Low level of antioxidant capacity biomarkers but not target overexpression predicts vulnerability to ROS-inducing drugs.

Despite a strong rationale for why cancer cells are susceptible to redox-targeting drugs, such drugs often face tumor resistance or dose-limiting toxicity in preclinical and clinical studies. An important reason is the lack of specific biomarkers to better select susceptible cancer entities and stratify patients. Using a large panel of lung cancer cell lines, we identified a set of "antioxidant-capacity" biomarkers (ACB), which were tightly repressed, partly by STAT3 and STAT5A/B in sensitive cells, rendering them susceptible to multiple redox-targeting and ferroptosis-inducing drugs. Contrary to expectation, constitutively low ACB expression was not associated with an increased steady state level of reactive oxygen species (ROS) but a high level of nitric oxide, which is required to sustain high replication rates. Using ACBs, we identified cancer entities with a high percentage of patients with favorable ACB expression pattern, making it likely that more responders to ROS-inducing drugs could be stratified for clinical trials.

Proteome-Wide Identification of RNA-Dependent Proteins in Lung Cancer Cells.

Following the concept of RNA dependence and exploiting its application in the R-DeeP screening approach, we have identified RNA-dependent proteins in A549 lung adenocarcinoma cells. RNA-dependent proteins are defined as proteins whose interactome depends on RNA and thus entails RNA-binding proteins (RBPs) as well as proteins in ribonucleoprotein complexes (RNPs) without direct RNA interaction. With this proteome-wide technique based on sucrose density gradient ultracentrifugation and fractionation followed by quantitative mass spectrometry and bioinformatic analysis, we have identified 1189 RNA-dependent proteins including 170 proteins which had never been linked to RNA before. R-DeeP provides quantitative information on the fraction of a protein being RNA-dependent as well as it allows the reconstruction of protein complexes based on co-segregation. The RNA dependence of three newly identified RNA-dependent proteins, DOCK5, ELMO2, also known as CED12A, and ABRAXAS1, also known as CCDC98, was validated using western blot analysis, and the direct RNA interaction was verified by iCLIP2 for the migration-related protein DOCK5 and the mitosis-related protein ABRAXAS1. The R-DeeP 2.0 database provides proteome-wide and cell line-specific information from A549 and HeLa S3 cells on proteins and their RNA dependence to contribute to understanding the functional role of RNA and RNA-binding proteins in cancer cells.

Systematic analysis of migration factors by MigExpress identifies essential cell migration control genes in non-small cell lung cancer.

Cell migration is an essential process in health and in disease, including cancer metastasis. A comprehensive inventory of migration factors is nonetheless lacking-in part due to the difficulty in assessing migration using high-throughput technologies. Hence, there are currently very few screens that systematically reveal factors controlling cell migration. Here, we introduce MigExpress as a platform for the 'identification of Migration control genes by differential Expression'. MigExpress exploits the combination of in-depth molecular profiling and the robust quantitative analysis of migration capacity in a broad panel of samples and identifies migration-associated genes by their differential expression in slow- versus fast-migrating cells. We applied MigExpress to investigate non-small cell lung cancer (NSCLC), which is the most frequent cause of cancer mortality mainly due to metastasis. In 54 NSCLC cell lines, we comprehensively determined mRNA and protein expression. Correlating the transcriptome and proteome profiles with the quantified migration properties led to the discovery and validation of FLNC, DSE, CPA4, TUBB6, and BICC1 as migration control factors in NSCLC cells, which were also negatively correlated with patient survival. Notably, FLNC was the least expressed filamin in NSCLC, but the only one controlling cell migration and correlating with patient survival and metastatic disease stage. In our study, we present MigExpress as a new method for the systematic analysis of migration factors and provide a comprehensive resource of transcriptomic and proteomic data of NSCLC cell lines related to cell migration.

A pan-cancer analysis reveals nonstop extension mutations causing SMAD4 tumour suppressor degradation.

