Paraspeckles are stress-induced nuclear RNA-protein condensates that assemble on the long non-coding RNA NEAT1. Their increased formation under certain cellular circumstances has gained growing interest due to their association with serious human diseases, such as neurodegenerative disorders and cancer. The biological functions of paraspeckles still appear obscure, but increasing evidence suggests that they contribute to regulation of gene expression by recruiting specific proteins and RNA molecules. Here, we have characterized and compared two stress-enriched interactomes of the essential paraspeckle protein NONO in both wild-type and paraspeckle-deficient NEAT1 knockout cells. We identified Hsp70 as part of stress-enriched NONO complexes in wild-type but not in NEAT1-depleted cells. We show that proteotoxic stress-induced paraspeckle formation and NEAT1 expression are strictly dependent on Hsp70 chaperone activity. Our data demonstrate that both NONO and Hsp70 transiently translocate to the nucleolus during heat shock and that paraspeckle formation during recovery follows Hsp70-dependent relocation of NONO from the nucleolus to the nucleoplasm. Taken together, we demonstrate an important role of Hsp70 in paraspeckle assembly and identify a possible link between the nuclear protein quality control system and paraspeckles.

Serglycin is a key hematopoietic proteoglycan that regulates the storage and retention of proteases, cytokines, and chemokines within immune-cell secretory granules. We previously identified serglycin as the predominant proteoglycan secreted by macrophages and a regulator of tumor necrosis factor-α (TNF-α) release following lipopolysaccharide (LPS) stimulation. Here, we analyzed serglycin expression in peritoneal macrophages from 92 mouse strains in the hybrid mouse diversity panel (HMDP) after LPS exposure and investigated its role in M1 polarization using serglycin-deficient murine bone marrow-derived macrophages and human THP-1 macrophages stimulated with LPS and interferon-γ (IFN-γ). Serglycin upregulation is strongly associated with M1 polarization and correlated with TNF-α and IFN-γ transcriptional signatures across models. Integrated transcriptomic and secretome analyses revealed a marked discordance between mRNA and protein output in serglycin-deficient macrophages, with impaired cytokine secretion despite elevated transcript levels, validated by RT-qPCR and ELISA. Loss of serglycin additionally affected macrophage metabolism, vesicle biogenesis, phagocytosis, and extracellular reactive oxygen species generation, identifying serglycin as a key regulator of macrophage polarization and functional programming.

Head and neck squamous cell carcinoma (HNSCC) is notoriously resistant to immunotherapy. The interplay between β-adrenergic signaling and p53 loss, both key regulators of immune responses, has remained largely unexplored in the setting of tumor-immune evasion. This study demonstrates that pharmacologic stimulation of β2-adrenergic receptors with isoprenaline significantly enhances cytotoxic T cell activity against p53-deficient HNSCC cells via a CXCL10-dependent paracrine mechanism. Comprehensive transcriptomic and co-culture assays reveal that p53-null cancer cells upregulate CXCL10, which promotes CD8+ T cell recruitment and activation. Neutralization of CXCL10 abolishes the β-adrenergic-induced cytotoxic T cell response, establishing this chemokine as a pivotal mediator. Using tyrosine hydroxylase knockout mouse models, we show that adrenergic innervation is essential for intra-tumoral CXCL10 expression and the infiltration of effector CXCR3+ T cells in vivo. Notably, the CXCL10-driven T cell response is associated with simultaneous upregulation of both activation and exhaustion markers, indicating a robust but transient effector state within the tumor microenvironment. Collectively, these findings uncover a neuro-immune axis that reverses immune escape in p53-deficient HNSCC and suggest novel therapeutic strategies targeting adrenergic signaling to convert immune "cold" tumors into "hot" ones more amenable to immunotherapy.

