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Background RNA-binding proteins (RBPs) function as master regulators of gene expression. Alterations in RBP expression and function are often observed in cancer and influence critical pathways implicated in tumor initiation and growth. Identification and characterization of oncogenic RBPs and their regulatory networks provide new opportunities for targeted therapy. Results We identify the RNA-binding protein SERBP1 as a novel regulator of glioblastoma (GBM) development. High SERBP1 expression is prevalent in GBMs and correlates with poor patient survival and poor response to chemo- and radiotherapy. SERBP1 knockdown causes delay in tumor growth and impacts cancer-relevant phenotypes in GBM and glioma stem cell lines. RNAcompete identifies a GC-rich region as SERBP1-binding motif; subsequent genomic and functional analyses establish SERBP1 regulation role in metabolic routes preferentially used by cancer cells. An important consequence of these functions is SERBP1 impact on methionine production. SERBP1 knockdown decreases methionine levels causing a subsequent reduction in histone methylation as shown for H3K27me3 and upregulation of genes associated with neurogenesis, neuronal differentiation, and function. Further analysis demonstrates that several of these genes are downregulated in GBM, potentially through epigenetic silencing as indicated by the presence of H3K27me3 sites. Conclusions SERBP1 is the first example of an RNA-binding protein functioning as a central regulator of cancer metabolism and indirect modulator of epigenetic regulation in GBM. By bridging these two processes, SERBP1 enhances glioma stem cell phenotypes and contributes to GBM poorly differentiated state.

RNA binding proteins (RBPs) are essential for critical biological processes such as translation regulation and mRNA processing, and misfunctions of these proteins are associated with diseases such as cancer and neurodegeneration. SERBP1 (SERPINE1 mRNA Binding Protein 1) is an RBP that comprises two RG/RGG repeat regions yet lacks other recognizable RNA-binding motifs. It is involved in mRNA maturation, and translational regulation. It was initially identified as a hyaluronic acid binding protein, but recent studies have identified central roles for SERBP1 in brain function and development, especially neurogenesis and synaptogenesis. SERBP1 regulates One-carbon metabolism and epigenetic modification of histones, and increased SERBP1 expression in cancers such as leukemia, ovarian, prostate, liver and glioblastoma is correlated with poor patient outcomes. Despite these important regulatory roles for SERBP1, little is known about its structural and dynamic properties, nor about the molecular mechanisms governing its interaction with mRNA. Here, we define SERBP1 as an intrinsically disordered protein, containing highly conserved elements that were shown to be functionally important. The RNA binding activity of SERBP1 was explored using solution NMR and other biophysical techniques. The outcome of these experiments revealed that SERBP1 preferentially samples compact conformations including a central, stable α-helix and show that SERBP1 recognizes G-rich RNA sequences at the C-terminus involving the RGG box and neighboring residues. Despite the role in RNA recognition, the RGG boxes do not seem to stabilize the central helix and the central helix does not participate in RNA binding. Further, SERBP1 undergoes liquid-liquid phase separation, mediated by salt and RNA, and both RGG boxes are necessary for the efficient formation of condensed phases. Together, these results provide a foundation for understanding the molecular mechanisms of SERBP1 functions in physiological and pathological processes.

RNA-binding proteins (RBPs) and miRNAs are critical gene expression regulators that interact with one another in cooperative and antagonistic fashions. We identified Musashi1 (Msi1) and miR-137 as regulators of a molecular switch between self-renewal and differentiation. Msi1 and miR-137 have opposite expression patterns and functions, and Msi1 is repressed by miR-137. Msi1 is a stem–cell protein implicated in self-renewal while miR-137 functions as a proneuronal differentiation miRNA. In gliomas, miR-137 functions as a tumor suppressor while Msi1 is a prooncogenic factor. We suggest that the balance between Msi1 and miR-137 is a key determinant in cell fate decisions and disruption of this balance could contribute to neurodegenerative diseases and glioma development. Genomic analyses revealed that Msi1 and miR-137 share 141 target genes associated with differentiation, development, and morphogenesis. Initial results pointed out that these two regulators have an opposite impact on the expression of their target genes. Therefore, we propose an antagonistic model in which this network of shared targets could be either repressed by miR-137 or activated by Msi1, leading to different outcomes (self-renewal, proliferation, tumorigenesis).

BackgroundGlioblastoma (GBM) is marked by cellular heterogeneity, including metabolic heterogeneity, that varies among cellular microenvironments in the same tumor. Altered cellular metabolism in cancer is well-established, but how lipid metabolism is altered to suit different microenvironmental conditions and cellular states within a tumor remains unexplored.MethodsWe assessed GBM organoid models that mimic the transition zone between nutrient-rich and nutrient-poor pseudopalisading/perinecrotic tumor zones and performed spatial RNA-sequencing of cells to interrogate lipid metabolism. Using targeted lipidomic analysis, we assessed differences in acutely enriched cancer stem cells (CSCs) and non-CSCs from multiple patient-derived models to explore the link between the stem cell state and lipid metabolism.ResultsSpatial analysis revealed a striking difference in lipid content between microenvironments, with lipid enrichment in the hypoxic organoid cores and the perinecrotic and pseudopalisading regions of primary patient tumors. This was accompanied by regionally restricted upregulation of hypoxia-inducible lipid droplet-associated (HILPDA) gene expression in organoid cores and in clinical GBM specimens, but not lower-grade brain tumors, that was specifically localized to pseudopalisading regions of patient tumors. CSCs have low lipid droplet accumulation compared to non-CSCs in organoid models and xenograft tumors, and prospectively sorted lipid-low GBM cells are functionally enriched for stem cell activity. Targeted lipidomic analysis revealed that CSCs had decreased levels of major classes of neutral lipids compared to non-CSCs but had significantly increased polyunsaturated fatty acid production due to high fatty acid desaturase (FADS1/2) expression.ConclusionsOur data demonstrate that lipid metabolism is differentially altered across GBM microenvironments and cellular hierarchies, providing guidance for targeting of these altered lipid metabolic pathways.Key pointsGBM cells in nutrient-poor tumor regions have increased accumulation of lipid droplets.CSCs have reduced lipid content compared to non-CSCs.GBM CSCs and non-CSCs have disparate lipid metabolisms that may be uniquely targetable.Importance of the StudyMetabolic targeting has long been advocated as a therapy against many tumors including GBM, and it remains an outstanding question whether cancer stem cells (CSCs) have altered lipid metabolism. We demonstrated striking differences in lipid metabolism between diverse cell populations from the same patient. These spatially and phenotypically distinct lipid phenotypes occur clinically in the majority of patients and can be recapitulated in laboratory models. Lipidomic analysis of multiple patient-derived models shows a significant shift in lipid metabolism between GBM CSCs and non-CSCs, suggesting that lipid levels may not be simply a product of the microenvironment but also may be a reflection of cellular state. Our results suggest that therapeutic targeting of GBM lipid metabolism must consider multiple separate tumor cell populations to be effective, and we provide a methodologic framework for studying these metabolically diverse cellular populations.

The thyroid hormone T3 and its nuclear receptor TRα1 control gut development and homeostasis through the modulation of intestinal crypt cell proliferation. Despite increasing data, in-depth analysis on their specific action on intestinal stem cells is lacking. By using ex vivo 3D organoid cultures and molecular approaches, we observed early responses to T3 involving the T3-metabolizing enzyme Dio1 and the transporter Mct10, accompanied by a complex response of stem cell- and progenitor-enriched genes. Interestingly, specific TRα1 loss-of-function (inducible or constitutive) was responsible for low ex vivo organoid development and impaired stem cell activity. T3 treatment of animals in vivo not only confirmed the positive action of this hormone on crypt cell proliferation but also demonstrated its key action in modulating the number of stem cells, the expression of their specific markers and the commitment of progenitors into lineage-specific differentiation. In conclusion, T3 treatment or TRα1 modulation has a rapid and strong effect on intestinal stem cells, broadening our perspectives in the study of T3/TRα1-dependent signaling in these cells.

