Noncoding RNA Contribution to Aging and Lifespan.

Aging is a multifactorial process characterized by an age-related decline in organismal fitness. This deterioration is the major risk factor for chronic diseases such as cardiovascular pathologies, neurodegeneration, or cancer, and it represents one of the main challenges of modern society. Therefore, understanding why and how we age would be a fundamental pillar to design strategies to promote a healthy aging. In the last decades, the study of the molecular bases of disease has been revolutionized by the discovery of different types of noncoding RNAs (ncRNAs) with regulatory potential. In this work, we will review the implication of ncRNAs in aging, with the aim to provide a first approach to the different aging-associated ncRNAs, their mechanism of action, and their potential relevance as therapeutic targets and disease biomarkers.

USP49 deubiquitinase regulates the mitotic spindle checkpoint and prevents aneuploidy.

The spindle assembly checkpoint (SAC) is an essential mechanism that ensures the accurate chromosome segregation during mitosis, thus preventing genomic instability. Deubiquitinases have emerged as key regulators of the SAC, mainly by determining the fate of proteins during cell cycle progression. Here, we identify USP49 deubiquitinase as a novel regulator of the spindle checkpoint. We show that loss of USP49 in different cancer cell lines impairs proliferation and increases aneuploidy. In addition, USP49-depleted cells overcome the arrest induced by the SAC in the presence of nocodazole. Finally, we report new binding partners of USP49, including ribophorin 1, USP44, and different centrins.

Healthspan and lifespan extension by fecal microbiota transplantation into progeroid mice.

The gut microbiome is emerging as a key regulator of several metabolic, immune and neuroendocrine pathways1,2. Gut microbiome deregulation has been implicated in major conditions such as obesity, type 2 diabetes, cardiovascular disease, non-alcoholic fatty acid liver disease and cancer3-6, but its precise role in aging remains to be elucidated. Here, we find that two different mouse models of progeria are characterized by intestinal dysbiosis with alterations that include an increase in the abundance of Proteobacteria and Cyanobacteria, and a decrease in the abundance of Verrucomicrobia. Consistent with these findings, we found that human progeria patients also display intestinal dysbiosis and that long-lived humans (that is, centenarians) exhibit a substantial increase in Verrucomicrobia and a reduction in Proteobacteria. Fecal microbiota transplantation from wild-type mice enhanced healthspan and lifespan in both progeroid mouse models, and transplantation with the verrucomicrobia Akkermansia muciniphila was sufficient to exert beneficial effects. Moreover, metabolomic analysis of ileal content points to the restoration of secondary bile acids as a possible mechanism for the beneficial effects of reestablishing a healthy microbiome. Our results demonstrate that correction of the accelerated aging-associated intestinal dysbiosis is beneficial, suggesting the existence of a link between aging and the gut microbiota that provides a rationale for microbiome-based interventions against age-related diseases.

Protease Silencing to Explore the Molecular Mechanisms of Cancer and Aging.

Proteases play key roles in the execution and regulation of most if not all biological functions, and alterations in their activity, expression, or location are associated with multiple pathological conditions, including cancer and aging. In this regard, the use of RNA interference-based approaches to specifically target the expression of individual proteases constitutes an invaluable tool to identify enzymes involved in central aspects of these processes and to explore their potential as targets of therapeutic interventions. Here we describe simple protocols to optimize and monitor the specific silencing of cancer- and aging-related proteases.

Loss of the deubiquitinase USP36 destabilizes the RNA helicase DHX33 and causes preimplantation lethality in mice.