Breast cancer, a prevalent malignancy worldwide, includes the triple‐negative subtype (TNBC) characterized by poor treatment outcomes. TNBC has been shown to be sensitive to ferroptotic cell death, an iron‐dependent cell death mechanism involving reactive oxygen species (ROS) and lipid peroxidation. Herein, biodegradable tetraphenylchlorin‐conjugated chitosan nanoparticles (TPC‐CS NPs) in combination with the free ferroptosis inducer RSL3 is used in MCF7 (hormone receptor‐positive, epithelial) and MDA‐MB‐231 (hormone receptor‐negative, mesenchymal‐like) breast cancer cell lines. The results show that RSL3 treatment has no cytotoxic effect in MCF7 and there is no enhanced sensitivity when combined with TPC‐CS NPs, while the combination sensitizes MDA‐MB‐231 cells. Western blot analysis reveals that the combined treatment decreases and differently affects GPX4 levels and the ratio between the two GPX isoforms in the two cell lines. In MDA‐MB‐231 cells, the combined treatment shows enhanced effects on lipid peroxidation, mitochondrial potential, and basal and maximal respiration, as compared to single treatments. Finally, ferroptosis expression signatures distinguish breast cancer cell lines with an increasing score in mesenchymal‐like cells. Moreover, the signatures correlate with breast cancer subtypes, exhibiting the highest scores in subtypes rich in mesenchymal‐like cells, particularly basal‐like and claudin‐low tumors, suggesting their susceptibility to ferroptosis induction.

Growth hormone (GH) is a neuromodulator that binds to receptors in the hippocampus and alters synaptic plasticity. A decline in GH levels is associated with normal aging, stress, and disease, and the mechanisms proposed involve the hippocampal circuit plasticity. To see how GH affects the hippocampal neural code, we recorded single neurons in the CA1 region of male Long-Evans rats with locally altered GH levels. Rats received injections of adeno-associated viruses into the hippocampus to make the cells overexpress either GH or an antagonizing mutated GH (aGH). Place cells were recorded in both familiar and novel environments to allow the assessment of pattern separation in the neural representations termed remapping. All the animals showed intact and stable place fields in the familiar environment. In the novel environment, aGH transfection increased the average firing rate, peak rate, and information density of the CA1 place fields. The tendency of global remapping increased in the GH animals compared with the controls, and only place cells of control animals showed significant rate remapping. Our results suggest that GH increases hippocampal sensitivity to novel information. Our findings show that GH is a significant neuromodulator in the hippocampus affecting how place cells represent the environment. These results could help us to understand the mechanisms behind memory impairments in GH deficiency as well as in normal aging.

Abstract Introduction: The metastasis of a pancreatic ductal adenocarcinoma (PAAD) is a diagnosis of exclusion and one of the most common causes of cancer of unknown primary (CUP). We have recently developed a genome-wide DNA methylation-based neural network classifier that can accurately differentiate between liver metastasis of a PAAD and intrahepatic cholangiocarcinoma (iCCA) (PAAD-iCCA-Classifier). Therefore, the aim of our study was to test whether our PAAD-iCCA-Classifier can be extended to be used to correctly diagnose PAAD metastases from other sites in CUP setting. Methods: For this purpose, we enhanced the anomaly detection layer of the classifier by incorporating ten mimicker carcinomas to be excluded by this layer. We used a validation set 1 (n=3786) including primary PAAD (n=242), PAAD liver metastases (n=20), iCCA (n=151) and 10 other mimicker carcinomas (n=3373) and a validation set 2 (n=26) including primary PAAD from a real-life clinical cohort from an independent institution to validate the classifier. Next, we tested the classifier on 16 PAAD initially considered CUP samples (test set) from different sites: peritoneum, lung, liver, and lymph node. The clinical history and diagnostic imaging of these samples were used to confirm PAAD as the most probable origin. We further performed differentially methylated probes (DMP) and copy number alterations (CNA) analysis of primary PAAD and metastatic PAAD from different locations. Results: The improved version of the PAAD-iCCA-Classifier achieved an accuracy of 98.43% on the validation set 1 and was able to exclude most of the mimicker carcinomas. On validation set 2, the classifier achieved an accuracy of 88.46%. Medical history, imaging and immunohistochemical analysis of the test set samples confirmed the diagnosis of PAAD. The DNA methylation classifier correctly labeled 15/16 PAAD metastatic samples as PAAD (93.75% accuracy). We observed that the classifier performance was negatively affected by a high CD3+ immune infiltrate and positively affected by high tumor purity and high proliferation rate. CNA revealed that PAAD liver metastases have a distinct CNA profile characterized by chromosome 6, 9 (CDKN2A/B) and 18q (SMAD4) deletions. DMP analysis showed that PAAD liver metastases have global hypomethylation of both promoter- and enhancer-associated CpGs compared to primary PAAD and PAAD peritoneal carcinomatosis. Finally, gene ontology analysis revealed that different epithelial-mesenchymal transition pathways are activated in PAAD liver metastases compared to PAAD peritoneal carcinomatosis. Conclusion: Our tool performs well in classifying metastatic PAAD samples and could be of great clinical use when a PAAD origin is suspected in the case of a CUP. DMP and CNA profiles show that PAAD liver metastases may have a distinct DNA methylation and copy number profile compared to primary and peritoneal carcinomatosis PAAD. Citation Format: Teodor G. Calina, Eilís Perez, Simon Schallenberg, Horst David, Erik Knutsen, David Capper, Mihnea P. Dragomir. Genome-wide DNA methylation classifier diagnoses pancreatic ductal adenocarcinoma in CUP setting [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4414.