Simple Summary Glioblastoma (GBM) is one of the most aggressive tumor types with no effective treatment options. To create new routes for therapy, it is necessary to continue mapping new pathways contributing to gliomagenesis. In this regard, there is growing evidence that RNA binding proteins (RBPs) are major contributors to expression alterations affecting genes in signaling pathways critical to GBM growth and response to therapy. We have established Musashi1 (Msi1) as a main player in GBM and medulloblastoma and as a marker of clinical outcome and response to therapy. Our genomic and functional analyses established that Msi1 directly and indirectly regulates the expression of a network of genes, promoting cell cycle progression and DNA replication. Ultimately, Msi1 impact on this network has important consequences in tumor initiation, growth and response to therapy. Abstract RNA-binding proteins (RBPs) function as master regulators of gene expression. Alterations in their levels are often observed in tumors with numerous oncogenic RBPs identified in recent years. Musashi1 (Msi1) is an RBP and stem cell gene that controls the balance between self-renewal and differentiation. High Msi1 levels have been observed in multiple tumors including glioblastoma and are often associated with poor patient outcomes and tumor growth. A comprehensive genomic analysis identified a network of cell cycle/division and DNA replication genes and established these processes as Msi1’s core regulatory functions in glioblastoma. Msi1 controls this gene network via two mechanisms: direct interaction and indirect regulation mediated by the transcription factors E2F2 and E2F8. Moreover, glioblastoma lines with Msi1 knockout (KO) displayed increased sensitivity to cell cycle and DNA replication inhibitors. Our results suggest that a drug combination strategy (Msi1 + cell cycle/DNA replication inhibitors) could be a viable route to treat glioblastoma.

Simple Summary miRNAs function as critical regulators of gene expression and have been defined as contributors of cancer phenotypes by acting as oncogenes or tumor suppressors. Based on these findings, miRNA-based therapies have been explored in the treatment of many different malignancies. The use of single miRNAs has faced some challenges and showed limited success. miRNAs cooperate to regulate distinct biological processes and pathways and, therefore, combination of related miRNAs could amplify the repression of oncogenic factors and the effect on cancer relevant pathways. We established that the combination of tumor suppressor miRNAs miR-124, miR-128, and miR-137 is much more effective than single miRNAs in disrupting proliferation and survival of glioma stem cells and neuroblastoma lines and promoting differentiation and response to radiation. Subsequent genomic analyses showed that other combinations of tumor suppressor miRNAs could be equally effective, and its use could provide new routes to target in special cancer-initiating cell populations. Abstract 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.

The stem cell marker Musashi1 (MSI1) is highly expressed during neurogenesis and in glioblastoma (GBM). MSI1 promotes self-renewal and impairs differentiation in cancer and non-malignant progenitor cells. However, a comprehensive understanding of its role in promoting GBM-driving networks remains to be deciphered. We demonstrate that MSI1 is highly expressed in GBM recurrences, an oncologist&rsquo, s major defiance. For the first time, we provide evidence that MSI1 promotes the expression of stem cell markers like CD44, co-expressed with MSI1 within recurrence-promoting cells at the migrating front of primary GBM samples. With GBM cell models of pediatric and adult origin, including isolated primary tumorspheres, we show that MSI1 promotes stem cell-like characteristics. Importantly, it impairs CD44 downregulation in a 3&prime, UTR- and miRNA-dependent manner by controlling mRNA turnover. This regulation is disturbed by the previously reported MSI1 inhibitor luteolin, providing further evidence for a therapeutic target potential of MSI1 in GBM treatment.

High-dose radiation is the main component of glioblastoma therapy. Unfortunately, radio-resistance is a common problem and a major contributor to tumor relapse. Understanding the molecular mechanisms driving response to radiation is critical for identifying regulatory routes that could be targeted to improve treatment response. We conducted an integrated analysis in the U251 and U343 glioblastoma cell lines to map early alterations in the expression of genes at three levels: transcription, splicing, and translation in response to ionizing radiation. Changes at the transcriptional level were the most prevalent response. Downregulated genes are strongly associated with cell cycle and DNA replication and linked to a coordinated module of expression. Alterations in this group are likely driven by decreased expression of the transcription factor FOXM1 and members of the E2F family. Genes involved in RNA regulatory mechanisms were affected at the mRNA, splicing, and translation levels, highlighting their importance in radiation-response. We identified a number of oncogenic factors, with an increased expression upon radiation exposure, including BCL6, RRM2B, IDO1, FTH1, APIP, and LRIG2 and lncRNAs NEAT1 and FTX. Several of these targets have been previously implicated in radio-resistance. Therefore, antagonizing their effects post-radiation could increase therapeutic efficacy. Our integrated analysis provides a comprehensive view of early response to radiation in glioblastoma. We identify new biological processes involved in altered expression of various oncogenic factors and suggest new target options to increase radiation sensitivity and prevent relapse.

Intestinal crypts are composed of heterogeneous and highly plastic cell populations. Lgr5high-stem cells (SC) are responsible for homeostatic renewal, but other cells can revert to an SC-like phenotype to maintain epithelial integrity. Despite their distinct roles in orchestrating homeostasis, both populations have been designated as the putative “cell-of-origin” of colorectal cancer. However, their respective involvement in the emergence of drug-resistant cancer SCs (CSC), responsible for tumor relapse and associated with poor outcome of colorectal cancer, remains elusive. In this context, the intestinal SC/progenitor-marker Musashi1 (MSI1) is interesting as it plays important functions in intestinal homeostasis and is frequently overexpressed in human colorectal cancer. Therefore, our aims were: (i) to study the impact of chemotherapy on Lgr5-expressing and MSI1-expressing cell populations, (ii) to explore the effect of increased MSI1 levels in response to treatment, and (iii) to evaluate the relevance in human colorectal cancer. Engineered mouse models treated with the therapeutic agent 5-fluorouracil showed that upon increased MSI1 levels, Lgr5high SCs remain sensitive while Lgr5low progenitors reprogram to a drug-resistant phenotype. This resulted in the expansion of an MSI1-expressing cell subpopulation with improved resistance to DNA damage and increased detoxification, typical properties of dormant-CSCs that can reactivate after chemotherapy. Analysis in patients with colorectal cancer revealed a correlation between MSI1 levels and tumor grading, CSC phenotype, and chemoresistance. Altogether, these results shed new light on the biology and plasticity of normal crypt and cancer cell populations and also open new perspectives to target MSI1 to improve chemotherapy outcome. Significance: This study unveils paradoxical roles for MSI1, underlining its importance in facilitating intestinal regeneration upon injury but also unraveling its new function in drug-resistant colorectal cancer stem cells.

The Flaviviridae virus family was named after the Yellow-fever virus, and the latin term flavi means “of golden color”. Dengue, caused by Dengue virus (DENV), is one of the most important infectious diseases worldwide. A sensitive and differential diagnosis is crucial for patient management, especially due to the occurrence of serological cross-reactivity to other co-circulating flaviviruses. This became particularly important with the emergence of Zika virus (ZIKV) in areas were DENV seroprevalence was already high. We developed a sensitive and specific diagnostic test based on gold nanorods (GNR) functionalized with DENV proteins as nanosensors. These were able to detect as little as one picogram of anti-DENV monoclonal antibodies and highly diluted DENV-positive human sera. The nanosensors could differentiate DENV-positive sera from other flavivirus-infected patients, including ZIKV, and were even able to distinguish which DENV serotype infected individual patients. Readouts are obtained in ELISA-plate spectrophotometers without the need of specific devices.

High-dose radiation is the main component of glioblastoma therapy. Unfortunately, radio-resistance is a common problem and a major contributor to tumor relapse. Understanding the molecular mechanisms driving response to radiation is critical for identifying regulatory routes that could be targeted to improve treatment response. We conducted an integrated analysis in the U251 and U343 glioblastoma cell lines to map early alterations in the expression of genes at three levels: transcription, splicing, and translation in response to ionizing radiation. Changes at the transcriptional level were the most prevalent response. Downregulated genes are strongly associated with cell cycle and DNA replication and linked to a coordinated module of expression. Alterations in this group are likely driven by decreased expression of the transcription factor FOXM1 and members of the E2F family. Genes involved in RNA regulatory mechanisms were affected at the mRNA, splicing, and translation levels, highlighting their importance in radiation-response. We identified a number of oncogenic factors, with an increased expression upon radiation exposure, including BCL6, RRM2B, IDO1, FTH1, APIP, and LRIG2 and lncRNAs NEAT1 and FTX. Several of these targets have been previously implicated in radio-resistance. Therefore, antagonizing their effects post-radiation could increase therapeutic efficacy. Our integrated analysis provides a comprehensive view of early response to radiation in glioblastoma. We identify new biological processes involved in altered expression of various oncogenic factors and suggest new target options to increase radiation sensitivity and prevent relapse.

