TATDN2 resolution of R-loops is required for survival of BRCA1-mutant cancer cells.

BRCA1-deficient cells have increased IRE1 RNase, which degrades multiple microRNAs. Reconstituting expression of one of these, miR-4638-5p, resulted in synthetic lethality in BRCA1-deficient cancer cells. We found that miR-4638-5p represses expression of TATDN2, a poorly characterized member of the TATD nuclease family. We discovered that human TATDN2 has RNA 3' exonuclease and endonuclease activity on double-stranded hairpin RNA structures. Given the cleavage of hairpin RNA by TATDN2, and that BRCA1-deficient cells have difficulty resolving R-loops, we tested whether TATDN2 could resolve R-loops. Using in vitro biochemical reconstitution assays, we found TATDN2 bound to R-loops and degraded the RNA strand but not DNA of multiple forms of R-loops in vitro in a Mg2+-dependent manner. Mutations in amino acids E593 and E705 predicted by Alphafold-2 to chelate an essential Mg2+ cation completely abrogated this R-loop resolution activity. Depleting TATDN2 increased cellular R-loops, DNA damage and chromosomal instability. Loss of TATDN2 resulted in poor replication fork progression in the presence of increased R-loops. Significantly, we found that TATDN2 is essential for survival of BRCA1-deficient cancer cells, but much less so for cognate BRCA1-repleted cancer cells. Thus, we propose that TATDN2 is a novel target for therapy of BRCA1-deficient cancers.

Identification of a novel mechanism for meso-tetra (4-carboxyphenyl) porphyrin (TCPP) uptake in cancer cells.

Porphyrins are used for cancer diagnostic and therapeutic applications, but the mechanism of how porphyrins accumulate in cancer cells remains elusive. Knowledge of how porphyrins enter cancer cells can aid the development of more accurate cancer diagnostics and therapeutics. To gain insight into porphyrin uptake mechanisms in cancer cells, we developed a flow cytometry assay to quantify cellular uptake of meso-tetra (4-carboxyphenyl) porphyrin (TCPP), a porphyrin that is currently being developed for cancer diagnostics. We found that TCPP enters cancer cells through clathrin-mediated endocytosis. The LDL receptor, previously implicated in the cellular uptake of other porphyrins, only contributes modestly to uptake. We report that TCPP instead binds strongly ( KD=42nM ) to CD320, the cellular receptor for cobalamin/transcobalamin II (Cbl/TCN2). Additionally, TCPP competes with Cbl/TCN2 for CD320 binding, suggesting that CD320 is a novel receptor for TCPP. Knockdown of CD320 inhibits TCPP uptake by up to 40% in multiple cancer cell lines, including lung, breast, and prostate cell lines, which supports our hypothesis that CD320 both binds to and transports TCPP into cancer cells. Our findings provide some novel insights into why porphyrins concentrate in cancer cells. Additionally, our study describes a novel function for the CD320 receptor which has been reported to transport only Cbl/TCN2 complexes.

GD2-directed CAR-T cells in combination with HGF-targeted neutralizing antibody (AMG102) prevent primary tumor growth and metastasis in Ewing sarcoma.

Ewing sarcoma (EWS) is the second most common and aggressive type of metastatic bone tumor in adolescents and young adults. There is unmet medical need to develop and test novel pharmacological targets and novel therapies to treat EWS. Here, we found that EWS expresses high levels of a p53 isoform, delta133p53. We further determined that aberrant expression of delta133p53 induced HGF secretion resulting in tumor growth and metastasis. Thereafter, we evaluated targeting EWS tumors with HGF receptor neutralizing antibody (AMG102) in preclinical studies. Surprisingly, we found that targeting EWS tumors with HGF receptor neutralizing antibody (AMG102) in combination with GD2-specific, CAR-reengineered T-cell therapy synergistically inhibited primary tumor growth and establishment of metastatic disease in preclinical models. Furthermore, our data suggested that AMG102 treatment alone might increase leukocyte infiltration including efficient CAR-T access into tumor mass and thereby improves its antitumor activity. Together, our findings warrant the development of novel CAR-T-cell therapies that incorporate HGF receptor neutralizing antibody to improve therapeutic potency, not only in EWS but also in tumors with aberrant activation of the HGF/c-MET pathway.

