IGF2BP1 induces neuroblastoma via a druggable feedforward loop with MYCN promoting 17q oncogene expression.

BACKGROUND: Neuroblastoma is the most common solid tumor in infants accounting for approximately 15% of all cancer-related deaths. Over 50% of high-risk neuroblastoma relapse, emphasizing the need of novel drug targets and therapeutic strategies. In neuroblastoma, chromosomal gains at chromosome 17q, including IGF2BP1, and MYCN amplification at chromosome 2p are associated with adverse outcome. Recent, pre-clinical evidence indicates the feasibility of direct and indirect targeting of IGF2BP1 and MYCN in cancer treatment. METHODS: Candidate oncogenes on 17q were identified by profiling the transcriptomic/genomic landscape of 100 human neuroblastoma samples and public gene essentiality data. Molecular mechanisms and gene expression profiles underlying the oncogenic and therapeutic target potential of the 17q oncogene IGF2BP1 and its cross-talk with MYCN were characterized and validated in human neuroblastoma cells, xenografts and PDX as well as novel IGF2BP1/MYCN transgene mouse models. RESULTS: We reveal a novel, druggable feedforward loop of IGF2BP1 (17q) and MYCN (2p) in high-risk neuroblastoma. This promotes 2p/17q chromosomal gains and unleashes an oncogene storm resulting in fostered expression of 17q oncogenes like BIRC5 (survivin). Conditional, sympatho-adrenal transgene expression of IGF2BP1 induces neuroblastoma at a 100% incidence. IGF2BP1-driven malignancies are reminiscent to human high-risk neuroblastoma, including 2p/17q-syntenic chromosomal gains and upregulation of Mycn, Birc5, as well as key neuroblastoma circuit factors like Phox2b. Co-expression of IGF2BP1/MYCN reduces disease latency and survival probability by fostering oncogene expression. Combined inhibition of IGF2BP1 by BTYNB, MYCN by BRD inhibitors or BIRC5 by YM-155 is beneficial in vitro and, for BTYNB, also. CONCLUSION: We reveal a novel, druggable neuroblastoma oncogene circuit settling on strong, transcriptional/post-transcriptional synergy of MYCN and IGF2BP1. MYCN/IGF2BP1 feedforward regulation promotes an oncogene storm harboring high therapeutic potential for combined, targeted inhibition of IGF2BP1, MYCN expression and MYCN/IGF2BP1-effectors like BIRC5.

Post-transcriptional regulation of MRTF-A by miRNAs during myogenic differentiation of myoblasts.

The differentiation and regeneration of skeletal muscle from myoblasts to myotubes involves myogenic transcription factors, such as myocardin-related transcription factor A (MRTF-A) and serum response factor (SRF). In addition, post-transcriptional regulation by miRNAs is required during myogenesis. Here, we provide evidence for novel mechanisms regulating MRTF-A during myogenic differentiation. Endogenous MRTF-A protein abundance and activity decreased during C2C12 differentiation, which was attributable to miRNA-directed inhibition. Conversely, overexpression of MRTF-A impaired differentiation and myosin expression. Applying miRNA trapping by RNA affinity purification (miTRAP), we identified miRNAs which directly regulate MRTF-A via its 3'UTR, including miR-1a-3p, miR-206-3p, miR-24-3p and miR-486-5p. These miRNAs were upregulated during differentiation and specifically recruited to the 3'UTR of MRTF-A. Concomitantly, Ago2 recruitment to the MRTF-A 3'UTR was considerably increased, whereas Dicer1 depletion or 3'UTR deletion elevated MRTF-A and inhibited differentiation. MRTF-A protein expression was inhibited by ectopic miRNA expression in murine C2C12 and primary human myoblasts. 3'UTR reporter activity diminished upon differentiation or miRNA expression, whereas deletion of the predicted binding sites reversed these effects. Furthermore, TGF-ß abolished MRTF-A reduction and decreased miR-486-5p expression. Our findings implicate miR-24-3p and miR-486-5p in the repression of MRTF-A and suggest a complex network of transcriptional and post-transcriptional mechanisms regulating myogenesis.

