Liquid Biopsy for Pancreatic Cancer by Serum Extracellular Vesicle-Encapsulated Long Noncoding RNA HEVEPA.

OBJECTIVES: The role of long noncoding RNAs (lncRNAs) in pancreatic ductal adenocarcinoma (PDAC) remain unclear. Extracellular vesicle (EV)-encapsulated RNAs could be effective targets for liquid biopsy. We aimed to identify previously unknown EV-encapsulated lncRNAs in PDAC and establish highly accurate methods for isolating EVs. MATERIALS AND METHODS: Extracellular vesicles were isolated using existing and newly developed methods, namely, PEViA-UC and PEViA-IP, from serum samples of 20 patients with PDAC, 22 patients with intraductal papillary mucinous neoplasms, and 21 healthy individuals. Extracellular vesicle lncRNA expression was analyzed using digital PCR. RESULTS: Gene expression analysis using cDNA microarray revealed a highly expressed lncRNA, HEVEPA , in serum EVs from patients with PDAC. We established PEViA-UC and PEViA-IP using PEViA reagent, ultracentrifugation, and immunoprecipitation. Although detection of EV-encapsulated HEVEPA using existing methods is challenging, PEViA-UC and PEViA-IP detected EV HEVEPA , which was highly expressed in patients with PDAC compared with non-PDAC patients. The detection sensitivity for discriminating PDAC from non-PDAC using the combination of HEVEPA and HULC , which are highly expressed lncRNAs in PDAC, and carbohydrate antigen 19-9 (CA19-9), was higher than that of HEVEPA , HULC , or CA19-9 alone. CONCLUSIONS: Extracellular vesicle lncRNAs isolated using PEViA-IP and CA19-9 together could be effective targets in liquid biopsy for PDAC diagnosis.

LncRNA ZNNT1 induces p53 degradation by interfering with the interaction between p53 and the SART3-USP15 complex.

Mammalian genomes encode large number of long noncoding RNAs (lncRNAs) that play key roles in various biological processes, including proliferation, differentiation, and stem cell pluripotency. Recent studies have addressed that some lncRNAs are dysregulated in human cancers and may play crucial roles in tumor development and progression. Here, we show that the lncRNA ZNNT1 is required for the proliferation and tumorigenicity of colon cancer cells with wild-type p53. ZNNT1 knockdown leads to decreased ubiquitination and stabilization of p53 protein. Moreover, we demonstrate that ZNNT1 needs to interact with SART3 to destabilize p53 and to promote the proliferation and tumorigenicity of colon cancer cells. We further show that SART3 is associated with the ubiquitin-specific peptidase USP15 and that ZNNT1 may induce p53 destabilization by inhibiting this interaction. These results suggest that ZNNT1 interferes with the SART3-USP15 complex-mediated stabilization of p53 protein and thereby plays important roles in the proliferation and tumorigenicity of colon cancer cells. Our findings suggest that ZNNT1 may be a promising molecular target for the therapy of colon cancer.

Multiplex Digital PCR Assay to Detect Multiple KRAS and GNAS Mutations Associated with Pancreatic Carcinogenesis from Minimal Specimen Amounts.

Digital PCR (dPCR) allows for highly sensitive quantification of low-frequency mutations and facilitates early detection of cancer. However, low-throughput targeting of single hotspots in dPCR hinders variant specification when multiple probes are used. We developed a dPCR method to simultaneously identify major variants related to pancreatic carcinogenesis. Using a two-dimensional plot of droplet fluorescence under the optimized concentration of two fluorescent probe pools, the absolute quantification of different KRAS and GNAS variants was determined. Successful detection of the multiple driver mutations was verified in 24 surgically resected tumor samples from 19 patients and 22 fine-needle aspiration samples from patients with pancreatic ductal adenocarcinoma. Precise quantification of the variant allele frequency was optimized by using template DNA at a concentration as low as 1 to 10 ng. Furthermore, amplicons targeting multiple hotspots were successfully enriched with fewer false-positive findings using high-fidelity polymerase, allowing for the detection of various KRAS and GNAS mutations with high probability in small amount of cell/tissue specimens. Using this target enrichment, mutations at a rate of 90% in small residual tissues, such as the fine-needle aspiration needle flush and microscopic lesions in resected specimens, were successfully identified. The proposed method allows for low-cost, accurate detection of driver mutations to diagnose cancers, even with minimal tissue collection.

