Maria Polycarpou-Schwarz

Transient cyclical methylation of promoter DNA.

Methylation of CpG dinucleotides is generally associated with epigenetic silencing of transcription and is maintained through cellular division. Multiple CpG sequences are rare in mammalian genomes, but frequently occur at the transcriptional start site of active genes, with most clusters of CpGs being hypomethylated. We reported previously that the proximal region of the trefoil factor 1 (TFF1, also known as pS2) and oestrogen receptor α (ERα) promoters could be partially methylated by treatment with deacetylase inhibitors, suggesting the possibility of dynamic changes in DNA methylation. Here we show that cyclical methylation and demethylation of CpG dinucleotides, with a periodicity of around 100 min, is characteristic for five selected promoters, including the oestrogen (E2)-responsive pS2 gene, in human cells. When the pS2 gene is actively transcribed, DNA methylation occurs after the cyclical occupancy of ERα and RNA polymerase II (polII). Moreover, we report conditions that provoke methylation cycling of the pS2 promoter in cell lines in which pS2 expression is quiescent and the proximal promoter is methylated. This coincides with a low-level re-expression of ERα and of pS2 transcripts.

The lncRNA VELUCT strongly regulates viability of lung cancer cells despite its extremely low abundance

Abstract Little is known about the function of most non-coding RNAs (ncRNAs). The majority of long ncRNAs (lncRNAs) is expressed at very low levels and it is a matter of intense debate whether these can be of functional relevance. Here, we identified lncRNAs regulating the viability of lung cancer cells in a high-throughput RNA interference screen. Based on our previous expression profiling, we designed an siRNA library targeting 638 lncRNAs upregulated in human cancer. In a functional siRNA screen analyzing the viability of lung cancer cells, the most prominent hit was a novel lncRNA which we called Viability Enhancing LUng Cancer Transcript (VELUCT). In silico analyses confirmed the non-coding properties of the transcript. Surprisingly, VELUCT was below the detection limit in total RNA from NCI-H460 cells by RT-qPCR as well as RNA-Seq, but was robustly detected in the chromatin-associated RNA fraction. It is an extremely low abundant lncRNA with an RNA copy number of less than one copy per cell. Blocking transcription with actinomycin D revealed that VELUCT RNA was highly unstable which may partially explain its low steady-state concentration. Despite its extremely low abundance, loss-of-function of VELUCT with three independent experimental approaches in three different lung cancer cell lines led to a significant reduction of cell viability: Next to four individual siRNAs, also two complex siPOOLs as well as two antisense oligonucleotides confirmed the strong and specific phenotype. In summary, the extremely low abundant lncRNA VELUCT is essential for regulation of cell viability in several lung cancer cell lines. Hence, VELUCT is the first example for a lncRNA that is expressed at a very low level, but has a strong loss-of-function phenotype. Thus, our study proves that at least individual low-abundant lncRNAs can play an important functional role.

LIMT is a novel metastasis inhibiting lncRNA suppressed by EGF and downregulated in aggressive breast cancer.

Long noncoding RNAs (lncRNAs) are emerging as regulators of gene expression in pathogenesis, including cancer. Recently, lncRNAs have been implicated in progression of specific subtypes of breast cancer. One aggressive, basal‐like subtype associates with increased EGFR signaling, while another, the HER2‐enriched subtype, engages a kin of EGFR. Based on the premise that EGFR‐regulated lncRNAs might control the aggressiveness of basal‐like tumors, we identified multiple EGFR‐inducible lncRNAs in basal‐like normal cells and overlaid them with the transcriptomes of over 3,000 breast cancer patients. This led to the identification of 11 prognostic lncRNAs. Functional analyses of this group uncovered LINC01089 (here renamed LncRNA Inhibiting Metastasis; LIMT), a highly conserved lncRNA, which is depleted in basal‐like and in HER2‐positive tumors, and the low expression of which predicts poor patient prognosis. Interestingly, EGF rapidly downregulates LIMT expression by enhancing histone deacetylation at the respective promoter. We also find that LIMT inhibits extracellular matrix invasion of mammary cells in vitro and tumor metastasis in vivo. In conclusion, lncRNAs dynamically regulated by growth factors might act as novel drivers of cancer progression and serve as prognostic biomarkers.

