Interplay between steroid signalling and microRNAs: implications for hormone-dependent cancers.

RNA silencing is an evolutionarily conserved system that functions as an antiviral mechanism in higher plants and insects. To counteract RNA silencing, viruses express silencing suppressors that interfere with both siRNA- and microRNA-guided silencing pathways. We used comparative in vitro and in vivo approaches to analyse the molecular mechanism of suppression by three well-studied silencing suppressors. We found that silencing suppressors p19, p21 and HC-Pro each inhibit the intermediate step of RNA silencing via binding to siRNAs, although the molecular features required for duplex siRNA binding differ among the three proteins. None of the suppressors affected the activity of preassembled RISC complexes. In contrast, each suppressor uniformly inhibited the siRNA-initiated RISC assembly pathway by preventing RNA silencing initiator complex formation.

Akt-Mediated Phosphorylation of Argonaute 2 Downregulates Cleavage and Upregulates Translational Repression of MicroRNA Targets

: One of the principal objectives of our laboratory is to understand the mechanisms by which hormones and growth factors regulate normal mammary gland development, and how these same regulatory pathways become altered in breast cancer. Therefore, specific interest has been placed upon studying the mechanisms by which the lactogenic hormones regulate milk protein gene expression. CAAT/enhancer binding protein beta(C/EBP(beta)), Yin Yang-1 (YY-1), signal transducers and activators or transcription 5 (STAT5), and the glucocorticoid receptor (GR) have been shown to mediate the hormonal and developmental regulation of a beta-casein gene. However, the mechanism by which these transcription factors as well as coactivator proteins coordinately function to promote normal mammary gland development and breast cancer remains undefined. In this study we tested the dynamic of assembly and disassembly of Stat5, GR, C/EBPb and YY-1 at the hormonally activated b-casein promoter as well as at the recently identified mouse b-casein enhancer located 6kb upstream of the transcription start site. Using ChIP analysis we examined the recruitment of coactivator p300 and determined chromatin acetylation and deacetylation status upon hormone treatments. Finally, we established the time course of pol II and phospho-pol II accumulation at the promoter and enhancer sites. Collectively, these data suggest a model for the assembly of multi-protein complex at the b-casein regulatory regions that helps to understand how the signaling pathways regulated by lactogenic hormones and local growth factors are integrated in the nucleus.

The class of persistent gamma-herpesviruses has developed a variety of strategies that exploit host-cell regulatory pathways to ensure a long-lasting, well-balanced infection of their host. However when these pathways are deregulated, an otherwise harmless infection can lead to disease including cancer. We recently demonstrated that the human herpes virus 4 (HHV4) also known as Epstein–Barr virus (EBV), encodes for small regulatory non-coding microRNAs (miRNAs) that can be transferred from an infected cell to uninfected neighboring cells. Upon arrival these miRNAs are functional in the recipient cell, in that they are able to down regulate specific target genes. These secreted miRNAs are transported to recipient cells via small nano-sized vesicles (known as exosomes) that are of endosomal origin, formed as intraluminal vesicles (ILV) inside multivesicular bodies (MVB). One question that needs to be addressed is how viral miRNAs are sorted into these exosomes. Mature miRNAs, including those of viral origin, are loaded into RNA-induced silencing complexes (RISC) for gene silencing via blocking mRNA translation and/or initiating mRNA decay. Recent insights indicate that cytoplasmic RNA granules rich in RISC complexes are closely associated with endosomes. In fact, selective components of RISC, including GW182 and Argonaut proteins, miRNAs and mRNAs are present in exosomes. Thus miRNA function, mRNA stability and exosome-mediated intercellular communication converge at the level of endosomes. Since endosomes can be considered as key intracellular cross-roads that regulate communication of cells with their exterior, including neighboring cells, it is perhaps not surprising that viruses have found means to exploit this pathway to their benefit. Little is known however, how and if (micro) RNA species are specifically sorted into ILVs and what (micro)RNA-binding proteins are involved. Here we discuss recent developments relating to intracellular trafficking and function of miRNA-containing protein complexes that EBV may exploit for promoting or restricting miRNAs sorting into exosomes for intercellular regulatory functions. This article is part of a Special Issue entitled: MicroRNAs in viral gene regulation.