Nonstop or stop-loss mutations convert a stop into a sense codon, resulting in translation into the 3' untranslated region as a nonstop extension mutation to the next in-frame stop codon or as a readthrough mutation into the poly-A tail. Nonstop mutations have been characterized in hereditary diseases, but not in cancer genetics. In a pan-cancer analysis, we curated and analysed 3,412 nonstop mutations from 62 tumour entities, generating a comprehensive database at http://NonStopDB.dkfz.de. Six different nonstop extension mutations affected the tumour suppressor SMAD4, extending its carboxy terminus by 40 amino acids. These caused rapid degradation of the SMAD4 mutants via the ubiquitin-proteasome system. A hydrophobic degron signal sequence of ten amino acids within the carboxy-terminal extension was required to induce complete loss of the SMAD4 protein. Thus, we discovered that nonstop mutations can be functionally important in cancer and characterize their loss-of-function impact on the tumour suppressor SMAD4.

The Circular RNA Landscape of Non-Small Cell Lung Cancer Cells.

The class of circular RNA (circRNA) is characterized by head-to-tail bonds between exons formed by backsplicing. Here, we provide a resource of circRNA expression in a comprehensive panel of 60 lung cancer and non-transformed cell lines (FL3C dataset). RNA sequencing after depletion of ribosomal RNA quantified the expression of circRNA and linear RNA. We detected 148,811 circular RNAs quantified by 2.8 million backsplicing reads originating from 12,251 genes. The number of identified circRNAs was markedly higher using rRNA depletion compared to public polyA-enriched RNA-seq datasets. CircRNAs almost never started in the first exon nor ended in the last exon and started more frequently in earlier exons. Most circRNAs showed high cell line specificity and correlated positively with their linear RNA counterpart. Known cancer genes produced more circRNAs than non-cancer genes. Subsets of circRNAs correlated with cell proliferation, histological subtype or genotype. CircTNFRSF21 was translated crossing the backsplice site in two different reading frames. Overexpression of circPVT1, circERBB2, circHIPK3, circCCNB1, circSMAD2, circTNFRSF21 and circKIF5B significantly increased colony formation. In conclusion, our data provide a comprehensive map of circRNA expression in lung cancer cells and global patterns of circRNA production as a useful resource for future research into lung cancer circRNAs.

Identification, quantification and bioinformatic analysis of RNA-dependent proteins by RNase treatment and density gradient ultracentrifugation using R-DeeP.

Analysis of RNA-protein complexes is central to understanding the molecular circuitry governing cellular processes. In recent years, several proteome-wide studies have been dedicated to the identification of RNA-binding proteins. Here, we describe in detail R-DeeP, an approach built on RNA dependence, defined as the ability of a protein to engage in protein complexes only in the presence of RNA, involving direct or indirect interaction with RNA. This approach provides-for the first time, to our knowledge-quantitative information on the fraction of a protein associated with RNA-protein complexes. R-DeeP is independent of any potentially biased purification procedures. It is based on cellular lysate fractionation by density gradient ultracentrifugation and subsequent analysis by proteome-wide mass spectrometry (MS) or individual western blotting. The comparison of lysates with and without previous RNase treatment enables the identification of differences in the apparent molecular weight and, hence, the size of the complexes. In combination with information from databases of protein-protein complexes, R-DeeP facilitates the computational reconstruction of protein complexes from proteins migrating in the same fraction. In addition, we developed a pipeline for the statistical analysis of the MS dataset to automatically identify RNA-dependent proteins (proteins whose interactome depends on RNA). With this protocol, the individual analysis of proteins of interest by western blotting can be completed within 1-2 weeks. For proteome-wide studies, additional time is needed for the integration of the proteomic and statistical analyses. In the future, R-DeeP can be extended to other fractionation techniques, such as chromatography.

R-DeeP: Proteome-wide and Quantitative Identification of RNA-Dependent Proteins by Density Gradient Ultracentrifugation.

The comprehensive but specific identification of RNA-binding proteins as well as the discovery of RNA-associated protein functions remain major challenges in RNA biology. Here we adapt the concept of RNA dependence, defining a protein as RNA dependent when its interactome depends on RNA. We converted this concept into a proteome-wide, unbiased, and enrichment-free screen called R-DeeP (RNA-dependent proteins), based on density gradient ultracentrifugation. Quantitative mass spectrometry identified 1,784 RNA-dependent proteins, including 537 lacking known links to RNA. Exploiting the quantitative nature of R-DeeP, proteins were classified as not, partially, or completely RNA dependent. R-DeeP identified the transcription factor CTCF as completely RNA dependent, and we uncovered that RNA is required for the CTCF-chromatin association. Additionally, R-DeeP allows reconstruction of protein complexes based on co-segregation. The whole dataset is available at http://R-DeeP.dkfz.de, providing proteome-wide, specific, and quantitative identification of proteins with RNA-dependent interactions and aiming at future functional discovery of RNA-protein complexes.