Predictors of Long-Term Survival in Pancreatic Ductal Adenocarcinoma after Pancreatectomy: TP53 and SMAD4 Mutation Scoring in Combination with CA19-9.

BACKGROUND: Pancreatic ductal adenocarcinoma (PDA) is a fatal cancer for which even unfavorable clinicopathological factors occasionally fail to preclude long-term survival. We sought to establish a scoring system that utilizes measurable pre-intervention factors for predicting survival following surgical resection. METHODS: We retrospectively analyzed 34 patients who died from short-term recurrences and 32 long-term survivors among 310 consecutively resected patients with PDA. A logistic regression model was used to define factors related to clinical parameters, molecular profiles of 18 pancreatic cancer-associated genes, and aberrant expression of major tumor suppressors. RESULTS: Carbohydrate antigen 19-9 (CA19-9) had the best ability to classify patients with short-term recurrence and long-term survivors [odds ratio 21.04, 95% confidence interval (CI) 4.612-96.019], followed by SMAD4 and TP53 mutation scoring (odds ratio 41.322, 95% CI 3.156-541.035). Missense TP53 mutations were strongly associated with the nuclear expression of p53, whereas truncating mutations were associated with the absence of nuclear p53. The former subset was associated with a worse prognosis. The combination of aberrant SMAD4 and mutation types of TP53 exhibited a better resolution for distinguishing patients with short-term recurrences from long-term survivors (compared with the assessment of the number of mutated KRAS, CDKN2A, TP53, and SMAD4 genes). Calibration of mutation scores combined with CA19-9 in a logistic regression model setting demonstrated a practical effect in classifying long survivors and patients with early recurrence (c-statistic = 0.876). CONCLUSIONS: Genetic information, i.e., TP53 mutation types and SMAD4 abnormalities, combined with CA19-9, will be a valuable tool for improving surgical strategies for pancreatic cancer.

Mutant GNAS limits tumor aggressiveness in established pancreatic cancer via antagonizing the KRAS-pathway.

BACKGROUND: Mutations in GNAS drive pancreatic tumorigenesis and frequently occur in intraductal papillary mucinous neoplasm (IPMN); however, their value as a therapeutic target is yet to be determined. This study aimed at evaluating the involvement of mutant GNAS in tumor aggressiveness in established pancreatic cancer. METHODS: CRISPR/Cas9-mediated GNAS R201H silencing was performed using human primary IPMN-associated pancreatic cancer cells. The role of oncogenic GNAS in tumor maintenance was evaluated by conducting cell culture and xenograft experiments, and western blotting and transcriptome analyses were performed to uncover GNAS-driven signatures. RESULTS: Xenografts of GNAS wild-type cells were characterized by a higher Ki-67 labeling index relative to GNAS-mutant cells. Phenotypic alterations in the GNAS wild-type tumors resulted in a significant reduction in mucin production accompanied by solid with massive stromal components. Transcriptional profiling suggested an apparent conflict of mutant GNAS with KRAS signaling. A significantly higher Notch intercellular domain (NICD) was observed in the nuclear fraction of GNAS wild-type cells. Meanwhile, inhibition of protein kinase A (PKA) induced NICD in GNAS-mutant IPMN cells, suggesting that NOTCH signaling is negatively regulated by the GNAS-PKA pathway. GNAS wild-type cells were characterized by a significant invasive property relative to GNAS-mutant cells, which was mediated through the NOTCH regulatory pathway. CONCLUSIONS: Oncogenic GNAS induces mucin production, not only via MUC2 but also via MUC5AC/B, which may enlarge cystic lesions in the pancreas. The mutation may also limit tumor aggressiveness by attenuating NOTCH signaling; therefore, such tumor-suppressing effects must be considered when therapeutically inhibiting the GNAS pathway.

RNA Exosome Component EXOSC4 Amplified in Multiple Cancer Types Is Required for the Cancer Cell Survival.

