LMO1 Synergizes with MYCN to Promotes Neuroblastoma Initiation and Metastasis
ABSTRACT: High levels of LMO1 expression synergizes with MYCN to accelerate neuroblastomagenesis, enhance disease penetrance and promote widespread metastasis in zebrafish. Transcriptomic analysis of human neuroblasotma cells with programed expression of LMO1 vs vector control or neuroblastoma cells with differential endogenous LMO1 expression revealed that gene signitures affecting tumor cell-extracellular matrix interaction are significantly associated with high levels of LMO1 expression. Our findings provide compelling evidence for a major pathogenic role of LMO1 in MYCN-driven neuroblastoma. Overall design: Examination of transcriptiome profiles in LMO1 overexpression neuroblastoma cells
Project description:A genome-wide association study identified LMO1, which encodes an LIM-domain-only transcriptional cofactor, as a neuroblastoma susceptibility gene that functions as an oncogene in high-risk neuroblastoma. Here we show that d?h promoter-mediated expression of LMO1 in zebrafish synergizes with MYCN to increase the proliferation of hyperplastic sympathoadrenal precursor cells, leading to a reduced latency and increased penetrance of neuroblastomagenesis. The transgenic expression of LMO1 also promoted hematogenous dissemination and distant metastasis, which was linked to neuroblastoma cell invasion and migration, and elevated expression levels of genes affecting tumor cell-extracellular matrix interaction, including loxl3, itga2b, itga3, and itga5. Our results provide in vivo validation of LMO1 as an important oncogene that promotes neuroblastoma initiation, progression, and widespread metastatic dissemination.
Project description:LMO1 is a high-risk neuroblastoma susceptibility gene, but how LMO1 cooperates with MYCN in neuroblastoma tumorigenesis is unclear. In this issue of Cancer Cell, Zhu et al. develop a novel zebrafish model that elucidates a mechanism by which LMO1 and MYCN synergistically initiate neuroblastoma and contribute to metastatic disease progression.
Project description:A heritable polymorphism within regulatory sequences of the LMO1 gene is associated with its elevated expression and increased susceptibility to develop neuroblastoma, but the oncogenic pathways downstream of the LMO1 transcriptional co-regulatory protein are unknown. Our ChIP-seq and RNA-seq analyses reveal that a key gene directly regulated by LMO1 and MYCN is ASCL1, which encodes a basic helix-loop-helix transcription factor. Regulatory elements controlling ASCL1 expression are bound by LMO1, MYCN and the transcription factors GATA3, HAND2, PHOX2B, TBX2 and ISL1-all members of the adrenergic (ADRN) neuroblastoma core regulatory circuitry (CRC). ASCL1 is required for neuroblastoma cell growth and arrest of differentiation. ASCL1 and LMO1 directly regulate the expression of CRC genes, indicating that ASCL1 is a member and LMO1 is a coregulator of the ADRN neuroblastoma CRC.
Project description:Neuroblastoma is a childhood cancer of the sympathetic nervous system that accounts for approximately 10% of all paediatric oncology deaths. To identify genetic risk factors for neuroblastoma, we performed a genome-wide association study (GWAS) on 2,251 patients and 6,097 control subjects of European ancestry from four case series. Here we report a significant association within LIM domain only 1 (LMO1) at 11p15.4 (rs110419, combined P = 5.2?×?10(-16), odds ratio of risk allele = 1.34 (95% confidence interval 1.25-1.44)). The signal was enriched in the subset of patients with the most aggressive form of the disease. LMO1 encodes a cysteine-rich transcriptional regulator, and its paralogues (LMO2, LMO3 and LMO4) have each been previously implicated in cancer. In parallel, we analysed genome-wide DNA copy number alterations in 701 primary tumours. We found that the LMO1 locus was aberrant in 12.4% through a duplication event, and that this event was associated with more advanced disease (P?<?0.0001) and survival (P = 0.041). The germline single nucleotide polymorphism (SNP) risk alleles and somatic copy number gains were associated with increased LMO1 expression in neuroblastoma cell lines and primary tumours, consistent with a gain-of-function role in tumorigenesis. Short hairpin RNA (shRNA)-mediated depletion of LMO1 inhibited growth of neuroblastoma cells with high LMO1 expression, whereas forced expression of LMO1 in neuroblastoma cells with low LMO1 expression enhanced proliferation. These data show that common polymorphisms at the LMO1 locus are strongly associated with susceptibility to developing neuroblastoma, but also may influence the likelihood of further somatic alterations at this locus, leading to malignant progression.
