RAVER1 hinders lethal EMT and modulates miR/RISC activity by the control of alternative splicing.

The RAVER1 protein serves as a co-factor in guiding the polypyrimidine tract-binding protein (PTBP)-dependent control of alternative splicing (AS). Whether RAVER1 solely acts in concert with PTBPs and how it affects cancer cell fate remained elusive. Here, we provide the first comprehensive investigation of RAVER1-controlled AS in cancer cell models. This reveals a pro-oncogenic role of RAVER1 in modulating tumor growth and epithelial-mesenchymal-transition (EMT). Splicing analyses and protein-association studies indicate that RAVER1 guides AS in association with other splicing regulators, including PTBPs and SRSFs. In cancer cells, one major function of RAVER1 is the stimulation of proliferation and restriction of apoptosis. This involves the modulation of AS events within the miR/RISC pathway. Disturbance of RAVER1 impairs miR/RISC activity resulting in severely deregulated gene expression, which promotes lethal TGFB-driven EMT. Among others, RAVER1-modulated splicing events affect the insertion of protein interaction modules in factors guiding miR/RISC-dependent gene silencing. Most prominently, in all three human TNRC6 proteins, RAVER1 controls AS of GW-enriched motifs, which are essential for AGO2-binding and the formation of active miR/RISC complexes. We propose, that RAVER1 is a key modulator of AS events in the miR/RISC pathway ensuring proper abundance and composition of miR/RISC effectors. This ensures balanced expression of TGFB signaling effectors and limits TGFB induced lethal EMT.

NANOS1 restricts oral cancer cell motility and TGF-ß signaling.

Oral squamous cell carcinoma (OSCC) is the most frequent type of cancer of the head and neck area accounting for approx. 377,000 new cancer cases every year. The epithelial-to-mesenchymal transition (EMT) program plays an important role in OSCC progression and metastasis therefore contributing to a poor prognosis in patients with advanced disease. Transforming growth factor beta (TGF-ß) is a powerful inducer of EMT thereby increasing cancer cell aggressiveness. Here, we aimed at identifying RNA-binding proteins (RBPs) that affect TGF-ß-induced EMT. To this end we treated oral cancer cells with TGF-ß and identified a total of 643 significantly deregulated protein-coding genes in response to TGF-ß. Of note, 19 genes encoded RBPs with NANOS1 being the most downregulated RBP. Subsequent cellular studies demonstrated a strong inhibitory effect of NANOS1 on migration and invasion of SAS oral cancer cells. Further mechanistic studies revealed an interaction of NANOS1 with the TGF-ß receptor 1 (TGFBR1) mRNA, leading to increased decay of this transcript and a reduced TGFBR1 protein expression, thereby preventing downstream TGF-ß/SMAD signaling. In summary, we identified NANOS1 as negative regulator of TGF-ß signaling in oral cancer cells.

Interferon signaling promotes tolerance to chromosomal instability during metastatic evolution in renal cancer.

Molecular routes to metastatic dissemination are critical determinants of aggressive cancers. Through in vivo CRISPR-Cas9 genome editing, we generated somatic mosaic genetically engineered models that faithfully recapitulate metastatic renal tumors. Disruption of 9p21 locus is an evolutionary driver to systemic disease through the rapid acquisition of complex karyotypes in cancer cells. Cross-species analysis revealed that recurrent patterns of copy number variations, including 21q loss and dysregulation of the interferon pathway, are major drivers of metastatic potential. In vitro and in vivo genomic engineering, leveraging loss-of-function studies, along with a model of partial trisomy of chromosome 21q, demonstrated a dosage-dependent effect of the interferon receptor genes cluster as an adaptive mechanism to deleterious chromosomal instability in metastatic progression. This work provides critical knowledge on drivers of renal cell carcinoma progression and defines the primary role of interferon signaling in constraining the propagation of aneuploid clones in cancer evolution.

Extravesicular TIMP-1 is a non-invasive independent prognostic marker and potential therapeutic target in colorectal liver metastases.