Identification of lymphocyte cell-specific protein-tyrosine kinase (LCK) as a driver for invasion and migration of oral cancer by tumor heterogeneity exploitation.

BACKGROUND: Cancer metastases are the main cause of lethality. The five-year survival rate for patients diagnosed with advanced stage oral cancer is 30%. Hence, the identification of novel therapeutic targets is an urgent need. However, tumors are comprised of a heterogeneous collection of cells with distinct genetic and molecular profiles that can differentially promote metastasis making therapy development a challenging task. Here, we leveraged intratumoral heterogeneity in order to identify drivers of cancer cell motility that might be druggable targets for anti-metastasis therapy. METHODS: We used 2D migration and 3D matrigel-based invasion assays to characterize the invasive heterogeneity among and within four human oral cancer cell lines in vitro. Subsequently, we applied mRNA-sequencing to map the transcriptomes of poorly and strongly invasive subclones as well as primary tumors and matched metastasis. RESULTS: We identified SAS cells as a highly invasive oral cancer cell line. Clonal analysis of SAS yielded a panel of 20 subclones with different invasive capacities. Integrative gene expression analysis identified the Lymphocyte cell-specific protein-tyrosine kinase (LCK) as a druggable target gene associated with cancer cell invasion and metastasis. Inhibition of LCK using A-770041 or dasatinib blocked invasion of highly aggressive SAS cells. Interestingly, reduction of LCK activity increased the formation of adherens junctions and induced cell differentiation. CONCLUSION: Analysis of invasive heterogeneity led to the discovery of LCK as an important regulator of motility in oral cancer cells. Hence, small molecule mediated inhibition of LCK could be a promising anti-metastasis therapy option for oral cancer patients.

IGF2BP1 is the first positive marker for anaplastic thyroid carcinoma diagnosis.

Anaplastic thyroid carcinomas (ATC) are rare, but represent the most lethal malignancy of the thyroid. Selective molecular markers and drivers distinguishing ATC from other thyroid carcinomas of follicular origin remain largely unknown, limiting advances in diagnosis and treatment. In a retrospective study, we analyzed gene expression in 36 ATC, 18 poorly differentiated, 132 papillary, and 55 follicular thyroid carcinoma, as well as 124 paired and unpaired normal thyroid tissues in three independent cohorts by RNA-sequencing and immunohistochemistry. RNA-sequencing data in the test cohort suggested selective ATC protein biomarkers. Evaluation of these revealed that ATCs are characterized by the de novo expression of various testis antigens, including melanoma-associated antigen A3 (MAGEA3), but most importantly the oncofetal IGF2 mRNA binding protein 1 (IGF2BP1). Shallow whole genome sequencing essentially excluded that IGF2BP1 upregulation results from gene copy number alterations. Immunohistochemical analyses in all three tumor cohorts confirmed the selective de novo expression of IGF2BP1 protein in ATC. In sum, 75% (27/36) of all tested ATC and 0.5% (1/204) of poorly and well-differentiated thyroid carcinoma tissue samples were positive for IGF2BP1 protein. This indicates that IGF2BP1 protein expression identifies ATC with a diagnostic odds ratio of 612 (95% CI: 74.6-5021). In addition, we found that MAGEA3 is exclusively, although less consistently upregulated in ATC, presenting with an odds ratio of 411 (95% CI: 23.8-7098.7). Importantly, we provide confirmatory evidence that IGF2BP1 and MAGEA3 expression distinguishes ATC from poorly differentiated thyroid carcinoma. IGF2BP1 furthermore identified ATC foci within low-grade follicular thyroid carcinoma. In conclusion, IGF2BP1 represents the most promising single-gene marker available for ATC, followed by MAGEA3, improving on current techniques. Robust markers are essential to help distinguish this high-grade malignancy from other thyroid carcinomas, to guide surgical decision making, therapy and post-resection/therapy monitoring strategies.

Identification and initial characterization of POLIII-driven transcripts by msRNA-sequencing.