Adipose tissue plays an essential role in metabolic health. Ames dwarf mice are exceptionally long-lived and display metabolically beneficial phenotypes in their adipose tissue, providing an ideal model for studying the intersection between adipose tissue and longevity. To this end, we assessed the metabolome and lipidome of adipose tissue in Ames dwarf mice. We observed distinct lipid profiles in brown versus white adipose tissue of Ames dwarf mice that are consistent with increased thermogenesis and insulin sensitivity, such as increased cardiolipin and decreased ceramide concentrations. Moreover, we identified 5-hydroxyeicosapentaenoic acid (5-HEPE), an ω-3 fatty acid metabolite, to be increased in Ames dwarf brown adipose tissue (BAT), as well as in circulation. Importantly, 5-HEPE is increased in other models of BAT activation and is negatively correlated with body weight, insulin resistance, and circulating triglyceride concentrations in humans. Together, these data represent a novel lipid signature of adipose tissue in a mouse model of extreme longevity. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (10.1007/s11357-020-00221-0) contains supplementary material, which is available to authorized users.

Brown and brown-like beige/brite adipocytes dissipate energy and have been proposed as therapeutic targets to combat metabolic disorders. However, the therapeutic effects of cell-based therapy in humans remain unclear. Here, we created human brown-like (HUMBLE) cells by engineering human white preadipocytes using CRISPR/Cas9-SAM-gRNA to activate endogenous uncoupling protein 1 expression. Obese mice that received HUMBLE cell transplants showed a sustained improvement in glucose tolerance and insulin sensitivity, as well as increased energy expenditure. Mechanistically, increased arginine/nitric oxide (NO) metabolism in HUMBLE adipocytes promoted the production of NO that was carried by S-nitrosothiols and nitrite in red blood cells to activate endogenous brown fat and improved glucose homeostasis in recipient animals. Taken together, these data demonstrate the utility of using CRISPR/Cas9 technology to engineer human white adipocytes to display brown fat-like phenotypes and may open up cell-based therapeutic opportunities to combat obesity and diabetes.

Therapy resistance and recurrence in high-grade gliomas are driven by their populations of glioma stem cells (GSCs). Thus, detailed molecular characterization of GSCs is needed to develop more effective therapies. We conducted a study to identify differences in the splicing profile and expression of long non-coding RNAs in proneural and mesenchymal GSC cell lines. Genes related to cell cycle, DNA repair, cilium assembly, and splicing showed the most differences between GSC subgroups. We also identified genes distinctly associated with survival among patients of mesenchymal or proneural subgroups. We determined that multiple long non-coding RNAs with increased expression in mesenchymal GSCs are associated with poor survival of glioblastoma patients. In summary, our study established critical differences between proneural and mesenchymal GSCs in splicing profiles and expression of long non-coding RNA. These splicing isoforms and lncRNA signatures may contribute to the uniqueness of GSC subgroups, thus contributing to cancer phenotypes and explaining differences in therapeutic responses. This study was supported by a grant from Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Brazil to PAFG and LOFP and by NIH 7R21CA175875-03. This study was partially supported by grants from Serrapilheira foundation and Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP; 2018/15579-8) to PAFG. GDAG and BRC were supported by fellowships from FAPESP (2017/19541-2) and (2013/25483-4 and 2013/07159-5), respectively. PRA was supported by CPRIT Training Grant - RP140105

RNA binding proteins have emerged as critical oncogenic factors and potential targets in cancer therapy. In this study, we evaluated Musashi1 (Msi1) targeting as a strategy to treat glioblastoma (GBM); the most aggressive brain tumor type. Msi1 expression levels are often high in GBMs and other tumor types and correlate with poor clinical outcome. Moreover, Msi1 has been implicated in chemo- and radio-resistance. Msi1 modulates a range of cancer relevant processes and pathways and regulates the expression of stem cell markers and oncogenic factors via mRNA translation/stability. To identify Msi1 inhibitors capable of blocking its RNA binding function, we performed a ~ 25,000 compound fluorescence polarization screen. NMR and LSPR were used to confirm direct interaction between Msi1 and luteolin, the leading compound. Luteolin displayed strong interaction with Msi1 RNA binding domain 1 (RBD1). As a likely consequence of this interaction, we observed via western and luciferase assays that luteolin treatment diminished Msi1 positive impact on the expression of pro-oncogenic target genes. We tested the effect of luteolin treatment on GBM cells and showed that it reduced proliferation, cell viability, colony formation, migration and invasion of U251 and U343 GBM cells. Luteolin also decreased the proliferation of patient-derived glioma initiating cells (GICs) and tumor-organoids but did not affect normal astrocytes. Finally, we demonstrated the value of combined treatments with luteolin and olaparib (PARP inhibitor) or ionizing radiation (IR). Our results show that luteolin functions as an inhibitor of Msi1 and demonstrates its potential use in GBM therapy.

BackgroundHigh-dose radiation is the main component of glioblastoma therapy. Unfortunately, radio-resistance is a common problem and a major contributor to tumor relapse. Understanding the molecular mechanisms driving response to radiation is critical for identifying regulatory routes that could be targeted to improve treatment response.MethodsWe conducted an integrated analysis in the U251 and U343 glioblastoma cell lines to map early alterations in the expression of genes at three levels: transcription, splicing, and translation in response to ionizing radiation.ResultsChanges at the transcriptional level were the most prevalent response. Downregulated genes are strongly associated with cell cycle and DNA replication and linked to a coordinated module of expression. Alterations in this group are likely driven by decreased expression of the transcription factor FOXM1 and members of the E2F family. Genes involved in RNA regulatory mechanisms were affected at the mRNA, splicing, and translation levels, highlighting their importance in radiation-response. We identified a number of oncogenic factors, with an increased expression upon radiation exposure, including BCL6, RRM2B, IDO1, FTH1, APIP, and LRIG2 and lncRNAs NEAT1 and FTX. Several of these targets have been previously implicated in radio-resistance. Therefore, antagonizing their effects post-radiation could increase therapeutic efficacy.ConclusionsOur integrated analysis provides a comprehensive view of early response to radiation in glioblastoma. We identify new biological processes involved in altered expression of various oncogenic factors and suggest new target options to increase radiation sensitivity and prevent relapse.

Our previous work demonstrated a key function of the thyroid hormone nuclear receptor TRα1, a T3-modulated transcription factor, in controlling intestinal development and homeostasis via the Wnt and Notch pathways. Importantly, increased expression of TRα1 in the intestinal epithelium in a mutated Apc genetic background (vil-TRα1/Apc+/1638N mice) accelerated tumorigenesis and contributed to a more aggressive tumor phenotype compared to that of the Apc mutants alone. Therefore, the aim of this study was to determine the relevance of this synergistic effect in human colorectal cancers and to gain insights into the mechanisms involved. We analyzed cohorts of patients by in silico and experimental approaches and observed increased TRα1 expression and a significant correlation between TRα1 levels and Wnt activity. TRα1 loss-of-function and gain-of-function in Caco2 cell lines not only confirmed that TRα1 levels control Wnt activity but also demonstrated the role of TRα1 in regulating cell proliferation and migration. Finally, upon investigation of the molecular mechanisms responsible for the Wnt-TRα1 association, we described the repression by TRα1 of several Wnt inhibitors, including Frzb, Sox17 and Wif1. In conclusion, our results underline an important functional interplay between the thyroid hormone nuclear receptor TRα1 and the canonical Wnt pathway in intestinal cancer initiation and progression. More importantly, we show for the first time that the expression of TRα1 is induced in human colorectal cancers.

Adipocytes possess remarkable adaptive capacity to respond to nutrient excess, fasting or cold exposure, and they are thus an important cell type for the maintenance of proper metabolic health. Although the endoplasmic reticulum (ER) is a critical organelle for cellular homeostasis, the mechanisms that mediate adaptation of the ER to metabolic challenges in adipocytes are unclear. Here we show that brown adipose tissue (BAT) thermogenic function requires an adaptive increase in proteasomal activity to secure cellular protein quality control, and we identify the ER-localized transcription factor nuclear factor erythroid 2-like 1 (Nfe2l1, also known as Nrf1) as a critical driver of this process. We show that cold adaptation induces Nrf1 in BAT to increase proteasomal activity and that this is crucial for maintaining ER homeostasis and cellular integrity, specifically when the cells are in a state of high thermogenic activity. In mice, under thermogenic conditions, brown-adipocyte-specific deletion of Nfe2l1 (Nrf1) resulted in ER stress, tissue inflammation, markedly diminished mitochondrial function and whitening of the BAT. In mouse models of both genetic and dietary obesity, stimulation of proteasomal activity by exogenously expressing Nrf1 or by treatment with the proteasome activator PA28α in BAT resulted in improved insulin sensitivity. In conclusion, Nrf1 emerges as a novel guardian of brown adipocyte function, providing increased proteometabolic quality control for adapting to cold or to obesity.

