Spliceosomal vulnerability of MYCN-amplified neuroblastoma is contingent on PRMT5-mediated regulation of epitranscriptomic and metabolomic pathways.

Approximately 50 % of poor prognosis neuroblastomas arise due to MYCN over-expression. We previously demonstrated that MYCN and PRMT5 proteins interact and PRMT5 knockdown led to apoptosis of MYCN-amplified (MNA) neuroblastoma. Here we evaluate the highly selective first-in-class PRMT5 inhibitor GSK3203591 and its in vivo analogue GSK3326593 as targeted therapeutics for MNA neuroblastoma. Cell-line analyses show MYCN-dependent growth inhibition and apoptosis, with approximately 200-fold greater sensitivity of MNA neuroblastoma lines. RNA sequencing of three MNA neuroblastoma lines treated with GSK3203591 reveal deregulated MYCN transcriptional programmes and altered mRNA splicing, converging on key regulatory pathways such as DNA damage response, epitranscriptomics and cellular metabolism. Stable isotope labelling experiments in the same cell lines demonstrate that glutamine metabolism is impeded following GSK3203591 treatment, linking with disruption of the MLX/Mondo nutrient sensors via intron retention of MLX mRNA. Interestingly, glutaminase (GLS) protein decreases after GSK3203591 treatment despite unchanged transcript levels. We demonstrate that the RNA methyltransferase METTL3 and cognate reader YTHDF3 proteins are lowered following their mRNAs undergoing GSK3203591-induced splicing alterations, indicating epitranscriptomic regulation of GLS; accordingly, we observe decreases of GLS mRNA m6A methylation following GSK3203591 treatment, and decreased GLS protein following YTHDF3 knockdown. In vivo efficacy of GSK3326593 is confirmed by increased survival of Th-MYCN mice, with drug treatment triggering splicing events and protein decreases consistent with in vitro data. Together our study demonstrates the PRMT5-dependent spliceosomal vulnerability of MNA neuroblastoma and identifies the epitranscriptome and glutamine metabolism as critical determinants of this sensitivity.

Anticancer Effects of the Novel Pyrazolyl-Urea GeGe-3.

In a screen of over 200 novel pyrazole compounds, ethyl 1-(2-hydroxypentyl)-5-(3-(3-(trifluoromethyl) phenyl)ureido)-1H-pyrazole-4-carboxylate (named GeGe-3) has emerged as a potential anticancer compound. GeGe-3 displays potent anti-angiogenic properties through the presumptive targeting of the protein kinase DMPK1 and the Ca2+-binding protein calreticulin. We further explored the anticancer potential of GeGe-3 on a range of established cancer cell lines, including PC3 (prostate adenocarcinoma), SKMEL-28 (cutaneous melanoma), SKOV-3 (ovarian adenocarcinoma), Hep-G2 (hepatocellular carcinoma), MDA-MB231, SKBR3, MCF7 (breast adenocarcinoma), A549 (lung carcinoma), and HeLa (cervix epithelioid carcinoma). At concentrations in the range of 10 µM, GeGe-3 significantly restricted cell proliferation and metabolism. GeGe-3 also reduced PC3 cell migration in a standard wound closure and trans-well assay. Together, these results confirm the anticancer potential of GeGe-3 and underline the need for more detailed pre-clinical investigations into its molecular targets and mechanisms of action.

Fluorescence-Activated Cell Sorting and Quantitative Real-Time PCR to Reveal VEGF-Expressing Macrophage Populations in the Zebrafish Larvae.

The transparent, genetically tractable zebrafish is increasingly recognized as a useful model to both live image and uncover mechanistic insight into cell interactions governing tissue homeostasis, pathology, and regeneration. Here, we describe a protocol for the isolation of macrophages from zebrafish wounds using fluorescence-activated cell sorting (FACS), and the identification of specific pro-angiogenic macrophage populations that express high levels of vascular endothelial growth factor (vegf) using quantitative real-time PCR (qPCR). The cell dissociation and FACS sorting techniques have been optimized for immune cells and successfully used to isolate other fluorescently marked populations within the wound such as neutrophils and endothelial cells. More broadly, this protocol can be easily adapted to other contexts where identification of pro-angiogenic immune cells is transformative for understanding, from development to pathologies such as infection, cancer, and diabetes.