A cautionary tale of sense-antisense gene pairs: independent regulation despite inverse correlation of expression

Abstract Long non-coding RNAs (lncRNAs) have been proven to play important roles in diverse cellular processes including the DNA damage response. Nearly 40% of annotated lncRNAs are transcribed in antisense direction to other genes and have often been implicated in their regulation via transcript- or transcription-dependent mechanisms. However, it remains unclear whether inverse correlation of gene expression would generally point toward a regulatory interaction between the genes. Here, we profiled lncRNA and mRNA expression in lung and liver cancer cells after exposure to DNA damage. Our analysis revealed two pairs of mRNA-lncRNA sense-antisense transcripts being inversely expressed upon DNA damage. The lncRNA NOP14-AS1 was strongly upregulated upon DNA damage, while the mRNA for NOP14 was downregulated, both in a p53-dependent manner. For another pair, the lncRNA LIPE-AS1 was downregulated, while its antisense mRNA CEACAM1 was upregulated. To test whether as expected the antisense genes would regulate each other resulting in this highly significant inverse correlation, we employed antisense oligonucleotides and RNAi to study transcript-dependent effects as well as dCas9-based transcriptional modulation by CRISPRi/CRISPRa for transcription-dependent effects. Surprisingly, despite the strong stimulus-dependent inverse correlation, our data indicate that neither transcript- nor transcription-dependent mechanisms explain the inverse regulation of NOP14-AS1:NOP14 or LIPE-AS1:CEACAM1 expression. Hence, sense-antisense pairs whose expression is strongly—positively or negatively—correlated can be nonetheless regulated independently. This highlights the requirement of individual experimental studies for each antisense pair and prohibits drawing conclusions on regulatory mechanisms from expression correlations.

The long non-coding RNA LINC00152 is essential for cell cycle progression through mitosis in HeLa cells

In recent years, long non-coding RNA (lncRNA) research has identified essential roles of these transcripts in virtually all physiological cellular processes including tumorigenesis, but their functions and molecular mechanisms are poorly understood. In this study, we performed a high-throughput siRNA screen targeting 638 lncRNAs deregulated in cancer entities to analyse their impact on cell division by using time-lapse microscopy. We identified 26 lncRNAs affecting cell morphology and cell cycle including LINC00152. This transcript was ubiquitously expressed in many human cell lines and its RNA levels were significantly upregulated in lung, liver and breast cancer tissues. A comprehensive sequence analysis of LINC00152 revealed a highly similar paralog annotated as MIR4435-2HG and several splice variants of both transcripts. The shortest and most abundant isoform preferentially localized to the cytoplasm. Cells depleted of LINC00152 arrested in prometaphase of mitosis and showed reduced cell viability. In RNA affinity purification (RAP) studies, LINC00152 interacted with a network of proteins that were associated with M phase of the cell cycle. In summary, we provide new insights into the properties and biological function of LINC00152 suggesting that this transcript is crucial for cell cycle progression through mitosis and thus, could act as a non-coding oncogene.

The cancer-associated microprotein CASIMO1 controls cell proliferation and interacts with squalene epoxidase modulating lipid droplet formation

Breast cancer is a leading cause of cancer-related death in women. Small open reading frame (sORF)-encoded proteins or microproteins constitute a new class of molecules often transcribed from presumed long non-coding RNA transcripts (lncRNAs). The translation of some of these sORFs has been confirmed, but their cellular function and importance remains largely unknown. Here, we report the identification and characterization of a novel microprotein of 10 kDa, which we named Cancer-Associated Small Integral Membrane Open reading frame 1 (CASIMO1). CASIMO1 RNA is overexpressed predominantly in hormone receptor-positive breast tumors. Its knockdown leads to decreased proliferation in multiple breast cancer cell lines. Its loss disturbs the organization of the actin cytoskeleton, leads to inhibition of cell motility, and causes a G0/G1 cell cycle arrest. The proliferation phenotype upon overexpression is observed only with CASIMO1 protein expression, but not with a non-translatable mutant attributing the effects to the sORF-derived protein rather than a lncRNA function. CASIMO1 microprotein interacts with squalene epoxidase (SQLE), a key enzyme in cholesterol synthesis and a known oncogene in breast cancer. Overexpression of CASIMO1 leads to SQLE protein accumulation without affecting its RNA levels and increased lipid droplet clustering, while knockdown of CASIMO1 decreased SQLE protein abundance and ERK phosphorylation downstream of SQLE. Importantly, SQLE knockdown mimicked the CASIMO1 knockdown phenotype and in turn SQLE overexpression fully rescued the effect of CASIMO1 knockdown. These findings establish CASIMO1 as the first functional microprotein that plays a role in carcinogenesis and is implicated in the cell lipid homeostasis.