Diverse pathways generate microRNA-like RNAs and Dicer-independent small interfering RNAs in fungi.

A variety of small RNAs, including the Dicer-dependent miRNAs and the Dicer-independent Piwi-interacting RNAs, associate with Argonaute family proteins to regulate gene expression in diverse cellular processes. These two species of small RNA have not been found in fungi. Here, by analyzing small RNAs associated with the Neurospora Argonaute protein QDE-2, we show that diverse pathways generate miRNA-like small RNAs (milRNAs) and Dicer-independent small interfering RNAs (disiRNAs) in this filamentous fungus. Surprisingly, milRNAs are produced by at least four different mechanisms that use a distinct combination of factors, including Dicers, QDE-2, the exonuclease QIP, and an RNase III domain-containing protein, MRPL3. In contrast, disiRNAs originate from loci producing overlapping sense and antisense transcripts, and do not require the known RNAi components for their production. Taken together, these results uncover several pathways for small RNA production in filamentous fungi, shedding light on the diversity and evolutionary origins of eukaryotic small RNAs.

Contextual extracellular cues promote tumor cell EMT and metastasis by regulating miR-200 family expression.

Metastatic disease is a primary cause of cancer-related death, and factors governing tumor cell metastasis have not been fully elucidated. Here, we address this question by using tumor cell lines derived from mice that develop metastatic lung adenocarcinoma owing to expression of mutant K-ras and p53. Despite having widespread somatic genetic alterations, the metastasis-prone tumor cells retained a marked plasticity. They transited reversibly between epithelial and mesenchymal states, forming highly polarized epithelial spheres in three-dimensional culture that underwent epithelial-to-mesenchymal transition (EMT) following treatment with transforming growth factor-beta or injection into syngeneic mice. This transition was entirely dependent on the microRNA (miR)-200 family, which decreased during EMT. Forced expression of miR-200 abrogated the capacity of these tumor cells to undergo EMT, invade, and metastasize, and conferred transcriptional features of metastasis-incompetent tumor cells. We conclude that tumor cell metastasis is regulated by miR-200 expression, which changes in response to contextual extracellular cues.

microRNA-449a functions as a tumor suppressor in neuroblastoma through inducing cell differentiation and cell cycle arrest.

microRNA-449a (miR-449a) has been identified to function as a tumor suppressor in several types of cancers. However, the role of miR-449a in neuroblastoma has not been intensively investigated. We recently found that the overexpression of miR-449a significantly induces neuroblastoma cell differentiation, suggesting its potential tumor suppressor function in neuroblastoma. In this study, we further investigated the mechanisms underlying the tumor suppressive function of miR-449a in neuroblastoma. We observed that miR-449a inhibits neuroblastoma cell survival and growth through 2 mechanisms--inducing cell differentiation and cell cycle arrest. Our comprehensive investigations on the dissection of the target genes of miR-449a revealed that 3 novel targets- MFAP4, PKP4 and TSEN15 -play important roles in mediating its differentiation-inducing function. In addition, we further found that its function in inducing cell cycle arrest involves down-regulating its direct targets CDK6 and LEF1. To determine the clinical significance of the miR-449a-mediated tumor suppressive mechanism, we examined the correlation between the expression of these 5 target genes in neuroblastoma tumor specimens and the survival of neuroblastoma patients. Remarkably, we noted that high tumor expression levels of all the 3 miR-449a target genes involved in regulating cell differentiation, but not the target genes involved in regulating cell cycle, are significantly correlated with poor survival of neuroblastoma patients. These results suggest the critical role of the differentiation-inducing function of miR-449a in determining neuroblastoma progression. Overall, our study provides the first comprehensive characterization of the tumor-suppressive function of miR-449a in neuroblastoma, and reveals the potential clinical significance of the miR-449a-mediated tumor suppressive pathway in neuroblastoma prognosis.

Low LDL cholesterol in individuals of African descent resulting from frequent nonsense mutations in PCSK9.