Project description:Neuroblastoma is an embryonal tumor of the peripheral sympathetic nervous system. Elevated expression of the transcription factor LMO1 and the polymorphisms within this gene are associated with the susceptibility to develop neuroblastoma. LMO1 has been implicated as an oncogene in T-cell acute lymphoblastic leukemia; however, the transcriptional targets regulated by this protein in neuroblastoma cells are unknown. Here, we identify the genes and molecular pathways controlled by LMO1 in neuroblastoma cells. ChIP-seq analysis revealed that LMO1-bound regions are frequently co-occupied by GATA3 and MYCN proteins and are associated with active histone marks in neuroblastoma cells. RNA-seq analysis demonstrated that LMO1 regulates gene expression in a tumor type-specific manner. One high-confidence target gene directly regulated by LMO1 and MYCN is ASCL1, which is more highly expressed in adrenergic subtype of neuroblastoma cells as compared to normal neuronal cells. High levels of ASCL1 expression are associated with inferior overall survival in primary human neuroblastoma cases. ChIP-seq analysis identified a regulatory element controlling ASCL1 expression that is bound by LMO1, MYCN and the members of the core regulatory circuitry in neuroblastoma cells. Furthermore, ASCL1 is required for neuroblastoma cell growth and regulates genes responsible for repression of neuronal cell differentiation. Taken together, our results implicate ASCL1 as a critical downstream target of LMO1 in the molecular pathogenesis of neuroblastoma. Overall design: ChIP-Seq for LMO1 in the Kelly neuroblastoma cell line
Project description:Neuroblastoma is a paediatric malignancy that typically arises in early childhood, and is derived from the developing sympathetic nervous system. Clinical phenotypes range from localized tumours with excellent outcomes to widely metastatic disease in which long-term survival is approximately 40% despite intensive therapy. A previous genome-wide association study identified common polymorphisms at the LMO1 gene locus that are highly associated with neuroblastoma susceptibility and oncogenic addiction to LMO1 in the tumour cells. Here we investigate the causal DNA variant at this locus and the mechanism by which it leads to neuroblastoma tumorigenesis. We first imputed all possible genotypes across the LMO1 locus and then mapped highly associated single nucleotide polymorphism (SNPs) to areas of chromatin accessibility, evolutionary conservation and transcription factor binding sites. We show that SNP rs2168101 G>T is the most highly associated variant (combined P?=?7.47?×?10(-29), odds ratio 0.65, 95% confidence interval 0.60-0.70), and resides in a super-enhancer defined by extensive acetylation of histone H3 lysine 27 within the first intron of LMO1. The ancestral G allele that is associated with tumour formation resides in a conserved GATA transcription factor binding motif. We show that the newly evolved protective TATA allele is associated with decreased total LMO1 expression (P?=?0.028) in neuroblastoma primary tumours, and ablates GATA3 binding (P?<?0.0001). We demonstrate allelic imbalance favouring the G-containing strand in tumours heterozygous for this SNP, as demonstrated both by RNA sequencing (P?<?0.0001) and reporter assays (P?=?0.002). These findings indicate that a recently evolved polymorphism within a super-enhancer element in the first intron of LMO1 influences neuroblastoma susceptibility through differential GATA transcription factor binding and direct modulation of LMO1 expression in cis, and this leads to an oncogenic dependency in tumour cells.
Project description:RNA-seq analysis was performed in Kelly neuroblastoma cell line to analyze gene expression changes after LMO1 or MYCN knockdown. Overall design: Lentiviral shRNA transduction was carried out in Kelly cells to knockdown control GFP, LMO1 or MYCN. One shRNA was used to target each gene. Experiments were done in biological duplicates. Total RNA was harvested 3 days after knockdown and RNA-seq analysis was performed.