BackgroundWe have recently constructed a DNA methylation classifier that can discriminate between pancreatic ductal adenocarcinoma (PAAD) liver metastasis and intrahepatic cholangiocarcinoma (iCCA) with high accuracy (PAAD-iCCA-Classifier). PAAD is one of the leading causes of cancer of unknown primary and diagnosis is based on exclusion of other malignancies. Therefore, our focus was to investigate whether the PAAD-iCCA-Classifier can be used to diagnose PAAD metastases from other sites.MethodsFor this scope, the anomaly detection filter of the initial classifier was expanded by 8 additional mimicker carcinomas, amounting to a total of 10 carcinomas in the negative class. We validated the updated version of the classifier on a validation set, which consisted of a biological cohort (n = 3579) and a technical one (n = 15). We then assessed the performance of the classifier on a test set, which included a positive control cohort of 16 PAAD metastases from various sites and a cohort of 124 negative control samples consisting of 96 breast cancer metastases from 18 anatomical sites and 28 carcinoma metastases to the brain.ResultsThe updated PAAD-iCCA-Classifier achieved 98.21% accuracy on the biological validation samples, and on the technical validation ones it reached 100%. The classifier also correctly identified 15/16 (93.75%) metastases of the positive control as PAAD, and on the negative control, it correctly classified 122/124 samples (98.39%) for a 97.85% overall accuracy on the test set. We used this DNA methylation dataset to explore the organotropism of PAAD metastases and observed that PAAD liver metastases are distinct from PAAD peritoneal carcinomatosis and primary PAAD, and are characterized by specific copy number alterations and hypomethylation of enhancers involved in epithelial-mesenchymal-transition.ConclusionsThe updated PAAD-iCCA-Classifier (available at https://classifier.tgc-research.de/) can accurately classify PAAD samples from various metastatic sites and it can serve as a diagnostic aid.

Accuracy and transparency of scientific data are becoming more and more relevant with the increasing concern regarding the evaluation of data reproducibility in many research areas. This concern is also true for quantifying coding and noncoding RNAs, with the remarkable increase in publications reporting RNA profiling and sequencing studies. To address the problem, we propose the following recommendations: (a) accurate documentation of experimental procedures in Materials and methods (and not only in the supplementary information, as many journals have a strict mandate for making Materials and methods as visible as possible in the main text); (b) submission of RT-qPCR raw data for all experiments reported; and (c) adoption of a unified, simple format for submitted RT-qPCR raw data. The Real-time PCR Data Essential Spreadsheet Format (RDES) was created for this purpose.