Molecular reprogramming of stromal microarchitecture by tumour-derived extracellular vesicles (EVs) is proposed to favour pre-metastatic niche formation. We elucidated the role of extravesicular tissue inhibitor of matrix metalloproteinase-1 (TIMP1EV) in pro-invasive extracellular matrix (ECM) remodelling of the liver microenvironment to aid tumour progression in colorectal cancer (CRC). Immunohistochemistry analysis revealed a high expression of stromal TIMP1 in the invasion front that was associated with poor progression-free survival in patients with colorectal liver metastases. Molecular analysis identified TIMP1EV enrichment in CRC-EVs as a major factor in the induction of TIMP1 upregulation in recipient fibroblasts. Mechanistically, we proved that EV-mediated TIMP1 upregulation in recipient fibroblasts induced ECM remodelling. This effect was recapitulated by human serum-derived EVs providing strong evidence that CRC release active EVs into the blood circulation of patients for the horizontal transfer of malignant traits to recipient cells. Moreover, EV-associated TIMP1 binds to HSP90AA, a heat-shock protein, and the inhibition of HSP90AA on human-derived serum EVs attenuates TIMP1EV-mediated ECM remodelling, rendering EV-associated TIMP1 a potential therapeutic target. Eventually, in accordance with REMARK guidelines, we demonstrated in three independent cohorts that EV-bound TIMP1 is a robust circulating biomarker for a non-invasive, preoperative risk stratification in patients with colorectal liver metastases.

The Cohesin Complex and Its Interplay with Non-Coding RNAs.

The cohesin complex is a multi-subunit protein complex initially discovered for its role in sister chromatid cohesion. However, cohesin also has several other functions and plays important roles in transcriptional regulation, DNA double strand break repair, and chromosome architecture thereby influencing gene expression and development in organisms from yeast to man. While most of these functions rely on protein-protein interactions, post-translational protein, as well as DNA modifications, non-coding RNAs are emerging as additional players that facilitate and modulate the function or expression of cohesin and its individual components. This review provides a condensed overview about the architecture as well as the function of the cohesin complex and highlights its multifaceted interplay with both short and long non-coding RNAs.

Identification of lymphocyte cell-specific protein-tyrosine kinase (LCK) as a driver for invasion and migration of oral cancer by tumor heterogeneity exploitation.

BACKGROUND: Cancer metastases are the main cause of lethality. The five-year survival rate for patients diagnosed with advanced stage oral cancer is 30%. Hence, the identification of novel therapeutic targets is an urgent need. However, tumors are comprised of a heterogeneous collection of cells with distinct genetic and molecular profiles that can differentially promote metastasis making therapy development a challenging task. Here, we leveraged intratumoral heterogeneity in order to identify drivers of cancer cell motility that might be druggable targets for anti-metastasis therapy. METHODS: We used 2D migration and 3D matrigel-based invasion assays to characterize the invasive heterogeneity among and within four human oral cancer cell lines in vitro. Subsequently, we applied mRNA-sequencing to map the transcriptomes of poorly and strongly invasive subclones as well as primary tumors and matched metastasis. RESULTS: We identified SAS cells as a highly invasive oral cancer cell line. Clonal analysis of SAS yielded a panel of 20 subclones with different invasive capacities. Integrative gene expression analysis identified the Lymphocyte cell-specific protein-tyrosine kinase (LCK) as a druggable target gene associated with cancer cell invasion and metastasis. Inhibition of LCK using A-770041 or dasatinib blocked invasion of highly aggressive SAS cells. Interestingly, reduction of LCK activity increased the formation of adherens junctions and induced cell differentiation. CONCLUSION: Analysis of invasive heterogeneity led to the discovery of LCK as an important regulator of motility in oral cancer cells. Hence, small molecule mediated inhibition of LCK could be a promising anti-metastasis therapy option for oral cancer patients.

Identification of a heat-inducible novel nuclear body containing the long noncoding RNA MALAT1.

The heat-shock response is critical for the survival of all organisms. Metastasis-associated long adenocarcinoma transcript 1 (MALAT1) is a long noncoding RNA localized in nuclear speckles, but its physiological role remains elusive. Here, we show that heat shock induces translocation of MALAT1 to a distinct nuclear body named the heat shock-inducible noncoding RNA-containing nuclear (HiNoCo) body in mammalian cells. MALAT1-knockout A549 cells showed reduced proliferation after heat shock. The HiNoCo body, which is formed adjacent to nuclear speckles, is distinct from any other known nuclear bodies, including the nuclear stress body, Cajal body, germs, paraspeckles, nucleoli and promyelocytic leukemia body. The formation of HiNoCo body is reversible and independent of heat shock factor 1, the master transcription regulator of the heat-shock response. Our results suggest the HiNoCo body participates in heat shock factor 1-independent heat-shock responses in mammalian cells.