To investigate the effects of high dose rate (HDR) brachytherapy on cellular progression of a radioresistant human squamous cell carcinoma in vitro, based on clinical parameters.An acrylic platform was designed to attach tissue culture flasks and assure source positioning during irradiation. At exponential phase, A431cells, a human squamous cell carcinoma, were irradiated twice up to 1100 cGy. Cellular proliferation was assessed by Trypan blue exclusion assay and survival fraction was calculated by clonogenic assay. DNA content analysis and cell cycle phases were assessed by flow cytometry and gel electrophoresis, respectively. Cellular death patterns were measured by HOPI double-staining method.Significant decreasing cellular proliferation rate (p 0.05) as well as reduced survival fraction (p 0.001) in irradiated cells were observed. Moreover, increased percentage of cells arrested in the GHDR brachytherapy led to a decreased proliferation rate and cell survival and also hampered cellular progression to mitosis suggesting that tumor cell death mainly occurred due to mitotic death and G

Genomic analyses have become an important tool to identify new avenues for therapy. This is especially true for cancer types with extremely poor outcomes, since our lack of effective therapies offers no tangible clinical starting point to build upon. The highly malignant brain tumor glioblastoma (GBM) exemplifies such a refractory cancer, with only 15 month average patient survival. Analyses of several hundred GBM samples compiled by the TCGA (The Cancer Genome Atlas) have produced an extensive transcriptomic map, identified prevalent chromosomal alterations, and defined important driver mutations. Unfortunately, clinical trials based on these results have not yet delivered an improvement on outcome. It is, therefore, necessary to characterize other regulatory routes known for playing a role in tumor relapse and response to treatment. Alternative splicing affects more than 90% of the human coding genes and it is an important source for transcript variation and gene regulation. Mutations and alterations in splicing factors are highly prevalent in multiple cancers, demonstrating the potential for splicing to act as a tumor driver. As a result, numerous genes are expressed as cancer-specific splicing isoforms that are functionally distinct from the canonical isoforms found in normal tissue. These include genes that regulate cancer-critical pathways such as apoptosis, DNA repair, cell proliferation, and migration. Splicing defects can even induce genomic instability, a common characteristic of cancer, and a driver of tumor evolution. Importantly, components of the splicing machinery are targetable; multiple drugs can inhibit splicing factors or promote changes in splicing which could be exploited to begin improving clinical outcomes. Here, we review the current literature and present a case for exploring RNA processing as therapeutic route for the treatment of GBM.

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.

Post-transcriptional regulation of gene expression is fundamental for all forms of life, as it critically contributes to the composition and quantity of a cell's proteome. These processes encompass splicing, polyadenylation, mRNA decay, mRNA editing and modification and translation and are modulated by a variety of RNA-binding proteins (RBPs). Alterations affecting RBP expression and activity contribute to the development of different types of cancer. In this chapter, we discuss current research shedding light on the role of different RBPs in gliomas. These studies place RBPs as modulators of critical signaling pathways, establish their relevance as prognostic markers and open doors for new therapeutic strategies.

The conserved RNA-binding protein Musashi1 (MSI1) has been characterized as a stem cell marker, controlling the balance between self-renewal and differentiation and as a key oncogenic factor in numerous solid tumors, including glioblastoma. To explore the potential use of MSI1 targeting in therapy, we studied MSI1 in the context of radiation sensitivity. Knockdown of MSI1 led to a decrease in cell survival and an increase in DNA damage compared to control in cells treated with ionizing radiation. We subsequently examined mechanisms of double-strand break repair and found that loss of MSI1 reduces the frequency of nonhomologous end-joining. This phenomenon could be attributed to the decreased expression of DNA-protein kinase catalytic subunit, which we have previously identified as a target of MSI1. Collectively, our results suggest a role for MSI1 in double-strand break repair and that its inhibition may enhance the effect of radiotherapy.

Obesity and insulin resistance have been associated with deterioration in asthma outcomes. High oxidative stress and deficient activation of AMP-activated protein kinase (AMPK) have emerged as important regulators linking insulin resistance and inflammation. This study aimed to evaluate the effects of resveratrol on obesity-associated allergic pulmonary inflammation. Male C57/Bl6 mice fed with high-fat diet to induce obesity (obese group) or standard-chow diet (lean group) were treated or not with resveratrol (100mg/kg/day, two weeks). Mice were sensitized and challenged with ovalbumin (OVA). At 48h thereafter, bronchoalveolar lavage fluid was performed, and lungs collected for morphological studies and Western blot analysis. Treatment of obese mice with resveratrol significantly reduced hyperglycemia and insulin resistance, as well as the body measures (body mass, fat mass, % fat, and body area). OVA-challenge promoted a higher increase in pulmonary eosinophil infiltration in obese compared with lean mice, which was nearly abrogated by resveratrol treatment. Resveratrol markedly increased the phosphorylated AMPK expression in lung tissues of obese compared with lean mice. Resveratrol reduced the p47phox expression and reactive-oxygen species (ROS) production, and elevated the superoxide dismutase (SOD) levels in lung tissues of obese mice. The increased pulmonary levels of TNF-α and inducible nitric oxide synthase (iNOS) in obese mice were also normalized after resveratrol treatment. In lean mice, resveratrol failed to affect the levels of fasting glucose, p47phox, ROS levels, TNF-α, iNOS and phosphorylated AMPK. Resveratrol exhibits protective effects in obesity-associated lung inflammation that is accompanied by local AMPK activation and antioxidant property.

Adipose tissue exerts multiple vital functions that critically maintain energy balance, including storing and expending energy, as well as secreting factors that systemically modulate nutrient metabolism. Since lipids are the major constituents of the adipocytes, it is unsurprising that the lipid composition of these cells plays a critical role in maintaining their functions and communicating with other organs and cells. In both positive and negative energy balance conditions, lipids and free fatty acids secreted from adipocytes exert either beneficial or detrimental effects in other tissues, such as the liver, pancreas and muscle. The way the adipocytes communicate with other organs tightly depends on the nature of their lipidome composition. Notwithstanding, the lipidome composition of the adipocytes is affected by physiological factors such as adipocyte type, gender and age, but also by environmental cues such as diet composition, thermal stress and physical activity. Here we provide an updated overview on how the adipose tissue lipidome profile is shaped by different physiological and environmental factors and how these changes impact the way the adipocytes regulate whole-body energy metabolism.

Opsin3 (Opn3) is a transmembrane heptahelical G protein–coupled receptor (GPCR) with the potential to produce a nonvisual photoreceptive effect. Interestingly, anatomical profiling of GPCRs reveals that Opn3 mRNA is highly expressed in adipose tissue. The photosensitive functions of Opn3 in mammals are poorly understood, and whether Opn3 has a role in fat is entirely unknown. In this study, we found that Opn3-knockout (Opn3-KO) mice were prone to diet-induced obesity and insulin resistance. At the cellular level, Opn3-KO brown adipocytes cultured in darkness had decreased glucose uptake and lower nutrient-induced mitochondrial respiration than wild-type (WT) cells. Light exposure promoted mitochondrial activity and glucose uptake in WT adipocytes but not in Opn3-KO cells. Brown adipocytes carrying a defective mutation in Opn3’s putative G protein–binding domain also exhibited a reduction in glucose uptake and mitochondrial respiration in darkness. Using RNA-sequencing, we identified several novel light-sensitive and Opn3-dependent molecular signatures in brown adipocytes. Importantly, direct exposure of brown adipose tissue (BAT) to light in living mice significantly enhanced thermogenic capacity of BAT, and this effect was diminished in Opn3-KO animals. These results uncover a previously unrecognized cell-autonomous, light-sensing mechanism in brown adipocytes via Opn3-GPCR signaling that can regulate fuel metabolism and mitochondrial respiration. Our work also provides a molecular basis for developing light-based treatments for obesity and its related metabolic disorders., Brown adipose tissue plays a pivotal role in energy homeostasis and serves as a metabolic sink for glucose and fatty acid. This study demonstrates a novel light-sensing mechanism in mice via the photoreceptor Opsin3 that regulates fuel utilization and mitochondrial activity of brown adipocytes.

Zika virus (ZIKV) causes microcephaly by killing neural precursor cells (NPCs) and other brain cells. ZIKV also displays therapeutic oncolytic activity against glioblastoma (GBM) stem cells (GSCs). Here we demonstrate that ZIKV preferentially infected and killed GSCs and stem-like cells in medulloblastoma and ependymoma in a SOX2-dependent manner. Targeting SOX2 severely attenuated ZIKV infection, in contrast to AXL. As mechanisms of SOX2-mediated ZIKV infection, we identified inverse expression of antiviral interferon response genes (ISGs) and positive correlation with integrin α(v) (ITGAV). ZIKV infection was disrupted by genetic targeting of ITGAV or its binding partner ITGB5 and by an antibody specific for integrin α(v)β(5). ZIKV selectively eliminated GSCs from species-matched human mature cerebral organoids and GBM surgical specimens, which was reversed by integrin α(v)β(5) inhibition. Collectively, our studies identify integrin α(v)β(5) as a functional cancer stem cell marker essential for GBM maintenance and ZIKV infection, providing potential brain tumor therapy.