Interacting Proteins, Polymorphisms and the Susceptibility of Animals to SARS-CoV-2.

COVID-19, caused by SARS-CoV-2, is a world-wide problem for the human population. It is known that some animal species, such as mink, can become infected and transmit the virus. However, the susceptibility of most animals is not known. Here, we review the use of sequence analysis of the proteins which are known to interact with SARS-CoV-2 as a way to estimate an animal's susceptibility. Although most such work concentrates on the angiotensin-converting enzyme 2 receptor (ACE2), here TMPRSS2 (Transmembrane Serine Protease 2), neuropilin-1 and furin are also considered. Polymorphisms, especially ones which are known to alter viral/host interactions are also discussed. Analysis of ACE2 and TMPRSS2 protein sequences across species suggests this approach may be of some utility in predicting susceptibility; however, this analysis fails to highlight some susceptible animals such as mink. However, combined with observational data which emerges over time about which animals actually become infected, this may, in the future, be a useful tool to assist the management of risks associated with human/animal contact and support conservation and animal welfare measures.

5-Aminosalicylic acid inhibits stem cell function in human adenoma-derived cells: implications for chemoprophylaxis in colorectal tumorigenesis.

BACKGROUND: Most colorectal cancers (CRC) arise sporadically from precursor lesions: colonic polyps. Polyp resection prevents progression to CRC. Risk of future polyps is proportional to the number and size of polyps detected at screening, allowing identification of high-risk individuals who may benefit from effective chemoprophylaxis. We aimed to investigate the potential of 5-aminosalicylic acid (5-ASA), a medication used in the treatment of ulcerative colitis, as a possible preventative agent for sporadic CRC. METHODS: Human colorectal adenoma (PC/AA/C1, S/AN/C1 and S/RG/C2), transformed adenoma PC/AA/C1/SB10 and carcinoma cell lines (LS174T and SW620) were treated with 5-ASA. The effect on growth in two- and three-dimensional (3D) culture, ß-catenin transcriptional activity and on cancer stemness properties of the cells were investigated. RESULTS: 5-ASA was shown, in vitro, to inhibit the growth of adenoma cells and suppress ß-catenin transcriptional activity. Downregulation of ß-catenin was found to repress expression of stem cell marker LGR5 (leucine-rich G protein-coupled receptor-5) and functionally suppress stemness in human adenoma and carcinoma cells using 3D models of tumorigenesis. CONCLUSIONS: 5-ASA can suppress the cancer stem phenotype in adenoma-derived cells. Affordable and well-tolerated, 5-ASA is an outstanding candidate as a chemoprophylactic medication to reduce the risk of colorectal polyps and CRC in those at high risk.

Transcriptomic Analyses of MYCN-Regulated Genes in Anaplastic Wilms' Tumour Cell Lines Reveals Oncogenic Pathways and Potential Therapeutic Vulnerabilities.

The MYCN proto-oncogene is deregulated in many cancers, most notably in neuroblastoma, where MYCN gene amplification identifies a clinical subset with very poor prognosis. Gene expression and DNA analyses have also demonstrated overexpression of MYCN mRNA, as well as focal amplifications, copy number gains and presumptive change of function mutations of MYCN in Wilms' tumours with poorer outcomes, including tumours with diffuse anaplasia. Surprisingly, however, the expression and functions of the MYCN protein in Wilms' tumours still remain obscure. In this study, we assessed MYCN protein expression in primary Wilms' tumours using immunohistochemistry of tissue microarrays. We found MYCN protein to be expressed in tumour blastemal cells, and absent in stromal and epithelial components. For functional studies, we used two anaplastic Wilms' tumour cell-lines, WiT49 and 17.94, to study the biological and transcriptomic effects of MYCN depletion. We found that MYCN knockdown consistently led to growth suppression but not cell death. RNA sequencing identified 561 MYCN-regulated genes shared by WiT49 and 17.94 cell-lines. As expected, numerous cellular processes were downstream of MYCN. MYCN positively regulated the miRNA regulator and known Wilms' tumour oncogene LIN28B, the genes encoding methylosome proteins PRMT1, PRMT5 and WDR77, and the mitochondrial translocase genes TOMM20 and TIMM50. MYCN repressed genes including the developmental signalling receptor ROBO1 and the stromal marker COL1A1. Importantly, we found that MYCN also repressed the presumptive Wilms' tumour suppressor gene REST, with MYCN knockdown resulting in increased REST protein and concomitant repression of RE1-Silencing Transcription factor (REST) target genes. Together, our study identifies regulatory axes that interact with MYCN, providing novel pathways for potential targeted therapeutics for poor-prognosis Wilms' tumour.