Quantitative Analysis of RNA Interference by mRNA Couting at Single-Cell Level

In this issue of Circulation , Glynn and colleagues1 make an important contribution to our understanding of the physiological and pathophysiological roles of late sodium current ( I Na) in the heart, with a focus on a key pathways regulating late I Na amplitude. They conducted well-designed and detailed studies with 2 new genetically engineered mouse lines: an S571A mouse that ablates phosphorylation by Ca2+/calmodulin-dependent kinase II (CaMKII)2 selectively at serine 571 (S571) in the cardiac Na channel pore-forming protein Scn5a and an S571E mouse that mimics phosphorylation at S571. S571 was shown previously to be a target for phosphorylation by CaMKII, and this phosphorylation enhanced late I Na.2 The present studies in “knock-in” mice expressing either S571A or S571E have distinct advantages over earlier studies in heterologous expression systems, including cultured myocyte models, because they allow the study of whole-animal and organ phenotypes and cellular and molecular biophysical properties in a more native environment. These new in vivo studies reveal that, despite the extensive network of CaMKII targets, phosphorylation of S571 selectively regulates late I Na and, in particular, enhanced late I Na in failing heart. Article see p 567 Peak I Na is the large inward current flowing mainly through the cardiac Na+ channel pore formed by Scn5a, which is part of a larger sodium channel macromolecular complex. Members of this macromolecular complex act to localize the complex and to regulate I Na.3 With the onset of the action potential (AP) in the myocardium, the peak I Na rapidly rises and decays to nearly zero over several milliseconds. This I Na spike underlies excitability and conduction in working myocardium and the Purkinje conduction system. In contrast to peak I Na, late I Na is a small inward current, usually …

In recent years, non-coding RNAs (ncRNAs) have been acknowledged as master regulators of various cellular behaviors, and their dysregulation contributes to the development, progression, and therapeutic resistance of various cancers, including gastric cancer (GC). The mitogen-activated protein kinases (MAPKs) signaling pathways are frequently dysregulated in GC, leading to tumor initiation, progression, cell death resistance, metastasis, and therapeutic resistance. Emerging evidence supports the critical role of non-coding RNAs (ncRNAs), including microRNAs (miRNAs) and long ncRNAs (lncRNAs), in the regulation of MAPK pathways. These molecules can act as oncogenes or tumor suppressors by affecting components of the MAPK pathway or regulating their upstream regulators/downstream effectors. MAPK-regulating ncRNAs can drive various behaviors of malignant cells, including tumor growth and progression, angiogenesis, metastasis, immune evasion, and drug resistance. Understanding the regulatory network of MAPK-related ncRNAs can help develop novel biomarkers for diagnosis, predict prognosis, and stratify patients before treatment. Moreover, MAPK-regulating ncRNAs can serve as potential therapeutic targets in GC. In this review, we present an overview of current insights into ncRNA-mediated regulation of the MAPK pathway in GC, emphasizing the clinical relevance of these molecules. Moreover, the paths and challenges of the clinical use of MAPK-regulating ncRNAs as diagnostic/predictive biomarkers and therapeutic targets are comprehensively discussed. By elucidating the mechanistic roles of MAPK-regulating ncRNAs in GC, this review provides valuable insights into novel molecular biomarkers and innovative ncRNA-based therapies in GC management.