The RNA exosome is a multi-subunit ribonuclease complex that is evolutionally conserved and the major cellular machinery for the surveillance, processing, degradation, and turnover of diverse RNAs essential for cell viability. Here we performed integrated genomic and clinicopathological analyses of 27 RNA exosome components across 32 tumor types using The Cancer Genome Atlas PanCancer Atlas Studies' datasets. We discovered that the EXOSC4 gene, which encodes a barrel component of the RNA exosome, was amplified across multiple cancer types. We further found that EXOSC4 alteration is associated with a poor prognosis of pancreatic cancer patients. Moreover, we demonstrated that EXOSC4 is required for the survival of pancreatic cancer cells. EXOSC4 also repressed BIK expression and destabilized SESN2 mRNA by promoting its degradation. Furthermore, knockdown of BIK and SESN2 could partially rescue pancreatic cells from the reduction in cell viability caused by EXOSC4 knockdown. Our study provides evidence for EXOSC4-mediated regulation of BIK and SESN2 mRNA in the survival of pancreatic tumor cells.

Aberrant phase separation and cancer.

Eukaryotic cells are intracellularly divided into numerous compartments or organelles, which coordinate specific molecules and biological reactions. Membrane-bound organelles are physically separated by lipid bilayers from the surrounding environment. Biomolecular condensates, also referred to membraneless organelles, are micron-scale cellular compartments that lack membranous enclosures but function to concentrate proteins and RNA molecules, and these are involved in diverse processes. Liquid-liquid phase separation (LLPS) driven by multivalent weak macromolecular interactions is a critical principle for the formation of biomolecular condensates, and a multitude of combinations among multivalent interactions may drive liquid-liquid phase transition (LLPT). Dysregulation of LLPS and LLPT leads to aberrant condensate and amyloid formation, which causes many human diseases, including neurodegeneration and cancer. Here, we describe recent findings regarding abnormal forms of biomolecular condensates and aggregation via aberrant LLPS and LLPT of cancer-related proteins in cancer development driven by mutation and fusion of genes. Moreover, we discuss the regulatory mechanisms by which aberrant LLPS and LLPT occur in cancer and the drug candidates targeting these mechanisms. Further understanding of the molecular events regulating how biomolecular condensates and aggregation form in cancer tissue is critical for the development of therapeutic strategies against tumorigenesis.

Pan-cancer methylome analysis for cancer diagnosis and classification of cancer cell of origin.

The accurate and early diagnosis and classification of cancer origin from either tissue or liquid biopsy is crucial for selecting the appropriate treatment and reducing cancer-related mortality. Here, we established the CAncer Cell-of-Origin (CACO) methylation panel using the methylation data of the 28 types of cancer in The Cancer Genome Atlas (7950 patients and 707 normal controls) as well as healthy whole blood samples (95 subjects). We showed that the CACO methylation panel had high diagnostic potential with high sensitivity and specificity in the discovery (maximum AUC = 0.998) and validation (maximum AUC = 1.000) cohorts. Moreover, we confirmed that the CACO methylation panel could identify the cancer cell type of origin using the methylation profile from liquid as well as tissue biopsy, including primary, metastatic, and multiregional cancer samples and cancer of unknown primary, independent of the methylation analysis platform and specimen preparation method. Together, the CACO methylation panel can be a powerful tool for the classification and diagnosis of cancer.

Long Non-Coding RNAs in Epithelial-Mesenchymal Transition of Pancreatic Cancer.

Non-coding RNAs (ncRNAs), or RNA molecules that do not code for proteins, are generally categorized as either small or long ncRNA (lncRNA) and are involved in the pathogenesis of several diseases including many cancers. Identification of a large number of ncRNAs could help to elucidate previously unknown mechanisms in phenotype regulation. Some ncRNAs are encapsulated by extracellular vesicles (EVs) and transferred to recipient cells to regulate cellular processes, including epigenetic and post-transcriptional regulations. Recent studies have uncovered novel molecular mechanisms and functions of lncRNAs in pancreatic ductal adenocarcinoma (PDAC), one of the most intractable cancers that is highly invasive and metastatic. As the epithelial-mesenchymal transition (EMT) triggers tumor cell invasion and migration, clarification of the roles of lncRNA in EMT and tumor cell stemness would be critical for improving diagnostic and therapeutic approaches in metastatic cancers. This review provides an overview of relevant studies on lncRNA and its involvement with EMT in PDAC. Emerging knowledge offers evidence for the dysregulated expression of lncRNAs and essential insights into the potential contribution of both lncRNAs and EVs in the pathogenesis of PDAC. Future directions and new clinical applications for PDAC are also discussed.

hnRNPH1-MTR4 complex-mediated regulation of NEAT1v2 stability is critical for IL8 expression.