The low-density lipoprotein receptor (LDLR) prevents hypercholesterolemia and atherosclerosis by removing low-density lipoprotein (LDL) from circulation. Mutations in the genes encoding either LDLR or its ligand (APOB) cause severe hypercholesterolemia. Missense mutations in PCSK9, encoding a serine protease in the secretory pathway, also cause hypercholesterolemia. These mutations are probably gain-of-function mutations, as overexpression of PCSK9 in the liver of mice produces hypercholesterolemia by reducing LDLR number. To test whether loss-of-function mutations in PCSK9 have the opposite effect, we sequenced the coding region of PCSK9 in 128 subjects (50% African American) with low plasma levels of LDL and found two nonsense mutations (Y142X and C679X). These mutations were common in African Americans (combined frequency, 2%) but rare in European Americans (<0.1%) and were associated with a 40% reduction in plasma levels of LDL cholesterol. These data indicate that common sequence variations have large effects on plasma cholesterol levels in selected populations.

A high-throughput screen identifies miRNA inhibitors regulating lung cancer cell survival and response to paclitaxel.

microRNAs (miRNAs) are small RNAs endogenously expressed in multiple organisms that regulate gene expression largely by decreasing levels of target messenger RNAs (mRNAs). Over the past few years, numerous studies have demonstrated critical roles for miRNAs in the pathogenesis of many cancers, including lung cancer. Cellular miRNA levels can be easily manipulated, showing the promise of developing miRNA-targeted oligos as next-generation therapeutic agents. In a comprehensive effort to identify novel miRNA-based therapeutic agents for lung cancer treatment, we combined a high-throughput screening platform with a library of chemically synthesized miRNA inhibitors to systematically identify miRNA inhibitors that reduce lung cancer cell survival and those that sensitize cells to paclitaxel. By screening three lung cancer cell lines with different genetic backgrounds, we identified miRNA inhibitors that potentially have a universal cytotoxic effect on lung cancer cells and miRNA inhibitors that sensitize cells to paclitaxel treatment, suggesting the potential of developing these miRNA inhibitors as therapeutic agents for lung cancer. We then focused on characterizing the inhibitors of three miRNAs (miR-133a/b, miR-361-3p, and miR-346) that have the most potent effect on cell survival. We demonstrated that two of the miRNA inhibitors (miR-133a/b and miR-361-3p) decrease cell survival by activating caspase-3/7-dependent apoptotic pathways and inducing cell cycle arrest in S phase. Future studies are certainly needed to define the mechanisms by which the identified miRNA inhibitors regulate cell survival and drug response, and to explore the potential of translating the current findings into clinical applications.

microRNAs and lung cancer: tumors and 22-mers.

Work over the last decade has revealed novel regulatory mechanisms in pathological disease states that are mediated by microRNAs and has inspired researchers to begin elucidating the specific roles of miRNAs in the regulation of genes involved in cancer development and progression. Recently, miRNAs have been explored as therapeutic targets and diagnostic markers of cancer. In this paper, we review recent advances in the study of miRNAs involved in tumorigenesis, focusing on miRNA regulation of genes that have been demonstrated to play critical roles in lung cancer development. We discuss miRNA regulation of genes that play critical roles in the process of malignant transformation, angiogenesis and tumor metastasis, the dysregulation of miRNA expression in cancer development, and the development of miRNA-based diagnostics and therapeutics.

Synergism of Proneurogenic miRNAs Provides a More Effective Strategy to Target Glioma Stem Cells.

Tumor suppressor microRNAs (miRNAs) have been explored as agents to target cancer stem cells. Most strategies use a single miRNA mimic and present many disadvantages, such as the amount of reagent required and the diluted effect on target genes. miRNAs work in a cooperative fashion to regulate distinct biological processes and pathways. Therefore, we propose that miRNA combinations could provide more efficient ways to target cancer stem cells. We have previously shown that miR-124, miR-128, and miR-137 function synergistically to regulate neurogenesis. We used a combination of these three miRNAs to treat glioma stem cells and showed that this treatment was much more effective than single miRNAs in disrupting cell proliferation and survival and promoting differentiation and response to radiation. Transcriptomic analyses indicated that transcription regulation, angiogenesis, metabolism, and neuronal differentiation are among the main biological processes affected by transfection of this miRNA combination. In conclusion, we demonstrated the value of using combinations of neurogenic miRNAs to disrupt cancer phenotypes and glioma stem cell growth. The synergistic effect of these three miRNA amplified the repression of oncogenic factors and the effect on cancer relevant pathways. Future therapeutic approaches would benefit from utilizing miRNA combinations, especially when targeting cancer-initiating cell populations.

