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Getah virus (GETV) belongs to the Alphavirus genus in the Togaviridae family and is a zoonotic arbovirus causing disease in both humans and animals. The capsid protein (CP) of GETV regulates the viral core assembly, but the mechanism underlying this process is poorly understood. In this study, we demonstrate that CP undergoes liquid-liquid phase separation (LLPS) with the GETV genome RNA (gRNA) in vitro and forms cytoplasmic puncta in cells. Two regions of GETV gRNA (nucleotides 1-4000 and 5000-8000) enhance CP droplet formation in vitro and the lysine-rich Link region of CP is essential for its phase separation. CP(K/R) mutant with all lysines in the Link region replaced by arginines exhibits improved LLPS versus wild type (WT) CP, but CP(K/E) mutant with lysines substituted by glutamic acids virtually loses condensation ability. Consistently, recombinant virus mutant with CP(K/R) possesses significantly higher gRNA binding affinity, virion assembly efficiency and infectivity than the virus with WT-CP. Overall, our findings provide new insights into the understanding of GETV assembly and development of new antiviral drugs against alphaviruses., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

As ubiquitously expressed transcripts in eukaryotes, circular RNAs (circRNAs) are covalently closed and lack a 5'-cap and 3'-polyadenylation (poly (A)) tail. Initially, circRNAs were considered non-coding RNA (ncRNA), and their roles as sponging molecules to adsorb microRNAs have been extensively reported. However, in recent years, accumulating evidence has demonstrated that circRNAs could encode functional polypeptides through the initiation of translation mediated by internal ribosomal entry sites (IRESs) or N 6 -methyladenosine (m 6 A). In this review, we collectively discuss the biogenesis, cognate mRNA products, regulatory mechanisms, aberrant expression and biological phenotypes or clinical relevance of all currently reported, cancer-relevant protein-coding circRNAs. Overall, we provide a comprehensive overview of circRNA-encoded proteins and their physiological and pathological functions., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023 Elsevier B.V. All rights reserved.)

Extraction processes significantly alter the structural and functional properties of polysaccharides. In this study, we extracted polysaccharides from Chroogomphis rutilus fruiting bodies (designated as CRP) using four methods, including hot water, ultrasound, microwave and sequential ultrasound-microwave, and designated these polysaccharides as CRP-H, CRP-M, CRP-U and CRP-UM, respectively. All CRPs were heteropolysaccharides with semblable monosaccharide types of glucose, mannose and galactose, mainly constituted of α-d-glucopyranosyl-(1 → 4). The extraction processes significantly affected the molecular weights, monosaccharide proportions, glycosidic bond ratios, branching degrees, triple-helix conformation and surface morphology of the CRPs. Among them, CRP-UM showed the highest yield and most potent antioxidative capacity in vitro and in HL-7702 cells, but the weakest activation of immunostimulatory response in RAW264.7 cells. In contrast, CRP-H exhibited the lowest yield but strongest immunostimulatory activity. Overall, microwave extraction could be utilized as a general and practical CRP extraction approach, based on its relatively high yield and bioactivities., (Copyright © 2021. Published by Elsevier B.V.)

AKT1 plays a key role in cell growth and survival, and its activation in cancers is mediated by different mechanisms. In this study, we investigated the potential of G-quadruplex (G4) formation by multiple consecutive G-tracts in the AKT1 promoter and its 3'-UTR. In circular dichroism analyses, synthetic oligonucleotides based on these G-tract regions showed molar ellipticity peaks at specific wavelengths of G4 structures. We verified G4 forming potential of these oligonucleotides using dimethyl sulfate footprinting, gel-shift and immunostaining assays. In reporter assays, mutations of the G-tracts in either the promoter or the 3'-UTR of AKT1 reduced expression mediated by these G-rich regions, suggesting positive regulation of AKT1 gene expression by these G4 structures. Furthermore, SP1 bound to its consensus sites regardless of the presence of G4 motifs in the AKT1 promoter, and both the G4 motifs and SP1 binding sites were needed to reach the strongest promoter strength., (Copyright © 2021 Elsevier Inc. All rights reserved.)