Many long noncoding RNAs (lncRNAs) are localized in the nucleus and play important roles in various biological processes, including cell proliferation, differentiation and antiviral response. Yet, it remains unclear how some nuclear lncRNAs are turned over. Here we show that the heterogeneous nuclear ribonucleoprotein H1 (hnRNPH1) controls expression levels of NEAT1v2, a lncRNA involved in the formation of nuclear paraspeckles. hnRNPH1 associates, in an RNA-independent manner, with the RNA helicase MTR4/MTREX, an essential co-factor of the nuclear ribonucleolytic RNA exosome. hnRNPH1 localizes in nuclear speckles and depletion of hnRNPH1 enhances NEAT1v2-mediated expression of the IL8 mRNA, encoding a cytokine involved in the innate immune response. Taken together, our results indicate that the hnRNPH1-MTR4 linkage regulates IL8 expression through the degradation of NEAT1v2 RNA.

Human U90926 orthologous long non-coding RNA as a novel biomarker for visual prognosis in herpes simplex virus type-1 induced acute retinal necrosis.

Acute retinal necrosis (ARN) is a form of infectious uveitis caused by alpha herpesviruses, including herpes simplex virus type 1 (HSV-1). We previously found that the long non-coding RNA (lncRNA) U90926 is upregulated in murine retinal photoreceptor cells following HSV-1 infection, leading to host cell death. However, to date, an orthologous transcript has not been identified in humans. We investigated U90926 orthologous transcript in humans and examined its utility as a prognostic marker for visual acuity in patients with ARN. We identified two human orthologous transcripts (1955 and 592 bases) of lncRNA U90926. The amount of the longer human U90926 transcript was approximately 30- and 40-fold higher in the vitreous fluid of patients with ARN than in those with sarcoidosis and intraocular lymphoma, respectively. Furthermore, the expression of the longer human U90926 transcript in the vitreous fluid was highly correlated with the final best-corrected logarithm of the minimum angle of resolution visual acuity in patients with ARN (r = 0.7671, p = 0.0079). This suggests higher expression of the longer human U90926 transcript in the vitreous fluid results in worse visual prognosis; therefore, expression of the longer human U90926 transcript is a potential negative prognostic marker for visual acuity in patients with ARN.

Fusion Genes and RNAs in Cancer Development.

Fusion RNAs are a hallmark of some cancers. They result either from chromosomal rearrangements or from splicing mechanisms that are non-chromosomal rearrangements. Chromosomal rearrangements that result in gene fusions are particularly prevalent in sarcomas and hematopoietic malignancies; they are also common in solid tumors. The splicing process can also give rise to more complex RNA patterns in cells. Gene fusions frequently affect tyrosine kinases, chromatin regulators, or transcription factors, and can cause constitutive activation, enhancement of downstream signaling, and tumor development, as major drivers of oncogenesis. In addition, some fusion RNAs have been shown to function as noncoding RNAs and to affect cancer progression. Fusion genes and RNAs will therefore become increasingly important as diagnostic and therapeutic targets for cancer development. Here, we discuss the function, biogenesis, detection, clinical relevance, and therapeutic implications of oncogenic fusion genes and RNAs in cancer development. Further understanding the molecular mechanisms that regulate how fusion RNAs form in cancers is critical to the development of therapeutic strategies against tumorigenesis.

The Functions and Unique Features of LncRNAs in Cancer Development and Tumorigenesis.

