Significance of signal recognition particle 9 nuclear translocation: Implications for pancreatic cancer prognosis and functionality.

Signal recognition particles (SRPs) are essential for regulating intracellular protein transport and secretion. Patients with tumors with high SRP9 expression tend to have a poorer overall survival. However, to the best of our knowledge, no reports have described the relationship between SRP9 localization and prognosis in pancreatic cancer. Thus, the present study aimed to investigate this relationship. Immunohistochemical staining for SRP9 using excised specimens from pancreatic cancer surgery cases without preoperative chemotherapy or radiotherapy showed that SRP9 was preferentially expressed in the nucleus of the cancerous regions in some cases, which was hardly detected in other cases, indicating that SRP9 was transported to the nucleus in the former cases. To compare the prognosis of patients with SRP9 nuclear translocation, patients were divided into two groups: Those with a nuclear translocation rate of >50% and those with a nuclear translocation rate of ≤50%. The nuclear translocation rate of >50% group had a significantly better recurrence­free survival than the nuclear translocation rate of ≤50% group (P=0.037). Subsequent in vitro experiments were conducted; notably, the nuclear translocation rate of SRP9 was reduced under amino acid­deficient conditions, suggesting that multiple factors are involved in this phenomenon. To further study the function of SRP9 nuclear translocation, in vitro experiments were performed by introducing SRP9 splicing variants (v1 and v2) and their deletion mutants lacking C­terminal regions into MiaPaCa pancreatic cancer cells. The results demonstrated that both splicing variants showed nuclear translocation regardless of the C­terminal deletions, suggesting the role of the N­terminal regions. Given that SRP9 is an RNA­binding protein, the study of RNA immunoprecipitation revealed that signaling pathways involved in cancer progression and protein translation were downregulated in nuclear­translocated v1 and v2. Undoubtedly, further studies of the nuclear translocation of SRP9 will open an avenue to optimize the precise evaluation and therapeutic control of pancreatic cancer.

High N6-methyladenosine-activated TCEAL8 mRNA is a novel pancreatic cancer marker.

N6-methyladenosine (m6A) is an RNA modification involved in RNA processing and widely found in transcripts. In cancer cells, m6A is upregulated, contributing to their malignant transformation. In this study, we analyzed gene expression and m6A modification in cancer tissues, ducts, and acinar cells derived from pancreatic cancer patients using MeRIP-seq. We found that dozens of RNAs highly modified by m6A were detected in cancer tissues compared with ducts and acinar cells. Among them, the m6A-activated mRNA TCEAL8 was observed, for the first time, as a potential marker gene in pancreatic cancer. Spatially resolved transcriptomic analysis showed that TCEAL8 was highly expressed in specific cells, and activation of cancer-related signaling pathways was observed relative to TCEAL8-negative cells. Furthermore, among TCEAL8-positive cells, the cells expressing the m6A-modifying enzyme gene METTL3 showed co-activation of Notch and mTOR signaling, also known to be involved in cancer metastasis. Overall, these results suggest that m6A-activated TCEAL8 is a novel marker gene involved in the malignant transformation of pancreatic cancer.

Role of RNA methylation in the regulation of pancreatic cancer stem cells (Review).

Pancreatic cancer stem cells (CSCs) play a key role in the initiation and progression of pancreatic adenocarcinoma (PDAC). CSCs are responsible for resistance to chemotherapy and radiation, and for cancer metastasis. Recent studies have indicated that RNA methylation, a type of RNA modification, predominantly occurring as m6A methylation, plays an important role in controlling the stemness of cancer cells, therapeutic resistance against chemotherapy and radiation therapy, and their overall relevance to a patient's prognosis. CSCs regulate various behaviors of cancer through cell-cell communication by secreting factors, through their receptors, and through signal transduction. Recent studies have shown that RNA methylation is involved in the biology of the heterogeneity of PDAC. The present review provides an update on the current understanding of RNA modification-based therapeutic targets against deleterious PDAC. Several key pathways and agents that can specifically target CSCs have been identified, thus providing novel insights into the early diagnosis and efficient treatment of PDAC.

Cancer-specific tissue-resident memory T-cells express ZNF683 in colorectal cancer.

