PanCancer analysis of somatic mutations in repetitive regions reveals recurrent mutations in snRNA U2.

Current somatic mutation callers are biased against repetitive regions, preventing the identification of potential driver alterations in these loci. We developed a mutation caller for repetitive regions, and applied it to study repetitive non protein-coding genes in more than 2200 whole-genome cases. We identified a recurrent mutation at position c.28 in the gene encoding the snRNA U2. This mutation is present in B-cell derived tumors, as well as in prostate and pancreatic cancer, suggesting U2 c.28 constitutes a driver candidate associated with worse prognosis. We showed that the GRCh37 reference genome is incomplete, lacking the U2 cluster in chromosome 17, preventing the identification of mutations in this gene. Furthermore, the 5'-flanking region of WDR74, previously described as frequently mutated in cancer, constitutes a functional copy of U2. These data reinforce the relevance of non-coding mutations in cancer, and highlight current challenges of cancer genomic research in characterizing mutations affecting repetitive genes.

Recurrent noncoding U1 snRNA mutations drive cryptic splicing in SHH medulloblastoma.

In cancer, recurrent somatic single-nucleotide variants-which are rare in most paediatric cancers-are confined largely to protein-coding genes1-3. Here we report highly recurrent hotspot mutations (r.3A>G) of U1 spliceosomal small nuclear RNAs (snRNAs) in about 50% of Sonic hedgehog (SHH) medulloblastomas. These mutations were not present across other subgroups of medulloblastoma, and we identified these hotspot mutations in U1 snRNA in only <0.1% of 2,442 cancers, across 36 other tumour types. The mutations occur in 97% of adults (subtype SHHδ) and 25% of adolescents (subtype SHHα) with SHH medulloblastoma, but are largely absent from SHH medulloblastoma in infants. The U1 snRNA mutations occur in the 5' splice-site binding region, and snRNA-mutant tumours have significantly disrupted RNA splicing and an excess of 5' cryptic splicing events. Alternative splicing mediated by mutant U1 snRNA inactivates tumour-suppressor genes (PTCH1) and activates oncogenes (GLI2 and CCND2), and represents a target for therapy. These U1 snRNA mutations provide an example of highly recurrent and tissue-specific mutations of a non-protein-coding gene in cancer.

The U1 spliceosomal RNA is recurrently mutated in multiple cancers.

Cancers are caused by genomic alterations known as drivers. Hundreds of drivers in coding genes are known but, to date, only a handful of noncoding drivers have been discovered-despite intensive searching1,2. Attention has recently shifted to the role of altered RNA splicing in cancer; driver mutations that lead to transcriptome-wide aberrant splicing have been identified in multiple types of cancer, although these mutations have only been found in protein-coding splicing factors such as splicing factor 3b subunit 1 (SF3B1)3-6. By contrast, cancer-related alterations in the noncoding component of the spliceosome-a series of small nuclear RNAs (snRNAs)-have barely been studied, owing to the combined challenges of characterizing noncoding cancer drivers and the repetitive nature of snRNA genes1,7,8. Here we report a highly recurrent A>C somatic mutation at the third base of U1 snRNA in several types of tumour. The primary function of U1 snRNA is to recognize the 5' splice site via base-pairing. This mutation changes the preferential A-U base-pairing between U1 snRNA and the 5' splice site to C-G base-pairing, and thus creates novel splice junctions and alters the splicing pattern of multiple genes-including known drivers of cancer. Clinically, the A>C mutation is associated with heavy alcohol use in patients with hepatocellular carcinoma, and with the aggressive subtype of chronic lymphocytic leukaemia with unmutated immunoglobulin heavy-chain variable regions. The mutation in U1 snRNA also independently confers an adverse prognosis to patients with chronic lymphocytic leukaemia. Our study demonstrates a noncoding driver in spliceosomal RNAs, reveals a mechanism of aberrant splicing in cancer and may represent a new target for treatment. Our findings also suggest that driver discovery should be extended to a wider range of genomic regions.

Pleiotropic functions of the tumor- and metastasis-suppressing matrix metalloproteinase-8 in mammary cancer in MMTV-PyMT transgenic mice.