A Combined Proteomics and Mendelian Randomization Approach to Investigate the Effects of Aspirin-Targeted Proteins on Colorectal Cancer.

BACKGROUND: Evidence for aspirin's chemopreventative properties on colorectal cancer (CRC) is substantial, but its mechanism of action is not well-understood. We combined a proteomic approach with Mendelian randomization (MR) to identify possible new aspirin targets that decrease CRC risk. METHODS: Human colorectal adenoma cells (RG/C2) were treated with aspirin (24 hours) and a stable isotope labeling with amino acids in cell culture (SILAC) based proteomics approach identified altered protein expression. Protein quantitative trait loci (pQTLs) from INTERVAL (N = 3,301) and expression QTLs (eQTLs) from the eQTLGen Consortium (N = 31,684) were used as genetic proxies for protein and mRNA expression levels. Two-sample MR of mRNA/protein expression on CRC risk was performed using eQTL/pQTL data combined with CRC genetic summary data from the Colon Cancer Family Registry (CCFR), Colorectal Transdisciplinary (CORECT), Genetics and Epidemiology of Colorectal Cancer (GECCO) consortia and UK Biobank (55,168 cases and 65,160 controls). RESULTS: Altered expression was detected for 125/5886 proteins. Of these, aspirin decreased MCM6, RRM2, and ARFIP2 expression, and MR analysis showed that a standard deviation increase in mRNA/protein expression was associated with increased CRC risk (OR: 1.08, 95% CI, 1.03-1.13; OR: 3.33, 95% CI, 2.46-4.50; and OR: 1.15, 95% CI, 1.02-1.29, respectively). CONCLUSIONS: MCM6 and RRM2 are involved in DNA repair whereby reduced expression may lead to increased DNA aberrations and ultimately cancer cell death, whereas ARFIP2 is involved in actin cytoskeletal regulation, indicating a possible role in aspirin's reduction of metastasis. IMPACT: Our approach has shown how laboratory experiments and population-based approaches can combine to identify aspirin-targeted proteins possibly affecting CRC risk.

BCL­3 promotes cyclooxygenase­2/prostaglandin E2 signalling in colorectal cancer.

First discovered as an oncogene in leukaemia, recent reports highlight an emerging role for the proto­oncogene BCL­3 in solid tumours. Importantly, BCL­3 expression is upregulated in >30% of colorectal cancer cases and is reported to be associated with a poor prognosis. However, the mechanism by which BCL­3 regulates tumorigenesis in the large intestine is yet to be fully elucidated. In the present study, it was shown for the first time that knocking down BCL­3 expression suppressed cyclooxygenase­2 (COX­2)/prostaglandin E2 (PGE2) signalling in colorectal cancer cells, a pathway known to drive several of the hallmarks of cancer. RNAi­mediated suppression of BCL­3 expression decreased COX­2 expression in colorectal cancer cells both at the mRNA and protein level. This reduction in COX­2 expression resulted in a significant and functional reduction (30­50%) in the quantity of pro­tumorigenic PGE2 produced by the cancer cells, as shown by enzyme linked immunoassays and medium exchange experiments. In addition, inhibition of BCL­3 expression also significantly suppressed cytokine­induced (TNF­α or IL­1ß) COX­2 expression. Taken together, the results of the present study identified a novel role for BCL­3 in colorectal cancer and suggested that expression of BCL­3 may be a key determinant in the COX­2­meditated response to inflammatory cytokines in colorectal tumour cells. These results suggest that targeting BCL­3 to suppress PGE2 synthesis may represent an alternative or complementary approach to using non­steroidal anti­inflammatory drugs [(NSAIDs), which inhibit cyclooxygenase activity and suppress the conversion of arachidonic acid to prostaglandin], for prevention and/or recurrence in PGE2­driven tumorigenesis.

