Expression of NORAD correlates with breast cancer aggressiveness and protects breast cancer cells from chemotherapy.

The recently discovered human lncRNA NORAD is induced after DNA damage in a p53-dependent manner. It plays a critical role in the maintenance of genomic stability through interaction with Pumilio proteins, limiting the repression of their target mRNAs. Therefore, NORAD inactivation causes chromosomal instability and aneuploidy, which contributes to the accumulation of genetic abnormalities and tumorigenesis. NORAD has been detected in several types of cancer, including breast cancer, which is the most frequently diagnosed and the second-leading cause of cancer death in women. In the present study, we confirmed upregulated NORAD expression levels in a set of human epithelial breast cancer cell lines (MDA-MB-231, MDA-MB-436, and MDA-MB-468), which belong to the most aggressive subtypes (triple-negative breast cancer). These results are in line with previous data showing that high NORAD expression levels in basal-like tumors were associated with poor prognosis. Here, we demonstrate that NORAD downregulation sensitizes triple-negative breast cancer cells to chemotherapy, through a potential accumulation of genomic aberrations and an impaired capacity to signal DNA damage. These results show that NORAD may represent an unexploited neoadjuvant therapeutic target for chemotherapy-unresponsive breast cancer.

Loss of dyskerin facilitates the acquisition of metastatic traits by altering the mevalonate pathway.

The initial dissemination of cancer cells from many primary tumors implies intravasation to lymphatic nodes or blood vessels. To investigate the mechanisms involved, we analyzed the expression of small non-coding RNAs in cutaneous squamous cell carcinoma (cSCC), a prevalent tumor that mainly spreads to lymph nodes. We report the reduced expression of small nucleolar RNAs in primary cSCCs that metastasized when compared to non-metastasizing cSCCs, and the progressive loss of DKC1 (dyskerin, which stabilizes the small nucleolar RNAs) along the metastasis. DKC1 depletion in cSCC cells triggered lipid metabolism by altering the mevalonate pathway and the acquisition of metastatic traits. Treatment of DKC1-depleted cells with simvastatin, an inhibitor of the mevalonate pathway, blocked the expression of proteins involved in the epithelial-to-mesenchymal transition. Consistently, the expression of the enzyme 3-hydroxy-3-methylglutaryl-CoA synthase 1 was associated with pathological features of high metastatic risk in cSCC patients. Our data underpin the relevance of the mevalonate metabolism in metastatic dissemination and pave the possible incorporation of therapeutic approaches among the antineoplastic drugs used in routine patient care.

High-performance nano-flow liquid chromatography column combined with high- and low-collision energy data-independent acquisition enables targeted and discovery identification of modified ribonucleotides by mass spectrometry.

Over 170 post-transcriptional RNA modifications have been described and are common in all kingdoms of life. These modifications range from methylation to complex chemical structures, with methylation being the most abundant. RNA modifications play a key role in RNA folding and function and their dysregulation in humans has been linked to several diseases such as cancer, metabolic diseases or neurological disorder. Nowadays, liquid chromatography-tandem mass spectrometry is considered the gold standard method for the identification and quantification of these modifications due to its sensitivity and accuracy. However, the analysis of modified ribonucleosides by mass spectrometry is complex due to the presence of positional isomers. In this scenario, optimal separation of these compounds by highly sensitive liquid chromatography combined with the generation of high-information spectra is critical to unequivocally identify them, especially in high-complex mixtures. Here we present an analytical method that comprises a new type of mixed-mode nano-flow liquid chromatography column combined with high- and low-collision energy data-independent mass spectrometric acquisition for the identification and quantitation of modified ribonucleosides. The method produces content-rich spectra and combines targeted and screening capabilities thus enabling the identification of a variety of modified nucleosides in biological matrices by single-shot liquid chromatographic analysis coupled to mass spectrometry.

The loss of DHX15 impairs endothelial energy metabolism, lymphatic drainage and tumor metastasis in mice.

DHX15 is a downstream substrate for Akt1, which is involved in key cellular processes affecting vascular biology. Here, we explored the vascular regulatory function of DHX15. Homozygous DHX15 gene deficiency was lethal in mouse and zebrafish embryos. DHX15-/- zebrafish also showed downregulation of VEGF-C and reduced formation of lymphatic structures during development. DHX15+/- mice depicted lower vascular density and impaired lymphatic function postnatally. RNAseq and proteome analysis of DHX15 silenced endothelial cells revealed differential expression of genes involved in the metabolism of ATP biosynthesis. The validation of these results demonstrated a lower activity of the Complex I in the mitochondrial membrane of endothelial cells, resulting in lower intracellular ATP production and lower oxygen consumption. After injection of syngeneic LLC1 tumor cells, DHX15+/- mice showed partially inhibited primary tumor growth and reduced lung metastasis. Our results revealed an important role of DHX15 in vascular physiology and pave a new way to explore its potential use as a therapeutical target for metastasis treatment.

Knock-down of AHCY and depletion of adenosine induces DNA damage and cell cycle arrest.