Deubiquitinases are proteases with a wide functional diversity that profoundly impact multiple biological processes. Among them, the ubiquitin-specific protease 36 (USP36) has been implicated in the regulation of nucleolar activity. However, its functional relevance in vivo has not yet been fully described. Here, we report the generation of an Usp36-deficient mouse model to examine the function of this enzyme. We show that Usp36 depletion is lethal in preimplantation mouse embryos, where it blocks the transition from morula to blastocyst during embryonic development. USP36 reduces the ubiquitination levels and increases the stability of the DEAH-box RNA helicase DHX33, which is critically involved in ribosomal RNA synthesis and mRNA translation. In agreement with this finding, O-propargyl-puromycin incorporation experiments, Northern blot, and electron microscopy analyses demonstrated the role of USP36 in ribosomal RNA and protein synthesis. Finally, we show that USP36 down-regulation alters cell proliferation in human cancer cells by inducing both apoptosis and cell cycle arrest, and that reducing DHX33 levels through short hairpin RNA interference has the same effect. Collectively, these results support that Usp36 is essential for cell and organism viability because of its role in ribosomal RNA processing and protein synthesis, which is mediated, at least in part, by regulating DHX33 stability.

USP39 Deubiquitinase Is Essential for KRAS Oncogene-driven Cancer.

KRAS is the most frequently mutated oncogene in human cancer, but its therapeutic targeting remains challenging. Here, we report a synthetic lethal screen with a library of deubiquitinases and identify USP39, which encodes an essential splicing factor, as a critical gene for the viability of KRAS-dependent cells. We show that splicing fidelity inhibitors decrease preferentially the proliferation rate of KRAS-active cells. Moreover, depletion of DHX38, encoding an USP39-interacting splicing factor, also reduces the viability of these cells. In agreement with these results, USP39 depletion caused a significant reduction in pre-mRNA splicing efficiency, as demonstrated through RNA-seq experiments. Furthermore, we show that USP39 is up-regulated in lung and colon carcinomas and its expression correlates with KRAS levels and poor clinical outcome. Accordingly, our work provides critical information for the development of splicing-directed antitumor treatments and supports the potential of USP39-targeting strategies as the basis of new anticancer therapies.

The deubiquitinase USP54 is overexpressed in colorectal cancer stem cells and promotes intestinal tumorigenesis.

Ubiquitin-Specific Proteases (USPs) are deubiquitinating enzymes frequently deregulated in human malignancies. Here, we show that USP54 is overexpressed in intestinal stem cells and demonstrate that its downregulation in colorectal carcinoma cells impedes tumorigenesis. We have generated mutant mice deficient for this deubiquitinase, which are viable and fertile, and protected against chemically-induced colorectal carcinoma. Furthermore, we show that USP54 is upregulated in human colon cancer and associates with poor prognosis. In agreement with these results, Usp54 downregulation in mouse melanoma cells inhibits lung metastasis formation. Collectively, this work has uncovered the pro-tumorigenic properties of USP54, highlighting the importance of deubiquitinating enzymes as promising targets for the development of specific anti-cancer therapies.

Loss of the proteostasis factor AIRAPL causes myeloid transformation by deregulating IGF-1 signaling.

AIRAPL (arsenite-inducible RNA-associated protein-like) is an evolutionarily conserved regulator of cellular proteostasis linked to longevity in nematodes, but its biological function in mammals is unknown. We show herein that AIRAPL-deficient mice develop a fully-penetrant myeloproliferative neoplastic process. Proteomic analysis of AIRAPL-deficient mice revealed that this protein exerts its antineoplastic function through the regulation of the insulin/insulin-like growth factor 1 (IGF-1) signaling pathway. We demonstrate that AIRAPL interacts with newly synthesized insulin-related growth factor-1 receptor (IGF1R) polypeptides, promoting their ubiquitination and proteasome-mediated degradation. Accordingly, genetic and pharmacological IGF1R inhibitory strategies prevent the hematological disease found in AIRAPL-deficient mice as well as that in mice carrying the Jak2(V617F) mutation, thereby demonstrating the causal involvement of this pathway in the pathogenesis of myeloproliferative neoplasms. Consistent with its proposed role as a tumor suppressor of myeloid transformation, AIRAPL expression is widely abrogated in human myeloproliferative disorders. Collectively, these findings support the oncogenic relevance of proteostasis deregulation in hematopoietic cells, and they unveil novel therapeutic targets for these frequent hematological neoplasias.