Over the past decades, research on cancer biology has focused on the involvement of protein-coding genes in cancer development. Long noncoding RNAs (lncRNAs), which are transcripts longer than 200 nucleotides that lack protein-coding potential, are an important class of RNA molecules that are involved in a variety of biological functions. Although the functions of a majority of lncRNAs have yet to be clarified, some lncRNAs have been shown to be associated with human diseases such as cancer. LncRNAs have been shown to contribute to many important cancer phenotypes through their interactions with other cellular macromolecules including DNA, protein and RNA. Here we describe the literature regarding the biogenesis and features of lncRNAs. We also present an overview of the current knowledge regarding the roles of lncRNAs in cancer from the view of various aspects of cellular homeostasis, including proliferation, survival, migration and genomic stability. Furthermore, we discuss the methodologies used to identify the function of lncRNAs in cancer development and tumorigenesis. Better understanding of the molecular mechanisms involving lncRNA functions in cancer is critical for the development of diagnostic and therapeutic strategies against tumorigenesis.

Identification and analysis of short open reading frames (sORFs) in the initially annotated noncoding RNA LINC00493 from human cells.

Whole transcriptome analyses have revealed that mammalian genomes are massively transcribed, resulting in the production of huge numbers of transcripts with unknown functions (TUFs). Previous research has categorized most TUFs as noncoding RNAs (ncRNAs) because most previously studied TUFs do not encode open reading frames (ORFs) with biologically significant lengths [>100 amino acids (AAs)]. Recent studies, however, have reported that several transcripts harbouring small ORFs that encode peptides shorter than 100 AAs are translated and play important biological functions. Here, we examined the translational capacity of transcripts annotated as ncRNAs in human cells, and identified several hundreds of ribosome-associated transcripts previously annotated as ncRNAs. Ribosome footprinting and polysome profiling analyses revealed that 61 of them are potentially translatable. Among them, 45 were nonnonsense-mediated mRNA decay targets, suggesting that they are productive mRNAs. We confirmed the translation of one ncRNA, LINC00493, by luciferase reporter assaying and western blotting of a FLAG-tagged LINC00493 peptide. While proteomic analysis revealed that the LINC00493 peptide interacts with many mitochondrial proteins, immunofluorescence assays showed that its peptide is mitochondrially localized. Our findings indicate that some transcripts annotated as ncRNAs encode peptides and that unannotated peptides may perform important roles in cells.

Long noncoding RNA U90926 is crucial for herpes simplex virus type 1 proliferation in murine retinal photoreceptor cells.

Long non-coding RNAs (lncRNAs) play vital roles in the pathogenesis of infectious diseases, but the role of lncRNAs in herpes simplex virus 1 (HSV-1) infection remains unknown. Using RNA sequencing analysis, we explored lncRNAs that were highly expressed in murine retinal photoreceptor cell-derived 661W cells infected with HSV-1. U90926 RNA (522 nucleotides) was the most upregulated lncRNA detected post HSV-1 infection. The level of U90926 RNA was continuously increased post HSV-1 infection, reaching a 100-fold increase at 24 h. Cellular fractionation showed that U90926 RNA was located in the nucleus post HSV-1 infection. Downregulation of U90926 expression by RNA interference markedly suppressed HSV-1 DNA replication (80% reduction at 12 h post infection) and HSV-1 proliferation (93% reduction at 12 h post infection) in 661W cells. The survival rates of U90926-knockdown cells were significantly increased compared to those of control cells (81% and 21%, respectively; p < 0.0001). Thus, lncRNA U90926 is crucial for HSV-1 proliferation in retinal photoreceptor cells and consequently leads to host cell death by promoting HSV-1 proliferation.

Digital PCR-based plasma cell-free DNA mutation analysis for early-stage pancreatic tumor diagnosis and surveillance.