Recently, functional connections between S-adenosylhomocysteine hydrolase (AHCY) activity and cancer have been reported. As the properties of AHCY include the hydrolysis of S-adenosylhomocysteine and maintenance of the cellular methylation potential, the connection between AHCY and cancer is not obvious. The mechanisms by which AHCY influences the cell cycle or cell proliferation have not yet been confirmed. To elucidate AHCY-driven cancer-specific mechanisms, we pursued a multi-omics approach to investigate the effect of AHCY-knockdown on hepatocellular carcinoma cells. Here, we show that reduced AHCY activity causes adenosine depletion with activation of the DNA damage response (DDR), leading to cell cycle arrest, a decreased proliferation rate and DNA damage. The underlying mechanism behind these effects might be applicable to cancer types that have either significant levels of endogenous AHCY and/or are dependent on high concentrations of adenosine in their microenvironments. Thus, adenosine monitoring might be used as a preventive measure in liver disease, whereas induced adenosine depletion might be the desired approach for provoking the DDR in diagnosed cancer, thus opening new avenues for targeted therapy. Additionally, including AHCY in mutational screens as a potential risk factor may be a beneficial preventive measure.

Proteome profiling of clear cell renal cell carcinoma in von Hippel-Lindau patients highlights upregulation of Xaa-Pro aminopeptidase-1, an anti-proliferative and anti-migratory exoprotease.

Patients of the von Hippel-Lindau (VHL) disease frequently develop clear cell renal cell carcinoma (ccRCC). Using archived, formalin-fixed, paraffin-embedded (FFPE) samples, we sought to determine global proteome alterations that distinguish ccRCC tissue from adjacent, non-malignant kidney tissue in VHL-patients. Our quantitative proteomic analysis clearly discriminated tumor and non-malignant tissue. Significantly dysregulated proteins were distinguished using the linear models for microarray data algorithm. In the ccRCC tissue, we noticed a predominant under-representation of proteins involved in the tricarboxylic acid cycle and an increase in proteins involved in glycolysis. This profile possibly represents a proteomic fingerprint of the "Warburg effect", which is a molecular hallmark of ccRCC. Furthermore, we observed an increase in proteins involved in extracellular matrix organization. We also noticed differential expression of many exoproteases in the ccRCC tissue. Of particular note were opposing alterations of Xaa-Pro Aminopeptidases-1 and -2 (XPNPEP-1 and -2): a strong decrease of XPNPEP-2 in ccRCC was accompanied by abundant presence of the related protease XPNPEP-1. In both cases, we corroborated the proteomic results by immunohistochemical analysis of ccRCC and adjacent, non-malignant kidney tissue of VHL patients. To functionally investigate the role of XPNPEP-1 in ccRCC, we performed small-hairpin RNA mediated XPNPEP-1 expression silencing in 786-O ccRCC cells harboring a mutated VHL gene. We found that XPNPEP-1 expression dampens cellular proliferation and migration. These results suggest that XPNPEP-1 is likely an anti-target in ccRCC. Methodologically, our work further validates the robustness of using FFPE material for quantitative proteomics.

Proteotoxicity in cardiac amyloidosis: amyloidogenic light chains affect the levels of intracellular proteins in human heart cells.

AL amyloidosis is characterized by widespread deposition of immunoglobulin light chains (LCs) as amyloid fibrils. Cardiac involvement is frequent and leads to life-threatening cardiomyopathy. Besides the tissue alteration caused by fibrils, clinical and experimental evidence indicates that cardiac damage is also caused by proteotoxicity of prefibrillar amyloidogenic species. As in other amyloidoses, the damage mechanisms at cellular level are complex and largely undefined. We have characterized the molecular changes in primary human cardiac fibroblasts (hCFs) exposed in vitro to soluble amyloidogenic cardiotoxic LCs from AL cardiomyopathy patients. To evaluate proteome alterations caused by a representative cardiotropic LC, we combined gel-based with label-free shotgun analysis and performed bioinformatics and data validation studies. To assess the generalizability of our results we explored the effects of multiple LCs on hCF viability and on levels of a subset of cellular proteins. Our results indicate that exposure of hCFs to cardiotropic LCs translates into proteome remodeling, associated with apoptosis activation and oxidative stress. The proteome alterations affect proteins involved in cytoskeletal organization, protein synthesis and quality control, mitochondrial activity and metabolism, signal transduction and molecular trafficking. These results support and expand the concept that soluble amyloidogenic cardiotropic LCs exert toxic effects on cardiac cells.

The oxidized thiol proteome in fission yeast--optimization of an ICAT-based method to identify H2O2-oxidized proteins.

Major intracellular disulfide formation is prevented in the cytosol by potent reducing systems. However, protein thiols can be oxidized as a consequence of redox-mediated physiological reactions or due to the unwanted toxicity of reactive oxygen species. In addition, the reactivity of cysteine residues towards peroxides is used by H(2)O(2) sensors in signal transduction pathways in a gain-of-function process to induce transcriptional antioxidant responses. Thus, the Schizosaccharomyces pombe peroxiredoxin Tpx1 and the transcription factor Pap1 are sensors of H(2)O(2) meant to promote cell survival. In an attempt to compare signaling events versus global thiol oxidation, we have optimized thiol-labeling approaches to characterize the disulfide proteome of fission yeast in response to added H(2)O(2). We propose a method based on (i) freezing the redox state of thiols with strong acids prior to cell lysis; (ii) blocking thiol groups with iodoacetamide, and reversibly oxidized thiols with heavy and light isotope-coded affinity tags (ICAT) reagents; and (iii) quantifying individual relative protein concentrations with stable-isotope dimethyl labeling. We have applied this highly sensitive strategy to provide a map of H(2)O(2)-dependent oxidized thiols in fission yeast, and found Tpx1 and Pap1 as some of the major targets.