Abstract High phylogenetic conservation of genomic loci is a potential marker of essential biological functionality. Ultraconserved elements (UCEs) are DNA sequences entirely conserved through millions of years of evolution, but their functions are not well understood. Several long non-coding RNAs (lncRNAs) are transcribed from UCEs and play active roles in cancer development. Using a combination of in vitro and in vivo experiments with two animal models and 4 patient cohorts, we discovered several remarkable aspects of ultraconservation. Using a multi-step genetically defined carcinogenesis model, we identified one UCE, uc.206, and its associated overexpressed lncRNA TRUC-16 as a regulator of cell proliferation in chronic lymphocytic leukemia (CLL). High levels of TRUC-16 inhibit p16INK4A translation, causing increased proliferation through an E2F1-mediated mechanism in CLL patients, as well as in zebrafish and murine models. uc.206/TRUC-16 function is extremely conserved throughout evolution. We found cell cycle-related effects of TRUC-16 in both mice and zebrafish, in which we observed increased proliferation of blood cells after overexpression of the human TRUC-16. High levels of CDK4/6 consequent to TRUC-16 overexpression and p16 decrease can be specifically targeted in both CLL and Richter’s Transformation (RT), a rare but deadly consequence of CLL. Inhibition of CDK6 is a therapeutic option never tested in CLL, but in use for another type of B cell malignancy, such as the mantle cell lymphoma. Our results highlight the multifaceted contribution of a UCE to human tumorigenesis and open new possibilities for the use of long-noncoding RNAs in cancer therapies. Citation Format: Linda Fabris, Erik Knutsen, Maria Teresa S. Bertilaccio, Steliana Calin, Recep Bayraktar, George T. Eisenhoffer, Leonard Girnita, George A. Calin. Regulation of p16-E2F1-CDK4/6 signaling by TRUC-16 ultraconserved long non-coding RNA. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3799.

Sepsis remains a leading cause of death for humans and currently has no pathogenesis-specific therapy. Hampered progress is partly due to a lack of insight into deep mechanistic processes. In the past decade, deciphering the functions of small noncoding miRNAs in sepsis pathogenesis became a dynamic research topic. To screen for new miRNA targets for sepsis therapeutics, we used samples for miRNA array analysis of PBMCs from patients with sepsis and control individuals, blood samples from 2 cohorts of patients with sepsis, and multiple animal models: mouse cecum ligation puncture-induced (CLP-induced) sepsis, mouse viral miRNA challenge, and baboon Gram+ and Gram- sepsis models. miR-93-5p met the criteria for a therapeutic target, as it was overexpressed in baboons that died early after induction of sepsis, was downregulated in patients who survived after sepsis, and correlated with negative clinical prognosticators for sepsis. Therapeutically, inhibition of miR-93-5p prolonged the overall survival of mice with CLP-induced sepsis, with a stronger effect in older mice. Mechanistically, anti-miR-93-5p therapy reduced inflammatory monocytes and increased circulating effector memory T cells, especially the CD4+ subset. AGO2 IP in miR-93-KO T cells identified important regulatory receptors, such as CD28, as direct miR-93-5p target genes. In conclusion, miR-93-5p is a potential therapeutic target in sepsis through the regulation of both innate and adaptive immunity, with possibly a greater benefit for elderly patients than for young patients.

The causes and consequences of abnormal biogenesis of extracellular vesicles (EVs) are not yet well understood in malignancies, including in breast cancers (BCs). Given the hormonal signaling dependence of estrogen receptor-positive (ER+) BC, we hypothesized that 17β-estradiol (estrogen) might influence EV production and microRNA (miRNA) loading. We report that physiological doses of 17β-estradiol promote EV secretion specifically from ER+ BC cells via inhibition of miR-149-5p, hindering its regulatory activity on SP1, a transcription factor that regulates the EV biogenesis factor nSMase2. Additionally, miR-149-5p downregulation promotes hnRNPA1 expression, responsible for the loading of let-7's miRNAs into EVs. In multiple patient cohorts, we observed increased levels of let-7a-5p and let-7d-5p in EVs derived from the blood of premenopausal ER+ BC patients, and elevated EV levels in patients with high BMI, both conditions associated with higher levels of 17β-estradiol. In brief, we identified a unique estrogen-driven mechanism by which ER+ BC cells eliminate tumor suppressor miRNAs in EVs, with effects on modulating tumor-associated macrophages in the microenvironment.