BACKGROUND: Cell-free DNA (cfDNA) shed from tumors into the circulation offers a tool for cancer detection. Here, we evaluated the feasibility of cfDNA measurement and utility of digital PCR (dPCR)-based assays, which reduce subsampling error, for diagnosing pancreatic ductal adenocarcinoma (PDA) and surveillance of intraductal papillary mucinous neoplasm (IPMN). METHODS: We collected plasma from seven institutions for cfDNA measurements. Hot-spot mutations in KRAS and GNAS in the cfDNA from patients with PDA (n = 96), undergoing surveillance for IPMN (n = 112), and normal controls (n = 76) were evaluated using pre-amplification dPCR. RESULTS: Upon Qubit measurement and copy number assessment of hemoglobin-subunit (HBB) and mitochondrially encoded NADH:ubiquinone oxidoreductase core subunit 1 (MT-ND1) in plasma cfDNA, HBB offered the best resolution between patients with PDA relative to healthy subjects [area under the curve (AUC) 0.862], whereas MT-ND1 revealed significant differences between IPMN and controls (AUC 0.851). DPCR utilizing pre-amplification cfDNA afforded accurate tumor-derived mutant KRAS detection in plasma in resectable PDA (AUC 0.861-0.876) and improved post-resection recurrence prediction [hazard ratio (HR) 3.179, 95% confidence interval (CI) 1.025-9.859] over that for the marker CA19-9 (HR 1.464; 95% CI 0.674-3.181). Capturing KRAS and GNAS could also provide genetic evidence in patients with IPMN-associated PDA and undergoing pancreatic surveillance. CONCLUSIONS: Plasma cfDNA quantification by distinct measurements is useful to predict tumor burden. Through appropriate methods, dPCR-mediated mutation detection in patients with localized PDA and IPMN likely to progress to invasive carcinoma is feasible and complements conventional biomarkers.

Metabolic labeling of RNA using multiple ribonucleoside analogs enables the simultaneous evaluation of RNA synthesis and degradation rates.

Gene expression is determined by a balance between RNA synthesis and RNA degradation. To elucidate the underlying regulatory mechanisms and principles of this, simultaneous measurements of RNA synthesis and degradation are required. Here, we report the development of "Dyrec-seq," which uses 4-thiouridine and 5-bromouridine to simultaneously quantify RNA synthesis and degradation rates. Dyrec-seq enabled the quantification of RNA synthesis and degradation rates of 4702 genes in HeLa cells. Functional enrichment analysis showed that the RNA synthesis and degradation rates of genes are actually determined by the genes' biological functions. A comparison of theoretical and experimental analyses revealed that the amount of RNA is determined by the ratio of RNA synthesis to degradation rates, whereas the rapidity of responses to external stimuli is determined only by the degradation rate. This study emphasizes that not only RNA synthesis but also RNA degradation is important in shaping gene expression patterns.

Systematic Analysis of Targets of Pumilio-Mediated mRNA Decay Reveals that PUM1 Repression by DNA Damage Activates Translesion Synthesis.

RNA-binding proteins (RBPs) play a pivotal role in gene expression by modulating the stability of transcripts. However, the identification of degradation target mRNAs of RBPs remains difficult. By the combined analysis of transcriptome-wide mRNA stabilities and the binding of mRNAs to human Pumilio 1 (PUM1), we identify 48 mRNAs that both bind to PUM1 and exhibit PUM1-dependent degradation. Analysis of changes in the abundance of PUM1 and its degradation target mRNAs in RNA-seq data indicate that DNA-damaging agents negatively regulate PUM1-mediated mRNA decay. Cells exposed to cisplatin have reduced PUM1 abundance and increased PCNA and UBE2A mRNAs encoding proteins involved in DNA damage tolerance by translesion synthesis (TLS). Cells overexpressing PUM1 exhibit impaired DNA synthesis and TLS and increased sensitivity to the cytotoxic effect of cisplatin. Thus, our method identifies target mRNAs of PUM1-mediated decay and reveals that cells respond to DNA damage by inhibiting PUM1-mediated mRNA decay to activate TLS.

UHRF1-KAT7-mediated regulation of TUSC3 expression via histone methylation/acetylation is critical for the proliferation of colon cancer cells.

The epigenetic factor UHRF1 regulates transcription by modulating DNA methylation and histone modification, and plays critical roles in proliferation, development, and tumorigenesis. Here, we show that Wnt/c-Myc signaling upregulates UHRF1, which in turn downregulates TUSC3, a candidate tumor suppressor gene that is frequently deleted or downregulated in several cancers. We also show that UHRF1-mediated downregulation of TUSC3 is required for the proliferation of colon cancer cells. Furthermore, we demonstrate that UHRF1 suppresses TUSC3 expression by interacting with methylated H3K14 and thereby suppressing the acetylation of H3K14 by the histone acetyltransferase KAT7. Our study provides evidence for the significance of UHRF1-KAT7-mediated regulation of histone methylation/acetylation in the proliferation of tumor cells and in a diverse set of biological processes controlled by Wnt/c-Myc signaling.