scholarly journals TEsmall identifies small RNAs associated with targeted inhibitor resistance in melanoma

2018 ◽  
Author(s):  
Kathryn O’Neill ◽  
Wen-Wei Liao ◽  
Ami Patel ◽  
Molly Gale Hammell
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
Target Genes ◽  
Mapk Pathway ◽  
Single Agent ◽  
Resistance Mechanisms ◽  
Melanoma Cells ◽  
Cancer Therapies ◽  
Mrna Targets ◽  
Inhibitor Resistance

MicroRNAs (miRNAs) are small 21-22nt RNAs that act to regulate the expression of mRNA target genes through direct binding to mRNA targets. While miRNAs typically dominate small RNA transcriptomes, many other classes are present including tRNAs, snoRNAs, snRNAs, Y-RNAs, piRNAs, and siRNAs. Interactions between processing machinery and targeting networks of these various small RNA classes remains unclear, largely because these small RNAs are typically analyzed separately. Here we present TEsmall, a tool that allows for the simultaneous processing and analysis of small RNAs from each annotated class in a single integrated workflow. The pipeline begins with raw fastq reads and proceeds all the way to producing count tables formatted for differential expression. Several interactive charts are also produced to look at overall distributions in length and annotation classes. We next applied the TEsmall pipeline to small RNA libraries generated from melanoma cells responding to targeted inhibitors of the MAPK pathway. Targeted oncogene inhibitors have emerged as way to tailor cancer therapies to the particular mutations present in a given tumor. While these targeted strategies are typically effective for short intervals, the emergence of resistance is extremely common, limiting the effectiveness of single-agent therapeutics and driving the need for a better understanding of resistance mechanisms. Using TEsmall, we identified several microRNAs and other small RNA classes that are enriched in inhibitor resistant melanoma cells in multiple melanoma cell lines and may be able to serve as markers of resistant populations more generally.

scholarly journals The Non-Canonical Aspects of MicroRNAs: Many Roads to Gene Regulation

Cells ◽  
2019 ◽  
Vol 8 (11) ◽  
pp. 1465 ◽  
Author(s):  
Christiaan J. Stavast ◽  
Stefan J. Erkeland
Keyword(s):  
Rna Polymerase Ii ◽  
Transcriptional Activation ◽  
Small Rnas ◽  
Target Genes ◽  
Biological Functions ◽  
Rnase Iii ◽  
Mrna Targets ◽  
Argonaute Proteins ◽  
Epigenetic Modifiers ◽  
Encoding Genes

MicroRNAs (miRNAs) are critical regulators of gene expression. As miRNAs are frequently deregulated in many human diseases, including cancer and immunological disorders, it is important to understand their biological functions. Typically, miRNA-encoding genes are transcribed by RNA Polymerase II and generate primary transcripts that are processed by RNase III-endonucleases DROSHA and DICER into small RNAs of approximately 21 nucleotides. All miRNAs are loaded into Argonaute proteins in the RNA-induced silencing complex (RISC) and act as post-transcriptional regulators by binding to the 3′- untranslated region (UTR) of mRNAs. This seed-dependent miRNA binding inhibits the translation and/or promotes the degradation of mRNA targets. Surprisingly, recent data presents evidence for a target-mediated decay mechanism that controls the level of specific miRNAs. In addition, several non-canonical miRNA-containing genes have been recently described and unexpected functions of miRNAs have been identified. For instance, several miRNAs are located in the nucleus, where they are involved in the transcriptional activation or silencing of target genes. These epigenetic modifiers are recruited by RISC and guided by miRNAs to specific loci in the genome. Here, we will review non-canonical aspects of miRNA biology, including novel regulators of miRNA expression and functions of miRNAs in the nucleus.

scholarly journals Analysis of miRNAs in the Heads of Different Castes of the Bumblebee Bombus lantschouensis (Hymenoptera: Apidae)

Insects ◽  
2019 ◽  
Vol 10 (10) ◽  
pp. 349 ◽  
Author(s):  
Liu ◽  
Huang ◽  
Zhang ◽  
Liu ◽  
An
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
Target Genes ◽  
Solexa Sequencing ◽  
Biological Functions ◽  
Putative Target ◽  
Differentially Expressed Mirnas ◽  
Rna Deep Sequencing ◽  
Small Rna Deep Sequencing ◽  
Go Terms