INTRODUCTION: Matrix metalloproteinase-8 (MMP-8; neutrophil collagenase) is an important regulator of innate immunity that has oncosuppressive actions in numerous tumor types. METHODS: We have intercrossed Mmp8-null mice with the Polyoma virus middle T oncogene-driven (MMTV-PyMT) mouse model of mammary cancer to explore the effects of loss of MMP-8 on the incidence and progression of mammary carcinomas. RESULTS: In this aggressive mouse model of breast cancer, loss of MMP-8 accelerated tumor onset even further, such that 90% of MMTV-PyMT; Mmp8-null female mice were tumor-bearing at the time of weaning. Throughout the 14 weeks of the model, tumor burden increased in homozygous Mmp8-null mice compared to Mmp8-wild-type and -heterozygote animals. Likewise, lung metastasis dramatically increased in the MMTV-PyMT; Mmp8-null mice. Immunohistochemistry revealed that tumors in wild-type, Mmp8-heterozygotes and -null animals had similar vascular density at 8 weeks, but at 10 weeks Mmp8-wild-type tumors had a lower vascularity than their heterozygote and null counterparts. No differences in macrophage infiltration were apparent throughout primary tumor development, though at 10 weeks a drop in neutrophil infiltrates was observed in Mmp8-wild-type tumors. Using quantitative real-time RT-PCR, we tracked the expression of the entire Mmp and Timp gene families, observing a significant decrease in Mmp3 expression in Mmp8-null tumors compared to wild-type and heterozygotes throughout the time course of the model, which was confirmed at the protein level. CONCLUSIONS: These findings provide novel insight into the suppressive action of MMP-8 on mammary tumorigenesis and metastasis, and indicate that the loss of MMP-8 likely has pleiotropic effects on innate immunity and angiogenesis that are reflected in changes in the protease web.

Effects of azacitidine on matrix metalloproteinase-9 in acute myeloid leukemia and myelodysplasia.

Matrix metalloprotease-9 (MMP9) plays a critical role in acute myeloid leukemia (AML) by increasing the invasive properties of malignant myeloblasts. The role of this enzyme in high-risk myelodysplastic diseases (MDS) and the effect of azacitidine on its expression in MDS and AML have not been studied in detail. In this work, we have analyzed the effect of different concentrations of azacitidine in two well-established, MDS-derived, acute myeloid leukemic cell lines: MOLM-13 and SKM-1. We have demonstrated that 1 µmol/L azacitidine decreases MMP9 DNA methylation levels and that this is correlated with a significant increase in messenger RNA expression in both cell lines. Surprisingly, changes in protein levels were minor. This paradoxic effect is explained by the drug-dependent induction of apoptosis that reduces the amount of active secreting cells. A balance between induced expression and apoptosis was established at an azacitidine concentration of 0.2 µmol/L in MOLM-13 cells. This dose significantly increased the invasive capacity of viable cells, as measured in the Matrigel assay. To evaluate the clinical relevance of this observation, we have examined the effect of azacitidine on MMP9 expression in bone marrow from five patients with MDS, with the finding that this drug significantly increased MMP9 protein levels in all analyzed patients after six cycles of treatment. Based on these results, we conclude that azacitidine increases MMP9 expression and may enhance invasiveness in vitro. Because all five patients relapsed, these findings might explain, at least partially, the clinical failure of the drug and the progression to a more aggressive disease.

New and paradoxical roles of matrix metalloproteinases in the tumor microenvironment.

Processes such as cell proliferation, angiogenesis, apoptosis, or invasion are strongly influenced by the surrounding microenvironment of the tumor. Therefore, the ability to change these surroundings represents an important property through which tumor cells are able to acquire specific functions necessary for tumor growth and dissemination. Matrix metalloproteinases (MMPs) constitute key players in this process, allowing tumor cells to modify the extracellular matrix (ECM) and release cytokines, growth factors, and other cell-surface molecules, ultimately facilitating protease-dependent tumor progression. Remodeling of the ECM by collagenolytic enzymes such as MMP1, MMP8, MMP13, or the membrane-bound MT1-MMP as well as by other membrane-anchored proteases is required for invasion and recruitment of novel blood vessels. However, the multiple roles of the MMPs do not all fit into a simple pattern. Despite the pro-tumorigenic function of certain metalloproteinases, recent studies have shown that other members of these families, such as MMP8 or MMP11, have a protective role against tumor growth and metastasis in animal models. These studies have been further expanded by large-scale genomic analysis, revealing that the genes encoding metalloproteinases, such as MMP8, MMP27, ADAM7, and ADAM29, are recurrently mutated in specific tumors, while several ADAMTSs are epigenetically silenced in different cancers. The importance of these proteases in modifying the tumor microenvironment highlights the need for a deeper understanding of how stroma cells and the ECM can modulate tumor progression.

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.

Chemokine monocyte chemoattractant protein-3 in progressive periodontal lesions in patients with chronic periodontitis.