A Wnt-BMP4 Signaling Axis Induces MSX and NOTCH Proteins and Promotes Growth Suppression and Differentiation in Neuroblastoma.

The Wnt and bone morphogenetic protein (BMP) signaling pathways are known to be crucial in the development of neural crest lineages, including the sympathetic nervous system. Surprisingly, their role in paediatric neuroblastoma, the prototypic tumor arising from this lineage, remains relatively uncharacterised. We previously demonstrated that Wnt/b-catenin signaling can have cell-type-specific effects on neuroblastoma phenotypes, including growth inhibition and differentiation, and that BMP4 mRNA and protein were induced by Wnt3a/Rspo2. In this study, we characterised the phenotypic effects of BMP4 on neuroblastoma cells, demonstrating convergent induction of MSX homeobox transcription factors by Wnt and BMP4 signaling and BMP4-induced growth suppression and differentiation. An immunohistochemical analysis of BMP4 expression in primary neuroblastomas confirms a striking absence of BMP4 in poorly differentiated tumors, in contrast to a high expression in ganglion cells. These results are consistent with a tumor suppressive role for BMP4 in neuroblastoma. RNA sequencing following BMP4 treatment revealed induction of Notch signaling, verified by increases of Notch3 and Hes1 proteins. Together, our data demonstrate, for the first time, Wnt-BMP-Notch signaling crosstalk associated with growth suppression of neuroblastoma.

Wnt Signaling Is a Major Determinant of Neuroblastoma Cell Lineages.

The neural crest (NC), which has been referred to as the fourth germ layer, comprises a multipotent cell population which will specify diverse cells and tissues, including craniofacial cartilage and bones, melanocytes, the adrenal medulla and the peripheral nervous system. These cell fates are known to be determined by gene regulatory networks (GRNs) acting at various stages of NC development, such as induction, specification, and migration. Although transcription factor hierarchies and some of their interplay with morphogenetic signaling pathways have been characterized, the full complexity of activities required for regulated development remains uncharted. Deregulation of these pathways may contribute to tumorigenesis, as in the case of neuroblastoma, a frequently lethal embryonic cancer thought to arise from the sympathoadrenal lineage of the NC. In this "Hypothesis and Theory" article, we utilize the next generation sequencing data from neuroblastoma cells and tumors to evaluate the possible influences of Wnt signaling on NC GRNs and on neuroblastoma cell lineages. We propose that Wnt signaling is a major determinant of regulatory networks that underlie mesenchymal/neural crest cell (NCC)-like cell identities through PRRX1 and YAP/TAZ transcription factors. Furthermore, Wnt may also co-operate with Hedgehog signaling in driving proneural differentiation programmes along the adrenergic (ADRN) lineage. Elucidation of Signaling Regulatory Networks can augment and complement GRNs in characterizing cell identities, which may in turn contribute to the design of improved therapeutics tailored to primary and relapsing neuroblastoma.

BCL-3 promotes a cancer stem cell phenotype by enhancing ß-catenin signalling in colorectal tumour cells.

To decrease bowel cancer incidence and improve survival, we need to understand the mechanisms that drive tumorigenesis. Recently, B-cell lymphoma 3 (BCL-3; a key regulator of NF-κB signalling) has been recognised as an important oncogenic player in solid tumours. Although reported to be overexpressed in a subset of colorectal cancers (CRCs), the role of BCL-3 expression in colorectal tumorigenesis remains poorly understood. Despite evidence in the literature that BCL-3 may interact with ß-catenin, it is perhaps surprising, given the importance of deregulated Wnt/ß-catenin/T-cell factor (TCF) signalling in colorectal carcinogenesis, that the functional significance of this interaction is not known. Here, we show for the first time that BCL-3 acts as a co-activator of ß-catenin/TCF-mediated transcriptional activity in CRC cell lines and that this interaction is important for Wnt-regulated intestinal stem cell gene expression. We demonstrate that targeting BCL-3 expression (using RNA interference) reduced ß-catenin/TCF-dependent transcription and the expression of intestinal stem cell genes LGR5 and ASCL2 In contrast, the expression of canonical Wnt targets Myc and cyclin D1 remained unchanged. Furthermore, we show that BCL-3 increases the functional stem cell phenotype, as shown by colorectal spheroid and tumoursphere formation in 3D culture conditions. We propose that BCL-3 acts as a driver of the stem cell phenotype in CRC cells, potentially promoting tumour cell plasticity and therapeutic resistance. As recent reports highlight the limitations of directly targeting cancer stem cells (CSCs), we believe that identifying and targeting drivers of stem cell plasticity have significant potential as new therapeutic targets.This article has an associated First Person interview with the first author of the paper.