Bumblebees are important insect pollinators for many wildflowers and crops. MicroRNAs (miRNAs) are endogenous non-coding small RNAs that regulate different biological functions in insects. In this study, the miRNAs in the heads of the three castes of the bumblebee Bombus lantschouensis were identified and characterized by small RNA deep sequencing. The significant differences in the expression of miRNAs and their target genes were analyzed. The results showed that the length of the small RNA reads from males, queens, and workers was distributed between 18 and 30 nt, with a peak at 22 nt. A total of 364 known and 89 novel miRNAs were identified from the heads of the three castes. The eight miRNAs with the highest expressed levels in males, queens, and workers were identical, although the order of these miRNAs based on expression differed. The male vs. queen, male vs. worker, and worker vs. queen comparisons identified nine, fourteen, and four miRNAs with significant differences in expression, respectively. The different castes were clustered based on the differentially expressed miRNAs (DE miRNAs), and the expression levels of the DE miRNAs obtained by RT-qPCR were consistent with the read counts obtained through Solexa sequencing. The putative target genes of these DE miRNAs were enriched in 29 Gene Ontology (GO) terms, and catalytic activity was the most enriched GO term, as demonstrated by its association with 2837 target genes in the male vs. queen comparison, 3535 target genes in the male vs. worker comparison, and 2185 target genes in the worker vs. queen comparison. This study highlights the characteristics of the miRNAs in the three B. lantschouensis castes and will aid further studies on the functions of miRNAs in bumblebees.

scholarly journals Small RNAs in parasitic nematodes – forms and functions

Parasitology ◽  
2019 ◽  
Vol 147 (8) ◽  
pp. 855-864
Author(s):  
Collette Britton ◽  
Roz Laing ◽  
Eileen Devaney
Keyword(s):  
Gene Expression ◽  
Small Rna ◽  
Small Rnas ◽  
Target Genes ◽  
Parasitic Nematodes ◽  
Small Rna Sequencing ◽  
Small Interfering Rnas ◽  
Rna Sequences ◽  
C Elegans

AbstractSmall RNAs are important regulators of gene expression. They were first identified in Caenorhabditis elegans, but it is now apparent that the main small RNA silencing pathways are functionally conserved across diverse organisms. Availability of genome data for an increasing number of parasitic nematodes has enabled bioinformatic identification of small RNA sequences. Expression of these in different lifecycle stages is revealed by small RNA sequencing and microarray analysis. In this review we describe what is known of the three main small RNA classes in parasitic nematodes – microRNAs (miRNAs), Piwi-interacting RNAs (piRNAs) and small interfering RNAs (siRNAs) – and their proposed functions. miRNAs regulate development in C. elegans and the temporal expression of parasitic nematode miRNAs suggest modulation of target gene levels as parasites develop within the host. miRNAs are also present in extracellular vesicles released by nematodes in vitro, and in plasma from infected hosts, suggesting potential regulation of host gene expression. Roles of piRNAs and siRNAs in suppressing target genes, including transposable elements, are also reviewed. Recent successes in RNAi-mediated gene silencing, and application of small RNA inhibitors and mimics will continue to advance understanding of small RNA functions within the parasite and at the host–parasite interface.

scholarly journals Co-targeting AR and HSP90 suppresses prostate cancer cell growth and prevents resistance mechanisms

2015 ◽  
Vol 22 (5) ◽  
pp. 805-818 ◽  
Author(s):  
Margaret M Centenera ◽  
Sarah L Carter ◽  
Joanna L Gillis ◽  
Deborah L Marrocco-Tallarigo ◽  
Randall H Grose ◽  
...  
Keyword(s):  
Prostate Cancer ◽  
Heat Shock ◽  
Combination Treatment ◽  
Target Genes ◽  
Single Agent ◽  
Heat Shock Protein 90 ◽  
Resistance Mechanisms ◽  
Castration Resistant Prostate Cancer ◽  
Hsp90 Inhibitors ◽  
Cancer Cell Growth