Non-coding RNAs (ncRNAs) are powerful regulators of gene expression but medium-sized (50-300 nts in length) ncRNAs (msRNAs) are barely picked-up precisely by RNA-sequencing. Here we describe msRNA-sequencing (msRNAseq), a modified protocol that associated with a computational analyses pipeline identified about ~1800 msRNA loci, including over 300 putatively novel msRNAs, in human and murine cells. We focused on the identification and initial characterization of three POLIII-derived transcripts. The validation of these uncharacterized msRNAs identified an ncRNA in antisense orientation from the POLR3E locus transcribed by POLIII. This msRNA, termed POLAR (POLR3E Antisense RNA), has a strikingly short half-life, localizes to paraspeckles (PSPs) and associates with PSP-associated proteins indicating that msRNAseq identifies functional msRNAs. Thus, our analyses will pave the way for analysing the roles of msRNAs in cells, development and diseases.

IGF2BP1 is a targetable SRC/MAPK-dependent driver of invasive growth in ovarian cancer.

Epithelial-to-mesenchymal transition (EMT) is a hallmark of aggressive, mesenchymal-like high-grade serous ovarian carcinoma (HGSOC). The SRC kinase is a key driver of cancer-associated EMT promoting adherens junction (AJ) disassembly by phosphorylation-driven internalization and degradation of AJ proteins. Here, we show that the IGF2 mRNA-binding protein 1 (IGF2BP1) is up-regulated in mesenchymal-like HGSOC and promotes SRC activation by a previously unknown protein-ligand-induced, but RNA-independent mechanism. IGF2BP1-driven invasive growth of ovarian cancer cells essentially relies on the SRC-dependent disassembly of AJs. Concomitantly, IGF2BP1 enhances ERK2 expression in an RNA-binding dependent manner. Together this reveals a post-transcriptional mechanism of interconnected stimulation of SRC/ERK signalling in ovarian cancer cells. The IGF2BP1-SRC/ERK2 axis is targetable by the SRC-inhibitor saracatinib and MEK-inhibitor selumetinib. However, due to IGF2BP1-directed stimulation, only combinatorial treatment effectively overcomes the IGF2BP1-promoted invasive growth in 3D culture conditions as well as intraperitoneal mouse models. In conclusion, we reveal an unexpected role of IGF2BP1 in enhancing SRC/MAPK-driven invasive growth of ovarian cancer cells. This provides a rationale for the therapeutic benefit of combinatorial SRC/MEK inhibition in mesenchymal-like HGSOC.

miR-221 and -222 target CACNA1C and KCNJ5 leading to altered cardiac ion channel expression and current density.

MicroRNAs (miRs) contribute to different aspects of cardiovascular pathology, among others cardiac hypertrophy and atrial fibrillation. The aim of our study was to evaluate the impact of miR-221/222 on cardiac electrical remodeling. Cardiac miR expression was analyzed in a mouse model with altered electrocardiography parameters and severe heart hypertrophy. Next generation sequencing revealed 14 differentially expressed miRs in hypertrophic hearts, with miR-221 and -222 being the strongest regulated miR-cluster. This increase was restricted to cardiomyocytes and not observed in cardiac fibroblasts. Additionally, we evaluated the change of miR-221/222 in vivo in two models of pharmacologically induced heart hypertrophy (angiotensin II, isoprenaline), thereby demonstrating a stimulus-induced increase in miR-221/222 in vivo by angiotensin II but not by isoprenaline. Whole transcriptome analysis by RNA-seq and qRT-PCR validation revealed an enriched number of downregulated mRNAs coding for proteins located in the T-tubule, which are also predicted targets for miR-221/222. Among those, mRNAs were the L-type Ca2+ channel subunits as well as potassium channel subunits. We confirmed that both miRs target the 3'-untranslated regions of Cacna1c and Kcnj5. Furthermore, enhanced expression of these miRs reduced L-type Ca2+ channel and Kcnj5 channel abundance and function, which was analyzed by whole-cell patch clamp recordings or Western blot and flux measurements, respectively. miR-221 and -222 contribute to the regulation of L-type Ca2+ channels as well as Kcnj5 channels and, therefore, potentially contribute to disturbed cardiac excitation generation and propagation. Future studies will have to evaluate the pathophysiological and clinical relevance of aberrant miR-221/222 expression for electrical remodeling.

Mud2 functions in transcription by recruiting the Prp19 and TREX complexes to transcribed genes.

