RNA damage compartmentalization by DHX9 stress granules.

Biomolecules incur damage during stress conditions, and damage partitioning represents a vital survival strategy for cells. Here, we identified a distinct stress granule (SG), marked by dsRNA helicase DHX9, which compartmentalizes ultraviolet (UV)-induced RNA, but not DNA, damage. Our FANCI technology revealed that DHX9 SGs are enriched in damaged intron RNA, in contrast to classical SGs that are composed of mature mRNA. UV exposure causes RNA crosslinking damage, impedes intron splicing and decay, and triggers DHX9 SGs within daughter cells. DHX9 SGs promote cell survival and induce dsRNA-related immune response and translation shutdown, differentiating them from classical SGs that assemble downstream of translation arrest. DHX9 modulates dsRNA abundance in the DHX9 SGs and promotes cell viability. Autophagy receptor p62 is activated and important for DHX9 SG disassembly. Our findings establish non-canonical DHX9 SGs as a dedicated non-membrane-bound cytoplasmic compartment that safeguards daughter cells from parental RNA damage.

Histone variant H2A.Z regulates zygotic genome activation.

During embryogenesis, the genome shifts from transcriptionally quiescent to extensively active in a process known as Zygotic Genome Activation (ZGA). In Drosophila, the pioneer factor Zelda is known to be essential for the progression of development; still, it regulates the activation of only a small subset of genes at ZGA. However, thousands of genes do not require Zelda, suggesting that other mechanisms exist. By conducting GRO-seq, HiC and ChIP-seq in Drosophila embryos, we demonstrate that up to 65% of zygotically activated genes are enriched for the histone variant H2A.Z. H2A.Z enrichment precedes ZGA and RNA Polymerase II loading onto chromatin. In vivo knockdown of maternally contributed Domino, a histone chaperone and ATPase, reduces H2A.Z deposition at transcription start sites, causes global downregulation of housekeeping genes at ZGA, and compromises the establishment of the 3D chromatin structure. We infer that H2A.Z is essential for the de novo establishment of transcriptional programs during ZGA via chromatin reorganization.

The secreted inhibitor of invasive cell growth CREG1 is negatively regulated by cathepsin proteases.

Previous clinical and experimental evidence strongly supports a breast cancer-promoting function of the lysosomal protease cathepsin B. However, the cathepsin B-dependent molecular pathways are not completely understood. Here, we studied the cathepsin-mediated secretome changes in the context of the MMTV-PyMT breast cancer mouse model. Employing the cell-conditioned media from tumor-macrophage co-cultures, as well as tumor interstitial fluid obtained by a novel strategy from PyMT mice with differential cathepsin B expression, we identified an important proteolytic and lysosomal signature, highlighting the importance of this organelle and these enzymes in the tumor micro-environment. The Cellular Repressor of E1A Stimulated Genes 1 (CREG1), a secreted endolysosomal glycoprotein, displayed reduced abundance upon over-expression of cathepsin B as well as increased abundance upon cathepsin B deletion or inhibition. Moreover, it was cleaved by cathepsin B in vitro. CREG1 reportedly could act as tumor suppressor. We show that treatment of PyMT tumor cells with recombinant CREG1 reduced proliferation, migration, and invasion; whereas, the opposite was observed with reduced CREG1 expression. This was further validated in vivo by orthotopic transplantation. Our study highlights CREG1 as a key player in tumor-stroma interaction and suggests that cathepsin B sustains malignant cell behavior by reducing the levels of the growth suppressor CREG1 in the tumor microenvironment.

MMP14 empowers tumor-initiating breast cancer cells under hypoxic nutrient-depleted conditions.