Epithelial-mesenchymal transition (EMT) is a cellular plasticity program critical for embryonic development and tissue regeneration, and aberrant EMT is associated with disease including cancer. The high degree of plasticity in the mammary epithelium is reflected in extensive heterogeneity among breast cancers. Here, we have analyzed RNA-sequencing data from three different mammary epithelial cell line-derived EMT models and identified a robust mammary EMT gene expression signature that separates breast cancers into distinct subgroups. Most strikingly, the basal-like breast cancers form two subgroups displaying partial-EMT and post-EMT gene expression patterns. We present evidence that key EMT-associated transcription factors play distinct roles at different stages of EMT in mammary epithelial cells.

The ultraconserved regions (UCRs) are the genomic segments manifesting perfect conservation between the orthologous genomes of humans, rats, and mice that get transcribed into mono-exonic long non-coding RNAs (lncRNAs) known as transcribed ultraconserved regions (T-UCRs). Increasing evidence demonstrates the importance of T-UCRs in human cancers, however, their involvement in the pathogenesis of Chronic Lymphocytic Leukemia (CLL) is poorly understood. Our lab has recently identified a lncRNA transcribed from a UCR and named it TRUC-16 (Translational Regulatory UltraConserved gene affecting p16), which is overexpressed in CLL and correlates with treatment-free survival. We have developed a transgenic C57BL6 mouse model to overexpress TRUC-16 in B cells. We monitored the number and development of B cell subsets over time in peripheral blood and lymphoid organs (lymph nodes, spleen, and bone marrow), and performed immunophenotyping to assess clonality and proportion of the cells considered to be the origin for CLL (i.e. B1 cells and IgM memory B cells that are CD5+). We followed mice survival (n=10 for each gender) and performed necropsy with histopathologic and immunophenotypic analysis. If blood sampling suggests developing a CLL-like state, we will monitor for evolution to Richter Syndrome by flow cytometry analysis and biochemistry markers. We have validated the TRUC-16 overexpression specific to the B-cell compartment in the mice cohorts and have observed lymphoma in several organs. Interestingly, TRUC-16 overexpressing mice showed significantly higher splenic weights compared to controls. The mice were found to develop adulthood lymphoma that we are now analyzing molecularly and phenotypically. We are planning to further characterize the subtype of lymphoma by utilizing specific diagnostic markers through immunohistochemistry and flow cytometry. This is the first-ever transgenic mice modeling overexpression of UCR in the context of cancer. Validation of the functional correlation between the TRUC-16 phenotype and CLL patients’ clinical data will provide a valuable resource to pursue a multitude of preclinical studies to find targets that might help better understand the disease pathophysiology. Citation Format: Swati Mohapatra, Erik Knutsen, Mihai Iurascu Gagea, Linda Fabris, George Adrian Calin. Deciphering the in vivo roles of a novel long non-coding RNA in chronic lymphocytic leukemia and Richter syndrome. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3795.

Serglycin is a proteoglycan highly expressed by immune cells, in which its functions are linked to storage, secretion, transport, and protection of chemokines, proteases, histamine, growth factors, and other bioactive molecules. In recent years, it has been demonstrated that serglycin is also expressed by several other cell types, such as endothelial cells, muscle cells, and multiple types of cancer cells. Here, we show that serglycin expression is upregulated in transforming growth factor beta (TGF-β) induced epithelial-mesenchymal transition (EMT). Functional studies provide evidence that serglycin plays an important role in the regulation of the transition between the epithelial and mesenchymal phenotypes, and it is a significant EMT marker gene. We further find that serglycin is more expressed by breast cancer cell lines with a mesenchymal phenotype as well as the basal-like subtype of breast cancers. By examining immune staining and single cell sequencing data of breast cancer tissue, we show that serglycin is highly expressed by infiltrating immune cells in breast tumor tissue.

