human biology. To understand better the complexity of proteases in human and other model organisms, we have used the available genome sequences of different mammalian organisms, including mouse, rat and chimpanzee, to identify and compare their degradomes, the complete set of protease genes in these species. Surprisingly, the rodent protease complement is more complex when compared with that of primates, mainly due to the expansion of protease families implicated in reproduction and host defence. Similarly, most differences between human and chimpanzee proteases are found in genes implicated in the immune system, which might explain some of the differences between both organisms. We have also found several genes implicated in reproduction, nutrition and the immune system, which are functional in rat, mouse or chimpanzee, but have been inactivated by mutations in the human lineage. These findings suggest that pseudogenization of specific protease genes has been a mechanism contributing to the evolution of the human genome. Finally, we found that proteases implicated in human hereditary diseases, and especially in neurodegenerative disorders, are highly conserved among mammals.

Comparative analysis of cancer genes in the human and chimpanzee genomes

BackgroundCancer is a major medical problem in modern societies. However, the incidence of this disease in non-human primates is very low. To study whether genetic differences between human and chimpanzee could contribute to their distinct cancer susceptibility, we have examined in the chimpanzee genome the orthologous genes of a set of 333 human cancer genes.ResultsThis analysis has revealed that all examined human cancer genes are present in chimpanzee, contain intact open reading frames and show a high degree of conservation between both species. However, detailed analysis of this set of genes has shown some differences in genes of special relevance for human cancer. Thus, the chimpanzee gene encoding p53 contains a Pro residue at codon 72, while this codon is polymorphic in humans and can code for Arg or Pro, generating isoforms with different ability to induce apoptosis or interact with p73. Moreover, sequencing of the BRCA1 gene has shown an 8 Kb deletion in the chimpanzee sequence that prematurely truncates the co-regulated NBR2 gene.ConclusionThese data suggest that small differences in cancer genes, as those found in tumor suppressor genes, might influence the differences in cancer susceptibility between human and chimpanzee. Nevertheless, further analysis will be required to determine the exact contribution of the genetic changes identified in this study to the different cancer incidence in non-human primates.

Collagenase-2 (MMP-8) and matrilysin-2 (MMP-26) expression in human wounds of different etiologies.

Wound healing involves highly controlled events including reepithelialization, neoangiogenesis, and reformation of the stromal compartment. Matrix metalloproteinases (MMPs) are a family of neutral zinc‐dependent endopeptidases known to be essential for the wound‐healing process. MMP‐8 (collagenase‐2) is a neutrophil‐derived highly effective type I collagenase, recently indicated to be important for acute wound healing. MMP‐26 is a more recent and less well‐studied member of the MMP family. Our aim was to study the expression of MMP‐8 and MMP‐26 in human cutaneous wound repair and chronic wounds using histological methods and cell culture. MMP‐8 expression was associated with epithelial cells, neutrophils, and other inflammatory cells in chronic human wounds. MMP‐26 was prominently expressed in the extracellular compartment of most chronic wounds in close vicinity to the basement membrane area. MMP‐26 was also expressed in acute day 1 wounds with declining expression thereafter. In vitro wound experiments showed that both MMP‐8 and MMP‐26 were expressed by migrating human mucosal keratinocytes. Inhibiting MMP‐26 resulted in aberrant keratinocyte migration and proliferation. We conclude that MMP‐8 and MMP‐26 are differentially expressed in acute and chronic wounds.

NF-κB activation impairs somatic cell reprogramming in ageing.

Ageing constitutes a critical impediment to somatic cell reprogramming. We have explored the regulatory mechanisms that constitute age-associated barriers, through derivation of induced pluripotent stem cells (iPSCs) from individuals with premature or physiological ageing. We demonstrate that NF-κB activation blocks the generation of iPSCs in ageing. We also show that NF-κB repression occurs during cell reprogramming towards a pluripotent state. Conversely, ageing-associated NF-κB hyperactivation impairs the generation of iPSCs by eliciting the reprogramming repressor DOT1L, which reinforces senescence signals and downregulates pluripotency genes. Genetic and pharmacological NF-κB inhibitory strategies significantly increase the reprogramming efficiency of fibroblasts from Néstor–Guillermo progeria syndrome and Hutchinson–Gilford progeria syndrome patients, as well as from normal aged donors. Finally, we demonstrate that DOT1L inhibition in vivo extends lifespan and ameliorates the accelerated ageing phenotype of progeroid mice, supporting the interest of studying age-associated molecular impairments to identify targets of rejuvenation strategies.

Collagenase-2 Deficiency or Inhibition Impairs Experimental Autoimmune Encephalomyelitis in Mice

Matrix metalloproteinases (MMPs) have been implicated in a variety of human diseases, including neuroimmunological disorders such as multiple sclerosis. However, the recent finding that some MMPs play paradoxical protective roles in these diseases has made necessary the detailed study of the specific function of each family member in their pathogenesis. To determine the relevance of collagenase-2 (MMP-8) in experimental autoimmune encephalomyelitis (EAE), an animal model for multiple sclerosis, we have performed two different analyses involving genetic and biochemical approaches. First, we have analyzed the development of EAE in mutant mouse deficient in MMP-8, with the finding that the absence of this proteolytic enzyme is associated with a marked reduction in the clinical symptoms of EAE. We have also found that MMP-8-/- mice exhibit a marked reduction in central nervous system-infiltrating cells and demyelinating lesions. As a second approach, we have carried out a pharmacological inhibition of MMP-8 with a selective inhibitor against this protease (IC50 = 0.4 nm). These studies have revealed that the administration of the MMP-8 selective inhibitor to mice with EAE also reduces the severity of the disease. Based on these findings, we conclude that MMP-8 plays an important role in EAE development and propose that this enzyme may be a novel therapeutic target in human neuro-inflammatory diseases such as multiple sclerosis.