Circular RNAs with protein-coding ability in oncogenesis.

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 N6-methyladenosine (m6A). 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.

A Faculty-Led, Community-Building Program That Enhances Student-Faculty Connections within Biology.

Connections between students and faculty on campus may influence students' sense of belonging, and a greater sense of belonging has a positive effect on student success. We developed a low-cost, faculty-led program of community-building events and implemented the program in the biology department at a small liberal-arts institution with the goal of improving students' sense of community. Student responses to surveys indicated that the majority of students felt connected to faculty and students in the department; however, Black or African American students initially felt a lower level of connection to faculty than did white students. After implementing our series of community-building events, students surveyed reported high levels of satisfaction with the events. Furthermore, there was a trend toward a higher percentage of Black or African American students than white students reporting that they were more likely to reach out to faculty after participating in the community-building events. Thus, our low-cost program improved connections between students and faculty in the biology department. Collectively, our results suggest that academic departments can implement community-building programs to improve students' sense of belonging.

Phase-Separated Subcellular Compartmentation and Related Human Diseases.

In live cells, proteins and nucleic acids can associate together through multivalent interactions, and form relatively isolated phases that undertake designated biological functions and activities. In the past decade, liquid-liquid phase separation (LLPS) has gradually been recognized as a general mechanism for the intracellular organization of biomolecules. LLPS regulates the assembly and composition of dozens of membraneless organelles and condensates in cells. Due to the altered physiological conditions or genetic mutations, phase-separated condensates may undergo aberrant formation, maturation or gelation that contributes to the onset and progression of various diseases, including neurodegenerative disorders and cancers. In this review, we summarize the properties of different membraneless organelles and condensates, and discuss multiple phase separation-regulated biological processes. Based on the dysregulation and mutations of several key regulatory proteins and signaling pathways, we also exemplify how aberrantly regulated LLPS may contribute to human diseases.

YY1 Oligomerization Is Regulated by Its OPB Domain and Competes with Its Regulation of Oncoproteins.

Yin Yang 1 (YY1) plays an oncogenic role through regulating the expression of various cancer-related genes and activating key oncoproteins. Previous research reported that YY1 protein formed dimers or oligomers without definite biological implications. In this study, we first demonstrated the oncoprotein binding (OPB) and zinc finger (ZF) domains of YY1 as the regions involved in its intermolecular interactions. ZFs are well-known for protein dimerization, so we focused on the OPB domain. After mutating three hydrophobic residues in the OPB to alanines, we discovered that YY1(F219A) and YY1(3A), three residues simultaneously replaced by alanines, were defective of intermolecular interaction. Meanwhile, the OPB peptide could robustly facilitate YY1 protein oligomerization. When expressed in breast cancer cells with concurrent endogenous YY1 knockdown, YY1(F219A) and (3A) mutants showed better capacity than wt in promoting cell proliferation and migration, while their interactions with EZH2, AKT and MDM2 showed differential alterations, especially with improved EZH2 binding affinity. Our study revealed a crucial role of the OPB domain in facilitating YY1 oligomerization and suggested a mutually exclusive regulation between YY1-mediated enhancer formation and its activities in promoting oncoproteins.

A histidine cluster determines YY1-compartmentalized coactivators and chromatin elements in phase-separated enhancer clusters.

As an oncogenic transcription factor, Yin Yang 1 (YY1) regulates enhancer and promoter connection. However, gaps still exist in understanding how YY1 coordinates coactivators and chromatin enhancer elements to assemble enhancers and super-enhancers. Here, we demonstrate that a histidine cluster in YY1's transactivation domain is essential for its formation of phase separation condensates, which can be extended to additional proteins. The histidine cluster is also required for YY1-promoted cell proliferation, migration, clonogenicity and tumor growth. YY1-rich nuclear puncta contain coactivators EP300, BRD4, MED1 and active RNA polymerase II, and colocalize with histone markers of gene activation, but not that of repression. Furthermore, YY1 binds to the consensus motifs in the FOXM1 promoter to activate its expression. Wild-type YY1, but not its phase separation defective mutant, connects multiple enhancer elements and the FOXM1 promoter to form an enhancer cluster. Consistently, fluorescent in situ hybridization (FISH) assays reveal the colocalization of YY1 puncta with both the FOXM1 gene locus and its nascent RNA transcript. Overall, this study demonstrates that YY1 activates target gene expression through forming liquid-liquid phase separation condensates to compartmentalize both coactivators and enhancer elements, and the histidine cluster of YY1 plays a determinant role in this regulatory mechanism.