Post-Transcriptional Expression Control in Platelet Biogenesis and Function.

Platelets are highly abundant cell fragments of the peripheral blood that originate from megakaryocytes. Beside their well-known role in wound healing and hemostasis, they are emerging mediators of the immune response and implicated in a variety of pathophysiological conditions including cancer. Despite their anucleate nature, they harbor a diverse set of RNAs, which are subject to an active sorting mechanism from megakaryocytes into proplatelets and affect platelet biogenesis and function. However, sorting mechanisms are poorly understood, but RNA-binding proteins (RBPs) have been suggested to play a crucial role. Moreover, RBPs may regulate RNA translation and decay following platelet activation. In concert with other regulators, including microRNAs, long non-coding and circular RNAs, RBPs control multiple steps of the platelet life cycle. In this review, we will highlight the different RNA species within platelets and their impact on megakaryopoiesis, platelet biogenesis and platelet function. Additionally, we will focus on the currently known concepts of post-transcriptional control mechanisms important for RNA fate within platelets with a special emphasis on RBPs.

RNA-Binding Proteins as Regulators of Migration, Invasion and Metastasis in Oral Squamous Cell Carcinoma.

Nearly 7.5% of all human protein-coding genes have been assigned to the class of RNA-binding proteins (RBPs), and over the past decade, RBPs have been increasingly recognized as important regulators of molecular and cellular homeostasis. RBPs regulate the post-transcriptional processing of their target RNAs, i.e., alternative splicing, polyadenylation, stability and turnover, localization, or translation as well as editing and chemical modification, thereby tuning gene expression programs of diverse cellular processes such as cell survival and malignant spread. Importantly, metastases are the major cause of cancer-associated deaths in general, and particularly in oral cancers, which account for 2% of the global cancer mortality. However, the roles and architecture of RBPs and RBP-controlled expression networks during the diverse steps of the metastatic cascade are only incompletely understood. In this review, we will offer a brief overview about RBPs and their general contribution to post-transcriptional regulation of gene expression. Subsequently, we will highlight selected examples of RBPs that have been shown to play a role in oral cancer cell migration, invasion, and metastasis. Last but not least, we will present targeting strategies that have been developed to interfere with the function of some of these RBPs.

Comprehensive Analysis of LincRNAs in Classical and Basal-Like Subtypes of Pancreatic Cancer.

Pancreatic ductal adenocarcinomas (PDAC) belong to the deadliest malignancies in the western world. Mutations in TP53 and KRAS genes along with some other frequent polymorphisms occur almost universally and are major drivers of tumour initiation. However, these mutations cannot explain the heterogeneity in therapeutic responses and differences in overall survival observed in PDAC patients. Thus, recent classifications of PDAC tumour samples have leveraged transcriptome-wide gene expression data to account for epigenetic, transcriptional and post-transcriptional mechanisms that may contribute to this deadly disease. Intriguingly, long intervening RNAs (lincRNAs) are a special class of long non-coding RNAs (lncRNAs) that can control gene expression programs on multiple levels thereby contributing to cancer progression. However, their subtype-specific expression and function as well as molecular interactions in PDAC are not fully understood yet. In this study, we systematically investigated the expression of lincRNAs in pancreatic cancer and its molecular subtypes using publicly available data from large-scale studies. We identified 27 deregulated lincRNAs that showed a significant different expression pattern in PDAC subtypes suggesting context-dependent roles. We further analyzed these lincRNAs regarding their common expression patterns. Moreover, we inferred clues on their functions based on correlation analyses and predicted interactions with RNA-binding proteins, microRNAs, and mRNAs. In summary, we identified several PDAC-associated lincRNAs of prognostic relevance and potential context-dependent functions and molecular interactions. Hence, our study provides a valuable resource for future investigations to decipher the role of lincRNAs in pancreatic cancer.