The different steps of gene expression are intimately linked to coordinate and regulate this complex process. During transcription, numerous RNA-binding proteins are already loaded onto the nascent mRNA and package the mRNA into a messenger ribonucleoprotein particle (mRNP). These RNA-binding proteins are often also involved in other steps of gene expression than mRNA packaging. For example, TREX functions in transcription, mRNP packaging and nuclear mRNA export. Previously, we showed that the Prp19 splicing complex (Prp19C) is needed for efficient transcription as well as TREX occupancy at transcribed genes. Here, we show that the splicing factor Mud2 interacts with Prp19C and is needed for Prp19C occupancy at transcribed genes in Saccharomyces cerevisiae. Interestingly, Mud2 is not only recruited to intron-containing but also to intronless genes indicating a role in transcription. Indeed, we show for the first time that Mud2 functions in transcription. Furthermore, these functions of Mud2 are likely evolutionarily conserved as Mud2 is also recruited to an intronless gene and interacts with Prp19C in Drosophila melanogaster. Taken together, we classify Mud2 as a novel transcription factor that is necessary for the recruitment of mRNA-binding proteins to the transcription machinery. Thus, Mud2 is a multifunctional protein important for transcription, splicing and most likely also mRNP packaging.

IGF2BP1 enhances an aggressive tumor cell phenotype by impairing miRNA-directed downregulation of oncogenic factors.

The oncofetal IGF2 mRNA binding proteins (IGF2BPs) are upregulated in most cancers but their paralogue-specific roles in tumor cells remain poorly understood. In a panel of five cancer-derived cell lines, IGF2BP1 shows highly conserved oncogenic potential. Consistently, the deletion of IGF2BP1 impairs the growth and metastasis of ovarian cancer-derived cells in nude mice. Gene expression analyses in ovarian cancer-derived cells reveal that the knockdown of IGF2BPs is associated with the downregulation of mRNAs that are prone to miRNA regulation. All three IGF2BPs preferentially associate upstream of miRNA binding sites (MBSs) in the 3'UTR of mRNAs. The downregulation of mRNAs co-regulated by miRNAs and IGF2BP1 is abrogated at low miRNA abundance or when miRNAs are depleted. IGF2BP1 associates with these target mRNAs in RISC-free complexes and its deletion enhances their association with AGO2. The knockdown of most miRNA-regulated target mRNAs of IGF2BP1 impairs tumor cell properties. In four primary cancers, elevated synthesis of these target mRNAs is largely associated with upregulated IGF2BP1 mRNA levels. In ovarian cancer, the enhanced expression of IGF2BP1 and most of its miRNA-controlled target mRNAs is associated with poor prognosis. In conclusion, these findings indicate that IGF2BP1 enhances an aggressive tumor cell phenotype by antagonizing miRNA-impaired gene expression.

Identification of RNA-Binding Proteins as Targetable Putative Oncogenes in Neuroblastoma.

Neuroblastoma is a common childhood cancer with almost a third of those affected still dying, thus new therapeutic strategies need to be explored. Current experimental therapies focus mostly on inhibiting oncogenic transcription factor signalling. Although LIN28B, DICER and other RNA-binding proteins (RBPs) have reported roles in neuroblastoma development and patient outcome, the role of RBPs in neuroblastoma is relatively unstudied. In order to elucidate novel RBPs involved in MYCN-amplified and other high-risk neuroblastoma subtypes, we performed differential mRNA expression analysis of RBPs in a large primary tumour cohort (n = 498). Additionally, we found via Kaplan-Meier scanning analysis that 685 of the 1483 tested RBPs have prognostic value in neuroblastoma. For the top putative oncogenic candidates, we analysed their expression in neuroblastoma cell lines, as well as summarised their characteristics and existence of chemical inhibitors. Moreover, to help explain their association with neuroblastoma subtypes, we reviewed candidate RBPs' potential as biomarkers, and their mechanistic roles in neuronal and cancer contexts. We found several highly significant RBPs including RPL22L1, RNASEH2A, PTRH2, MRPL11 and AFF2, which remain uncharacterised in neuroblastoma. Although not all RBPs appear suitable for drug design, or carry prognostic significance, we show that several RBPs have strong rationale for inhibition and mechanistic studies, representing an alternative, but nonetheless promising therapeutic strategy in neuroblastoma treatment.