Tumor-initiating cells (TICs) existing in breast cancer are thought to be involved in initiation, progression, and relapse of tumors. In these processes, the epithelial-to-mesenchymal transition (EMT) and proteases are crucial factors that also dependent on the tumor milieu, including hypoxic nutrient-deprived, as well as normoxic nutrient-rich, environments. Therefore, we investigated EMT and proteases in TICs and their response to different environments by means of a newly generated immortalized TIC (iTIC) line. With the use of primary CD24+CD90+CD45- TICs from the mouse mammary tumor virus-polyoma middle T mouse breast cancer model, iTICs were generated by single cell-initiated sphere and subsequent 2-dimensional monolayer culture. Our data demonstrate the possibility to generate iTICs that are highly tumorigenic in culture and in mouse mammary fat pad. Contrasting environmental conditions provide these cells with a phenotypic and molecular plasticity that has a growth-promoting character in nutrient-rich normoxia and a motile character in nutrient-deprived hypoxia. Expression profiling revealed partial and dynamically changing EMT states, as well as a significantly up-regulated proteolytic signature, including many metalloproteinases, such as matrix metalloproteinase 14 ( Mmp14). Inhibitor treatment of metalloproteinases, as well as short hairpin RNA-mediated knockdown of Mmp14 strongly impacted TIC characteristics, including tumor initiation, cell growth, migration, and invasion, especially in starved environments. We conclude that metalloproteinases empower TICs to adapt to changing environments.-Hillebrand, L. E., Wickberg, S. M., Gomez-Auli, A., Follo, M., Maurer, J., Busch, H., Boerries, M., Reinheckel, T. MMP14 empowers tumor-initiating breast cancer cells under hypoxic nutrient-depleted conditions.

Proteomic analysis of lung metastases in a murine breast cancer model reveals divergent influence of CTSB and CTSL overexpression.

Studies in the MMTV-PyMT (PyMT) breast cancer mouse model have shown a strong influence of the lysosomal cysteine cathepsins B or L on lung metastasis formation. Transgenic expression of human CTSB (tgCTSB) or CTSL (tgCTSL) both led to similar metastatic phenotypes with increased metastatic burden in the PyMT mice. However, recent studies in other tumor models proved marked differences in effects of either cathepsin on the proteome composition. We sought to analyze and compare proteome changes in the metastatic proteome of PyMT mice expressing either tgCTSB or tgCTSL to evaluate similarities and differences in those models. Performing an explorative, quantitative proteome comparison based on LC-MS/MS, we identified up to 3,000 proteins from murine lung metastases in three independent biological replicates per genotype. In both cases, when compared to wild-type (WT) mice, we noticed a pronounced impact of transgene cathepsin expression on the metastasis proteome. Highlights include increased moesin, integrin beta 1 and vinexin levels in the tgCTSB dataset and increased saposin and granulin levels in the tgCTSL dataset. Importantly, non-supervised hierarchical clustering clearly separated tgCTSB vs. tgCTSL induced proteome changes. In summary, tgCTSB and tgCTSL both display a strong and distinct impact on proteome composition of lung macrometastases in the PyMT model. Our observations suggest that they impact malignant behavior in distinct ways, thus further emphasizing interest into their tumor-contextual functionality.

Inherited diseases caused by mutations in cathepsin protease genes.

Lysosomal cathepsins are proteolytic enzymes increasingly recognized as prognostic markers and potential therapeutic targets in a variety of diseases. In those conditions, the cathepsins are mostly overexpressed, thereby driving the respective pathogenic processes. Although less known, there are also diseases with a genetic deficiency of cathepsins. In fact, nowadays 6 of the 15 human proteases called 'cathepsins' have been linked to inherited syndromes. However, only three of these syndromes are typical lysosomal storage diseases, while the others are apparently caused by defective cleavage of specific protein substrates. Here, we will provide an introduction on lysosomal cathepsins, followed by a brief description of the clinical symptoms of the various genetic diseases. For each disease, we focus on the known mutations of which many have been only recently identified by modern genome sequencing approaches. We further discuss the effect of the respective mutation on protease structure and activity, the resulting pathogenesis, and possible therapeutic strategies.