Brown and brown-like beige adipocytes dissipate energy for thermogenesis and have been proposed to combat obesity and metabolic disorders. Uncoupling protein 1 (UCP1) is uniquely expressed in brown and beige adipocytes, and facilitates fuel utilization and energy expenditure. Ectopic UCP1 overexpression improves obesity and insulin resistance in mice. Recently, we utilized the CRISPR/Cas9 synergistic activation mediator and specific gRNAs to boost endogenous UCP1 expression in human white adipocytes. The CRISPR/Cas9 engineered human brown fat-like (referred to as HUMBLE, hereafter) cells had acquired human brown fat features when comparing bona fide human brown adipocytes derived from the same individual. Obese mice receiving a transplantation of HUMBLE cells showed improved glucose tolerance and insulin sensitivity, as well as increased energy expenditure. This was mediated, at least in part, via activation of endogenous brown fat, which showed an increase in heat production and glucose uptake in mice receiving HUMBLE cells. Co-culture with HUMBLE cells or treatment of conditional medium from HUMBLE cells promoted glucose uptake and thermogenesis in brown adipocytes. Metabolomic analysis identified an increased ratio of arginine and asymmetrical dimethylarginine (ADMA) in the sera of mice receiving HUMBLE cell transplantation. Arginine is known to promote nitric oxide biosynthesis, which can activate thermogenic function of brown fat. Consistently, the level of nitric oxide was increased in the endogenous brown fat of mice receiving HUMBLE cells. Inhibition of nitric oxide synthase abolished the HUMBLE cells-mediated activation of brown adipocytes in vitro and in vivo. In summary, CRISPR/Cas9-mediated activation of endogenous UCP1 expression in human white adipocytes creates brown fat-like features, and activates endogenous brown fat, potentially through arginine-related metabolites to improve the systemic glucose utilization. Disclosure C. Wang: None. M. Lundh: None. R. Kriszt: Employee; Self; QuintilesIMS. T. Huang: None. L.O. Leiria: None. F. Shamsi: None. M. Lynes: None. J. Darcy: None. B. Emanuelli: None. M. Kiebish: None. Y. Tseng: Other Relationship; Self; Chugai Pharmaceutical Co., Ltd.. Research Support; Self; MedImmune.

The conserved RNA-binding protein Musashi1 (MSI1) has emerged as a key oncogenic factor in numerous solid tumors, including glioblastoma. However, its mechanism of action has not yet been established comprehensively. To identify its target genes comprehensively and determine the main routes by which it influences glioblastoma phenotypes, we conducted individual-nucleotide resolution cross-linking and immunoprecipitation (iCLIP) experiments. We confirmed that MSI1 has a preference for UAG sequences contained in a particular structural context, especially in 3′ untranslated regions. Although numerous binding sites were also identified in intronic sequences, our RNA transcriptome sequencing analysis does not favor the idea that MSI1 is a major regulator of splicing in glioblastoma cells. MSI1 target mRNAs encode proteins that function in multiple pathways of cell proliferation and cell adhesion. Since these associations indicate potentially new roles for MSI1, we investigated its impact on glioblastoma cell adhesion, morphology, migration, and invasion. These processes are known to underpin the spread and relapse of glioblastoma, in contrast to other tumors where metastasis is the main driver of recurrence and progression.

SummaryObesity is a major risk factor for asthma, which is characterized by airway hyperreactivity (AHR). In obesity-associated asthma, AHR may be regulated by non-TH2 mechanisms. We hypothesized that airway reactivity is regulated by insulin in the CNS, and that the high levels of insulin associated with obesity contribute to AHR. We found that intracerebroventricular (ICV)-injected insulin increases airway reactivity in wild-type, but not in vesicle acetylcholine transporter knockdown (VAChT KDHOM−/−), mice. Either neutralization of central insulin or inhibition of extracellular signal-regulated kinases (ERK) normalized airway reactivity in hyperinsulinemic obese mice. These effects were mediated by insulin in cholinergic nerves located at the dorsal motor nucleus of the vagus (DMV) and nucleus ambiguus (NA), which convey parasympathetic outflow to the lungs. We propose that increased insulin-induced activation of ERK in parasympathetic pre-ganglionic nerves contributes to AHR in obese mice, suggesting a drug-treatable link between obesity and asthma.

Obesity is a major risk factor for asthma. Likewise, obesity is known to increase disease severity in asthmatic subjects and also to impair the efficacy of first-line treatment medications for asthma, worsening asthma control in obese patients. This concept is in agreement with the current understanding that some asthma phenotypes are not accompanied by detectable inflammation, and may not be ameliorated by classical anti-inflammatory therapy. There are growing evidences suggesting that the obesity-related asthma phenotype does not necessarily involve the classical T(H)2-dependent inflammatory process. Hormones involved in glucose homeostasis and in the pathogeneses of obesity likely directly or indirectly link obesity and asthma through inflammatory and non-inflammatory pathways. Furthermore, the endocrine regulation of the airway-related pre-ganglionic nerves likely contributes to airway hyperreactivity (AHR) in obese states. In this review, we focused our efforts on understanding the mechanism underlying obesity-related asthma by exploring the T(H)2-independent mechanisms leading to this disease.

Co- and post-transcriptional regulation of gene expression is complex and multifaceted, spanning the complete RNA lifecycle from genesis to decay. High-throughput profiling of the constituent events and processes is achieved through a range of technologies that continue to expand and evolve. Fully leveraging the resulting data is nontrivial, and requires the use of computational methods and tools carefully crafted for specific data sources and often intended to probe particular biological processes. Drawing upon databases of information pre-compiled by other researchers can further elevate analyses. Within this review, we describe the major co- and post-transcriptional events in the RNA lifecycle that are amenable to high-throughput profiling. We place specific emphasis on the analysis of the resulting data, in particular the computational tools and resources available, as well as looking toward future challenges that remain to be addressed.

High-throughput protein-RNA interaction data generated by CLIP-seq has provided an unprecedented depth of access to the activities of RNA-binding proteins (RBPs), the key players in co- and post-transcriptional regulation of gene expression. Motif discovery forms part of the necessary follow-up data analysis for CLIP-seq, both to refine the exact locations of RBP binding sites, and to characterize them. The specific properties of RBP binding sites, and the CLIP-seq methods, provide additional information not usually present in the classic motif discovery problem: the binding site structure, and cross-linking induced events in reads. We show that CLIP-seq data contains clear secondary structure signals, as well as technology- and RBP-specific cross-link signals. We introduce Zagros, a motif discovery algorithm specifically designed to leverage this information and explore its impact on the quality of recovered motifs. Our results indicate that using both secondary structure and cross-link modifications can greatly improve motif discovery on CLIP-seq data. Further, the motifs we recover provide insight into the balance between sequence- and structure-specificity struck by RBP binding.

A new study investigates how microRNAs affect the binding of proteins to RNA.

Background Melanoma is the most lethal type of skin cancer. Since chemoresistance is a significant barrier, identification of regulators affecting chemosensitivity is necessary in order to create new forms of intervention. Prohibitin 1 (PHB1) can act as anti-apoptotic or tumor suppressor molecule, depending on its subcellular localization. Our recent data shown that accumulation of PHB1 protects melanoma cells from chemotherapy-induced cell death. Lacking of post-transcriptional regulation of PHB1 could explain this accumulation. Interestingly, most of melanoma patients have down-regulation of microRNA-195. Here, we investigate the role of miR-195, its impact on PHB1 expression, and on chemosensitivity in melanoma cells. Methods TCGA-RNAseq data obtained from 341 melanoma patient samples as well as a panel of melanoma cell lines were used in an expression correlation analysis between PHB1 and predicted miRNAs. miR-195 impact on PHB1 mRNA and protein levels and relevance of this regulation were investigated in UACC-62 and SK-MEL-5 melanoma lines by RT-qPCR and western blot, luciferase reporter and genetic rescue experiments. Cell proliferation, cell-cycle analysis and caspase 3/7 assay were performed to investigate the potential action of miR-195 as chemosensitizer in melanoma cells treated with cisplatin and temozolomide. Results Analysis of the TCGA-RNAseq revealed a significant negative correlation (Pearson) between miR-195 and PHB1 expression. Moreover, RT-qPCR data showed that miR-195 is down-regulated while PHB1 is up-regulated in a collection of melanoma cells. We demonstrated that miR-195 regulates PHB1 directly by RT-qPCR and western blot in melanoma cells and luciferase assays. To establish PHB1 as a relevant target of miR-195, we conducted rescue experiments in which we showed that PHB1 transgenic expression could antagonize the suppressive effect miR-195 on the proliferation of melanoma cells. Finally, transfection experiments combined with drug treatments performed in the UACC-62 and SK-MEL-5 melanoma cells corroborated miR-195 as potential anti-proliferative agent, with potential impact in sensitization of melanoma cell death. Conclusions This study support the role of miR-195 as anti-proliferative miRNA via targeting of PHB1 in melanoma cells. Electronic supplementary material The online version of this article (10.1186/s12885-017-3721-7) contains supplementary material, which is available to authorized users.