LEF-1 drives aberrant ß-catenin nuclear localization in myeloid leukemia cells.

Canonical Wnt/ß-catenin signaling is frequently dysregulated in myeloid leukemias and is implicated in leukemogenesis. Nuclear-localized ß-catenin is indicative of active Wnt signaling and is frequently observed in acute myeloid leukemia (AML) patients; however, some patients exhibit little or no nuclear ß-catenin even where cytosolic ß-catenin is abundant. Control of the subcellular localization of ß-catenin therefore represents an additional mechanism regulating Wnt signaling in hematopoietic cells. To investigate the factors mediating the nuclear-localization of ß-catenin, we carried out the first nuclear/cytoplasmic proteomic analysis of the ß-catenin interactome in myeloid leukemia cells and identified putative novel ß-catenin interactors. Comparison of interacting factors between Wnt-responsive cells (high nuclear ß-catenin) versus Wnt-unresponsive cells (low nuclear ß-catenin) suggested the transcriptional partner, LEF-1, could direct the nuclear-localization of ß-catenin. The relative levels of nuclear LEF-1 and ß-catenin were tightly correlated in both cell lines and in primary AML blasts. Furthermore, LEF-1 knockdown perturbed ß-catenin nuclear-localization and transcriptional activation in Wnt-responsive cells. Conversely, LEF-1 overexpression was able to promote both nuclear-localization and ß-catenin-dependent transcriptional responses in previously Wnt-unresponsive cells. This is the first ß-catenin interactome study in hematopoietic cells and reveals LEF-1 as a mediator of nuclear ß- catenin level in human myeloid leukemia.

Cancer cell adaptation to hypoxia involves a HIF-GPRC5A-YAP axis.

Hypoxia is a hallmark of solid tumours and a key physiological feature distinguishing cancer from normal tissue. However, a major challenge remains in identifying tractable molecular targets that hypoxic cancer cells depend on for survival. Here, we used SILAC-based proteomics to identify the orphan G protein-coupled receptor GPRC5A as a novel hypoxia-induced protein that functions to protect cancer cells from apoptosis during oxygen deprivation. Using genetic approaches in vitro and in vivo, we reveal HIFs as direct activators of GPRC5A transcription. Furthermore, we find that GPRC5A is upregulated in the colonic epithelium of patients with mesenteric ischaemia, and in colorectal cancers high GPRC5A correlates with hypoxia gene signatures and poor clinical outcomes. Mechanistically, we show that GPRC5A enables hypoxic cell survival by activating the Hippo pathway effector YAP and its anti-apoptotic target gene BCL2L1 Importantly, we show that the apoptosis induced by GPRC5A depletion in hypoxia can be rescued by constitutively active YAP. Our study identifies a novel HIF-GPRC5A-YAP axis as a critical mediator of the hypoxia-induced adaptive response and a potential target for cancer therapy.

Live imaging of wound angiogenesis reveals macrophage orchestrated vessel sprouting and regression.

Wound angiogenesis is an integral part of tissue repair and is impaired in many pathologies of healing. Here, we investigate the cellular interactions between innate immune cells and endothelial cells at wounds that drive neoangiogenic sprouting in real time and in vivo Our studies in mouse and zebrafish wounds indicate that macrophages are drawn to wound blood vessels soon after injury and are intimately associated throughout the repair process and that macrophage ablation results in impaired neoangiogenesis. Macrophages also positively influence wound angiogenesis by driving resolution of anti-angiogenic wound neutrophils. Experimental manipulation of the wound environment to specifically alter macrophage activation state dramatically influences subsequent blood vessel sprouting, with premature dampening of tumour necrosis factor-α expression leading to impaired neoangiogenesis. Complementary human tissue culture studies indicate that inflammatory macrophages associate with endothelial cells and are sufficient to drive vessel sprouting via vascular endothelial growth factor signalling. Subsequently, macrophages also play a role in blood vessel regression during the resolution phase of wound repair, and their absence, or shifted activation state, impairs appropriate vessel clearance.