Proteomic analysis of silenced cathepsin B expression suggests non-proteolytic cathepsin B functionality.

Cathepsin B (CTSB) is a lysosomal endo- and exopeptidase that is also secreted in high amounts by malignant and non-malignant cells. We determined the effect of CTSB on the tumor cell secretome by shRNA-mediated silencing of CTSB mRNA expression and subsequent proteomic LC-MS/MS analysis of the cell supernatants. We identified significant protein changes of 17 secreted or shed proteins. Notably, we found a general reduction in protein abundance of ADAM10 substrates and lysosomal proteins. We corroborated reduced amounts of soluble ADAM10 (sADAM10) and soluble APP (sAPP) in the two cancer cell lines MDA-MB-231 and U2OS by immunoblotting. Interestingly, reductions in sADAM10 and sAPP could be reversed by re-introducing a catalytically inactive variant of CTSB, suggesting a formerly unknown non-catalytic function of the protease.

Proteomic Profiling of Cardiomyocyte-Specific Cathepsin A Overexpression Links Cathepsin A to the Oxidative Stress Response.

Cathepsin A (CTSA) is a lysosomal carboxypeptidase present at the cell surface and secreted outside the cell. Additionally, CTSA binds to ß-galactosidase and neuraminidase 1 to protect them from degradation. CTSA has gained attention as a drug target for the treatment of cardiac hypertrophy and heart failure. Here, we investigated the impact of CTSA on the murine cardiac proteome in a mouse model of cardiomyocyte-specific human CTSA overexpression using liquid chromatography-tandem mass spectrometry in conjunction with an isotopic dimethyl labeling strategy. We identified up to 2000 proteins in each of three biological replicates. Statistical analysis by linear models for microarray data (limma) found >300 significantly affected proteins (moderated p-value ≤0.01), thus establishing CTSA as a key modulator of the cardiac proteome. CTSA strongly impaired the balance of the proteolytic system by upregulating several proteases such as cathepsin B, cathepsin D, and cathepsin Z while down-regulating numerous protease inhibitors. Moreover, cardiomyocyte-specific human CTSA overexpression strongly reduced the levels of numerous antioxidative stress proteins, i.e., peroxiredoxins and protein deglycase DJ-1. In vitro, using cultured rat cardiomyocytes, ectopic overexpression of CTSA resulted in accumulation of reactive oxygen species. Collectively, our proteomic and functional data strengthen an association of CTSA with the cellular oxidative stress response.

Comprehensive Proteomic Analysis of Nitrogen-Starved Mycobacterium smegmatis Δpup Reveals the Impact of Pupylation on Nitrogen Stress Response.

Pupylation is a bacterial ubiquitin-like protein modification pathway, which results in the attachment of the small protein Pup to specific lysine residues of cellular targets. Pup was shown to serve as a degradation signal, directing proteins toward the bacterial proteasome for turnover. Recently, it was hypothesized that pupylation and proteasomal protein degradation support the survival of Mycobacterium smegmatis (Msm) during nitrogen starvation by supplying recycled amino acids. In the present study we generated a Pup deletion strain to investigate the influence of pupylation on Msm proteome in the absence of nitrogen sources. Quantitative proteomic analyses revealed a relatively low impact of Pup on MsmΔpup proteome immediately after exposure to growth medium lacking nitrogen. Less than 5.4% of the proteins displayed altered cellular levels when compared to Msm wild type. In contrast, post 24 h of nitrogen starvation 501 proteins (41% of the total quantified proteome) of Msm pup deletion strain showed significant changes in abundance. Noteworthy, important players involved in nitrogen assimilation were significantly affected in MsmΔpup. Furthermore, we quantified pupylated proteins of nitrogen-starved Msm to gain more detailed insights in the role of pupylation in surviving and overcoming the lack of nitrogen.

Impact of cathepsin B on the interstitial fluid proteome of murine breast cancers.