Increased Level of Long Non-Coding RNA MALAT1 is a Common Feature of Amoeboid Invasion.

The ability of cancer cells to adopt various migration modes (the plasticity of cancer cell invasiveness) is a substantive obstacle in the treatment of metastasis, yet still an incompletely understood process. We performed a comparison of publicly available transcriptomic datasets from various cell types undergoing a switch between the mesenchymal and amoeboid migration modes. Strikingly, lncRNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) was one of three genes that were found upregulated in all amoeboid cells analyzed. Accordingly, downregulation of MALAT1 in predominantly amoeboid cell lines A375m2 and A2058 resulted in decrease of active RhoA (Ras homolog family member A) and was accompanied by the amoeboid-mesenchymal transition in A375m2 cells. Moreover, MALAT1 downregulation in amoeboid cells led to increased cell proliferation. Our work is the first to address the role of MALAT1 in MAT/AMT (mesenchymal to amoeboid transition/amoeboid to mesenchymal transition) and suggests that increased MALAT1 expression is a common feature of amoeboid cells.

LINC00261 Is Differentially Expressed in Pancreatic Cancer Subtypes and Regulates a Pro-Epithelial Cell Identity.

Pancreatic adenocarcinoma (PDAC) is one of the major causes of cancer-associated deaths worldwide, with a dismal prognosis that has not significantly changed over the last decades. Transcriptional analysis has provided valuable insights into pancreatic tumorigenesis. Specifically, pancreatic cancer subtypes were identified, characterized by specific mutations and gene expression changes associated with differences in patient survival. In addition to differentially regulated mRNAs, non-coding RNAs, including long non-coding RNAs (lncRNAs), were shown to have subtype-specific expression patterns. Hence, we aimed to characterize prognostic lncRNAs with deregulated expression in the squamous subtype of PDAC, which has the worst prognosis. Extensive in silico analyses followed by in vitro experiments identified long intergenic non-coding RNA 261 (LINC00261) as a downregulated lncRNA in the squamous subtype of PDAC, which is generally associated with transforming growth factor ß (TGFß) signaling in human cancer cells. Its genomic neighbor, the transcription factor forkhead box protein A2 (FOXA2), regulated LINC00261 expression by direct binding of the LINC00261 promoter. CRISPR-mediated knockdown and promoter knockout validated the importance of LINC00261 in TGFß-mediated epithelial-mesenchymal transition (EMT) and established the epithelial marker E-cadherin, an important cell adhesion protein, as a downstream target of LINC00261. Consequently, depletion of LINC00261 enhanced motility and invasiveness of PANC-1 cells in vitro. Altogether, our data suggest that LINC00261 is an important tumor-suppressive lncRNA in PDAC that is involved in maintaining a pro-epithelial state associated with favorable disease outcome.

MALAT1: a therapeutic candidate for a broad spectrum of vascular and cardiorenal complications.

Cardiovascular and renal complications cover a wide array of diseases. The most commonly known overlapping complications include cardiac and renal fibrosis, cardiomyopathy, cardiac hypertrophy, hypertension, and cardiorenal failure. The known or reported causes for the abovementioned complications include injury, ischemia, infection, and metabolic stress. To date, various targets have been reported and investigated in detail that are considered to be the cause of these complications. In the past 5 years, the role of noncoding RNAs has emerged in the area of cardiovascular and renal research, especially in relation to metabolic stress. The long noncoding RNA MALAT1 (metastasis-associated lung adenocarcinoma transcript 1) has shown immense promise among the long noncoding RNA targets for treating cardiorenal complications. In this review, we shed light on the role of MALAT1 as a primary and novel target in treating cardiovascular and renal diseases as a whole.

Involvement of long non-coding RNA HULC (highly up-regulated in liver cancer) in pathogenesis and implications for therapeutic intervention.