Preclinical in vitro models provide an essential tool to study cancer cell biology as well as aid in translational research, including drug target identification and drug discovery efforts. For any model to be clinically relevant, it needs to recapitulate the biology and cell heterogeneity of the primary tumor. We recently developed and described a conditional reprogramming (CR) cell technology that addresses many of these needs and avoids the deficiencies of most current cancer cell lines, which are usually clonal in origin. Here, we used the CR cell method to generate a collection of patient-derived cell cultures from non-small cell lung cancers (NSCLC). Whole exome sequencing and copy number variations are used for the first time to address the capability of CR cells to keep their tumor-derived heterogeneity. Our results indicated that these primary cultures largely maintained the molecular characteristics of the original tumors. Using a mutant-allele tumor heterogeneity (MATH) score, we showed that CR cells are able to keep and maintain most of the intra-tumoral heterogeneity, suggesting oligoclonality of these cultures. CR cultures therefore represent a pre-clinical lung cancer model for future basic and translational studies.

Brown adipose tissue (BAT) and beige adipose tissue combust fuels for heat production in adult humans, and so constitute an appealing target for the treatment of metabolic disorders such as obesity, diabetes and hyperlipidemia1,2. Cold exposure can enhance energy expenditure by activating BAT, and it has been shown to improve nutrient metabolism3–5. These therapies, however, are time consuming and uncomfortable, demonstrating the need for pharmacological interventions. Recently, lipids have been identified that are released from tissues and act locally or systemically to promote insulin sensitivity and glucose tolerance; as a class, these lipids are referred to as ‘lipokines’6–8. Because BAT is a specialized metabolic tissue that takes up and burns lipids and is linked to systemic metabolic homeostasis, we hypothesized that there might be thermogenic lipokines that activate BAT in response to cold. Here we show that the lipid 12,13-dihydroxy-9Z-octadecenoic acid (12,13-diHOME) is a stimulator of BAT activity, and that its levels are negatively correlated with body-mass index and insulin resistance. Using a global lipidomic analysis, we found that 12,13-diHOME was increased in the circulation of humans and mice exposed to cold. Furthermore, we found that the enzymes that produce 12,13-diHOME were uniquely induced in BAT by cold stimulation. The injection of 12,13-diHOME acutely activated BAT fuel uptake and enhanced cold tolerance, which resulted in decreased levels of serum triglycerides. Mechanistically, 12,13-diHOME increased fatty acid (FA) uptake into brown adipocytes by promoting the translocation of the FA transporters FATP1 and CD36 to the cell membrane. These data suggest that 12,13-diHOME, or a functional analog, could be developed as a treatment for metabolic disorders.

ABCA1 is a major regulator of cellular cholesterol efflux and plasma HDL biogenesis. Even though the transcriptional activation of ABCA1 is well established, the posttranscriptional regulation of ABCA1 expression is poorly understood. Here, we investigate the potential contribution of the RNA binding protein (RBP) human antigen R (HuR) on the posttranscriptional regulation of ABCA1 expression. RNA immunoprecipitation assays demonstrate a direct interaction between HuR and ABCA1 mRNA. We found that HuR binds to the 3′ untranslated region of ABCA1 and increases ABCA1 translation, while HuR silencing reduces ABCA1 expression and cholesterol efflux to ApoA1 in human hepatic (Huh-7) and monocytic (THP-1) cells. Interestingly, cellular cholesterol levels regulate the expression, intracellular localization, and interaction between HuR and ABCA1 mRNA. Finally, we found that HuR expression was significantly increased in macrophages from human atherosclerotic plaques, suggesting an important role for this RBP in controlling macrophage cholesterol metabolism in vivo. In summary, we have identified HuR as a novel posttranscriptional regulator of ABCA1 expression and cellular cholesterol homeostasis, thereby opening new avenues for increasing cholesterol efflux from atherosclerotic foam macrophages and raising circulat­ing HDL cholesterol levels.