BACKGROUND: Tissue-resident memory T (Trm) cells are associated with cytotoxicity not only in viral infection and autoimmune disease pathologies but also in many cancers. Tumour-infiltrating CD103+ Trm cells predominantly comprise CD8 T cells that express cytotoxic activation and immune checkpoint molecules called exhausted markers. This study aimed to investigate the role of Trm in colorectal cancer (CRC) and characterise the cancer-specific Trm. METHODS: Immunochemical staining with anti-CD8 and anti-CD103 antibodies for resected CRC tissues was used to identify the tumour-infiltrating Trm cells. The Kaplan-Meier estimator was used to evaluate the prognostic significance. Cells immune to CRC were targeted for single-cell RNA-seq analysis to characterise cancer-specific Trm cells in CRC. RESULTS: The number of CD103+/CD8+ tumour-infiltrating lymphocytes (TILs) was a favourable prognostic and predictive factor of the overall survival and recurrence-free survival in patients with CRC. Single-cell RNA-seq analysis of 17,257 CRC-infiltrating immune cells revealed a more increased zinc finger protein 683 (ZNF683) expression in cancer Trm cells than in noncancer Trm cells and in high-infiltrating Trm cells than low-infiltrating Trm in cancer, with an upregulated T-cell receptor (TCR)- and interferon-γ (IFN-γ) signalling-related gene expression in ZNF683+ Trm cells. CONCLUSIONS: The number of CD103+/CD8+ TILs is a prognostic predictive factor in CRC. In addition, we identified the ZNF683 expression as one of the candidate markers of cancer-specific Trm cells. IFN-γ and TCR signalling and ZNF683 expression are involved in Trm cell activation in tumours and are promising targets for cancer immunity regulation.

RNA Modification in Inflammatory Bowel Diseases.

Inflammatory bowel disease (IBD) is a chronic inflammatory disorder characterized by damage to the intestinal mucosa, which is caused by a combination of factors. These include genetic and epigenetic alterations, environmental influence, microorganism interactions, and immune conditions. Some populations with IBD show a cancer-prone phenotype. Recent studies have provided insight into the involvement of RNA modifications in the specific pathogenesis of IBD through regulation of RNA biology in epithelial and immune cells. Studies of several RNA modification-targeting reagents have shown preferable outcomes in patients with colitis. Here, we note a new awareness of RNA modification in the targeting of IBD and related diseases, which will contribute to early diagnosis, disease monitoring, and possible control by innovative therapeutic approaches.

Cloaking the ACE2 receptor with salivary cationic proteins inhibits SARS-CoV-2 entry.

Saliva contributes to the innate immune system, which suggests that it can prevent SARS-CoV-2 entry. We studied the ability of healthy salivary proteins to bind to angiotensin-converting enzyme 2 (ACE2) using biolayer interferometry and pull-down assays. Their effects on binding between the receptor-binding domain of the SARS-CoV-2 spike protein S1 (S1) and ACE2 were determined using an enzyme-linked immunosorbent assay. Saliva bound to ACE2 and disrupted the binding of S1 to ACE2 and four ACE2-binding salivary proteins were identified, including cationic histone H2A and neutrophil elastase, which inhibited the S1-ACE2 interaction. Calf thymus histone (ct-histone) also inhibited binding as effectively as histone H2A. The results of a cell-based infection assay indicated that ct-histone suppressed SARS-CoV-2 pseudoviral invasion into ACE2-expressing host cells. Manufactured polypeptides, such as ε-poly-L-lysine, also disrupted S1-ACE2 binding, indicating the importance of the cationic properties of salivary proteins in ACE2 binding. Overall, we demonstrated that positively charged salivary proteins are a barrier against SARS-CoV-2 entry by cloaking the negatively charged surface of ACE2 and provided a view that the cationic polypeptides represent a preventative and therapeutic treatment against COVID-19.

EpisomiR, a New Family of miRNAs, and Its Possible Roles in Human Diseases.

MicroRNAs (miRNAs) are synthesized through a canonical pathway and play a role in human diseases, such as cancers and cardiovascular, neurodegenerative, psychiatric, and chronic inflammatory diseases. The development of sequencing technologies has enabled the identification of variations in noncoding miRNAs. These miRNA variants, called isomiRs, are generated through a non-canonical pathway, by several enzymes that alter the length and sequence of miRNAs. The isomiR family is, now, expanding further to include episomiRs, which are miRNAs with different modifications. Since recent findings have shown that isomiRs reflect the cell-specific biological function of miRNAs, knowledge about episomiRs and isomiRs can, possibly, contribute to the optimization of diagnosis and therapeutic technology for precision medicine.

Direct observation of DNA alterations induced by a DNA disruptor.