BackgroundUrothelial cancer (UC) and prostate cancer (PCa) are the most common cancers among men with a high ratio of mortality in advanced-stages. The higher risk of these malignancies among men can be associated with higher carcinogens exposure. Molecular pathology of UC and PCa is related to the specific mutations and aberrations in some signaling pathways. WNT signaling is a highly regulated pathway that has a pivotal role during urothelial and prostate development and homeostasis. This pathway also plays a vital role in adult stem cell niches to maintain a balance between stemness and differentiation. Deregulation of the WNT pathway is frequently correlated with tumor progression and metastasis in urothelial and prostate tumors. Therefore, regulatory factors of WNT pathways are being investigated as diagnostic or prognostic markers and novel therapeutic targets during urothelial and prostate tumorigenesis. MicroRNAs (miRNAs) have a pivotal role in WNT signaling regulation in which there are interactions between miRNAs and WNT signaling pathway during tumor progression. Since, the miRNAs are sensitive, specific, and noninvasive, they can be introduced as efficient biomarkers of tumor progression.Main bodyIn present review, we have summarized all of the miRNAs that have been involved in regulation of WNT signaling pathway in urothelial and prostate cancers.ConclusionsIt was observed that miRNAs were mainly involved in regulation of WNT signaling in bladder cancer cells through targeting the WNT ligands and cytoplasmic WNT components such as WNT5A, WNT7A, CTNNB1, GSK3β, and AXIN. Whereas, miRNAs were mainly involved in regulation of WNT signaling in prostate tumor cells via targeting the cytoplasmic WNT components and WNT related transcription factors such as CTNNB1, GSK3β, AXIN, TCF7, and LEF1. MiRNAs mainly functioned as tumor suppressors in bladder and prostate cancers through the WNT signaling inhibition. This review paves the way of introducing a noninvasive diagnostic panel of WNT related miRNAs in urothelial and prostate tumors.

BackgroundBasic leucine zipper (bZIP) gene family members represent one of the most diverse and largest groups of transcription factors in eukaryotes. Research has demonstrated that bZIP transcription factors play crucial roles not only in plant growth and development but also in response to various abiotic stresses. However, studies focusing on bZIP factors in Rhododendron delavayi (RdbZIPs) remain limited.ResultIn this study, a total of 59 RdbZIPs were identified using bioinformatics approaches, and these could be classified into 13 subfamilies based on the genomic data of R. delavayi. Members of the same RdbZIP subfamily exhibited similar gene structures and conserved motifs, and were unevenly distributed across the 13 chromosomes of R. delavayi. Collinearity analysis revealed a total of 20 duplication events, comprising 3 pairs of tandem duplications and 17 pairs of segmental duplications. Additionally, cis-acting element analysis indicated that RdbZIP family members may be involved in various biological processes, including transcription, development, hormone regulation, and responses to biotic and abiotic stresses. Transcriptomic analysis revealed that RdbZIP family genes were highly expressed in reproductive tissues. RT-qPCR expression analysis revealed that many selected RdbZIP genes were significantly upregulated under high salinity and drought conditions, suggesting their potential involvement in stress-responsive regulatory networks.ConclusionThis study provides the first comprehensive characterization of the bZIP transcription factor family in Rhododendron delavayi, laying a foundational framework for functional studies of individual RdbZIP genes. The results highlight the pivotal role of RdbZIP genes in abiotic stress tolerance, which is crucial for understanding the adaptive mechanisms of R. delavayi. Future research should focus on the functional validation of key RdbZIP genes and elucidation of their regulatory pathways, which may contribute to the genetic improvement of Rhododendron species under adverse environmental conditions.Clinical trialNot applicable.

Metastatic cells migrate from primary tumors and persist as dormant clusters. The mechanisms that allow these cells to escape their dormant state are an area of intense investigation. Protein phosphatase 2 (PPP2) is a tumor suppressor that dephosphorylates protein kinases involved in proliferation and metastasis. Increased PPP2 expression has been associated with progression-free survival in head and neck cancer (HNSC). A frequently altered epigenetic modifier in HNSC is the lysine demethylase KDM5A. KDM5A catalyzes demethylation of histone H3K4, resulting in chromatin compaction and repression of target genes. Despite the frequency of altered KDM5A expression in HNSC, the mechanism by which it regulates the progression of these cancers is unclear. Our present study demonstrates, using novel genetically engineered models, that PPP2 inhibition results in escape from metastatic dormancy via increased target protein phosphorylation, IL10 expression, and lymphocyte depletion. Genomic analysis demonstrated that KDM5A directly targets the PPP2 catalytic subunit. Genetic inhibition of KDM5A expression in vivo promotes HNSC metastatic dormancy via decreased IL10 expression. CRISPR-mediated ablation of KDM5A expression inhibits the proliferation of HNSC lines, and treatment with a small molecule KDM5A inhibitor results in the death of these cells. We concluded that PPP2 is required for HNSC metastatic dormancy and is regulated by KDM5A.