Cathepsins Drive Anti-Inflammatory Activity by Regulating Autophagy and Mitochondrial Dynamics in Macrophage Foam Cells.

BACKGROUND/AIMS: Atherosclerosis underlies the majority of cardiovascular events, consequent to non-resolving inflammation. Considerable evidence implicates autophagy dysfunction at the core of this inflammatory condition, but the basis of this dysfunction is not fully understood. METHODS: Using an in vitro model of lipid-laden macrophages, activity-based probes and high-throughput techniques, we studied the role of the cysteine proteases cathepsins in autophagy. RESULTS: We showed that cathepsin activity is suppressed by oxidized lipids and that cathepsin has an indispensable role in the autophagy-lysosomal degradation pathway. Accordingly, loss of cathepsin function resulted in autophagy derangement. Shotgun proteomics confirmed autophagy dysfunction and unveiled a pivotal role of cathepsin L in a putative cathepsin degradation network. At the physiological level, cathepsin inhibition resulted in mitochondrial stress, which translated into impaired oxidative metabolism, excessive production of reactive oxygen species and activation of the cellular stress response, driven by ATF4-CHOP transcription factors. In addition, transcriptomic analysis of these cells uncovered some genetic similarities with the inflammatory macrophage phenotype (a.k.a M1 macrophages) and increased expression of inflammatory cytokines. CONCLUSION: Our data highlight the importance of cathepsins for mitochondrial quality control mechanisms and amelioration of vascular inflammation.

The oncogenic triangle of HMGA2, LIN28B and IGF2BP1 antagonizes tumor-suppressive actions of the let-7 family.

The tumor-suppressive let-7 microRNA family targets various oncogene-encoding mRNAs. We identify the let-7 targets HMGA2, LIN28B and IGF2BP1 to form a let-7 antagonizing self-promoting oncogenic triangle. Surprisingly, 3'-end processing of IGF2BP1 mRNAs is unaltered in aggressive cancers and tumor-derived cells although IGF2BP1 synthesis was proposed to escape let-7 attack by APA-dependent (alternative polyadenylation) 3' UTR shortening. However, the expression of the triangle factors is inversely correlated with let-7 levels and promoted by LIN28B impairing let-7 biogenesis. Moreover, IGF2BP1 enhances the expression of all triangle factors by recruiting the respective mRNAs in mRNPs lacking AGO proteins and let-7 miRNAs. This indicates that the downregulation of let-7, largely facilitated by LIN28B upregulation, and the protection of let-7 target mRNAs by IGF2BP1-directed shielding in mRNPs synergize in enhancing the expression of triangle factors. The oncogenic potential of this triangle was confirmed in ovarian cancer (OC)-derived ES-2 cells transduced with let-7 targeting decoys. In these, the depletion of HMGA2 only diminishes tumor cell growth under permissive conditions. The depletion of LIN28B and more prominently IGF2BP1 severely impairs tumor cell viability, self-renewal and 2D as well as 3D migration. In conclusion, this suggests the targeting of the HMGA2-LIN28B-IGF2BP1 triangle as a promising strategy in cancer treatment.

Rapid identification of regulatory microRNAs by miTRAP (miRNA trapping by RNA in vitro affinity purification).

MicroRNAs (miRNAs) control gene expression at the post-transcriptional level. However, the identification of miRNAs regulating the fate of a specific messenger RNA remains limited due to the imperfect complementarity of miRNAs and targeted transcripts. Here, we describe miTRAP (miRNA trapping by RNA in vitro affinity purification), an advanced protocol of previously reported MS2-tethering approaches. MiTRAP allows the rapid identification of miRNAs targeting an in vitro transcribed RNA in cell lysates. Selective co-purification of regulatory miRNAs was confirmed for the MYC- as well as ZEB2-3'UTR, two well-established miRNA targets in vivo. Combined with miRNA-sequencing, miTRAP identified in addition to miRNAs reported to control MYC expression, 18 novel candidates including not in silico predictable miRNAs. The evaluation of 10 novel candidate miRNAs confirmed 3'UTR-dependent regulation of MYC expression as well as putative non-canonical targeting sites for the not in silico predictable candidates. In conclusion, miTRAP provides a rapid, cost-effective and easy-to-handle protocol allowing the identification of regulatory miRNAs for RNAs of choice in a cellular context of interest. Most notably, miTRAP not only identifies in silico predictable but also unpredictable miRNAs regulating the expression of a specific target RNA.