INTRODUCTION: HULC (highly upregulated in liver cancer) is a long non-coding RNA (lncRNA) which is, as its name suggests, highly upregulated in hepatocellular carcinoma and in several other cancers. Increased HULC expression levels are strongly associated with clinicopathologic features such as tumor stages and overall survival and is a driver of tumor proliferation, migration, and invasion. Areas covered: This review addresses the discovery of HULC and discusses the consequences of HULC deregulation in cancer, the underlying molecular mechanisms and the potential of HULC as a biomarker and therapeutic target. Expert opinion: HULC is a promising candidate as a therapeutic target in cancer; however, more studies are necessary to further elucidate the underlying molecular mechanism(s), especially in cancer types other than hepatocellular carcinomas. Future studies that focus on an optimized HULC-targeting approach are necessary to clarify the best strategy to target this lncRNA in vivo and in patients.

Evaluation of fluorescence in situ hybridization techniques to study long non-coding RNA expression in cultured cells.

Deciphering the functions of long non-coding RNAs (lncRNAs) is facilitated by visualization of their subcellular localization using in situ hybridization (ISH) techniques. We evaluated four different ISH methods for detection of MALAT1 and CYTOR in cultured cells: a multiple probe detection approach with or without enzymatic signal amplification, a branched-DNA (bDNA) probe and an LNA-modified probe with enzymatic signal amplification. All four methods adequately stained MALAT1 in the nucleus in all of three cell lines investigated, HeLa, NHDF and T47D, and three of the methods detected the less expressed CYTOR. The sensitivity of the four ISH methods was evaluated by image analysis. In all three cell lines, the two methods involving enzymatic amplification gave the most intense MALAT1 signal, but the signal-to-background ratios were not different. CYTOR was best detected using the bDNA method. All four ISH methods showed significantly reduced MALAT1 signal in knock-out cells, and siRNA-induced knock-down of CYTOR resulted in significantly reduced CYTOR ISH signal, indicating good specificity of the probe designs and detection systems. Our data suggest that the ISH methods allow detection of both abundant and less abundantly expressed lncRNAs, although the latter required the use of the most specific and sensitive probe detection system.

From biomarkers to therapeutic targets-the promises and perils of long non-coding RNAs in cancer.

Biomarker-driven personalized cancer therapy is a field of growing interest, and several molecular tests have been developed to detect biomarkers that predict, e.g., response of cancers to particular therapies. Identification of these molecules and understanding their molecular mechanisms is important for cancer prognosis and the development of therapeutics for late stage diseases. In the past, significant efforts have been placed on the discovery of protein or DNA-based biomarkers while only recently the class of long non-coding RNA (lncRNA) has emerged as a new category of biomarker. The mammalian genome is pervasively transcribed yielding a vast amount of non-protein-coding RNAs including lncRNAs. Hence, these transcripts represent a rich source of information that has the potential to significantly contribute to precision medicine in the future. Importantly, many lncRNAs are differentially expressed in carcinomas and they are emerging as potent regulators of tumor progression and metastasis. Here, we will highlight prime examples of lncRNAs that serve as marker for cancer progression or therapy response and which might represent promising therapeutic targets. Furthermore, we will introduce lncRNA targeting tools and strategies, and we will discuss potential pitfalls in translating these into clinical trials.

The Dark Side of the Epitranscriptome: Chemical Modifications in Long Non-Coding RNAs.

The broad application of next-generation sequencing technologies in conjunction with improved bioinformatics has helped to illuminate the complexity of the transcriptome, both in terms of quantity and variety. In humans, 70-90% of the genome is transcribed, but only ~2% carries the blueprint for proteins. Hence, there is a huge class of non-translated transcripts, called long non-coding RNAs (lncRNAs), which have received much attention in the past decade. Several studies have shown that lncRNAs are involved in a plethora of cellular signaling pathways and actively regulate gene expression via a broad selection of molecular mechanisms. Only recently, sequencing-based, transcriptome-wide studies have characterized different types of post-transcriptional chemical modifications of RNAs. These modifications have been shown to affect the fate of RNA and further expand the variety of the transcriptome. However, our understanding of their biological function, especially in the context of lncRNAs, is still in its infancy. In this review, we will focus on three epitranscriptomic marks, namely pseudouridine (Ψ), N6-methyladenosine (m6A) and 5-methylcytosine (m5C). We will introduce writers, readers, and erasers of these modifications, and we will present methods for their detection. Finally, we will provide insights into the distribution and function of these chemical modifications in selected, cancer-related lncRNAs.