DNA alterations, such as base modifications and mutations, are closely related to the activity of transcription factors and the corresponding cell functions; therefore, detection of DNA alterations is important for understanding their relationships. Particularly, DNA alterations caused by exposure to exogenous molecules, such as nucleic acid analogues for cancer therapy and the corresponding changes in cell functions, are of interest in medicine for drug development and diagnosis purposes. However, detection of comprehensive direct evidence for the relationship of DNA modifications/mutations in genes, their effect on transcription factors, and the corresponding cell functions have been limited. In this study, we utilized a single-molecule electrical detection method for the direct observation of DNA alterations on transcription factor binding motifs upon exposure to a nucleic acid analogue, trifluridine (FTD), and evaluated the effects of the DNA alteration on transcriptional activity in cancer cell line cells. We found ~ 10% FTD incorporation at the transcription factor p53 binding regions in cancer cells exposed to FTD for 5 months. Additionally, through single-molecule analysis of p53-enriched DNA, we found that the FTD incorporation at the p53 DNA binding regions led to less binding, likely due to weaken the binding of p53. This work suggests that single-molecule detection of DNA sequence alterations is a useful methodology for understanding DNA sequence alterations.

Detection techniques for epitranscriptomic marks.

Similar to epigenetic DNA modification, RNA can be methylated and altered for stability and processing. RNA modifications, namely, epitranscriptomes, involve the following three functions: writing, erasing, and reading of marks. Methods for measurement and position detection are useful for the assessment of cellular function and human disease biomarkers. After pyrimidine 5-methylcytosine was reported for the first time a hundred years ago, numerous techniques have been developed for studying nucleotide modifications, including RNAs. Recent studies have focused on high-throughput and direct measurements for investigating the precise function of epitranscriptomes, including the characterization of severe acute respiratory syndrome coronavirus 2. The current study presents an overview of the development of detection techniques for epitranscriptomic marks and briefs about the recent progress in this field.

Cancer metabolism challenges genomic instability and clonal evolution as therapeutic targets.

Although cancer precision medicine has improved diagnosis and therapy, refractory cancers such as pancreatic cancer remain to be challenging targets. Clinical sequencing has identified the significant alterations in driver genes and traced their clonal evolutions. Recent studies indicated that the tumor microenvironment elicits alterations in cancer metabolism, although its involvement in the cause and development of genomic alterations has not been established. Genomic abnormalities can contribute to the survival of selected subpopulations, recently recognized as clonal evolution, and dysfunction can lead to DNA mutations. Here, we present the most recent studies on the mechanisms of cancer metabolism involved in the maintenance of genomic stability to update current understanding of such processes. Sirtuins, which are NAD+-dependent protein deacetylases, appear to be involved in the control of genomic stability. Alterations of deleterious subpopulations would be exposed to selective pressure for cell survival. Recent studies indicated that a new type of cell death, ferroptosis, determines the survival of clones and exert cancer-restricting or -promoting effects to surrounding cells in the tumor microenvironment. Suppressing genomic instability and eliminating deleterious clones by cell death will contribute to the improvement of cancer medicine. Furthermore, the elucidation of the mechanisms involved is seen as a bridgehead to the pharmacologic suppression of such refractory cancers as pancreatic cancer.

Methylosystem for Cancer Sieging Strategy.

As cancer is a genetic disease, methylation defines a biologically malignant phenotype of cancer in the association of one-carbon metabolism-dependent S-adenosylmethionine (SAM) as a methyl donor in each cell. Methylated substances are involved in intracellular metabolism, but via intercellular communication, some of these can also be secreted to affect other substances. Although metabolic analysis at the single-cell level remains challenging, studying the "methylosystem" (i.e., the intercellular and intracellular communications of upstream regulatory factors and/or downstream effectors that affect the epigenetic mechanism involving the transfer of a methyl group from SAM onto the specific positions of nucleotides or other metabolites in the tumor microenvironment) and tracking these metabolic products are important research tasks for understanding spatial heterogeneity. Here, we discuss and highlight the involvement of RNA and nicotinamide, recently emerged targets, in SAM-producing one-carbon metabolism in cancer cells, cancer-associated fibroblasts, and immune cells. Their significance and implications will contribute to the discovery of efficient methods for the diagnosis of and therapeutic approaches to human cancer.

Single-molecule RNA sequencing for simultaneous detection of m6A and 5mC.

Epitranscriptomics is the study of RNA base modifications involving functionally relevant changes to the transcriptome. In recent years, epitranscriptomics has been an active area of research. However, a major issue has been the development of sequencing methods to map transcriptome-wide RNA base modifications. We have proposed a single-molecule quantum sequencer for mapping RNA base modifications in microRNAs (miRNAs), such as N6-methyladenosine (m6A) or 5-methylcytidine (5mC), which are related to cancer cell propagation and suppression. Here, we investigated 5mC and m6A in hsa-miR-200c-5p extracted from colorectal cancer cells and determined their methylation sites and rates; the data were comparable to those determined by mass spectrometry. Furthermore, we evaluated the methylation ratio of cytidine and adenosine at each site in the sequences and its relationship. These results suggest that the methylation ratio of cytidine and adenosine is facilitated by the presence of vicinal methylation. Our work provides a robust new tool for sequencing various types of RNA base modifications in their RNA context.