The biology of acute myelogenous leukemia (AML) is characterized by a block in differentiation, increase in proliferation and inhibition of apoptosis, all of which when combined lead to an expansion of leukemic blasts.1 AML therapy cures ~20% of those affected,2 highlighting the need for a better understanding of leukemia biology in order to identify novel therapeutic targets. The Wilms’ tumor 1 (WT1) gene has an oncogenic role in leukemogenesis and its overexpression correlates with a poor prognosis.3 WTAP is a highly conserved protein that partners with WT14,5 to function as a switch gene, regulating the balance between quiescence and proliferation.6 WTAP-null mice exhibit embryonic lethality.7 WTAP has been recently described as an oncogenic factor in gliomas.8 We hypothesized that alterations in WTAP, whose role in leukemogenesis is unknown, might provide an alternate means of modulating the WT1 pathway in AML. We performed a series of experiments to test this possibility. First, we examined WTAP expression in several primary AML samples observing increased levels of WTAP in AML compared with normal peripheral blood mononuclear cells (Figure 1a) as well as in AML cell lines (data not shown). Next, the WTAP expression levels were determined in 511 newly diagnosed AML patients using the reverse-phase protein array (RPPA) technique. In comparison with normal bone marrow CD34+ cells, WTAP expression in bulk AML cells was above normal levels in 32% of patients (Figure 1b). Although WTAP levels were not associated with individual cytogenetic abnormalities, some specific molecular mutations such as NPM1 and FLT3-ITD were found to have significant correlation (P ≤ 0.05) with WTAP expression (Supplementary Figure 1A). In addition, RPPA analysis showed that WTAP levels were positively correlated (R > |0.2|) with levels of various cell proliferation-related proteins (cyclins and Hsp90), antiapoptotic proteins (Bcl-2 and Bax), oncoproteins (FLI1) and proteins important for stem cell functions such as Myc and Ash2L (Supplementary Figure 1B). To assess the functional significance of increased WTAP expression, its expression was silenced in K562 and HL-60 cells, leading to a significant reduction (P ≤ 0.05) in proliferation (Figure 1c), and clonogenic survival (P ≤ 0.01) (Figure 1d). Similar effects on proliferation were observed in the AML cell line OCI-AML3 and in primary AML cells (Supplementary Figure 1C), suggesting a pro-proliferative role for WTAP in AML. WTAP knockdown alone did not induce apoptosis but markedly increased (P ≤ 0.01) the extent of apoptosis following etoposide treatment (Figure 1e). These results provide evidence for an association between the increased expression of WTAP and chemoresistance in AML. Figure 1 Expression of WTAP in AML and effect of WTAP silencing on AML cell behavior. (a) Peripheral blood mononuclear cells from normal donors (NL) and AML patients (AML) were obtained by Ficoll–Paque density centrifugation, and protein extracts were ... To examine the role of WTAP in AML progression in vivo, we performed tumor xenograft experiments in nude mice. As shown in Figure 1f, the growth rates and masses of tumors derived from WTAP-knockdown cells were significantly reduced (P ≤ 0.01) compared with control. To complement this analysis, the in vitro transforming activity of WTAP was examined by investigating its effects on growth of the Ba/F3 cell line. This line depends on interleukin 3 (IL-3) for survival and proliferation, but this dependence can be released by the transgenic expression of suitable oncogenes.9 Whereas control Ba/F3 cells were not viable in the absence of IL-3 at 72 h, WTAP-expressing Ba/F3 cells were able to maintain growth factor-independent proliferation, as demonstrated by significantly higher (P ≤ 0.01) number of viable cells (Figure 1g), suggesting that WTAP harbors oncogenic activity. The aberrant cellular proliferation and terminal differentiation block of myeloid cells are two hallmarks of AML.10 Having shown that WTAP regulates growth and survival, we investigated whether WTAP has a role in myeloid cell differentiation. As shown in Figure 1h, knockdown of WTAP promoted phorbol 12-myristate 13-acetate (PMA)-induced myeloid differentiation, as revealed by an increase in the expression of myeloid differentiation markers CD11b and CD14 compared with control cells. These results suggest that increased expression of WTAP in AML not only supports cell proliferation but also induces the differentiation block. Our RPPA analysis suggested a link between WTAP and mammalian target of rapamycin (mTOR) expression; and given that the mTOR pathway is deregulated in a number of cancers including AML,11 we hypothesized a putative regulatory role of WTAP on mTOR activity in AML. As shown in Figure 1i, WTAP knockdown induced a decrease in the phosphorylation levels of mTOR and its downstream effector p70 ribosomal subunit 6 kinase (pS6K) compared with control shRNA. To further understand the participation of WTAP in leukemogenesis, we performed transcriptomic analysis with RNA-Seq on WTAP knockdown in K562 cells. Gene ontology analysis indicated that cell adhesion and regulation of cell proliferation are the most enriched functionalities (Supplementary Figure 1D and Supplementary Table 2). Among the most relevant genes affected by WTAP with recognized roles in leukemia are CD4, CD44, CEBPA, CSF1R, MPO, ABCG2, TCL1A, CYP1A1, CYP3A4, FGFR1, PTPRC (CD45), CD83, CD86, CD9 and CCR4. Consistent with its described role in RNA processing, we determined that WTAP knockdown affected exon usage of 93 genes, including important factors such as MLL and MSI2. Interestingly, we also observed that WTAP affects the processing of its own transcript, very likely via polyA site selection. Complete analysis can be accessed in GEO ({"type":"entrez-geo","attrs":{"text":"GSE46718","term_id":"46718"}}GSE46718—http://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?token=jbkjjswaqscegjsa Fisher’s exact test) in their mRNA levels as determined by RNA-Seq. Mutations of WTAP were not observed in the TCGA analysis of AML.12 Therefore, the etiology of increased WTAP expression in AML remains unexplained. We next sought to determine the potential mechanism that may contribute to an increase in WTAP expression in AML. The molecular chaperone Hsp90 maintains the stability of many tumor-promoting oncoproteins,13 including WT1.14 Keeping in mind the connection between WT1 and WTAP, we investigated the potential interaction between Hsp90 and WTAP. First, we determined that WTAP co-immunoprecipitates with Hsp90 (Figure 2a), whereas treatment with the Hsp90 inhibitor ganetespib significantly reduced the binding of Hsp90 to WTAP. Therefore, formation of the WTAP–Hsp90 complex is dependent on the chaperoning activity of Hsp90. Studies have shown that Hsp90 client proteins shift the primary chaperone association from Hsp90 to Hsp70 following inhibition of Hsp90 activity.15 Accordingly, our results showed that ganetespib treatment increased WTAP association with Hsp70 (Figure 2a). Furthermore, the GST pull-down assay showed a direct interaction between WTAP and Hsp90 (Figure 2b). To understand the functional significance of the association of Hsp90 with WTAP, we investigated the effects of Hsp90 inhibitors on WTAP protein stability. Ganetespib treatment promoted the degradation of WTAP in K562 (CML), MV4-11 (AML) and Kasumi-1 (AML) cell lines, previously shown to be highly sensitive to ganetespib (Figure 2c).16 Marked reduction in WTAP expression was similarly observed in blasts exposed to ganetespib (Figure 2d). Furthermore, ganetespib inhibited AML tumor growth in nude mice, as we reported previously,14 and WTAP expression in xenograft tumors (Figure 2e). Inhibition of Hsp90 induces polyubiquitination and proteasomal degradation of Hsp90 client proteins.17 We confirmed that pretreatment with proteasomal inhibitor bortezomib largely prevented ganetespib-induced degradation of WTAP (Figure 2f), and higher levels of ubiquitinated WTAP were observed in the presence of the combination of bortezomib and ganetespib compared with either agent alone (Figure 2g). Together, these data show that ganetespib-mediated degradation of WTAP is dependent on the ubiquitin-proteasome pathway, very similar to other bonafide Hsp90 client proteins. Figure 2 Hsp90 associates with WTAP and is necessary for its stability. (a) The K562 cells were treated with vehicle (−) or Hsp90 inhibitor, ganetespib (1 µm for 6 h) followed by immunoprecipitation (IP) with IgG and WTAP antibody. The immunoprecipitates ... In summary, our study shows that WTAP has an important role in abnormal proliferation and arrested differentiation of leukemia cells, and WTAP is a novel client protein of Hsp90. Therefore, WTAP may be a promising novel therapeutic target in AML. Further investigation aimed to elucidate the mechanism of action of WTAP is warranted.

The fate of cellular RNA is largely determined by complex networks of protein-RNA interactions through ribonucleoprotein (RNP) complexes. Despite their relatively short half-life, transcripts associate with many different proteins that process, modify, translate, and degrade the RNA. Following biogenesis some mRNPs are immediately directed to translation and produce proteins, but many are diverted and regulated by processes including miRNA-mediated mechanisms, transport and localization, as well as turnover. Because of this complex interplay estimates of steady-state expression by methods such as RNAseq alone cannot capture critical aspects of cellular fate, environmental response, tumorigenesis, or gene expression regulation. More selective and integrative tools are needed to measure protein-RNA complexes and the regulatory processes involved. One focus area is measurements of the transcriptome associated with ribosomes and translation. These so-called polysome or ribosome profiling techniques can evaluate translation efficiency as well as the interplay between translation initiation, elongation, and termination-subject areas not well understood at a systems biology level. Ribosome profiling is a highly promising technique that provides mRNA positional information of ribosome occupancy, potentially bridging the gap between gene expression (i.e., RNAseq and microarray analysis) and protein quantification (i.e., mass spectrometry). In combination with methods such as RNA immunoprecipitation, miRNA profiling, or proteomics, we obtain a fresh view of global post-transcriptional and translational gene regulation. In addition, these techniques also provide new insight into new regulatory elements, such as alternative open reading frames, and translation regulation under different conditions.

Lung cancer remains one of the leading causes of cancer-related deaths worldwide with a 5-year survival rate of less than 20%. One approach to improving survival is the identification of biomarkers to detect early stage disease. In this study, we investigated the potential of the stem cell and progenitor cell marker, Musashi1 (Msi1), as a diagnostic marker and potential therapeutic target for lung cancer. Functional studies in A549 bronchioalveolar carcinoma and NCI-H520 squamous cell carcinoma cells revealed that Msi1 was enriched in spheroid cultures of tumor cells and in the CD133+ cell population. Downregulation of Msi1 by lentivirus-mediated expression of an Msi1 shRNA reduced spheroid colony proliferation. Growth inhibition was associated with reduced nuclear localization of β-catenin and inhibition of the processing of intracellular Notch. In primary lung cancer, Msi1 protein expression was elevated in 86% of 202 tissue microarray specimens, and Msi1 mRNA was increased in 80% of 118 bronchoscopic biopsies, including metastatic disease, but was rarely detected in adjacent normal lung tissue and in non-malignant diseased tissue. Msi1 was expressed in a diffuse pattern in most tumor subtypes, except in squamous cell carcinomas, where it appeared in a focal pattern in 50% of specimens. Thus, Msi1 is a sensitive and specific diagnostic marker for all lung cancer subtypes.