Background: Ellagic acid (EA) possesses prominent inhibitory activities against various cancers, including hepatocellular carcinoma (HCC). Our recent study demonstrated EA's activities in reducing HCC cell proliferation and tumor formation. However, the mechanisms of EA to exert its anticancer activities and its primary targets in cancer cells have not been systematically explored., Methods: Cell proliferation assay and flow cytometric analysis were used to examine the effects of EA treatment on viability and apoptosis, respectively, of HepG2 cells. RNA-seq studies and associated pathway analyses by Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) were employed to determine EA's primary targets. Differentially expressed genes (DEG) in EA-treated HepG2 cells were verified by RT-qPCR and Western blot. Integrative analyses of the RNA-seq dataset with a TCGA dataset derived from HCC patients were conducted to verify EA-targeted genes and signaling pathways. Interaction network analysis of the DEGs, shRNA-mediated knockdown, cell viability assay, and colony formation assay were used to validate EA's primary targets., Results: EA reduced cell viability, caused DNA damage, and induced cell cycle arrest at G1 phase of HepG2 cells. We identified 5765 DEGs encoding proteins with over 2.0-fold changes in EA-treated HepG2 cells by DESeq2. These DEGs showed significant enrichment in the pathways regulating DNA replication and cell cycle progression. As primary targets, p21 was significantly upregulated, while MCM2-7 were uniformly downregulated in response to EA treatment. Consistently, p21 knockdown desensitized liver cells to EA in cell viability and colony formation assays., Conclusion: EA induced G1 phase arrest and promoted apoptosis of HCC cells through activating the p21 gene and downregulating the MCM2-7 genes, respectively., General Significance: The discoveries in this study provide helpful insights into developing novel strategies in the therapeutic treatment of HCC patients., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Triple negative breast cancer (TNBC) has poor prognosis due to lack of biomarker and therapeutic target. Emerging research has revealed long noncoding RNAs (lncRNAs) are involved in breast cancer progression, but their functions and regulatory mechanisms remain poorly understood, especially in TNBC. In this study, we performed lncRNA microarray analysis of five TNBC samples and their matched normal tissues, and discovered a number of differentially expressed lncRNAs. We identified an antisense lncRNA, HYOU1-AS, which is transcribed from the opposite strand of the hypoxia up-regulated 1 (HYOU1) gene, enriched in the nucleus and highly expressed in TNBC. HYOU1-AS knockdown could inhibit the proliferation and migration of the TNBC MDA-MB-231 cells, and reduce their xenograft tumor formation in nude mice. In mechanistic studies, we found that HYOU1-AS could promote the expression of HYOU1, a proliferative gene, through competitively binding to hnRNPA1, an RNA-binding protein, to relieve its post-transcriptional inhibition of the HYOU1 mRNA. Consistently, increased HYOU1 levels correlated with poor clinical outcomes of breast cancer patients based on our study of the TCGA database. Overall, our data indicated that the lncRNA HYOU1-AS promoted TNBC progression through upregulating HYOU1., (Copyright © 2021 Elsevier B.V. All rights reserved.)

Overexpression of the MYC oncogene is a molecular hallmark of both cancer initiation and progression. Targeting MYC is a logical and effective cancer therapeutic strategy. A special DNA secondary structure, the G-quadruplex (G4), is formed within the nuclease hypersensitivity element III 1 (NHE III 1 ) region, located upstream of the MYC gene's P1 promoter that drives the majority of its transcription. Targeting such G4 structures has been a focus of anticancer therapies in recent decades. Thus, a comprehensive review of the MYC G4 structure and its role as a potential therapeutic target is timely. In this review, we first outline the discovery of the MYC G4 structure and evidence of its formation in vitro and in cells. Then, we describe the functional role of G4 in regulating MYC gene expression. We also summarize three types of MYC G4-interacting proteins that can promote, stabilize and unwind G4 structures. Finally, we discuss G4-binding molecules and the anticancer activities of G4-stabilizing ligands, including small molecular compounds and peptides, and assess their potential as novel anticancer therapeutics., (Copyright © 2020 Elsevier B.V. All rights reserved.)