Emerging Roles of LncRNAs in the EZH2-regulated Oncogenic Network.

Cancer is a life-threatening disease, but cancer therapies based on epigenetic mechanisms have made great progress. Enhancer of zeste homolog 2 (EZH2) is the key catalytic component of Polycomb repressive complex 2 (PRC2) that mediates the tri-methylation of lysine 27 on histone 3 (H3K27me3), a well-recognized marker of transcriptional repression. Mounting evidence indicates that EZH2 is elevated in various cancers and associates with poor prognosis. In addition, many studies revealed that EZH2 is also involved in transcriptional repression dependent or independent of PRC2. Meanwhile, long non-coding RNAs (lncRNAs) have been reported to regulate numerous and diverse signaling pathways in oncogenesis. In this review, we firstly discuss functional interactions between EZH2 and lncRNAs that determine PRC2-dependent and -independent roles of EZH2. Secondly, we summarize the lncRNAs regulating EZH2 expression at transcription, post-transcription and post-translation levels. Thirdly, we review several oncogenic pathways cooperatively regulated by lncRNAs and EZH2, including the Wnt/ß-catenin and p53 pathways. In conclusion, lncRNAs play a key role in the EZH2-regulated oncogenic network with many fertile directions to be explored.

SRSF5 regulates alternative splicing of DMTF1 pre-mRNA through modulating SF1 binding.

ABBREVIATIONS: ARF: alternative reading frame, that is, p14ARF, or CDKN2A (cyclin-dependent kinase inhibitor 2A); ß-gal: ß-galactosidase; CLIP-seq: crosslinking and immunoprecipitation-sequencing; DMTF1: the cyclin D binding myb-like transcription factor 1; ESS/ESE: exonic splicing silencer/enhancer; Ex: exon; FBS: fetal bovine serum; Gluc: Gaussia luciferase; hnRNPs: heterogeneous nuclear ribonucleoproteins; In: intron; ISS/ISE: intronic splicing silencer/enhancer; PBS: phosphate-buffered saline; PCR: polymerase chain reaction; PSI: percent-splice-in; qPCR: quantitative real-time PCR; RIP: RNA immunoprecipitation; RNAseq: RNA sequencing; RT: reverse transcription; SF1: splicing factor 1; SR: serine/arginine-rich proteins; SRSF5: serine and arginine-rich splicing factor 5; TCGA: the cancer genome atlas; UCSC: University of California, Santa Cruz. WT: Wild type.

Disruption of YY1-EZH2 Interaction Using Synthetic Peptides Inhibits Breast Cancer Development.

Enhancer of zeste homolog 2 (EZH2) is a methyltransferase to mediate lysine 27 trimethylation in histone H3 (i.e., H3K27me3) and repress gene expression. In solid tumors, EZH2 promotes oncogenesis and is considered a therapeutic target. As a transcription factor, Yin Yang 1 (YY1) recruits EZH2 through its oncoprotein binding (OPB) domain to establish gene repression. In this study, we mapped the YY1 protein binding (YPB) domain on EZH2 to a region of 27 amino acids. Both YPB and OPB domain synthetic peptides could disrupt YY1EZH2 interaction, markedly reduce breast cancer cell viability, and efficiently inhibit tumor growth in a xenograft mouse model. We analyzed MDA-MB-231 cells treated with YPB, OPB, and control peptides by chromatin immunoprecipitation DNA sequencing (ChIP-seq) using an antibody against H3K27me3. YPB and OPB treatments altered H3K27me3 on 465 and 1137 genes, respectively, compared to the control. Of these genes, 145 overlapped between the two peptides. Among them, PTENP1, the PTEN pseudogene, showed reduced H3K27me3 signal when treated by either YPB or OPB peptide. Consistently, the two peptides enhanced both PTENP1 and PTEN expression with concomitantly reduced AKT activation. Further studies validated PTENP1's contribution to the anticancer activity of YPB and OPB peptides.

AKT1 is positively regulated by G-quadruplexes in its promoter and 3'-UTR.

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.

Human MYC G-quadruplex: From discovery to a cancer therapeutic target.

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 III1 (NHE III1) 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.

The SNAIL1 promoter contains G-quadruplex structures regulating its gene expression and DNA replication.