Persistent androgen receptor (AR) signaling in castration resistant prostate cancer (CRPC) underpins the urgent need for therapeutic strategies that better target this pathway. Combining classes of agents that target different components of AR signaling has the potential to delay resistance and improve patient outcomes. Many oncoproteins, including the AR, rely on the molecular chaperone heat shock protein 90 (Hsp90) for functional maturation and stability. In this study, enhanced anti-proliferative activity of the Hsp90 inhibitors 17-allylamino-demethoxygeldanamycin (17-AAG) and AUY922 in androgen-sensitive and CRPC cells was achieved when the agents were used in combination with AR antagonists bicalutamide or enzalutamide. Moreover, significant caspase-dependent cell death was achieved using sub-optimal agent doses that individually have no effect. Expression profiling demonstrated regulation of a broadened set of AR target genes with combined 17-AAG and bicalutamide compared with the respective single agent treatments. This enhanced inhibition of AR signaling was accompanied by impaired chromatin binding and nuclear localization of the AR. Importantly, expression of the AR variant AR-V7 that is implicated in resistance to AR antagonists was not induced by combination treatment. Likewise, the heat shock response that is typically elicited with therapeutic doses of Hsp90 inhibitors, and is a potential mediator of resistance to these agents, was significantly reduced by combination treatment. In summary, the co-targeting strategy in this study more effectively inhibits AR signaling than targeting AR or HSP90 alone and prevents induction of key resistance mechanisms in prostate cancer cells. These findings merit further evaluation of this therapeutic strategy to prevent CRPC growth.

scholarly journals Comparative profiling of small RNAs of pig seminal plasma and ejaculated and epididymal sperm

Reproduction ◽  
2017 ◽  
Vol 153 (6) ◽  
pp. 785-796 ◽  
Author(s):  
Cai Chen ◽  
Han Wu ◽  
Dan Shen ◽  
Saisai Wang ◽  
Li Zhang ◽  
...  
Keyword(s):  
Seminal Plasma ◽  
Small Rna ◽  
Small Rnas ◽  
Target Genes ◽  
Large Population ◽  
Small Nuclear Rna ◽  
Epididymal Sperm ◽  
Zygote Development ◽  
Similarities And Differences ◽  
Small Rna Species

The similarities and differences of small RNAs in seminal plasma, epididymal sperm and ejaculated sperm remain largely undefined. We conducted a systematic comparative analysis of small RNA profiles in pig ejaculated sperm, epididymal sperm and seminal plasma and found that the diversity distribution of small RNA species was generally similar, whereas the abundance of small RNAs is dramatically different across the three libraries; miRNAs and small RNAs derived from rRNA, tRNA, small nuclear RNA, 7SK RNA, NRON RNA and cis-regulatory RNA were enriched in the three libraries, but piRNA was absent. A large population of small RNAs from ejaculated sperm are ejaculated sperm specific, and only 8–30% of small RNAs overlapped with those of epididymal sperm or seminal plasma and a small proportion (5–18%) of small RNAs were shared in the three libraries, suggesting that, in addition to the testes, sperm RNAs may also originate from seminal plasma, epididymis as well as other resources. Most miRNAs were co-distributed but differentially expressed across the three libraries, with epididymal sperm exhibiting the highest abundance, followed by ejaculated sperm and seminal plasma. The prediction of target genes of the top 10 highly expressed miRNAs across the three libraries revealed that these miRNAs may be involved in spermatogenesis, zygote development and the interaction between the environment and animals. Our study provides the first description of the similarities and differences of small RNA profiles in ejaculated sperm, epididymal sperm and seminal plasma and indicates that sperm RNA may have origins other than the testes.

A phase I, pharmacokinetic and pharmacodynamic study of sorafenib (S), a multi-targeted kinase inhibitor in combination with temsirolimus (T), an mTOR inhibitor in patients with advanced solid malignancies

2007 ◽  
Vol 25 (18_suppl) ◽  
pp. 3512-3512 ◽  
Author(s):  
A. Patnaik ◽  
A. Ricart ◽  
J. Cooper ◽  
K. Papadopoulos ◽  
M. Beeram ◽  
...  
Keyword(s):  
Kinase Inhibitor ◽  
Mapk Pathway ◽  
Single Agent ◽  
Resistance Mechanisms ◽  
Survival Pathway ◽  
Solid Malignancies ◽  
Hand Foot Syndrome ◽  
Grade 3 ◽  
Pathway Inhibition ◽  
Oral Doses