As a member of the SWI/SNF family, ARID1A plays an essential role in modulating chromatin structure and gene expression. The tumor suppressive function of ARID1A has been well-defined and its downregulation in cancers is attributed to genomic deletion, DNA methylation and microRNA-mediated inhibition. In this study, we demonstrated that the negative strand of a C-rich region in the upstream vicinity of the human ARID1A transcription start site could form G-quadruplexes. Synthesized oligonucleotides based on the sequence of this region exhibited molar ellipticity at specific wavelengths characteristic of G-quadruplex structures in circular dichroism analyses. The formation of G-quadruplexes by these oligonucleotides were also proved by native polyacrylamide gel electrophoresis, DNA synthesis block assays, immunofluorescent staining and dimethyl sulfate footprinting studies. In reporter assays, mutations of the G-quadruplex forming sequence reduced ARID1A promoter-mediated transcription. Transfection of the oligonucleotide with the full length of G-quadruplex motif region, but not its partial sequences or the mutants, could both promote endogenous ARID1A expression and reduce cell proliferation., Competing Interests: Declaration of competing interest We declare that we have no conflicts of interest., (Copyright © 2020 Elsevier B.V. All rights reserved.)

As a member of the homeobox gene family, HOXC10 plays an important role in cell differentiation and embryonic development of mammals. Increasing evidence also suggests a regulatory role of HOXC10 in oncogenesis, but the regulation of HOXC10 gene expression is relatively understudied. In this report, we revealed that the HOXC10 promoter contains multiple G-tracts in its negative strand and has high potential of forming G-quadruplex structures. In circular dichroism studies, synthesized oligonucleotides based on the G-rich region of 241-297 base pairs upstream of the HOXC10 transcription start site showed molar ellipticity at specific wavelengths characteristic of G-quadruplex structures. Analyses of these oligonucleotides by native polyacrylamide gel electrophoresis, gel-shift assay, immunostaining and replication stop assay revealed formation of multiple types of G-quadruplex structures in presence of potassium and lithium ions. In reporter assays, mutations or deletion of the G-tracts could differentially impact the expression of Gaussia luciferase downstream of the HOXC10 promoter. Additionally, CHD7, a chromatin remodeling protein with DNA helicase activity, could associate with the HOXC10 promoter and likely unwind the G-quadruplex structures to enhance its gene expression., (Copyright © 2018. Published by Elsevier B.V.)

The mechanisms for regulation of the Inhibitor of Apoptosis (IAP) Survivin in cells undergoing stress associated with tumor development and the tumor microenvironment are not well understood. The stress response transcription factors HIF-1α and Yin Yang 1 (YY1) were hypothesized to contribute to the upregulation of Survivin in tumor cells. As expected, U2OS cells overexpressing HIF-1α showed a 2- to 3-fold transactivation when transfected. Surprisingly, when YY1 was overexpressed in this survivin promoter reporter system, luciferase expression was repressed 30- to 40-fold. YY1 involvement in survivin promoter repression was confirmed using siRNA directed against YY1. These studies showed that knockdown of YY1 releases the survivin promoter from the observed repression and leads to a 3- to 5-fold increase in promoter activity above basal levels. A U2OS cell line containing a stable YY1 Tet-off system was used to determine whether a temporal increase in YY1 expression affects Survivin protein levels. A low to moderate decrease in Survivin protein was observed 24h and 48h after Tet removal. Studies also confirmed that YY1 is capable of directly binding to the survivin promoter. Collectively, these findings identify novel basal transcriptional requirements of survivin gene expression which are likely to play important roles in the development of cancer and resistance to its treatment., (Copyright © 2014 Elsevier Inc. All rights reserved.)

Both epigenetic silencing and genetic deletion of tumor suppressors contribute to the development and progression of breast cancer. SOX7 is a transcription factor important to development, and its down-regulation has been reported in tumor tissues and cell lines of prostate, colon, and lung cancers. However, the regulation of SOX7 expression and its functional role in breast cancer have not been reported. The current study demonstrates that SOX7 mRNA and protein expression are down-regulated in breast cancer tissues and cell lines compared with adjacent normal tissues and nontumorigenic cells, respectively. The SOX7 promoter is hypermethylated in breast cancer cell lines compared with nontumorigenic cells, and the inhibition of DNA methylation increases SOX7 mRNA levels. With shRNA-mediated SOX7 silencing, nontumorigenic immortal breast cells display increased proliferation, migration, and invasion and form structures that resemble that of breast cancer cells in a three-dimensional culture system. Conversely, ectopic SOX7 expression inhibits proliferation, migration, and invasion of breast cancer cells in vitro and tumor growth in vivo. Importantly, we discovered that SOX7 transcript levels positively correlated with clinical outcome of 674 breast cancer patients. Overall, our data suggest that SOX7 acts as a tumor suppressor in breast cancer. SOX7 expression is likely regulated by multiple mechanisms and potentially serves as a prognostic marker for breast cancer patients., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