LINC00261 and the Adjacent Gene FOXA2 Are Epithelial Markers and Are Suppressed during Lung Cancer Tumorigenesis and Progression.

Lung cancer continues to be the leading cause of cancer-related deaths worldwide, with little improvement in patient survival rates in the past decade. Long non-coding RNAs (lncRNAs) are gaining importance as possible biomarkers with prognostic potential. By large-scale data mining, we identified LINC00261 as a lncRNA which was significantly downregulated in lung cancer. Low expression of LINC00261 was associated with recurrence and poor patient survival in lung adenocarcinoma. Moreover, the gene pair of LINC00261 and its neighbor FOXA2 were significantly co-regulated. LINC00261 as well as FOXA2 negatively correlated with markers for epithelial-to-mesenchymal transition (EMT) and were suppressed by the EMT inducer TGFß. Hierarchical clustering of gene expression data from lung cancer cell lines could further verify the association of high LINC00261/FOXA2 expression to an epithelial gene signature. Furthermore, higher expression of the LINC00261/FOXA2 locus was associated with lung cancer cell lines with lower migratory capacity. All these data establish LINC00261 and FOXA2 as an epithelial-specific marker pair, downregulated during EMT and lung cancer progression, and associated with lower cell migration potential in lung cancer cells.

Alu element-containing RNAs maintain nucleolar structure and function.

Non-coding RNAs play a key role in organizing the nucleus into functional subcompartments. By combining fluorescence microscopy and RNA deep-sequencing-based analysis, we found that RNA polymerase II transcripts originating from intronic Alu elements (aluRNAs) were enriched in the nucleolus. Antisense-oligo-mediated depletion of aluRNAs or drug-induced inhibition of RNA polymerase II activity disrupted nucleolar structure and impaired RNA polymerase I-dependent transcription of rRNA genes. In contrast, overexpression of a prototypic aluRNA sequence increased both nucleolus size and levels of pre-rRNA, suggesting a functional link between aluRNA, nucleolus integrity and pre-rRNA synthesis. Furthermore, we show that aluRNAs interact with nucleolin and target ectopic genomic loci to the nucleolus. Our study suggests an aluRNA-based mechanism that links RNA polymerase I and II activities and modulates nucleolar structure and rRNA production.

Cooperative Action of Cdk1/cyclin B and SIRT1 Is Required for Mitotic Repression of rRNA Synthesis.

Mitotic repression of rRNA synthesis requires inactivation of the RNA polymerase I (Pol I)-specific transcription factor SL1 by Cdk1/cyclin B-dependent phosphorylation of TAF(I)110 (TBP-associated factor 110) at a single threonine residue (T852). Upon exit from mitosis, T852 is dephosphorylated by Cdc14B, which is sequestered in nucleoli during interphase and is activated upon release from nucleoli at prometaphase. Mitotic repression of Pol I transcription correlates with transient nucleolar enrichment of the NAD(+)-dependent deacetylase SIRT1, which deacetylates another subunit of SL1, TAFI68. Hypoacetylation of TAFI68 destabilizes SL1 binding to the rDNA promoter, thereby impairing transcription complex assembly. Inhibition of SIRT1 activity alleviates mitotic repression of Pol I transcription if phosphorylation of TAF(I)110 is prevented. The results demonstrate that reversible phosphorylation of TAF(I)110 and acetylation of TAFI68 are key modifications that regulate SL1 activity and mediate fluctuations of pre-rRNA synthesis during cell cycle progression.

Repression of RNA polymerase I upon stress is caused by inhibition of RNA-dependent deacetylation of PAF53 by SIRT7.