Carcinomas establish a molecular cross talk between malignant tumor cells and the activated non-malignant cells of the tumor stroma. This cell-cell communication in tumor-stroma interaction includes soluble, secreted proteins that act in a paracrine or autocrine manner. Proteases are crucial factors in tumor-stroma interaction by degrading or truncating secreted bioactive proteins. The cysteine protease cathepsin B is frequently overexpressed in several cancer types, including breast cancer. Its abundance often correlates with poor prognosis. In the murine polyoma virus middle T oncogene (PyMT) breast cancer model, cathepsin B is equally pro-tumorigenic. In this study, we investigate how cathepsin B shapes the secreted proteome of PyMT breast cancers. We employed a novel strategy to harvest tumor interstitial fluid (IF) in combination with chemical stable isotope tagging for quantitative proteomic comparison of IF stemming from PyMT tumors from wild-type mice, mice lacking cathepsin B, and mice over-expressing human cathepsin B. In three biological replicates, we achieve good proteome coverage (∼1700 proteins), with a large content (>70%) of secreted proteins. This characterizes IF as a robust source for the investigation of cancer secretomes. We also identified a large number of shed ectodomains, thus highlighting the importance of tumor-contextual cell surface proteolysis. Furthermore, IF contained >190 proteases and protease inhibitors, which span the entire range of absolute protein abundances; an observation testifying for an important role of proteolysis in tumor-stroma interaction. The cathepsin B genotype consistently affected proteins including alpha-1B-glycoprotein and major urinary proteins 11 and 8 (MUP8). Our study establishes tumor IF as a rich source for the investigation of secreted proteins in tumor biology and sheds light on complex proteolytic networks in the breast cancer secretome.

Enrichment of protein N-termini by charge reversal of internal peptides.

Protein N-termini provide useful information for the understanding of posttranslational processing of proteins. The majority of proteins undergo N-terminal processing, such as proteolytic truncation or modifications like acetylation. Multiple methods currently exist for the enrichment of N-terminal peptides for proteomic analyses. Here, we report a novel, simple, and straightforward N-terminomic strategy, based on charge reversal of internal peptides followed by their removal through strong cation exchange chromatography. Our initial proof-of-concept study shows the feasibility of this technique, yielding over 3000 identifications of protein N-termini. We further show the application of this strategy in investigating the N-terminome of mouse embryonic fibroblasts cells deficient for both cathepsin B and L in comparison to wild type) control cells. Finally, we demonstrate that this workflow can be used in combination with a gel-based strategy, allowing preseparation of proteins and thus providing an estimate of the molecular weight of the identified cleavage products.

Deletion of cysteine cathepsins B or L yields differential impacts on murine skin proteome and degradome.

Numerous studies highlight the fact that concerted proteolysis is essential for skin morphology and function. The cysteine protease cathepsin L (Ctsl) has been implicated in epidermal proliferation and desquamation, as well as in hair cycle regulation. In stark contrast, mice deficient in cathepsin B (Ctsb) do not display an overt skin phenotype. To understand the systematic consequences of deleting Ctsb or Ctsl, we determined the protein abundances of >1300 proteins and proteolytic cleavage events in skin samples of wild-type, Ctsb(-/-), and Ctsl(-/-) mice via mass-spectrometry-based proteomics. Both protease deficiencies revealed distinct quantitative changes in proteome composition. Ctsl(-/-) skin revealed increased levels of the cysteine protease inhibitors cystatin B and cystatin M/E, increased cathepsin D, and an accumulation of the extracellular glycoprotein periostin. Immunohistochemistry located periostin predominantly in the hypodermal connective tissue of Ctsl(-/-) skin. The proteomic identification of proteolytic cleavage sites within skin proteins revealed numerous processing sites that are underrepresented in Ctsl(-/-) or Ctsb(-/-) samples. Notably, few of the affected cleavage sites shared the canonical Ctsl or Ctsb specificity, providing further evidence of a complex proteolytic network in the skin. Novel processing sites in proteins such as dermokine and Notch-1 were detected. Simultaneous analysis of acetylated protein N termini showed prototypical mammalian N-alpha acetylation. These results illustrate an influence of both Ctsb and Ctsl on the murine skin proteome and degradome, with the phenotypic consequences of the absence of either protease differing considerably.