APOBEC3C-mediated NF-κB activation enhances clear cell renal cell carcinoma progression.

Renowned as the predominant form of kidney cancer, clear cell renal cell carcinoma (ccRCC) exhibits susceptibility to immunotherapies due to its specific expression profile as well as notable immune cell infiltration. Despite this, effectively treating metastatic ccRCC remains a significant challenge, necessitating a more profound comprehension of the underlying molecular mechanisms governing its progression. Here, we unveil that the enhanced expression of the RNA-binding protein DNA dC → dU-editing enzyme APOBEC-3C (APOBEC3C; also known as A3C) in ccRCC tissue and ccRCC-derived cell lines serves as a catalyst for tumor growth by amplifying nuclear factor-kappa B (NF-κB) activity. By employing RNA-sequencing and cell-based assays in ccRCC-derived cell lines, we determined that A3C is a stress-responsive factor and crucial for cell survival. Furthermore, we identified that A3C binds and potentially stabilizes messenger RNAs (mRNAs) encoding positive regulators of the NF-κB pathway. Upon A3C depletion, essential subunits of the NF-κB family are abnormally restrained in the cytoplasm, leading to deregulation of NF-κB target genes. Our study illuminates the pivotal role of A3C in promoting ccRCC tumor development, positioning it as a prospective target for future therapeutic strategies.

IGF2BP1 Promotes Proliferation of Neuroendocrine Neoplasms by Post-Transcriptional Enhancement of EZH2.

Neuroendocrine neoplasms (NENs) represent a heterogenous class of highly vascularized neoplasms that are increasing in prevalence and are predominantly diagnosed at a metastatic state. The molecular mechanisms leading to tumor initiation, metastasis, and chemoresistance are still under investigation. Hence, identification of novel therapeutic targets is of great interest. Here, we demonstrate that the RNA-binding Protein IGF2BP1 is a post-transcriptional regulator of components of the Polycomb repressive complex 2 (PRC2), an epigenic modifier affecting transcriptional regulation and proliferation: Comprehensive in silico analyses along with in vitro experiments showed that IGF2BP1 promotes neuroendocrine tumor cell proliferation by stabilizing the mRNA of Enhancer of Zeste 2 (EZH2), the catalytic subunit of PRC2, which represses gene expression by tri-methylation of histone H3 at lysine 27 (H3K27me3). The IGF2BP1-driven stabilization and protection of EZH2 mRNA is m6A-dependent and enhances EZH2 protein levels which stimulates cell cycle progression by silencing cell cycle arrest genes through enhanced H3K27 tri-methylation. Therapeutic inhibition of IGF2BP1 destabilizes EZH2 mRNA and results in a reduced cell proliferation, paralleled by an increase in G1 and sub-G1 phases. Combined targeting of IGF2BP1, EZH2, and Myc, a transcriptional activator of EZH2 and well-known target of IGF2BP1 cooperatively induces tumor cell apoptosis. Our data identify IGF2BP1 as an important driver of tumor progression in NEN, and indicate that disruption of the IGF2BP1-Myc-EZH2 axis represents a promising approach for targeted therapy of neuroendocrine neoplasms.

MiRNA Deregulation Distinguishes Anaplastic Thyroid Carcinoma (ATC) and Supports Upregulation of Oncogene Expression.