microRNAs (miRs) are small non-coding RNA molecules that have recently emerged as critical regulators of gene expression and are often deregulated in cancer. In particular, miRs encoded by the miR-15a, miR-16-1 cluster are frequently deleted in certain cancers and appear to act as tumor suppressors. Furthermore, miR-21 is over-expressed in a wide variety of human tumors and functions as an oncogene. Here, we provide evidence that miR-21, the miR-15a, miR-16-1 cluster and related miR-15b, miR-16-2 cluster comprise miRs regulated by E2F1, a pivotal transcription factor that can induce both proliferation and cell death. E2F1 is a critical downstream target of the tumor suppressor RB. The retinoblastoma pathway is often inactivated in human tumors resulting in deregulated E2F activity. We show that expression levels of the five mature miRs, miR-15a, miR-16-1, miR-15b, miR-16-2 and miR-21 are elevated upon activation of ectopic E2F1. Moreover, activation of endogenous E2Fs up-regulates expression of these miRs and endogenous E2F1 binds their respective promoters. Importantly, we corroborate that miR-15a/b inhibits expression of cyclin E, a key direct transcriptional target of E2F pivotal for the G1/S transition. Similarly, miR-21 inhibits the expression of the pro-apoptotic tumor suppressor gene maspin, another direct transcriptional target of E2F. Therefore, our data suggest the existence of feed forward loops that modulate E2F activity and consist of E2F1; E2F1-regulated miRs -miR-15 and miR-21; and E2F1-regulated genes-cyclin E and maspin. In support of this, ectopic expression of miR-15 inhibits G1/S transition and, conversely, inhibition of miR-15 expression enhances E2F1-induced G1/S transition and up-regulation of cyclin E levels. Also, inhibition of miR-21 expression enhances E2F1-induced up regulation of maspin, as well as E2F1-mediated apoptosis. In summary, our data identify miR-15, miR-16 and miR-21 as novel transcriptional targets of E2F that, in turn, modulate E2F activity. Citation Format: Matan Ofir, Berta Ben-Shachar, Dalia Hacohen, Doron Ginsberg. MiR-15, miR-16 and miR-21 are direct transcriptional targets of E2F1 that limit E2F-induced proliferation and apoptosis [abstract]. In: Proceedings of the AACR Special Conference on Noncoding RNAs and Cancer; 2012 Jan 8-11; Miami Beach, FL. Philadelphia (PA): AACR; Cancer Res 2012;72(2 Suppl):Abstract nr A17.