Many severe inflammation conditions are complement-dependent with the complement component C5a-C5aR1 axis as an important driver. At the RNA level, the blood transcriptome undergoes programmed expression of coding and long non-coding RNAs to combat invading microorganisms. Understanding the expression of long non-coding RNAs containing Alu elements in inflammation is important for reconstructing cell fate trajectories leading to severe disease. We have assembled a pipeline for computation mining of new Alu-containing long non-coding RNAs by intersecting immune genes with known Alu coordinates in the human genome. By applying the pipeline to patient bulk RNA-seq data with sepsis, we found immune genes containing 48 Alu insertion as robust candidates for further study. Interestingly, 1 of the 48 candidates was located within the complement system receptor gene C5aR1 and holds promise as a target for RNA therapeutics.

Proteoglycans (PGs) are pivotal components of extracellular matrices, involved in a variety of processes such as migration, invasion, morphogenesis, differentiation, drug resistance, and epithelial-to-mesenchymal transition (EMT). Cellular plasticity is a crucial intermediate phenotypic state acquired by cancer cells, which can modulate EMT and the generation of cancer stem cells (CSCs). PGs affect cell plasticity, stemness, and EMT, altering the cellular shape and functions. PGs control these functions, either by direct activation of signaling cascades, acting as co-receptors, or through regulation of the availability of biological compounds such as growth factors and cytokines. Differential expression of microRNAs is also associated with the expression of PGs and their interplay is implicated in the fine tuning of cancer cell phenotype and potential. This review summarizes the involvement of PGs in the regulation of EMT and stemness of cancer cells and highlights the molecular mechanisms.

The RNA-binding protein ALYREF (THOC4) is involved in transcriptional regulation and nuclear mRNA export, though its role and molecular mode of action in breast carcinogenesis are completely unknown. Here, we identified high ALYREF expression as a factor for poor survival in breast cancer patients. ALYREF significantly influenced cellular growth, apoptosis and mitochondrial energy metabolism in breast cancer cells as well as breast tumorigenesis in orthotopic mouse models. Transcriptional profiling, phenocopy and rescue experiments identified the short isoform of the lncRNA NEAT1 as a molecular trigger for ALYREF effects in breast cancer. Mechanistically, we found that ALYREF binds to the NEAT1 promoter region to enhance the global NEAT1 transcriptional activity. Importantly, by stabilizing CPSF6, a protein that selectively activates the post-transcriptional generation of the short isoform of NEAT1 , as well as by direct binding and stabilization of the short isoform of NEAT1, ALYREF selectively fine-tunes the expression of the short NEAT1 isoform. Overall, our study describes ALYREF as a novel factor contributing to breast carcinogenesis and identifies novel molecular mechanisms of regulation the two isoforms of NEAT1 .

Alterations in microRNAs (miRNAs) expression are causative in the initiation and progression of human cancers. The molecular events responsible for the widespread differential expression of miRNAs in malignancy are exemplified by their location in cancer-associated genomic regions, epigenetic mechanisms, transcriptional dysregulation, chemical modifications and editing, and alterations in miRNA biogenesis proteins. The classical miRNA function is synonymous with post-transcriptional repression of target protein genes. However, several studies have reported miRNAs functioning outside this paradigm and some of these novel modes of regulation of gene expression have been implicated in cancers. Here, we summarize key aspects of miRNA involvement in cancer, with a special focus on these lesser-studied mechanisms of action.

Knockout (KO) of long non-coding RNAs (lncRNAs) enables functional characterization of this still poorly described group of transcripts. One of the most efficient and simplest methods to achieve complete KO of a lncRNA is by employing CRISPR/Cas gene editing. As most lncRNAs are not well annotated, their individual functional regions are often not defined, and the majority of the transcripts are not affected by single nucleotide mutations. Therefore, CRISPR/Cas KO is more challenging for lncRNAs as compared to KO of protein coding genes. Strategies for lncRNAs KO include complete removal of the entire gene, removal of the promoter and transcriptional start site, abolishing exon-exon junctions, or removing the transcriptional termination site. Here, we describe the methodology to perform CRISPR/Cas9 KO of lncRNAs in vitro using electroporation as the method of transfection of presynthesized single guide RNAs (sgRNAs) and Cas9 enzyme.