Loss of GLUT4 induces metabolic reprogramming and impairs viability of breast cancer cells.

Metabolic reprogramming strategies focus on the normalization of metabolism of cancer cells and constitute promising targets for cancer treatment. Here, we demonstrate that the glucose transporter 4 (GLUT4) has a prominent role in basal glucose uptake in MCF7 and MDA-MB-231 breast cancer cells. We show that shRNA-mediated down-regulation of GLUT4 diminishes glucose uptake and induces metabolic reprogramming by reallocating metabolic flux to oxidative phosphorylation. This reallocation is reflected on an increased activity of the mitochondrial oxidation of pyruvate and lower lactate release. Altogether, GLUT4 inhibition compromises cell proliferation and critically affects cell viability under hypoxic conditions, providing proof-of-principle for the feasibility of using pharmacological approaches to inhibit GLUT4 in order to induce metabolic reprogramming in vivo in breast cancer models.

Identification of novel tumor suppressor proteases by degradome profiling of colorectal carcinomas.

Proteolytic enzymes play important roles during tumor development and progression through their ability to promote cell growth or by facilitating the invasion of surrounding tissues. The human genome contains more than 570 protease-coding genes, many of them forming functional networks, which has forced the use of global strategies for the analysis of this group of enzymes. In this study, we have designed a new quantitative PCR-based device for profiling the entire degradome in human malignancies. We have used this method to evaluate protease expression levels in colorectal carcinomas with the finding that most proteases with altered expression in these tumors exert their function in the extracellular compartment. In addition, we have found that among genes encoding repressed proteases there was a higher proportion with somatic mutations in colorectal cancer when compared to genes coding for upregulated proteases (14% vs. 4%, p<0.05). One of these genes, MASP3, is consistently repressed in colorectal carcinomas as well as in colorectal cancer cell lines when compared to normal colonic mucosa. Functional analysis of this gene revealed that ectopic expression of MASP3 reduces cell proliferation in vitro and restrains subcutaneous tumor growth, whereas its downregulation induces an increase in the tumorigenic potential of colorectal cancer cells. These results provide new insights into the diversity of proteases associated with cancer and support the utility of degradome profiling to identify novel proteases with tumor-defying functions.

Prelamin A causes progeria through cell-extrinsic mechanisms and prevents cancer invasion.

Defining the relationship between ageing and cancer is a crucial but challenging task. Mice deficient in Zmpste24, a metalloproteinase mutated in human progeria and involved in nuclear prelamin A maturation, recapitulate multiple features of ageing. However, their short lifespan and serious cell-intrinsic and cell-extrinsic alterations restrict the application and interpretation of carcinogenesis protocols. Here we present Zmpste24 mosaic mice that lack these limitations. Zmpste24 mosaic mice develop normally and keep similar proportions of Zmpste24-deficient (prelamin A-accumulating) and Zmpste24-proficient (mature lamin A-containing) cells throughout life, revealing that cell-extrinsic mechanisms are preeminent for progeria development. Moreover, prelamin A accumulation does not impair tumour initiation and growth, but it decreases the incidence of infiltrating oral carcinomas. Accordingly, silencing of ZMPSTE24 reduces human cancer cell invasiveness. Our results support the potential of cell-based and systemic therapies for progeria and highlight ZMPSTE24 as a new anticancer target.

ATG4B/autophagin-1 regulates intestinal homeostasis and protects mice from experimental colitis.