Simple SummaryKrüppel-like Factor-9 (KLF9) and Krüppel-like Factor-13 (KLF13) are highly related proteins that function as inducers and/or repressors of specific target gene repertoires in a variety of tissues and in diverse pathophysiological states, including neoplasia. Here, we describe the salient features of KLF9 and KLF13, the current state-of-the-art research regarding both protein’s actions in cancer development and response to therapies, and where the field requires further exploration. These paralogous proteins warrant further study for multiple cancers, and with respect to their multiplicities of action in suppression or promotion of proliferative and metastatic phenotypes, and likely involvement in immune cell biology within the tumor microenvironment.Specificity Proteins/Krüppel-like Factors (SP/KLF family) are a conserved family of transcriptional regulators. These proteins share three highly conserved, contiguous zinc fingers in their carboxy-terminus, requisite for binding to cis elements in DNA. Each SP/KLF protein has unique primary sequence within its amino-terminal and carboxy-terminal regions, and it is these regions which interact with co-activators, co-repressors, and chromatin-modifying proteins to support the transcriptional activation and repression of target genes. Krüppel-like Factor 9 (KLF9) and Krüppel-like Factor 13 (KLF13) are two of the smallest members of the SP/KLF family, are paralogous, emerged early in metazoan evolution, and are highly conserved. Paradoxically, while most similar in primary sequence, KLF9 and KLF13 display many distinct roles in target cells. In this article, we summarize the work that has identified the roles of KLF9 (and to a lesser degree KLF13) in tumor suppression or promotion via unique effects on differentiation, pro- and anti-inflammatory pathways, oxidative stress, and tumor immune cell infiltration. We also highlight the great diversity of miRNAs, lncRNAs, and circular RNAs which provide mechanisms for the ubiquitous tumor-specific suppression of KLF9 mRNA and protein. Elucidation of KLF9 and KLF13 in cancer biology is likely to provide new inroads to the understanding of oncogenesis and its prevention and treatments.

Protein kinase C (PKC) isozymes are key signal transducers involved in normal physiology and disease and have been widely implicated in cancer progression. Despite our extensive knowledge of the signaling pathways regulated by PKC isozymes and their effectors, there is essentially no information on how individual members of the PKC family regulate gene transcription. Here, we report the first PKC isozyme-specific analysis of global gene expression by microarray using RNAi depletion of diacylglycerol/phorbol ester-regulated PKCs. A thorough analysis of this microarray data revealed unique patterns of gene expression controlled by PKCα, PKCδ, and PKCε, which are remarkably different in cells growing in serum or in response to phorbol ester stimulation. PKCδ is the most relevant isoform in controlling the induction of genes by phorbol ester stimulation, whereas PKCε predominantly regulates gene expression in serum. We also established that two PKCδ-regulated genes, FOSL1 and BCL2A1, mediate the apoptotic effect of phorbol esters or the chemotherapeutic agent etoposide in prostate cancer cells. Our studies offer a unique opportunity for establishing novel transcriptional effectors for PKC isozymes and may have significant functional and therapeutic implications.

Micro-RNAs are approximately 21-25-nucleotide-long noncoding RNAs that regulate gene expression primarily at the post-transcriptional level in animals. Here, we report that micro-RNA-1 (miR-1), abundant in the cardiac and smooth muscles, is expressed in the lung and is down-regulated in human primary lung cancer tissues and cell lines. In situ hybridization demonstrated localization of miR-1 in bronchial epithelial cells. The tumor suppressor C/EBPalpha, frequently suppressed in lung cancer, reactivated miR-1 expression in the lung cancer cells. Repressed miR-1 was also activated in lung cancer cells upon treatment with a histone deacetylase inhibitor. These observations led us to examine the antitumorigenic potential of miR-1 in lung cancer cells. Expression of miR-1 in nonexpressing A549 and H1299 cells reversed their tumorigenic properties, such as growth, replication potential, motility/migration, clonogenic survival, and tumor formation in nude mice. Exogenous miR-1 significantly reduced expression of oncogenic targets, such as MET, a receptor tyrosine kinase, and Pim-1, a Ser/Thr kinase, frequently up-regulated in lung cancer. Similarly, the levels of two additional targets, FoxP1, a transcription factor with oncogeneic property, and HDAC4 that represses differentiation-promoting genes, were reduced in miR-1-expressing cells. Conversely, depletion of miR-1 facilitated N417 cell growth with concomitant elevation of these targets. Further, ectopic miR-1 induced apoptosis in A549 cells in response to the potent anticancer drug doxorubicin. Enhanced activation of caspases 3 and 7, cleavage of their substrate PARP-1, and depletion of anti-apoptotic Mcl-1 contributed to the sensitivity of miR-1-expressing cells to doxorubicin. Thus, miR-1 has potential therapeutic application against lung cancers.

hnRNP K Is a Novel Haploinsufficient Tumor Suppressor at the 9q21.32 Locus That Defines a Subset of AML