Medición de la calidad de vida en ensayos clínicos de pacientes con cáncer. Un estudio bibliométrico / Measuring Quality of Life among Cancer Patients in Clinical Trials. A Bibliometric Study

La calidad de vida relacionada con salud (CVRS) se refiere a la evaluación que hace el paciente de su estado actual de funcionamiento y satisfacción comparado con aquel que se percibe como ideal. La medición de la calidad de vida en ensayos clínicos provee una evaluación mas precisa de qué tan bien se sienten los individuos o grupos de pacientes y los beneficios y eventos adversos que puedan ser resultado de la intervención médica. En la investigación oncológica, la calidad de vida ha sido identificada como el segundo desenlace primario más importante luego de la supervivencia. Para determinar cómo se está evaluando la calidad de vida en ensayos clínicos en cáncer, se realizó una búsqueda en las bases de datos MedLine y LILACS. Luego de una selección basada en criterios de elegibilidad, se escogieron 192 artículos. El primer ensayo clínico que incluye una medición de calidad de vida se publicó en 1992; la neoplasia más frecuentemente estudiada fue el cáncer de mama, mientras que en el cáncer colo-rectal se observa una tendencia hacia la disminución de estudios que miden la calidad de vida. Se encontraron cinco trabajos en los que se utilizaron instrumentos no específicos para medir el constructo. Debido a la relevancia que tiene la calidad de vida, es necesario generar investigaciones similares, con el fin de evaluar estas mediciones en otros tipos de estudios epidemiológicos y en otras patologías.

Health Related Quality of Life (HRQL) refers to the evaluation a patient makes of his/her current functional state and sense of well-being compared to the ideal. The measurement of quality of life in clinical trials (CT) provides a more accurate evaluation on how well individual patients or groups of patients feel, and what benefits and adverse events might occur as a result of medical intervention. In oncological research, quality of life has been identified as the second most important outcome, preceded only by survival. In order to establish how quality of life evaluation was being carried out on clinical trials for cancer, we conducted a data base search at MEDLINE and LILACS. Following selection based upon elegibility criteria, 192 articles were chosen. The first CT to measure quality of life were published in 1992; the most frequently studied neoplasia was breast cancer; in the case of colorectal cancer, the trend was towards fewer quality of life measurement studies. Five studies were found in which non-specific instruments were utilized to measure the construct. Due to the relevance of quality of life, it is necessary to carry out similar searches to evaluate these measurements in other kinds of epidemilogical and pathological studies.

Psoriasis y cáncer / Psoriasis and Cancer

La psoriasis es una enfermedad dermatólogica crónica, caracterizada por hiperproliferación celular, anormalidades en la diferenciación celular y en la expresión de algunas moléculas e infiltrado inflamatorio, lo que produce unas características patognomónicas. El objetivo de este artículo es exponer las posibles relaciones existentes entre estas dos entidades. Luego de efectuar la revisión de la literatura, se concluye que existe una relación entre la psoriasis y el cáncer, al menos desde el punto de vista teórico, pues se encuentran similitudes estructurales y moleculares en las dos patologías. A pesar de esto, y aunque el paciente con psoriasis tiene mayor riesgo de desarrollar cáncer que la población general, es intereseante ver que es muy poco frecuente una transformación cancerígena de las placas psoriáticas. L a teoría de la senescencia podría ayudar a resolver esta paradoja, aunque también puede existir un factor de su supresión tumoral desconocido o no estudiado, así como alteraciones en la señalización celular que crearían un ambiebnte resistente a tumores en una forma "paracrina".