Project description:Neuroblastoma is one of the most commonly diagnosed extracranial solid tumors in infancy; however, the etiology of neuroblastoma remains largely unknown. Previous genome-wide association study (GWAS) indicated that several common genetic variations (rs110419 A > G, rs4758051 G > A, rs10840002 A > G and rs204938 A > G) in the LIM domain only 1 (LMO1) gene were associated with neuroblastoma susceptibility. The aim of this study was to evaluate the correlation between the four GWAS-identified LMO1 gene polymorphisms and neuroblastoma risk in a Southern Chinese population. We genotyped the four polymorphisms in 256 neuroblastoma cases and 531 controls. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to evaluate the strength of the associations. False-positive report probability was calculated for all significant findings. We found that the rs110419 A > G polymorphism was associated with a significantly decreased neuroblastoma risk (AG vs. AA: adjusted OR = 0.65, 95% CI = 0.47-0.91; GG vs. AA: adjusted OR = 0.58, 95% CI = 0.36-0.91; AG/GG vs. AA: adjusted OR = 0.63, 95% CI = 0.46-0.86), and the protective effect was more predominant in children of age > 18 months, males, subgroups with tumor in adrenal gland and mediastinum, and patients in clinical stages III/IV. These results suggested that LMO1 gene rs110419 A > G polymorphism may contribute to protection against neuroblastoma. Our findings call for further validation studies with larger sample size.
Project description:Previous genome-wide association and validation studies suggest that LIM domain only 1 (LMO1) gene polymorphisms affect neuroblastoma susceptibility. In this work, we used Taqman methodology to genotype four LMO1 polymorphisms (rs110419 A > G, rs4758051 G > A, rs10840002 A > G and rs204938 A > G) in 118 neuroblastoma cases and 281 controls from Northern China. Odds ratios (ORs) and 95% confidence intervals (CIs) were used to evaluate the association. We found that rs4758051 G > A was associated with a decreased neuroblastoma risk (AA vs. GG: adjusted OR = 0.28, 95% CI = 0.13-0.62; AG/AA vs. GG: adjusted OR = 0.62, 95% CI = 0.40-0.97; AA vs. GG/AG: adjusted OR = 0.33, 95% CI = 0.15-0.69). Likewise, carrying the rs10840002 G allele was also associated with a decreased neuroblastoma risk in this Northern Chinese population. In a combination analysis using Southern and Northern Chinese populations, we found that those carrying the rs110419 G, rs4758051 A or rs10840002 G allele were at decreased neuroblastoma risk, and this finding was supported by a false-positive report probability analysis. These results further verify that LMO1 polymorphisms are protective against neuroblastoma. Case-control studies with larger samples and using other ethnicities are still needed to confirm our conclusion.
Project description:Background: Neuroblastoma, a neuroendocrine tumor, stems from the developing sympathetic nervous system. Previous genome-wide association studies (GWASs) have discovered a number of neuroblastoma susceptibility genes in Caucasians including LIM domain only 1 (LMO1). Objective: We conducted a three-center case-control study including 313 cases and 716 controls with the purpose to evaluate the association between five GWAS-identified LMO1 variants (rs110419 A>G, rs4758051 G>A, rs10840002 A>G, rs204938 A>G, and rs2168101 G>T) and neuroblastoma susceptibility in eastern Chinese children. Methods: Odds ratios (ORs) and 95% confidence intervals (CIs) were calculated to evaluate the strength of the associations. False positive report possibility (FPRP) analysis was performed to check whether significant results were noteworthy. Results: Significant associations with neuroblastoma risk were found for four (rs110419, rs4758051, rs10840002, and rs2168101) out of the five polymorphisms. Combined analysis demonstrated that carriers of 4-5 protective genotypes had a significantly decreased risk of neuroblastoma in comparison those with 0-3 protective genotypes (adjusted OR = 0.51, 95% CI = 0.39-0.68, P < 0.0001). Haplotype analysis of the five SNPs yield four significant haplotypes associated with neuroblastoma susceptibility. Conclusion: In conclusion, we confirmed LMO1 polymorphisms may reduce neuroblastoma risk in eastern Chinese populations.