SNAIL1 is a key regulator of epithelial-mesenchymal transition (EMT) and its expression is associated with tumor progression and poor clinical prognosis of cancer patients. Compared to the studies of SNAIL1 stability and its transcriptional regulation, very limited knowledge is available regarding effective approaches to directly target SNAIL1. In this study, we revealed the potential regulation of SNAIL1 gene expression by G-quadruplex structures in its promoter. We first revealed that the negative strand of the SNAIL1 promoter contained a multi-G-tract region with high potential of forming G-quadruplex structures. In circular dichroism studies, the oligonucleotide based on this region showed characteristic molar ellipticity at specific wavelengths of G-quadruplex structures. We also utilized native polyacrylamide gel electrophoresis, gel-shift assays, immunofluorescent staining, dimethyl sulfate footprinting and chromatin immunoprecipitation studies to verify the G-quadruplex structures formed by the oligonucleotide. In reporter assays, disruption of G-quadruplex potential increased SNAIL1 promoter-mediated transcription, suggesting that G-quadruplexes played a negative role in SNAIL1 expression. In a DNA synthesis study, we detected G-quadruplex-mediated retardation in the SNAIL1 promoter replication. Consistently, we discovered that the G-quadruplex region of the SNAIL1 promoter is highly enriched for mutations, implicating the clinical relevance of G-quadruplexes to the altered SNAIL1 expression in cancer cells.

Characterization of G-quadruplex formation in the ARID1A promoter.

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.

Advances of Zinc Signaling Studies in Prostate Cancer.

Prostate cancer (PCa) is one of the most common cancers and the second leading cause of cancer-related death among men worldwide. Despite progresses in early diagnosis and therapeutic strategies, prognosis for patients with advanced PCa remains poor. Noteworthily, a unique feature of healthy prostate is its highest level of zinc content among all soft tissues in the human body, which dramatically decreases during prostate tumorigenesis. To date, several reviews have suggested antitumor activities of zinc and its potential as a therapeutic strategy of PCa. However, an overview about the role of zinc and its signaling in PCa is needed. Here, we review literature related to the content, biological function, compounds and clinical application of zinc in PCa. We first summarize zinc content in prostate tissue and sera of PCa patients with their clinical relevance. We then elaborate biological functions of zinc signaling in PCa on three main aspects, including cell proliferation, death and tumor metastasis. Finally, we discuss clinical applications of zinc-containing compounds and proteins involved in PCa signaling pathways. Based on currently available studies, we conclude that zinc plays a tumor suppressive role and can serve as a biomarker in PCa diagnosis and therapies.

Functional analysis of YY1 zinc fingers through cysteine mutagenesis.

As a transcription factor, Yin Yang 1 (YY1) either activates or represses gene expression depending on its recruited cofactors. The YY1 C-terminal consists of four zinc fingers (ZF) that are responsible for its DNA binding. However, the contribution of each YY1 ZF to its functions have not been fully elucidated. In this study, we used alanines to replace YY1 cysteines that are crucial to ZFs in binding to DNA. We characterized these YY1 mutants for their DNA binding, transcriptional activity, and functional role in maintaining MDA-MB-231 cell proliferation. We demonstrated that ZFs 2 and 3 are essential to the general biological activity of YY1. ZF 1 showed relatively low importance, while ZF 4 is virtually dispensable for YY1 function.

SOX7 Target Genes and Their Contribution to Its Tumor Suppressive Function.

SOX7 is a transcription factor and acts as a tumor suppressor, but its target genes in cancers are poorly explored. We revealed SOX7-mediated gene expression profile in breast cancer cells using microarray chips and discovered multiple altered signaling pathways. When combinatorially analyzing the microarray data with a gene array dataset from 759 breast cancer patients, we identified four genes as potential targets of SOX7 and validated them by quantitative PCR and chromatin immunoprecipitation assays. Among these four genes, we determined that SOX7-activated SPRY1 and SLIT2, and SOX7-repressed TRIB3 and MTHFD2 could all differentially contribute to SOX7-mediated tumor suppression. Overall, we identified multiple cancer-related pathways mediated by SOX7 and for the first time revealed SOX7-regulated target genes in a cancer-relevant context.

DMP1ß, a splice isoform of the tumour suppressor DMP1 locus, induces proliferation and progression of breast cancer.