Power of genetic association studies with fixed and random genotype frequencies.

When estimating the power of genetic association studies, the allele and genotype frequencies are often assumed to be known, and the numbers of individuals with each genotype are set equal to their expectations under Hardy-Weinberg equilibrium. In fact, both allele and genotype frequencies are unknown and thus random. It has previously been suggested that ignoring uncertainty in these parameters could lead to inflated power expectations. To overcome the problem, one can average power estimates over the distributions of unknown frequencies. We investigate the power-averaging method and find that, despite the intuitive appeal, it may not improve accuracy in practice, while significantly increasing computational time. For a fixed allele frequency, we show that the amount of overestimation diminishes rapidly with sample size and is completely negligible for N > 200. For an unknown frequency, the result of averaging depends on the genetic model, and may not always provide a more conservative estimate of power. We explore the effect of uncertainty in the factors that determine statistical power of association studies and propose a more economical approach to the power analysis.

Common single-nucleotide polymorphisms act in concert to affect plasma levels of high-density lipoprotein cholesterol.

The identification of DNA sequence variants underlying human complex phenotypes remains a significant challenge for several reasons: individual variants can have small phenotypic effects or low population frequencies, and multiple allelic variants may act in concert to affect a trait. We evaluated the combined effect of allelic variants in seven genes involved in high-density lipoprotein (HDL) metabolism, using forward stepwise regression. Analysis of all known common single-nucleotide polymorphisms (SNPs) in the seven candidate genes revealed four variants that were associated with incremental changes in HDL cholesterol levels in three independent samples. Conversely, analysis of 660 polymorphisms in eight genes that do not appear to be involved in HDL metabolism did not identify any associations with plasma HDL-cholesterol levels. These data indicate that several common SNPs act in concert to influence plasma levels of HDL cholesterol.

Integrative transcriptome and chromatin landscape analysis reveals distinct epigenetic regulations in human memory B cells.

Memory B cells (MBCs) are long-lived and produce high-affinity, generally, class-switched antibodies. Here, we use a multiparameter approach involving CD27 to segregate naïve B cells (NBC), IgD+ unswitched (unsw)MBCs and IgG+ or IgA+ class-switched (sw)MBCs from humans of different age, sex and race. Conserved antibody variable gene expression indicates that MBCs emerge through unbiased selection from NBCs. Integrative analyses of mRNAs, miRNAs, lncRNAs, chromatin accessibility and cis-regulatory elements uncover a core mRNA-ncRNA transcriptional signature shared by IgG+ and IgA+ swMBCs and distinct from NBCs, while unswMBCs display a transitional transcriptome. Some swMBC transcriptional signature loci are accessible but not expressed in NBCs. Profiling miRNAs reveals downregulated MIR181, and concomitantly upregulated MIR181 target genes such as RASSF6, TOX, TRERF1, TRPV3 and RORα, in swMBCs. Finally, lncRNAs differentially expressed in swMBCs cluster proximal to the IgH chain locus on chromosome 14. Our findings thus provide new insights into MBC transcriptional programs and epigenetic regulation, opening new investigative avenues on these critical cell elements in human health and disease.

ELF4 Is a Target of miR-124 and Promotes Neuroblastoma Proliferation and Undifferentiated State.