The identification of inflammatory bowel disease (IBD) susceptibility genes by genome-wide association has linked this pathology to autophagy, a lysosomal degradation pathway that is crucial for cell and tissue homeostasis. Here, we describe autophagy-related 4B, cysteine peptidase/autophagin-1 (ATG4B) as an essential protein in the control of inflammatory response during experimental colitis. In this pathological condition, ATG4B protein levels increase in parallel with the induction of autophagy. Moreover, ATG4B expression is significantly reduced in affected areas of the colon from IBD patients. Consistently, atg4b (-/-) mice present Paneth cell abnormalities, as well as an increased susceptibility to DSS-induced colitis. atg4b-deficient mice exhibit significant alterations in proinflammatory cytokines and mediators of the immune response to bacterial infections, which are reminiscent of those found in patients with Crohn disease or ulcerative colitis. Additionally, antibiotic treatments and bone marrow transplantation from wild-type mice reduced colitis in atg4b (-/-) mice. Taken together, these results provided additional evidence for the importance of autophagy in intestinal pathologies and describe ATG4B as a novel protective protein in inflammatory colitis. Finally, we propose that atg4b-null mice are a suitable model for in vivo studies aimed at testing new therapeutic strategies for intestinal diseases associated with autophagy deficiency.

Mutational analysis of BRCA1 and BRCA2 in hereditary breast and ovarian cancer families from Asturias (Northern Spain).

BACKGROUND: The prevalence of BRCA1 and BRCA2 mutations in Spain is heterogeneous and varies according to geographical origin of studied families. The contribution of these mutations to hereditary breast and ovarian cancer has not been previously investigated in Asturian populations (Northern Spain). METHODS: In the present work, 256 unrelated high-risk probands with breast and/or ovarian cancer from families living in Asturias were analyzed for the presence of a BRCA1 or BRCA2 gene mutation from October 2007 to May 2012. The entire coding sequences and each intron/exon boundaries of BRCA1/2 genes were screened both by direct sequencing and Multiplex Ligation-dependent Probe Amplification (MLPA). RESULTS: A total of 59 families (23%) were found to carry a pathogenic germ line mutation, 39 in BRCA1 and 20 in BRCA2. Twenty nine additional families (12%) carried an unknown significance variant. We detected 28 distinct pathogenic mutations (16 in BRCA1 and 12 in BRCA2), of which 3 mutations in BRCA1 (c.1674delA, c.1965C>A and c.2900_2901dupCT) and 5 in BRCA2 (c.262_263delCT, c.2095C>T, c.3263dupC, c.4030_4035delinsC, c.8042_8043delCA) had not been previously described.The novel mutations c.2900_2901dupCT in BRCA1 and c.4030_4035delinsC in BRCA2 occurred in 8 and 6 families respectively and clustered in two separated small geographically isolated areas suggesting a founder effect. These 2 mutations, together with the Galician BRCA1 mutation c.211A>G (9 families), and the common BRCA1 mutation c.3331_3334delCAAG (6 families), account for approximately 50% of all affected families. By contrast, very frequent mutations in other Spanish series such as the BRCA1 Ashkenazi founder mutation c.68_69delAG, was found in only one family. CONCLUSIONS: In this study we report the BRCA1 and BRCA2 spectrum of mutations and their geographical distribution in Asturias, which largely differ from other areas of Spain. Our findings may help design a first step recurrent mutation panel for screening high-risk breast and/or ovarian cancer families from this specific area.

Matrix metalloproteinase Mmp-1a is dispensable for normal growth and fertility in mice and promotes lung cancer progression by modulating inflammatory responses.