Our recent work has indicated that the DMP1 locus on 7q21, encoding a haplo-insufficient tumour suppressor, is hemizygously deleted at a high frequency in breast cancer. The locus encodes DMP1α protein, an activator of the p53 pathway leading to cell cycle arrest and senescence, and two other functionally undefined isoforms, DMP1ß and DMP1γ. In this study, we show that the DMP1 locus is alternatively spliced in ∼30% of breast cancer cases with relatively decreased DMP1α and increased DMP1ß expression. RNA-seq analyses of a publicly available database showed significantly increased DMP1ß mRNA in 43-55% of human breast cancers, dependent on histological subtypes. Similarly, DMP1ß protein was found to be overexpressed in ∼60% of tumours relative to their surrounding normal tissue. Importantly, alteration of DMP1 splicing and DMP1ß overexpression were associated with poor clinical outcomes of the breast cancer patients, indicating that DMP1ß may have a biological function. Indeed, DMP1ß increased proliferation of non-tumourigenic mammary epithelial cells and knockdown of endogenous DMP1 inhibited breast cancer cell growth. To determine DMP1ß's role in vivo, we established MMTV-DMP1ß transgenic mouse lines. DMP1ß overexpression was sufficient to induce mammary gland hyperplasia and multifocal tumour lesions in mice at 7-18 months of age. The tumours formed were adenosquamous carcinomas with evidence of transdifferentiation and keratinized deposits. Overall, we identify alternative splicing as a mechanism utilized by cancer cells to modulate the DMP1 locus through diminishing DMP1α tumour suppressor expression, while simultaneously up-regulating the tumour-promoting DMP1ß isoform.

SOX7: from a developmental regulator to an emerging tumor suppressor.

SOX7 belongs to the SOX (SRY-related HMG-box) family of transcription factors that have been shown to regulate multiple biological processes, such as hematopoiesis, vasculogenesis and cardiogenesis during embryonic development. Recent studies indicate that several SOX family members play important roles in tumorigenesis. In this review, we introduce SOX7 gene and protein structures, and discuss its expression and functional role in cancer development and progression. SOX7 is frequently downregulated in many human cancers and its reduced expression correlates with poor prognoses of several cancers. Functional studies reveal many tumor suppressive properties of SOX7 in prostate, colon, lung, and breast cancers. To date, although a few target genes of SOX7 have been identified, SOX7-mediated gene expression has not been investigated in a cancer-relevant context. Our recent studies not only for the first time demonstrate a tumor suppressive role of SOX7 in a xenograft mouse model, but also unravel that many genes regulating cell death, growth and apoptosis are affected by SOX7, strongly supporting a pivotal role of SOX7 in tumorigenesis. Thus, currently available data clearly indicate a tumor suppressive role of SOX7, but the mechanisms underlying its gene expression and tumor suppressive activity remain undetermined. The research of SOX7 in cancers remains a fertile area to be explored.

The regulation of SOX7 and its tumor suppressive role in breast cancer.

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.

DNA vector-based RNA interference to study gene function in cancer.

RNA interference (RNAi) inhibits gene expression by specifically degrading target mRNAs. Since the discovery of double-stranded small interference RNA (siRNA) in gene silencing, RNAi has become a powerful research tool in gene function studies. Compared to genetic deletion, RNAi-mediated gene silencing possesses many advantages, such as the ease with which it is carried out and its suitability to most cell lines. Multiple studies have demonstrated the applications of RNAi technology in cancer research. In particular, the development of the DNA vector-based technology to produce small hairpin RNA (shRNA) driven by the U6 or H1 promoter has made long term and inducible gene silencing possible. Its use in combination with genetically engineered viral vectors, such as lentivirus, facilitates high efficiencies of shRNA delivery and/or integration into genomic DNA for stable shRNA expression. We describe a detailed procedure using the DNA vector-based RNAi technology to determine gene function, including construction of lentiviral vectors expressing shRNA, lentivirus production and cell infection, and functional studies using a mouse xenograft model. Various strategies have been reported in generating shRNA constructs. The protocol described here employing PCR amplification and a 3-fragment ligation can be used to directly and efficiently generate shRNA-containing lentiviral constructs without leaving any extra nucleotide adjacent to a shRNA coding sequence. Since the shRNA-expression cassettes created by this strategy can be cut out by restriction enzymes, they can be easily moved to other vectors with different fluorescent or antibiotic markers. Most commercial transfection reagents can be used in lentivirus production. However, in this report, we provide an economic method using calcium phosphate precipitation that can achieve over 90% transfection efficiency in 293T cells. Compared to constitutive shRNA expression vectors, an inducible shRNA system is particularly suitable to knocking down a gene essential to cell proliferation. We demonstrate the gene silencing of Yin Yang 1 (YY1), a potential oncogene in breast cancer, by a Tet-On inducible shRNA system and its effects on tumor formation. Research using lentivirus requires review and approval of a biosafety protocol by the Biosafety Committee of a researcher's institution. Research using animal models requires review and approval of an animal protocol by the Animal Care and Use Committee (ACUC) of a researcher's institution.

Yin Yang 1 plays an essential role in breast cancer and negatively regulates p27.

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.