13-Cis-retinoic acid (RA) is typically used in postremission maintenance therapy in patients with neuroblastoma. However, side effects and recurrence are often observed. We investigated the use of miRNAs as a strategy to replace RA as promoters of differentiation. miR-124 was identified as the top candidate in a functional screen. Genomic target analysis indicated that repression of a network of transcription factors (TF) could be mediating most of miR-124's effect in driving differentiation. To advance miR-124 mimic use in therapy and better define its mechanism of action, a high-throughput siRNA morphologic screen focusing on its TF targets was conducted and ELF4 was identified as a leading candidate for miR-124 repression. By altering its expression levels, we showed that ELF4 maintains neuroblastoma in an undifferentiated state and promotes proliferation. Moreover, ELF4 transgenic expression was able to counteract the neurogenic effect of miR-124 in neuroblastoma cells. With RNA sequencing, we established the main role of ELF4 to be regulation of cell-cycle progression, specifically through the DREAM complex. Interestingly, several cell-cycle genes activated by ELF4 are repressed by miR-124, suggesting that they might form a TF-miRNA regulatory loop. Finally, we showed that high ELF4 expression is often observed in neuroblastomas and is associated with poor survival. IMPLICATIONS: miR-124 induces neuroblastoma differentiation partially through the downregulation of TF ELF4, which drives neuroblastoma proliferation and its undifferentiated phenotype.

Predicting potential miRNA target sites within gene promoters.

Synthetic small duplex RNAs that are complementary to gene promoters can activate or inhibit target gene expression. The potency and robustness of gene modulation by these RNAs suggests that natural mechanisms may exist to facilitate recognition of sequences within gene promoters by endogenous small RNAs. Here, we describe computational methods for identifying potential miRNA target sites within gene promoters. These methods will facilitate investigations of whether miRNAs interact with sequences outside of 3'-untranslated regions and suggest new targets for the design of synthetic modulators of gene expression.

The miR-195 Axis Regulates Chemoresistance through TUBB and Lung Cancer Progression through BIRC5.

Chemoresistance and metastasis are the major reasons for non-small cell lung cancer (NSCLC) treatment failure and patient deaths. We and others have shown that miR-195 regulates the sensitivity of NSCLC to microtubule-targeting agents (MTAs) in vitro and in vivo and that miR-195 represses the migration and invasion of NSCLC cells in vitro. However, the relationship between miR-195 and microtubule structure and function and whether miR-195 represses NSCLC metastasis in vivo remain unknown. We assessed the correlation between tumor levels of TUBB and patient survival, the effect of TUBB on drug response, and the effect of miR-195 on migration, invasion, and metastasis in vitro and in vivo. We found that miR-195 directly targets TUBB; knockdown of TUBB sensitizes cells to MTAs, while overexpression confers resistance; high expression of TUBB is correlated with worse survival of lung adenocarcinoma; TUBB is also regulated by CHEK1, which has been shown to regulate chemoresistance; and miR-195 targets BIRC5 to repress migration and invasion in vitro and metastasis in vivo. Our findings highlight the relevance of the miR-195/TUBB axis in regulating the response of NSCLC to MTAs and the importance of the miR-195/BIRC5 axis in regulating NSCLC metastasis.

miR-195 potentiates the efficacy of microtubule-targeting agents in non-small cell lung cancer.

Microtubule-targeting agents (MTAs) are widely used for the treatment of non-small cell lung cancer (NSCLC). The response rate is only ∼25%, mainly attributable to drug resistance. To identify determinants of resistance in NSCLC, we performed a high-throughput screen using a library of miRNA mimics. Here we report that miR-195 synergizes with MTAs to inhibit the growth of NSCLC cells in vitro, that increased expression of miR-195 sensitizes NSCLC cells to MTAs and that repression of miR-195 confers resistance to MTAs. We show that NSCLC tumors over-expressing miR-195 are more sensitive to MTA treatment and that induced expression of miR-195 in NSCLC tumors potentiates the anti-tumor effect of MTAs. Additionally, we demonstrate that miR-195 targets checkpoint kinase 1 (CHEK1) to regulate the response of NSCLC cells to MTAs, that over-expression of CHEK1 contributes to resistance to MTAs and that knock-down of CHEK1 synergizes with MTAs to repress cell growth. Our results highlight the importance of miR-195 in regulating the response of NSCLC cells to MTAs and underline the potential application of miR-195 as a biomarker for response to MTAs, and as a therapeutic adjuvant to MTA treatment.