The Long Noncoding RNA Cancer Susceptibility 9 and RNA Binding Protein Heterogeneous Nuclear Ribonucleoprotein L Form a Complex and Coregulate Genes Linked to AKT Signaling

The identification of viability-associated long non-coding RNAs (lncRNA) might be a promising rationale for new therapeutic approaches in liver cancer. Here, we applied the first RNAi screening approach in hepatocellular carcinoma (HCC) cell lines to find viabilityassociated lncRNAs. Among the multiple identified lncRNAs with a significant impact on HCC cell viability, we selected CASC9 (Cancer Susceptibility 9) due to the strength of its phenotype, expression, and upregulation in HCC versus normal liver. CASC9 regulated viability across multiple HCC cell lines as shown by CRISPR interference, single siRNAand siPOOL-mediated depletion of CASC9. Further, CASC9 depletion caused an increase in apoptosis and decrease of proliferation. We identified the RNA binding protein heterogeneous nuclear ribonucleoprotein L (HNRNPL) as a CASC9 interacting protein by RNA affinity purification (RAP) and validated it by native RNA immunoprecipitation (RIP). Knockdown of HNRNPL mimicked the loss-of-viability phenotype observed upon CASC9 depletion. Analysis of the proteome (SILAC) of CASC9and HNRNPL-depleted cells revealed a set of co-regulated genes which implied a role of the CASC9:HNRNPL complex in AKTsignaling and DNA damage sensing. CASC9 expression levels were elevated in patientderived tumor samples compared to normal control tissue and had a significant association with overall survival of HCC patients. In a xenograft chicken chorioallantoic membrane model, we measured a decreased tumor size after knockdown of CASC9. Conclusion: Taken together, we provide a comprehensive list of viability-associated lncRNAs in HCC. We identified the CASC9:HNRNPL complex as a clinically relevant viability-associated lncRNA/protein complex which affects AKT-signaling and DNA damage sensing in HCC. Introductory Statement Liver cancer is one of the most frequent cancer diseases worldwide and causes more than 750,000 deaths annually. It is ranked second among the most lethal cancer entities, with very limited treatment options (1). The most common subtype (90%) of liver cancer is hepatocellular carcinoma (HCC). The characterization of specific changes in the cancer transcriptome will facilitate the development of new therapeutic strategies in HCC. One surprising finding of the last decade of transcriptome profiling was that most of the human genes encode for non-coding RNA (ncRNA) genes and not for protein-coding genes (2). As a result, the ncRNA genes, Page 4 of 115 Hepatology Hepatology This article is protected by copyright. All rights reserved.The identification of viability‐associated long noncoding RNAs (lncRNAs) might be a promising rationale for new therapeutic approaches in liver cancer. Here, we applied an RNA interference screening approach in hepatocellular carcinoma (HCC) cell lines to find viability‐associated lncRNAs. Among the multiple identified lncRNAs with a significant impact on HCC cell viability, we selected cancer susceptibility 9 (CASC9) due to the strength of its phenotype, expression, and up‐regulation in HCC versus normal liver. CASC9 regulated viability across multiple HCC cell lines as shown by clustered regularly interspaced short palindromic repeats interference and single small interfering RNA (siRNA)–mediated and siRNA pool–mediated depletion of CASC9. Further, CASC9 depletion caused an increase in apoptosis and a decrease of proliferation. We identified the RNA binding protein heterogeneous nuclear ribonucleoprotein L (HNRNPL) as a CASC9 interacting protein by RNA affinity purification and validated it by native RNA immunoprecipitation. Knockdown of HNRNPL mimicked the loss‐of‐viability phenotype observed upon CASC9 depletion. Analysis of the proteome (stable isotope labeling with amino acids in cell culture) of CASC9‐depleted and HNRNPL‐depleted cells revealed a set of coregulated genes which implied a role of the CASC9:HNRNPL complex in AKT signaling and DNA damage sensing. CASC9 expression levels were elevated in patient‐derived tumor samples compared to normal control tissue and had a significant association with overall survival of HCC patients. In a xenograft chicken chorioallantoic membrane model, we measured decreased tumor size after knockdown of CASC9. Conclusion: Taken together, we provide a comprehensive list of viability‐associated lncRNAs in HCC; we identified the CASC9:HNRNPL complex as a clinically relevant viability‐associated lncRNA/protein complex which affects AKT signaling and DNA damage sensing in HCC.