Anaplastic thyroid carcinoma (ATC) is the most fatal and rapidly evolving endocrine malignancy invading the head and neck region and accounts for up to 50% of thyroid cancer-associated deaths. Deregulation of the microRNA (miRNA) expression promotes thyroid carcinoma progression by modulating the reorganization of the ATC transcriptome. Here, we applied comparative miRNA-mRNA sequencing on a cohort of 28 thyroid carcinomas to unravel the association of deregulated miRNA and mRNA expression. This identified 85 miRNAs significantly deregulated in ATC. By establishing a new analysis pipeline, we unraveled 85 prime miRNA-mRNA interactions supporting the downregulation of candidate tumor suppressors and the upregulation of bona fide oncogenes such as survivin (BIRC5) in ATC. This miRNA-dependent reprogramming of the ATC transcriptome provided an mRNA signature comprising 65 genes sharply distinguishing ATC from other thyroid carcinomas. The validation of the deregulated protein expression in an independent thyroid carcinoma cohort demonstrates that miRNA-dependent oncogenes comprised in this signature, the transferrin receptor TFRC (CD71) and the E3-ubiquitin ligase DTL, are sharply upregulated in ATC. This upregulation is sufficient to distinguish ATC even from poorly differentiated thyroid carcinomas (PDTC). In sum, these findings provide new diagnostic tools and a robust resource to explore the key miRNA-mRNA regulation underlying the progression of thyroid carcinoma.

IGF2BP1, a Conserved Regulator of RNA Turnover in Cancer.

The oncofetal IGF2 mRNA-binding protein 1 (IGF2BP1) promotes tumor progression in a variety of solid tumors and its expression is associated with adverse prognosis. The main role proposed for IGF2BP1 in cancer cells is the stabilization of mRNAs encoding pro-oncogenic factors. Several IGF2BP1-RNA association studies, however, revealed a plethora of putative IGF2BP1-RNA targets. Thus, at present the main conserved target RNAs and pathways controlled by IGF2BP1 in cancer remain elusive. In this study, we present a set of genes and cancer hallmark pathways showing a conserved pattern of deregulation in dependence of IGF2BP1 expression in cancer cell lines. By the integrative analysis of these findings with publicly available cancer transcriptome and IGF2BP1-RNA association data, we compiled a set of prime candidate target mRNAs. These analyses confirm a pivotal role of IGF2BP1 in controlling cancer cell cycle progression and reveal novel cancer hallmark pathways influenced by IGF2BP1. For three novel target mRNAs identified by these studies, namely AURKA, HDLBP and YWHAZ, we confirm IGF2BP1 mRNA stabilization. In sum our findings confirm and expand previous findings on the pivotal role of IGF2BP1 in promoting oncogenic gene expression by stabilizing target mRNAs in a mainly 3'UTR, m6A-, miRNA-, and potentially AU-rich element dependent manner.

The MicroRNA Landscape of MYCN-Amplified Neuroblastoma.

MYCN gene amplification and upregulated expression are major hallmarks in the progression of high-risk neuroblastoma. MYCN expression and function in modulating gene synthesis in neuroblastoma is controlled at virtually every level, including poorly understood regulation at the post-transcriptional level. MYCN modulates the expression of various microRNAs including the miR-17-92 cluster. MYCN mRNA expression itself is subjected to the control by miRNAs, most prominently the miR-17-92 cluster that balances MYCN expression by feed-back regulation. This homeostasis seems disturbed in neuroblastoma where MYCN upregulation coincides with severely increased expression of the miR-17-92 cluster. In the presented study, we applied high-throughput next generation sequencing to unravel the miRNome in a cohort of 97 neuroblastomas, representing all clinical stages. Aiming to reveal the MYCN-dependent miRNome, we evaluate miRNA expression in MYCN-amplified as well as none amplified tumor samples. In correlation with survival data analysis of differentially expressed miRNAs, we present various putative oncogenic as well as tumor suppressive miRNAs in neuroblastoma. Using microRNA trapping by RNA affinity purification, we provide a comprehensive view of MYCN-regulatory miRNAs in neuroblastoma-derived cells, confirming a pivotal role of the miR-17-92 cluster and moderate association by the let-7 miRNA family. Attempting to decipher how MYCN expression escapes elevated expression of inhibitory miRNAs, we present evidence that RNA-binding proteins like the IGF2 mRNA binding protein 1 reduce miRNA-directed downregulation of MYCN in neuroblastoma. Our findings emphasize the potency of post-transcriptional regulation of MYCN in neuroblastoma and unravel new avenues to pursue inhibition of this potent oncogene.