Key points • Metabolic syndrome, obesity and insulin resistance are risk factors for overactive bladder, but little is known about the impact of insulin resistance on bladder functioning. • We aimed to investigate the insulin effects in murine and human bladder, and whether its defective action contributes to the bladder dysfunction associated with obesity. • Under physiological conditions, insulin relaxes urinary bladder through the release of nitric oxide from the mucosal layer, and this mechanism is impaired in bladders from insulin-resistant obese mice. • The defective insulin action in bladder mucosa from obese mice is due to endoplasmic reticulum stress, which remarkably contributes to the bladder overactivity in obesity conditions. • Our results enable a better understanding of the mechanism of action of insulin in the urinary bladder and how its defective action in mucosa contributes to bladder dysfunction in conditions of obesity-associated insulin resistance. Abstract We aimed to investigate the role of insulin in the bladder and its relevance for the development of overactive bladder (OAB) in insulin-resistant obese mice. Bladders from male individuals who were involved in multiple organ donations were used. C57BL6/J mice were fed with a high-fat diet for 10 weeks to induce insulin-resistant obesity. Concentration–response curves to insulin were performed in human and mouse isolated mucosa-intact and mucosa-denuded bladders. Cystometric study was performed in terminally anaesthetized mice. Western blot was performed in bladders to detect phosphorylated endothelial NO synthase (eNOS) (Ser1177) and the phosphorylated protein kinase AKT (Ser473), as well as the unfolded protein response (UPR) markers TRIB3, CHOP and ATF4. Insulin (1–100 nm) produced concentration-dependent mouse and human bladder relaxations that were markedly reduced by mucosal removal or inhibition of the PI3K/AKT/eNOS pathway. In mouse bladders, insulin produced a 3.0-fold increase in cGMP levels (P < 0.05) that was prevented by PI3K/AKT/eNOS pathway inhibition. Phosphoinositide 3-kinase (PI3K) inhibition abolished insulin-induced phosphorylation of AKT and eNOS in bladder mucosa. Obese mice showed greater voiding frequency and non-voiding contractions, indicating overactive detrusor smooth muscle. Insulin failed to relax the bladder or to increase cGMP in the obese group. Insulin-stimulated AKT and eNOS phosphorylation in mucosa was also impaired in obese mice. The UPR markers TRIB3, CHOP and ATF4 were increased in the mucosa of obese mice. The UPR inhibitor 4-phenyl butyric acid normalized all the functional and molecular parameters in obese mice. Our data show that insulin relaxes human and mouse bladder via activation of the PI3K/AKT/eNOS pathway in the bladder mucosa. Endoplasmic reticulum stress-dependent insulin resistance in bladder contributes to OAB in obese mice.

Background Glioblastoma (GBM) is the most common and aggressive type of brain tumor. Currently, GBM has an extremely poor outcome and there is no effective treatment. In this context, genomic and transcriptomic analyses have become important tools to identify new avenues for therapies. RNA-binding proteins (RBPs) are master regulators of co- and post-transcriptional events; however, their role in GBM remains poorly understood. To further our knowledge of novel regulatory pathways that could contribute to gliomagenesis, we have conducted a systematic study of RBPs in GBM. Results By measuring expression levels of 1542 human RBPs in GBM samples and glioma stem cell samples, we identified 58 consistently upregulated RBPs. Survival analysis revealed that increased expression of 21 RBPs was also associated with a poor prognosis. To assess the functional impact of those RBPs, we modulated their expression in GBM cell lines and performed viability, proliferation, and apoptosis assays. Combined results revealed a prominent oncogenic candidate, SNRPB, which encodes core spliceosome machinery components. To reveal the impact of SNRPB on splicing and gene expression, we performed its knockdown in a GBM cell line followed by RNA sequencing. We found that the affected genes were involved in RNA processing, DNA repair, and chromatin remodeling. Additionally, genes and pathways already associated with gliomagenesis, as well as a set of general cancer genes, also presented with splicing and expression alterations. Conclusions Our study provides new insights into how RBPs, and specifically SNRPB, regulate gene expression and directly impact GBM development. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-0990-4) contains supplementary material, which is available to authorized users.

© 2016 The Author(s). Insulin-like growth factor 2 mRNA binding protein 3 (IGF2BP3) expression correlates with malignancy, but its role(s) in pathogenesis remains enigmatic. We interrogated the IGF2BP3-RNA interaction network in pancreatic ductal adenocarcinoma (PDAC) cells. Using a combination of genome-wide approaches, we have identified 164 direct mRNA targets of IGF2BP3. These transcripts encode proteins enriched for functions such as cell migration, proliferation, and adhesion. Loss of IGF2BP3 reduced PDAC cell invasiveness and remodeled focal adhesion junctions. Individual nucleotide resolution crosslinking immunoprecipitation (iCLIP) revealed significant overlap of IGF2BP3 and microRNA (miRNA) binding sites. IGF2BP3 promotes association of the RNA-induced silencing complex (RISC) with specific transcripts. Our results show that IGF2BP3 influences a malignancy-associated RNA regulon by modulating miRNA-mRNA interactions.

hnRNPs are polyvalent RNA binding proteins that have been implicated in a range of regulatory roles including splicing, mRNA decay, translation, and miRNA metabolism. A variety of genome wide studies have taken advantage of methods like CLIP and RIP to identify the targets and binding sites of RNA binding proteins. However, due to the complex nature of RNA-binding proteins, these studies are incomplete without assays that characterize the impact of RBP binding on mRNA target expression. Here we used a suite of high-throughput approaches (RIP-Seq, iCLIP, RNA-Seq and shotgun proteomics) to provide a comprehensive view of hnRNP H1s ensemble of targets and its role in splicing, mRNA decay, and translation. The combination of RIP-Seq and iCLIP allowed us to identify a set of 1,086 high confidence target transcripts. Binding site motif analysis of these targets suggests the TGGG tetramer as a prevalent component of hnRNP H1 binding motif, with particular enrichment around intronic hnRNP H1 sites. Our analysis of the target transcripts and binding sites indicates that hnRNP H1s involvement in splicing is 2-fold: it directly affects a substantial number of splicing events, but also regulates the expression of major components of the splicing machinery and other RBPs with known roles in splicing regulation. The identified mRNA targets displayed function enrichment in MAPK signaling and ubiquitin mediated proteolysis, which might be main routes by which hnRNP H1 promotes tumorigenesis.

Aberrant gene expression is the cause and the consequence of tumorigenesis. A major component of gene expression is translation regulation; a process whose main players are RNA-binding-proteins (RBPs). More than 800 RBPs have been identified in the human genome and several of them have been shown to control gene networks associated with relevant cancer processes. A more systematic characterization of RBPs starts to reveal that similar to transcription factors, they can function as tumor suppressors or oncogenes. A relevant example is Musashi1 (Msi1), which is emerging as a critical regulator of tumorigenesis in multiple cancer types, including colon cancer. Msi1 is a stem marker in several tissues and is critical in maintaining the balance between self-renewal and differentiation. However, a boost in Msi1 expression can most likely lead cells towards an oncogenic pathway. In this article, we discuss the parallels between Msi1 function in normal renewal of intestinal epithelium and in colon cancer.

Musashi1 (Msi1) is an evolutionarily conserved RNA-binding protein (RBP) that has profound implications in cellular processes such as stem cell maintenance, nervous system development, and tumorigenesis. Msi1 is highly expressed in many cancers, including glioblastoma, whereas in normal tissues, its expression is restricted to stem cells. Unfortunately, the factors that modulate Msi1 expression and trigger high levels in tumors are largely unknown. The Msi1 mRNA has a long 3′ untranslated region (UTR) containing several AU- and U-rich sequences. This type of sequence motif is often targeted by HuR, another important RBP known to be highly expressed in tumor tissue such as glioblastoma and to regulate a variety of cancer-related genes. In this report, we show an interaction between HuR and the Msi1 3′-UTR, resulting in a positive regulation of Msi1 expression. We show that HuR increased MSI1 mRNA stability and promoted its translation. We also present evidence that expression of HuR and Msi1 correlate positively in clinical glioblastoma samples. Finally, we show that inhibition of cell proliferation, increased apoptosis, and changes in cell-cycle profile as a result of silencing HuR are partially rescued when Msi1 is ectopically expressed. In summary, our results suggest that HuR is an important regulator of Msi1 in glioblastoma and that this regulation has important biological consequences during gliomagenesis. Mol Cancer Res; 10(1); 143–55. ©2012 AACR.

Cellular imbalances of cholesterol and fatty acid metabolism result in pathological processes, including atherosclerosis and metabolic syndrome. Recent work from our group and others has shown that the intronic microRNAs hsa-miR-33a and hsa-miR-33b are located within the sterol regulatory element-binding protein-2 and -1 genes, respectively, and regulate cholesterol homeostasis in concert with their host genes. Here, we show that miR-33a and -b also regulate genes involved in fatty acid metabolism and insulin signaling. miR-33a and -b target key enzymes involved in the regulation of fatty acid oxidation, including carnitine O -octaniltransferase, carnitine palmitoyltransferase 1A, hydroxyacyl-CoA-dehydrogenase, Sirtuin 6 (SIRT6), and AMP kinase subunit-α. Moreover, miR-33a and -b also target the insulin receptor substrate 2, an essential component of the insulin-signaling pathway in the liver. Overexpression of miR-33a and -b reduces both fatty acid oxidation and insulin signaling in hepatic cell lines, whereas inhibition of endogenous miR-33a and -b increases these two metabolic pathways. Together, these data establish that miR-33a and -b regulate pathways controlling three of the risk factors of metabolic syndrome, namely levels of HDL, triglycerides, and insulin signaling, and suggest that inhibitors of miR-33a and -b may be useful in the treatment of this growing health concern.