3512 Background: The combination of S and T is hypothesized to maximize pathway inhibition by concurrently targeting parallel signaling mechanisms and will abrogate potential resistance mechanisms directed towards the MAPK pathway through increased signaling via the survival pathway involving PI3K/Akt. Methods: Eligible patients (pts) were treated with escalating continuous oral doses of S (200 and 400 mg BID) and weekly T IV (15 mg, 25 mg). S began on day 8 of course 1 to permit PK evaluations of T. PD studies in PBMCs were performed serially in pts. Results: To date, 24 evaluable pts have received 85 courses [median 3;range1–12] in the following S/T dose cohorts; cohort 1: 200 mg/15 mg (n=6), cohort 2: 400 mg/15 mg (n=11), cohort 3: 400 mg/25 mg (n=6), and cohort 4: 200 mg/25 mg (n=1). Patients demographics were males/female 11/13, median age 54.5 [range 27–71] and PS of 0/1/2 : n=9/13/2. Dose limiting toxicities (DLT) were grade 3 typhlitis in 1/6 pts in cohort 1, and mucositis in 1/6 pts in cohort 2. Expansion of cohort 2 to 11 patients resulted in 4 additional DLTs (hand foot syndrome (HFS) x 2 pts, thrombycytopenia/rash x 1 pt, creatinine elevation x 1 pt). 3 of 6 pts in cohort 3 experienced DLT (HFS x 2 pts, thrombocytopenia x 1 pt). PK analyses show no evidence of S effect on T kinetics while Css,min values of S are consistent with those reported in single-agent studies (S 200mg BID median Css,min 4.42 μg/mL [SD 3.05], S 400 mg BID median Css,min 5.11 μg/mL [SD 4.35]). Apparent downregulation of 4E-BP1 and activated forms of p70S6 kinase and ERK was observed in some patients following treatment. Partial responses have occurred in NHL (1 pt) and papillary thyroid cancer (1 pt) and prolonged SD (> 12 mo) has been observed in RCC (1 pt). Conclusions: The combination of S and T demonstrate significant mucocutaneous toxicity at full doses of S, however preliminary PK analyses show no evidence of drug-drug interactions. Characterization of an intermediate dose of S 200 mg BID/T 25 mg IV is ongoing. [Table: see text]

MAPK pathway inhibition in melanoma: resistance three ways

2014 ◽  
Vol 42 (4) ◽  
pp. 727-732 ◽  
Author(s):  
Claudia Wellbrock
Keyword(s):  
Mapk Pathway ◽  
Acquired Resistance ◽  
Tumour Microenvironment ◽  
Resistance Mechanisms ◽  
Melanoma Cells ◽  
Advanced Melanoma ◽  
Mitogen Activated Protein Kinase ◽  
Compensatory Mechanisms ◽  
Stromal Fibroblasts ◽  
Paracrine Signalling

The serine threonine kinases BRAF and MEK [MAPK (mitogen-activated protein kinase)/ERK (extracellular-signal-regulated kinase) kinase] are major regulators of the ERK/MAPK pathway, which is deregulated in the majority of melanomas. Targeting BRAF is an effective therapy for advanced melanoma, but patients progress due to the development of resistance. This ‘acquired resistance’ is thought to be based on a minority of tumour cell populations that are resistant and will eventually re-establish tumour growth even in the presence of drug. In particular, mutations, amplifications or overexpression of genes encoding regulators of the MAPK pathway can confer this resistance, because it allows the melanoma cells to bypass inhibitor action by stimulating ERK activation through alternative routes. Furthermore, there are mechanisms that produce resistance by enhancing the tolerance of melanoma cells to the cytotoxic effects of the drug. These compensatory mechanisms can activate survival signals in the melanoma cells without reactivating ERK. Besides these cell-autonomous resistance mechanisms, stromal fibroblasts in the tumour microenvironment have been identified as a potential source of resistance, because these cells can produce growth factors that reactivate ERK through paracrine signalling. Understanding and further identifying mechanisms of resistance is crucial for the future treatment of advanced melanoma, because this can inform the design of improved therapies with more durable responses.

scholarly journals Melanoma subpopulations that rapidly escape MAPK pathway inhibition incur DNA damage and rely on stress signalling

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Chen Yang ◽  
Chengzhe Tian ◽  
Timothy E. Hoffman ◽  
Nicole K. Jacobsen ◽  
Sabrina L. Spencer
Keyword(s):  
Dna Damage ◽  
Drug Resistance ◽  
Mapk Pathway ◽  
Melanoma Cells ◽  
Cancer Therapies ◽  
Braf Inhibitors ◽  
Anti Cancer ◽  
Genetic Mechanisms ◽  
Pathway Inhibition ◽  
Stress Signalling

AbstractDespite the increasing number of effective anti-cancer therapies, successful treatment is limited by the development of drug resistance. While the contribution of genetic factors to drug resistance is undeniable, little is known about how drug-sensitive cells first evade drug action to proliferate in drug. Here we track the responses of thousands of single melanoma cells to BRAF inhibitors and show that a subset of cells escapes drug via non-genetic mechanisms within the first three days of treatment. Cells that escape drug rely on ATF4 stress signalling to cycle periodically in drug, experience DNA replication defects leading to DNA damage, and yet out-proliferate other cells over extended treatment. Together, our work reveals just how rapidly melanoma cells can adapt to drug treatment, generating a mutagenesis-prone subpopulation that expands over time.