Wnt Signalling Drives Context-Dependent Differentiation or Proliferation in Neuroblastoma.

Neuroblastoma is one of the commonest and deadliest solid tumours of childhood, and is thought to result from disrupted differentiation of the developing sympathoadrenergic lineage of the neural crest. Neuroblastoma exhibits intra- and intertumoural heterogeneity, with high risk tumours characterised by poor differentiation, which can be attributable to MYCN-mediated repression of genes involved in neuronal differentiation. MYCN is known to co-operate with oncogenic signalling pathways such as Alk, Akt and MEK/ERK signalling, and, together with c-MYC has been shown to be activated by Wnt signalling in various tissues. However, our previous work demonstrated that Wnt3a/Rspo2 treatment of some neuroblastoma cell lines can, paradoxically, decrease c-MYC and MYCN proteins. This prompted us to define the neuroblastoma-specific Wnt3a/Rspo2-driven transcriptome using RNA sequencing, and characterise the accompanying changes in cell biology. Here we report the identification of ninety Wnt target genes, and show that Wnt signalling is upstream of numerous transcription factors and signalling pathways in neuroblastoma. Using live-cell imaging, we show that Wnt signalling can drive differentiation of SK-N-BE(2)-C and SH-SY5Y cell-lines, but, conversely, proliferation of SK-N-AS cells. We show that cell-lines that differentiate show induction of pro-differentiation BMP4 and EPAS1 proteins, which is not apparent in the SK-N-AS cells. In contrast, SK-N-AS cells show increased CCND1, phosphorylated RB and E2F1 in response to Wnt3a/Rspo2, consistent with their proliferative response, and these proteins are not increased in differentiating lines. By meta-analysis of the expression of our 90 genes in primary tumour gene expression databases, we demonstrate discrete expression patterns of our Wnt genes in patient cohorts with different prognosis. Furthermore our analysis reveals interconnectivity within subsets of our Wnt genes, with one subset comprised of novel putative drivers of neuronal differentiation repressed by MYCN. Assessment of ß-catenin immunohistochemistry shows high levels of ß-catenin in tumours with better differentiation, further supporting a role for canonical Wnt signalling in neuroblastoma differentiation.

BCL-3 expression promotes colorectal tumorigenesis through activation of AKT signalling.

OBJECTIVE: Colorectal cancer remains the fourth most common cause of cancer-related mortality worldwide. Here we investigate the role of nuclear factor-κB (NF-κB) co-factor B-cell CLL/lymphoma 3 (BCL-3) in promoting colorectal tumour cell survival. DESIGN: Immunohistochemistry was carried out on 47 tumour samples and normal tissue from resection margins. The role of BCL-3/NF-κB complexes on cell growth was studied in vivo and in vitro using an siRNA approach and exogenous BCL-3 expression in colorectal adenoma and carcinoma cells. The question whether BCL-3 activated the AKT/protein kinase B (PKB) pathway in colorectal tumour cells was addressed by western blotting and confocal microscopy, and the ability of 5-aminosalicylic acid (5-ASA) to suppress BCL-3 expression was also investigated. RESULTS: We report increased BCL-3 expression in human colorectal cancers and demonstrate that BCL-3 expression promotes tumour cell survival in vitro and tumour growth in mouse xenografts in vivo, dependent on interaction with NF-κB p50 or p52 homodimers. We show that BCL-3 promotes cell survival under conditions relevant to the tumour microenvironment, protecting both colorectal adenoma and carcinoma cells from apoptosis via activation of the AKT survival pathway: AKT activation is mediated via both PI3K and mammalian target of rapamycin (mTOR) pathways, leading to phosphorylation of downstream targets GSK-3ß and FoxO1/3a. Treatment with 5-ASA suppressed BCL-3 expression in colorectal cancer cells. CONCLUSIONS: Our study helps to unravel the mechanism by which BCL-3 is linked to poor prognosis in colorectal cancer; we suggest that targeting BCL-3 activity represents an exciting therapeutic opportunity potentially increasing the sensitivity of tumour cells to conventional therapy.