Sirtuins are NAD(+)-dependent protein deacetylases that connect metabolism and cellular homeostasis. Here we show that the nuclear Sirtuin SIRT7 targets PAF53, a subunit of RNA polymerase I (Pol I). Acetylation of PAF53 at lysine 373 by CBP and deacetylation by SIRT7 modulate the association of Pol I with DNA, hypoacetylation correlating with increased rDNA occupancy of Pol I and transcription activation. SIRT7 is released from nucleoli in response to different stress conditions, leading to hyperacetylation of PAF53 and decreased Pol I transcription. Nucleolar detention requires binding of SIRT7 to nascent pre-rRNA, linking the spatial distribution of SIRT7 and deacetylation of PAF53 to ongoing transcription. The results identify a nonhistone target of SIRT7 and uncover an RNA-mediated mechanism that adapts nucleolar transcription to stress signaling.

Proteasomal and genetic inactivation of the NF1 tumor suppressor in gliomagenesis.

Loss-of-function mutations in the NF1 tumor suppressor result in deregulated Ras signaling and drive tumorigenesis in the familial cancer syndrome neurofibromatosis type I. However, the extent to which NF1 inactivation promotes sporadic tumorigenesis is unknown. Here we report that NF1 is inactivated in sporadic gliomas via two mechanisms: excessive proteasomal degradation and genetic loss. NF1 protein destabilization is triggered by the hyperactivation of protein kinase C (PKC) and confers sensitivity to PKC inhibitors. However, complete genetic loss, which only occurs when p53 is inactivated, mediates sensitivity to mTOR inhibitors. These studies reveal an expanding role for NF1 inactivation in sporadic gliomagenesis and illustrate how different mechanisms of inactivation are utilized in genetically distinct tumors, which consequently impacts therapeutic sensitivity.

Catalytic core of a membrane-associated eukaryotic polyphosphate polymerase.

Polyphosphate (polyP) occurs ubiquitously in cells, but its functions are poorly understood and its synthesis has only been characterized in bacteria. Using x-ray crystallography, we identified a eukaryotic polyphosphate polymerase within the membrane-integral vacuolar transporter chaperone (VTC) complex. A 2.6 angstrom crystal structure of the catalytic domain grown in the presence of adenosine triphosphate (ATP) reveals polyP winding through a tunnel-shaped pocket. Nucleotide- and phosphate-bound structures suggest that the enzyme functions by metal-assisted cleavage of the ATP gamma-phosphate, which is then in-line transferred to an acceptor phosphate to form polyP chains. Mutational analysis of the transmembrane domain indicates that VTC may integrate cytoplasmic polymer synthesis with polyP membrane translocation. Identification of the polyP-synthesizing enzyme opens the way to determine the functions of polyP in lower eukaryotes.

Function and regulation of Maskin, a TACC family protein, in microtubule growth during mitosis.

The Xenopus protein Maskin has been previously identified and characterized in the context of its role in translational control during oocyte maturation. Maskin belongs to the TACC protein family. In other systems, members of this family have been shown to localize to centrosomes during mitosis and play a role in microtubule stabilization. Here we have examined the putative role of Maskin in spindle assembly and centrosome aster formation in the Xenopus egg extract system. Depletion and reconstitution experiments indicate that Maskin plays an essential role for microtubule assembly during M-phase. We show that Maskin interacts with XMAP215 and Eg2, the Xenopus Aurora A kinase in vitro and in the egg extract. We propose that Maskin and XMAP215 cooperate to oppose the destabilizing activity of XKCM1 therefore promoting microtubule growth from the centrosome and contributing to the determination of microtubule steady-state length. Further more, we show that Maskin localization and function is regulated by Eg2 phosphorylation.

Synthesis and biological evaluation of new tetrahydro-beta-carbolines as inhibitors of the mitotic kinesin Eg5.

The mitotic kinesin Eg5 (or KSP) is a crucial player in the development and function of the mitotic spindle. Inhibition of this protein leads to cell cycle arrest and apoptosis without interfering with other microtubule-dependent processes. Therefore, it is a potential target in cancer therapy. Here, we report the synthesis and biological evaluation of a small library of molecules based on the structure of the known Eg5 inhibitor HR22C16. One of these derivatives (compound trans-24) proved to be a potent and specific Eg5 inhibitor.