scholarly journals Stress Resets Transgenerational Small RNA Inheritance

2019 ◽  
Author(s):  
Leah Houri-Ze’evi ◽  
Guy Teichman ◽  
Hila Gingold ◽  
Oded Rechavi
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
Memory Trace ◽  
Mapk Pathway ◽  
Transgenerational Inheritance ◽  
Cultivation Conditions ◽  
C Elegans ◽  
Genome Wide ◽  
A Genome ◽  
Basic Concepts

AbstractTransgenerational inheritance of small RNAs is challenging basic concepts of heredity and achieving control over such responses is of great interest. InC. elegansnematodes, small RNAs are transmitted across generations to establish a transgenerational memory trace of ancestral environments and distinguish self from non-self genes. Inheritance of small RNAs is regulated by dedicated machinery and carryover of aberrant heritable small RNA responses was shown to be maladaptive and to induce sterility. Here we show that various types of stress (starvation, high temperatures, and high osmolarity) but not non-stressful changes in cultivation conditions, lead to resetting of small RNA inheritance. We found that stress leads to a genome-wide reduction in heritable small RNA levels and that mutants defective in different stress pathways exhibit irregular RNAi inheritance dynamics. Moreover, we discovered that resetting of heritable RNAi is orchestrated by MAPK pathway factors, the transcription factor SKN-1, and the MET-2 methyltransferase. Termination of small RNA inheritance, and the fact that this process depends on stress, could protect from run-on of environment-irrelevant heritable gene regulation.

scholarly journals Non-Templated Oligo-Adenylation of Small RNAs in the Parasite Entamoeba: Potential Roles for Small RNA Control in Single Celled Eukaryotic Pathogen

2020 ◽  
Author(s):  
Hanbang Zhang ◽  
Gretchen M Ehrenkaufer ◽  
Neil Hall ◽  
Upinder Singh
Keyword(s):  
Small Rna ◽  
Small Rnas ◽  
Cellular Localization ◽  
High Throughput Sequencing ◽  
Target Genes ◽  
Protozoan Parasite ◽  
Rnai Pathway ◽  
Regulation Mechanism ◽  
Protozoan Parasites ◽  
Modification Process

Abstract Background: The RNA interference (RNAi) pathway is a gene regulation mechanism that uitilizes small RNA (sRNA) and Argonaute (Ago) proteins to silence target genes. Our previous work identified a functional RNAi pathway in the protozoan parasite Entamoeba histolytica, including abundant 27nt antisense sRNA populations derived from the secondary RNAi pathway which associate with EhAgo2-2 protein. However, there is lack of understanding about sRNAs that are bound to two other EhAgos (EhAgo2-1 and 2-3), and the mechanism of sRNA regulation itself is unclear in this parasite. Results: In the present study, we sequenced sRNA libraries from both total RNAs and EhAgo bound RNAs. We identified a new population of 31nt sRNAs that results from the addition of a non-templated 3-4 adenosine nucleotides at the 3´-end of the 27nt sRNA populations, indicating a non-templated RNA-tailing event in the parasite. We found that both sRNA populations (27nt and 31nt) are unchanged during the development of E. invadens. However, we detected an alteration in their relative abundance for the targeted gene in parasites transfected with a trigger-gene silencing construct, indicating that non-templated RNA-tailing is likely a pathway for sRNA turnover when the targeted gene is unable to be silenced in this parasite. In sequencing the sRNAs associating with the three EhAgo proteins, we observed that despite distinct cellular localization, all three EhAgo sRNA libraries contain 27nt sRNAs with 5´-polyphosphate (5´-polyP) structure and a largely overlapping sRNA repertoire, mainly targeting retrotransposons and a subset of ~226 genes that are endogenously silenced. Furthermore, our data show that 31nt sRNA populations paritally associate with wildtype EhAgo2-2 but not with its mutant protein (EhAgo2-2 C-terminal deletion), indicating an intact RISC is essential for the sRNA modification process.Conclusion: High-throughput sequencing of sRNA in Entamoeba has identified a new population of sRNA with non-templated adenylation modification, which is the first such observation amongst single cell protozoan parasites. Our sRNA sequencing libraries provide the first comprehensive sRNA dataset for all three Entamoeba Ago proteins, which can serve as a useful database for the amoeba community.

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