BACKGROUND: Chemokines are central in the activation and direction of leukocyte subsets to target tissues. However, the monocyte chemoattractant protein-3 (MCP-3) has not been associated with chronic periodontitis. Chronic periodontitis is an infection showing episodic supporting tissue destruction. The aim of this study is to determine the levels and expression of MCP-3 in periodontal sites characterized by active periodontal connective tissue destruction. METHODS: The study population consisted of 15 patients with a progression of periodontitis (15 of 56 patients), 18 patients with chronic periodontitis, and 10 healthy subjects without periodontal disease. As determined by the tolerance method, the 15 patients with moderate to advanced chronic periodontitis showed a progression of periodontitis over a 4-month period. Periodontitis was characterized by at least six sites with a probing depth >or=5 mm, clinical attachment level >or=3 mm, and radiographic bone loss. Gingival crevicular fluid was collected using a paper strip. The total protein concentration was determined. An enzyme-linked immunosorbent assay was performed to determine the total amount of MCP-3, and an immunoWestern blot was conducted to assess molecular MCP-3 forms. To determine the MCP-3 expression by immunohistochemistry, gingival biopsies were obtained from patients with chronic periodontitis and healthy subjects during third-molar extraction surgery. Statistical analyses were performed using statistical software. Data were expressed as subject means +/- SD, using the chi(2) and Student t tests. RESULTS: The total amount and concentration of chemokine MCP-3 were significantly higher in patients with chronic periodontitis than in healthy subjects (8.25 pg versus 0.53 pg, P = 0.006 and 2.95 pg/microl versus 0.45 pg/microl, P = 0.04, respectively). Active sites showed a significantly higher total amount and concentration of MCP-3 than inactive sites (11.12 versus 2.88 pg, P value = 0.005 and 3.95 versus 1.02, P value = 0.005, respectively). Western blot and immunohistochemical staining confirmed the presence of MCP-3 in periodontal disease, with observable differences between patients with chronic periodontitis and healthy subjects. CONCLUSIONS: MCP-3 was highly expressed in patients with chronic periodontitis, particularly in those with progressive periodontal lesions. MCP-3 could be involved in the recruitment of inflammatory cells toward periodontal tissues during the progression of the disease.

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.

Human beta-defensin-1 and -2 and matrix metalloproteinase-25 and -26 expression in chronic and aggressive periodontitis and in peri-implantitis.

OBJECTIVE: Aberrant matrix metalloproteinase (MMP) and human beta-defensin (HBD) functions have been found in inflammatory diseases. The objectives of this study were to investigate the immunolocalisation, mRNA expression and molecular forms of MMP-25, MMP-26, HBD-1 and HBD-2 in chronic and aggressive periodontitis and in peri-implantitis. The expression of MMP-25 by cultured human plasmacytoma cells and macrophages, and the effects of MMP-26 and Porphyromonas gingivalis trypsin-like proteinase on HBD-1 and -2 were also studied. DESIGN: Immunohistochemistry, immunofluorescent analysis, reverse transcriptase polymerase chain reaction and immunoblotting were used to assess localisation, mRNA expression and molecular forms of MMP-25, MMP-26, HBD-1 and HBD-2. HBD degradation by MMP-26 and P. gingivalis proteinase was studied by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. RESULTS: MMP-25 was present in plasma cells and polymorphonuclear leucocytes, and MMP-26 was present in oral and sulcular basement membrane zones. HBD-1 was distributed perivasculary in gingival connective tissue and in oral and sulcular epithelium, and HBD-2 was found to a lesser extent in the perivascular space. Low MMP-25, MMP-26, HBD-1 and HBD-2 mRNA expression was found. Immunoblot revealed 29-57-kDa MMP-25 in myeloma cell lysates, but not in macrophages, and partly activated MMP-25 and -26 in diseased gingival crevicular fluid and peri-implant sulcular fluid. P. gingivalis trypsin-like proteinase degraded HBD-1 and -2. CONCLUSIONS: Both MMP-25 and -26 were expressed more strongly in extensively inflamed gingiva compared with healthy gingiva. The expression of HBD-1 was stronger than that of HBD-2 in periodontitis and peri-implantitis. De-novo expression of MMP-25 and -26 is associated with periodontal and peri-implant inflammation. Furthermore, P. gingivalis trypsin-like proteinase, but not MMP-26, can degrade HBD-1 and -2, which could lead to a weakened innate immune response.

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-beta1 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 coordinately 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.

Comparative analysis of cancer genes in the human and chimpanzee genomes.

BACKGROUND: Cancer 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. RESULTS: This 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. CONCLUSION: These 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.

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.

Cloning and enzymatic analysis of 22 novel human ubiquitin-specific proteases.

We have identified and cloned 22 human cDNAs encoding novel members of the ubiquitin-specific protease (USP) family. Eighteen of the identified proteins contain all structural features characteristic of these cysteine proteinases, whereas four of them have been classified as non-peptidase homologues. Northern blot analysis demonstrated that the identified USPs are broadly and differentially distributed in human tissues, some of them being especially abundant in skeletal muscle or testis. Enzymatic studies performed with the identified USPs revealed that at least twelve of them are deubiquitylating enzymes based on their ability to cleave ubiquitin from a ubiquitin-beta-galactosidase fusion protein. These results provide additional evidence of the extreme complexity and diversity of the USP proteolytic system in human tissues and open the possibility to explore the relevance of their multiple components in the regulation of ubiquitin-mediated pathways in normal and pathological functions.