LGR5 regulates pro-survival MEK/ERK and proliferative Wnt/ß-catenin signalling in neuroblastoma.

LGR5 is a marker of normal and cancer stem cells in various tissues where it functions as a receptor for R-spondins and increases canonical Wnt signalling amplitude. Here we report that LGR5 is also highly expressed in a subset of high grade neuroblastomas. Neuroblastoma is a clinically heterogenous paediatric cancer comprising a high proportion of poor prognosis cases (~40%) which are frequently lethal. Unlike many cancers, Wnt pathway mutations are not apparent in neuroblastoma, although previous microarray analyses have implicated deregulated Wnt signalling in high-risk neuroblastoma. We demonstrate that LGR5 facilitates high Wnt signalling in neuroblastoma cell lines treated with Wnt3a and R-spondins, with SK-N-BE(2)-C, SK-N-NAS and SH-SY5Y cell-lines all displaying strong Wnt induction. These lines represent MYCN-amplified, NRAS and ALK mutant neuroblastoma subtypes respectively. Wnt3a/R-Spondin treatment also promoted nuclear translocation of ß-catenin, increased proliferation and activation of Wnt target genes. Strikingly, short-interfering RNA mediated knockdown of LGR5 induces dramatic Wnt-independent apoptosis in all three cell-lines, accompanied by greatly diminished phosphorylation of mitogen/extracellular signal-regulated kinases (MEK1/2) and extracellular signal-regulated kinases (ERK1/2), and an increase of BimEL, an apoptosis facilitator downstream of ERK. Akt signalling is also decreased by a Rictor dependent, PDK1-independent mechanism. LGR5 expression is cell cycle regulated and LGR5 depletion triggers G1 cell-cycle arrest, increased p27 and decreased phosphorylated retinoblastoma protein. Our study therefore characterises new cancer-associated pathways regulated by LGR5, and suggest that targeting of LGR5 may be of therapeutic benefit for neuroblastomas with diverse etiologies, as well as other cancers expressing high LGR5.

Protein arginine methyltransferase 5 is a key regulator of the MYCN oncoprotein in neuroblastoma cells.

Approximately half of poor prognosis neuroblastomas (NBs) are characterized by pathognomonic MYCN gene amplification and MYCN over-expression. Here we present data showing that short-interfering RNA mediated depletion of the protein arginine methyltransferase 5 (PRMT5) in cell-lines representative of NBs with MYCN gene amplification leads to greatly impaired growth and apoptosis. Growth suppression is not apparent in the MYCN-negative SH-SY5Y NB cell-line, or in two immortalized human fibroblast cell-lines. Immunoblotting of NB cell-lines shows that high PRMT5 expression is strongly associated with MYCN-amplification (P < 0.004, Mann-Whitney U-test) and immunohistochemical analysis of primary NBs reveals that whilst PRMT5 protein is ubiquitously expressed in the cytoplasm of most cells, MYCN-amplified tumours exhibit pronounced nuclear PRMT5 staining. PRMT5 knockdown in MYCN-overexpressing cells, including the SHEP-21N cell-line with inducible MYCN expression leads to a dramatic decrease in MYCN protein and MYCN-associated cell-death in SHEP-21N cells. Quantitative gene expression analysis and cycloheximide chase experiments suggest that PRMT5 regulates MYCN at a post-transcriptional level. Reciprocal co-immunoprecipitation experiments demonstrated that endogenous PRMT5 and MYCN interact in both SK-N-BE(2)C and NGP cell lines. By using liquid chromatography - tandem mass spectrometry (LC-MS/MS) analysis of immunoprecipitated MYCN protein, we identified several potential sites of arginine dimethylation on the MYCN protein. Together our studies implicate PRMT5 in a novel mode of MYCN post-translational regulation and suggest PRMT5 plays a major role in NB tumorigenesis. Small-molecule inhibitors of PRMT5 may therefore represent a novel therapeutic strategy for neuroblastoma and other cancers driven by the MYCN oncogene.