Cyclin D1 is a component of the core cell-cycle machinery and is frequently overexpressed in breast cancer. It physically interacts with the tumor suppressor Dmp1 that attenuates the oncogenic signals from Ras and HER2 by inducing Arf/p53-dependent cell-cycle arrest. Currently, the biological significance of Dmp1-cyclin D1 interplay in breast cancer has not been determined. Here, we show that cyclin D1 bound to Dmp1 to activate both Arf and Ink4a promoters and, consequently, induced apoptosis or G2/M cell-cycle delay in normal cells to protect them from neoplastic transformation. The cyclin D1-induced Ink4a/Arf gene expression was dependent on Dmp1 because the induction was not detected in Dmp1-deficient or DMP1-depleted cells. Arf/Ink4a expression was increased in pre-malignant mammary glands from Dmp1(+/+);MMTV-cyclin D1 and Dmp1(+/+);MMTV-D1T286A mice but significantly down-regulated in those from Dmp1-deficient mice. Selective Dmp1 deletion was found in 21% of the MMTV-D1 and D1T286A mammary carcinomas, and the Dmp1 heterozygous status significantly accelerated mouse mammary tumorigenesis with reduced apoptosis and increased metastasis. Overall, our study reveals a pivotal role of combined Dmp1 loss and cyclin D1 overexpression in breast cancer., (Copyright © 2013 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)

The phosphoinositide 3-kinase (PI3K) pathway is activated in most advanced prostate cancers, yet so far treatments with PI3K inhibitors have been at best tumorostatic in preclinical cancer models and do not show significant antitumor efficacy in clinical trials. Results from tissue culture experiments in prostate cancer cells suggest that PI3K inhibitors should be combined with other cytotoxic agents; however, the general toxicity of such combinations prevents translating these experimental data into preclinical and clinical models. We investigated the emerging concept of tumor-targeted synthetic lethality in prostate cancer cells by using the pan-PI3K inhibitor ZSTK474 and the immunotoxin J591PE, a protein chimera between the single-chain variable fragment of the monoclonal antibody J591 against the prostate-specific membrane antigen (PSMA) and the truncated form of the Pseudomonas aeruginosa exotoxin A (PE38QQR). The combination of ZSTK474 and J591PE increased apoptosis within 6 hours and cell death (monitored at 24-48 hours) in the PSMA-expressing cells LNCaP, C4-2, and C4-2Luc but not in control cells that do not express PSMA (PC3 and BT549 cells). Mechanistic analysis suggested that induction of apoptosis requires Bcl-2-associated death promoter (BAD) dephosphorylation and decreased expression of myeloid leukemia cell differentiation protein 1 (MCL-1). A single injection of ZSTK474 and J591PE into engrafted prostate cancer C4-2Luc cells led to consistent and stable reduction of luminescence within 6 days. These results suggest that the combination of a PI3K inhibitor and a PSMA-targeted protein synthesis inhibitor toxin represents a promising novel strategy for advanced prostate cancer therapy that should be further investigated.

Yin Yang 1 (YY1) is highly expressed in various types of cancers and regulates tumorigenesis through multiple pathways. In the present study, we evaluated YY1 expression levels in breast cancer cell lines, a breast cancer TMA, and two gene arrays. We observed that, compared with normal samples, YY1 is generally overexpressed in breast cancer cells and tissues. In functional studies, depletion of YY1 inhibited the clonogenicity, migration, invasion, and tumor formation of breast cancer cells, but did not affect the clonogenicity of nontumorigenic cells. Conversely, ectopically expressed YY1 enhanced the migration and invasion of nontumorigenic MCF-10A breast cells. In both a monolayer culture condition and a three-dimensional Matrigel system, silenced YY1 expression changed the architecture of breast cancer MCF-7 cells to that resembling MCF-10A cells, whereas ectopically expressed YY1 in MCF-10A cells had the opposite effect. Furthermore, we detected an inverse correlation between YY1 and p27 expression in both breast cancer cells and xenograft tumors with manipulated YY1 expression. Counteracting the changes in p27 expression attenuated the effects of YY1 alterations on these cells. In addition, YY1 promoted p27 ubiquitination and physically interacted with p27. In conclusion, our data suggest that YY1 is an oncogene and identify p27 as a new target of YY1., (Copyright © 2012 American Society for Investigative Pathology. Published by Elsevier Inc. All rights reserved.)