Human MMP-1 is a matrix metalloproteinase repeatedly associated with many pathological conditions, including cancer. Thus, MMP1 overexpression is a poor prognosis marker in a variety of advanced cancers, including colorectal, breast, and lung carcinomas. Moreover, MMP-1 plays a key role in the metastatic behavior of melanoma, breast, and prostate cancer cells. However, functional and mechanistic studies on the relevance of MMP-1 in cancer have been hampered by the absence of an in vivo model. In this work, we have generated mice deficient in Mmp1a, the murine ortholog of human MMP1. Mmp1a(-/-) mice are viable and fertile and do not exhibit obvious abnormalities, which has facilitated studies of cancer susceptibility. These studies have shown a decreased susceptibility to develop lung carcinomas induced by chemical carcinogens in Mmp1a(-/-) mice. Histopathological analysis indicated that tumors generated in Mmp1a(-/-) mice are smaller than those of wild-type mice, consistently with the idea that the absence of Mmp-1a hampers tumor progression. Proteomic analysis revealed decreased levels of chitinase-3-like 3 and accumulation of the receptor for advanced glycation end-products and its ligand S100A8 in lung samples from Mmp1a(-/-) mice compared with those from wild-type. These findings suggest that Mmp-1a could play a role in tumor progression by modulating the polarization of a Th1/Th2 inflammatory response to chemical carcinogens. On the basis of these results, we propose that Mmp1a knock-out mice provide an excellent in vivo model for the functional analysis of human MMP-1 in both physiological and pathological conditions.

Functional analysis of sucrase-isomaltase mutations from chronic lymphocytic leukemia patients.

Next-generation sequencing techniques have emerged as powerful tools for the understanding of cancer genomes. In recent years, whole-exome and whole-genome sequencing strategies have enabled the annotation of a comprehensive mutation landscape of chronic lymphocytic leukemia (CLL), the most frequent leukemia in western countries. Several recurrently mutated genes have been identified, with a subset being validated as neoplastic drivers. Still, a main challenge remains for the differentiation between driver and passenger mutations among candidates as well as for the functional description of the newly discovered leukemogenic genes that could be utilized for personalized anti-tumor strategies. In this scenario, we have identified the metabolic enzyme sucrase-isomaltase (SI) as one of the most frequently mutated genes in a cohort of 105 CLL patients. Here, we demonstrate that these SI mutations result in loss of enzyme function by preventing the biosynthesis of catalytically competent SI at the cell surface. Transcriptome analyses of RNA from CLL patients with SI loss-of-function mutations have uncovered gene expression patterns that depict ample metabolic reprogramming, pinpointing SI as a putative player in the cancer-associated metabolic switch. These results highlight SI as a relevant target for clinical evaluation in future CLL studies.

Nuclear lamina defects cause ATM-dependent NF-κB activation and link accelerated aging to a systemic inflammatory response.

Alterations in the architecture and dynamics of the nuclear lamina have a causal role in normal and accelerated aging through both cell-autonomous and systemic mechanisms. However, the precise nature of the molecular cues involved in this process remains incompletely defined. Here we report that the accumulation of prelamin A isoforms at the nuclear lamina triggers an ATM- and NEMO-dependent signaling pathway that leads to NF-κB activation and secretion of high levels of proinflammatory cytokines in two different mouse models of accelerated aging (Zmpste24(-/-) and Lmna(G609G/G609G) mice). Causal involvement of NF-κB in accelerated aging was demonstrated by the fact that both genetic and pharmacological inhibition of NF-κB signaling prevents age-associated features in these animal models, significantly extending their longevity. Our findings provide in vivo proof of principle for the feasibility of pharmacological modulation of the NF-κB pathway to slow down the progression of physiological and pathological aging.

Exome sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia.

Here we perform whole-exome sequencing of samples from 105 individuals with chronic lymphocytic leukemia (CLL), the most frequent leukemia in adults in Western countries. We found 1,246 somatic mutations potentially affecting gene function and identified 78 genes with predicted functional alterations in more than one tumor sample. Among these genes, SF3B1, encoding a subunit of the spliceosomal U2 small nuclear ribonucleoprotein (snRNP), is somatically mutated in 9.7% of affected individuals. Further analysis in 279 individuals with CLL showed that SF3B1 mutations were associated with faster disease progression and poor overall survival. This work provides the first comprehensive catalog of somatic mutations in CLL with relevant clinical correlates and defines a large set of new genes that may drive the development of this common form of leukemia. The results reinforce the idea that targeting several well-known genetic pathways, including mRNA splicing, could be useful in the treatment of CLL and other malignancies.