CRISPR-Based Editing Reveals Edge-Specific Effects in Biological Networks.

Unraveling the properties of biological networks is central to understanding both normal and disease cellular phenotypes. Networks consist of functional elements (nodes) that form a variety of diverse connections (edges), with each node being a hub for multiple edges. Herein, in contrast to node-centric network perturbation and analysis approaches, we present a high-throughput CRISPR-based methodology for delineating the role of network edges. Ablation of network edges using a library targeting 93 miRNA target sites in 71 genes reveals numerous edges that control, with variable importance, cellular growth and survival under stress. To compare the impact of removing nodes versus edges in a biological network, we dissect a specific p53-microRNA pathway. We show that removal of the miR-34a target site from the anti-apoptotic gene BCL2 desensitizes the cell to ectopic delivery of miR-34a in a p53-dependent manner. In summary, we demonstrate that network edges are critical to the function and stability of biological networks. Our results introduce a novel genetic screening opportunity via edge ablation and highlight a new dimension in biological network analysis.

miR-195 targets cyclin D3 and survivin to modulate the tumorigenesis of non-small cell lung cancer.

miR-195 has recently been reported to function as a tumor suppressor in various cancers, including non-small cell lung cancer (NSCLC). However, the mechanisms by which miR-195 represses the tumorigenesis of NSCLC cells are not fully understood. We performed a high-throughput screen using an miRNA mimic library and confirmed the identification of miR-195 as a tumor suppressor in NSCLC. We demonstrated that overexpression or induced expression of miR-195 in lung tumors slows tumor growth and that repression of miR-195 accelerates tumor growth. In addition, we found that knockout of miR-195 promotes cancer cell growth. We demonstrated that miR-195 targets cyclin D3 to cause cell cycle arrest at the G1 phase and that miR-195 targets survivin to induce apoptosis and senescence in NSCLC cells. Overexpression of cyclin D3 or survivin reverses the effects of miR-195 in NSCLC cells. Through the analysis of data from The Cancer Genome Atlas, we confirmed that the expression of miR-195 is lower in tumors than in adjacent normal tissues and that low expression of miR-195 is associated with poor survival in both lung adenocarcinoma and squamous cell carcinoma patients. Specifically, we found that BIRC5, which codes for survivin, is upregulated in both adenocarcinoma and squamous cell carcinoma tissues and that high expression of BIRC5 is associated with poor survival in adenocarcinoma, but not squamous cell carcinoma. In addition, the ratio of miR-195 level to BIRC5 level is associated with both recurrence-free and overall survival in lung adenocarcinoma. Our results suggest that the miR-195/BIRC5 axis is a potential target for treatment of lung adenocarcinoma specifically, and NSCLC in general.

microRNA-2110 functions as an onco-suppressor in neuroblastoma by directly targeting Tsukushi.

microRNA-2110 (miR-2110) was previously identified as inducing neurite outgrowth in a neuroblastoma cell lines BE(2)-C, suggesting its differentiation-inducing and oncosuppressive function in neuroblastoma. In this study, we demonstrated that synthetic miR-2110 mimic had a generic effect on reducing cell survival in neuroblastoma cell lines with distinct genetic backgrounds, although the induction of cell differentiation traits varied between cell lines. In investigating the mechanisms underlying such functions of miR-2110, we identified that among its predicted target genes down-regulated by miR-2110, knockdown of Tsukushi (TSKU) expression showed the most potent effect in inducing cell differentiation and reducing cell survival, suggesting that TSKU protein plays a key role in mediating the functions of miR-2110. In investigating the clinical relevance of miR-2110 and TSKU expression in neuroblastoma patients, we found that low tumor miR-2110 levels were significantly correlated with high tumor TSKU mRNA levels, and that both low miR-2110 and high TSKU mRNA levels were significantly correlated with poor patient survival. These findings altogether support the oncosuppressive function of miR-2110 and suggest an important role for miR-2110 and its target TSKU in neuroblastoma tumorigenesis and in determining patient prognosis.