Impact of One-Carbon Metabolism-Driving Epitranscriptome as a Therapeutic Target for Gastrointestinal Cancer.

One-carbon (1C) metabolism plays a key role in biological functions linked to the folate cycle. These include nucleotide synthesis; the methylation of DNA, RNA, and proteins in the methionine cycle; and transsulfuration to maintain the redox condition of cancer stem cells in the tumor microenvironment. Recent studies have indicated that small therapeutic compounds affect the mitochondrial folate cycle, epitranscriptome (RNA methylation), and reactive oxygen species reactions in cancer cells. The epitranscriptome controls cellular biochemical reactions, but is also a platform for cell-to-cell interaction and cell transformation. We present an update of recent advances in the study of 1C metabolism related to cancer and demonstrate the areas where further research is needed. We also discuss approaches to therapeutic drug discovery using animal models and propose further steps toward developing precision cancer medicine.

Immuno-Surgical Management of Pancreatic Cancer with Analysis of Cancer Exosomes.

Exosomes (EXs), a type of extracellular vesicles secreted from various cells and especially cancer cells, mesenchymal cells, macrophages and other cells in the tumor microenvironment (TME), are involved in biologically malignant behaviors of cancers. Recent studies have revealed that EXs contain microRNAs on their inside and express proteins and glycolipids on their outsides, every component of which plays a role in the transmission of genetic and/or epigenetic information in cell-to-cell communications. It is also known that miRNAs are involved in the signal transduction. Thus, EXs may be useful for monitoring the TME of tumor tissues and the invasion and metastasis, processes that are associated with patient survival. Because several solid tumors secrete immune checkpoint proteins, including programmed cell death-ligand 1, the EX-mediated mechanisms are suggested to be potent targets for monitoring patients. Therefore, a companion therapeutic approach against cancer metastasis to distant organs is proposed when surgical removal of the primary tumor is performed. However, EXs and immune checkpoint mechanisms in pancreatic cancer are not fully understood, we provide an update on the recent advances in this field and evidence that EXs will be useful for maximizing patient benefit in precision medicine.

State-of-the-Art Technology of Model Organisms for Current Human Medicine.

Since the 1980s, molecular biology has been used to investigate medical field mechanisms that still require the use of crude biological materials in order to achieve their necessary goals. Transcription factor-induced pluripotent stem cells are used in regenerative medicine to screen drugs and to support lost tissues. However, these cells insufficiently reconstruct whole organs and require various intact cells, such as damaged livers and diabetic pancreases. For efficient gene transfer in medical use, virally mediated gene transfers are used, although immunogenic issues are investigated. To obtain efficient detective and diagnostic power in intractable diseases, biological tools such as roundworms and zebrafish have been found to be useful for high-throughput screening (HST) and diagnosis. Taken together, this biological approach will help to fill the gaps between medical needs and novel innovations in the field of medicine.

COVID-19 Drug Discovery Using Intensive Approaches.

Since the infectious disease caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was reported in China during December 2019, the coronavirus disease 2019 (COVID-19) has spread on a global scale, causing the World Health Organization (WHO) to issue a warning. While novel vaccines and drugs that target SARS-CoV-2 are under development, this review provides information on therapeutics which are under clinical trials or are proposed to antagonize SARS-CoV-2. Based on the information gained from the responses to other RNA coronaviruses, including the strains that cause severe acute respiratory syndrome (SARS)-coronaviruses and Middle East respiratory syndrome (MERS), drug repurposing might be a viable strategy. Since several antiviral therapies can inhibit viral replication cycles or relieve symptoms, mechanisms unique to RNA viruses will be important for the clinical development of antivirals against SARS-CoV-2. Given that several currently marketed drugs may be efficient therapeutic agents for severe COVID-19 cases, they may be beneficial for future viral pandemics and other infections caused by RNA viruses when standard treatments are unavailable.

Distinct methylation levels of mature microRNAs in gastrointestinal cancers.

The biological significance of micro (mi)RNAs has traditionally been evaluated according to their RNA expression levels based on the assumption that miRNAs recognize and regulate their targets in an unvarying fashion. Here we show that a fraction of mature miRNAs including miR-17-5p, -21-5p, and -200c-3p and let-7a-5p harbor methyl marks that potentially alter their stability and target recognition. Importantly, methylation of these miRNAs was significantly increased in cancer tissues as compared to paired normal tissues. Furthermore, miR-17-5p methylation level in serum samples distinguished early pancreatic cancer patients from healthy controls with extremely high sensitivity and specificity. These findings provide a basis for diagnostic strategies for early-stage cancer and add a dimension to our understanding of miRNA biology.