MIR100 host gene-encoded lncRNAs regulate cell cycle by modulating the interaction between HuR and its target mRNAs

Abstract Long non-coding RNAs (lncRNAs) regulate vital biological processes, including cell proliferation, differentiation and development. A subclass of lncRNAs is synthesized from microRNA (miRNA) host genes (MIRHGs) due to pre-miRNA processing, and are categorized as miRNA-host gene lncRNAs (lnc-miRHGs). Presently, the cellular function of most lnc-miRHGs is not well understood. We demonstrate a miRNA-independent role for a nuclear-enriched lnc-miRHG in cell cycle progression. MIR100HG produces spliced and stable lncRNAs that display elevated levels during the G1 phase of the cell cycle. Depletion of MIR100HG-encoded lncRNAs in human cells results in aberrant cell cycle progression without altering the levels of miRNA encoded within MIR100HG. Notably, MIR100HG interacts with HuR/ELAVL1 as well as with several HuR-target mRNAs. Further, MIR100HG-depleted cells show reduced interaction between HuR and three of its target mRNAs, indicating that MIR100HG facilitates interaction between HuR and target mRNAs. Our studies have unearthed novel roles played by a MIRHG-encoded lncRNA in regulating RNA binding protein activity, thereby underscoring the importance of determining the function of several hundreds of lnc-miRHGs that are present in human genome.

Abstract 1824: The non-coding RNA landscape of lung, liver and breast cancer reveals novel tumor-associated ncRNAs, concerted regulation and unexpected tissue specificity.

Proceedings: AACR 104th Annual Meeting 2013; Apr 6-10, 2013; Washington, DC Non-coding RNA profiles in cancer are largely unknown which greatly impedes the discovery of functionally important ncRNAs in tumorigenesis as well as the generation of genome-wide libraries. Here, we define the ncRNA expression landscape of lung, breast and liver cancer as well as normal tissue from the respective organs in a large set of primary patient samples (N=150). These samples were carefully selected to have a long patient follow-up and complete clinical datasets to allow an in-depth analysis. We provide the first comprehensive map of 17000+ long ncRNAs in a broad range of human tumor and normal tissues and discovered hundreds of new ncRNAs associated with three major tumor entities. Importantly, we uncovered that ncRNA profiles are significantly more specific to the tissue of origin than patterns of protein-coding mRNAs. Also, the significant ncRNA signatures in this study demonstrate specific ncRNA patterns that are unlikely to be transcriptional background but rather the result of concerted regulation. To mimic the response to cytotoxic chemotherapy, we have also treated lung and liver cancer cells with the DNA damaging agents Cisplatin and Etoposide and found significant deregulation of long ncRNAs - reversing in part the differences seen between normal and malignant lung tissue. Importantly, one of the Cisplatin-regulated ncRNAs interacts with DNA repair factors linking it immediately to the DNA damage response. To elucidate the molecular functions of the novel ncRNAs, we used RNA affinity purification to identify the protein interaction partners. In addition, we create human knockout cells for lncRNAs using ZFN and TALEN technology to integrate RNA destabilizing elements into the human genome which yields more than 1000-fold lncRNA silencing. Our so far largest ncRNA expression map is also exploited in another way: We generate an siRNA library specifically targeting over 600 tumor-associated ncRNAs based on our profiling landscape. This comprehensive but focused library will elucidate the role of ncRNAs in tumorigenesis, viability, apoptosis and the DNA damage response. In summary, we provide the first global comprehensive map of long ncRNA expression in a broad range of human tumor and normal tissue samples and discovered many new lncRNAs associated with cancer as well as tissue-, histology- and prognosis-specific ncRNA signatures. Citation Format: Maria Polycarpou-Schwarz, Tony Gutschner, Monika Hammerle, Anna Roth, Ashish Goyal, Stefanie Grund, Catherina Hildenbrand, Arne Warth, Thomas Longerich, Sebastian Aulmann, Joachim Rom, Michael Meister, Thomas Muley, Heike Zabeck, Sabine Schmidt, Tomi Ivacevic, Vladimir Benes, Kai Breuhahn, Philipp Schnabel, Peter Sinn, Hans Hoffmann, Peter Schirmacher, Sven Diederichs. The non-coding RNA landscape of lung, liver and breast cancer reveals novel tumor-associated ncRNAs, concerted regulation and unexpected tissue specificity. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 1824. doi:10.1158/1538-7445.AM2013-1824