HACE1 Gene Research Articles

Magnolol promotes the autophagy of esophageal carcinoma cells by upregulating HACE1 gene expression.

Esophagus cancer (EC) is one of the most aggressive malignant digestive system tumors and has a high clinical incidence worldwide. Magnolol, a natural compound, has anticancer effects on many cancers, including esophageal carcinoma, but the underlying mechanism has not been fully elucidated. Here, we first find that magnolol inhibits the proliferation of esophageal carcinoma cells and enhances their autophagy activity in a dose- and time-dependent manner. This study demonstrates that magnolol increases the protein levels of LC3 II, accompanied by increased HACE1 protein levels in both esophageal carcinoma cells and xenograft tumors. HACE1-knockout (KO) cell lines are generated, and the ablation of HACE1 eliminates the anti-proliferative and autophagy-inducing effects of magnolol on esophageal carcinoma cells. Additionally, our results show that magnolol primarily promotes HACE1 expression at the transcriptional level. Therefore, this study shows that magnolol primarily exerts its antitumor effect by activating HACE1-OPTN axis-mediated autophagy. It can be considered a promising therapeutic drug for esophageal carcinoma.

Previously Undescribed Gross HACE1 Deletions as a Cause of Autosomal Recessive Spastic Paraplegia

Spastic paraplegia and psychomotor retardation with or without seizures (SPPRS, OMIM 616756) is a rare genetic disease caused by biallelic pathogenic variants in the HACE1 gene. Originally, these mutations have been reported to be implicated in tumor predisposition. Nonetheless, via whole exome sequencing in 2015, HACE1 mutations were suggested to be the cause of a new autosomal recessive neurodevelopmental disorder, which is characterized by spasticity, muscular hypotonia, and intellectual disability. To date, 14 HACE1 pathogenic variants have been described; these variants have a loss-of-function effect that leads to clinical presentations with variable severities. However, gross deletions in the HACE1 gene have not yet been mentioned as a cause of spastic paraplegia. Here, we report a clinical case involving a 2-year-old male presenting with spasticity, mainly affecting the lower limbs, and developmental delay. Exome sequencing, chromosomal microarray analysis, and mRNA analysis were used to identify the causative gene. We revealed that the clinical findings were due to previously undescribed HACE1 biallelic deletions. We identified the deletion of exon 7: c.(534+1_535-1)_(617+1_618-1)del (NM_020771.4) and the gross deletion in the 6q16.3 locus, which affected the entire HACE1 gene: g.105018931_105337494del, (GRCh37). A comprehensive diagnostic approach for the patients with originally homozygous mutations in HACE1 is required since false homozygosity results are possible. More than 80% of the described mutations were reported to be homozygous. Initial hemizygosity is hard to detect by quantitative methods, and this may challenge molecular diagnostic identification in patients with spastic paraplegia.

An Asymptomatic Patient of Phenylketonuria: A Case Report of 2 Siblings

: A 6-year-old girl presented to our hospital with a genetic result indicating a homozygous pathogenic variant (c.G898T) in the phenylalanine hydroxylase (PAH) gene and a heterozygote variant (c.94dupT) in the HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1) gene. The study was performed due to her brother’s earlier diagnosis of phenylketonuria (PKU) through a genetic analysis (homozygote for PAH). Her 4-year-old brother was also admitted to our hospital with symptoms of hypotonicity, which started at birth and deteriorated when he was 6 months old. He developed a prolonged fever from the age of 8 months until the age of 3 years. All infectious and rheumatologic workups were normal. He was screened for PKU twice at birth, both showing negative results. The plasma phenylalanine (Phe) level was checked several times in the first 2 years of his life, and all of them were in the borderline range (2 - 4 mg/dL). He was tested again at the age of 2 years for the plasma Phe level twice, both showing positive results (14 and 8 mg/dL, respectively). Both positive results for the plasma Phe level led to a genetic study, indicating that this case is homozygote for both variants, c.G898T in the PAH gene and c.94dupT in the HACE1 gene. Then, a Phe-restricted diet was given. At the age of 3 years, a Kuvan test was performed on the patient, indicating a non-BH4-responsive PKU (classic type of PKU). However, to reduce diet restriction, he was treated with Kuvan and responded to the treatment. The symptoms (such as hypotonia and developmental retardation) improved after treatment with Kuvan, probably due to HACE1 gene dysfunction.

HACE1, GLRX5, and ELP2 gene variant cause spastic paraplegies.

Hereditary spastic paraplegias (HSPs) are a clinically and genetically heterogeneous group of conditions that are characterized by lower limb spasticity and weakness. Considering the clinical overlap between metabolic causes, genetic diseases, and autosomal recessive HSP, differentiation between these types can be difficult based solely on their clinical characteristics. This study aimed to investigate the genetic etiology of patients with clinically suspected HSP. The study group was composed of seven Turkish families who each had two affected children and three families who each had a single affected child (17 total patients). The 17 probands (14 males, 3 females) underwent whole exome sequencing. Five typical HSP genes (FA2H, AP4M1, AP4E1, CYP7B1, and MAG) and three genes not previously related to HSP (HACE1, GLRX5, ad ELP2) were identified in 14 probands. Eight novel variants were identified in seven families: c.653T > C (p.Leu218Pro) in the FA2H gene, c.347G > A (p.Gly116Asp) in the GLRX5 gene, c.2581G > C (p.Ala861Pro) in the HACE1 gene, c.1580G > A (p.Arg527Gln) and c.1189-1G > A in the ELP2 gene, c.10C > T (p.Gln4*) and c.1025 + 1G > A in the AP4M1 gene, c.1291delG (p.Gly431Alafs*3) and c.3250delA (p.Ile1084*) in the AP4E1 gene, and c.475T > G (p.Cys159Gly) in the MAG gene. The growing use of next-generation sequencing improved diagnosis but also led to the continual identification of new causal genes for neurogenetic diseases associated with lower limb spasticity. The increasing number of HSP genes identified thus far highlights the extreme genetic heterogeneity of these disorders and their clinical and functional overlap with other neurological conditions. Our findings suggest that the HACE1, GLRX5, and ELP2 genes are genetic causes of HSP.

HACE1 blocks HIF1\u03b1 accumulation under hypoxia in a RAC1 dependent manner

Uncovering the mechanisms that underpin how tumor cells adapt to microenvironmental stress is essential to better understand cancer progression. The HACE1 (HECT domain and ankyrin repeat-containing E3 ubiquitin-protein ligase) gene is a tumor suppressor that inhibits the growth, invasive capacity, and metastasis of cancer cells. However, the direct regulatory pathways whereby HACE1 confers this tumor-suppressive effect remain to be fully elucidated. In this report, we establish a link between HACE1 and the major stress factor, hypoxia-inducible factor 1 alpha (HIF1α). We find that HACE1 blocks the accumulation of HIF1α during cellular hypoxia through decreased protein stability. This property is dependent on HACE1 E3 ligase activity and loss of Ras-related C3 botulinum toxin substrate 1 (RAC1), an established target of HACE1 mediated ubiquitinylation and degradation. In vivo, genetic deletion of Rac1 reversed the increased HIF1α expression observed in Hace1–/– mice in murine KRasG12D-driven lung tumors. An inverse relationship was observed between HACE1 and HIF1α levels in tumors compared to patient-matched normal kidney tissues, highlighting the potential pathophysiological significance of our findings. Together, our data uncover a previously unrecognized function for the HACE1 tumor suppressor in blocking HIF1α accumulation under hypoxia in a RAC1-dependent manner.

Demethylation of HACE1 gene promoter by propofol promotes autophagy of human A549 cells.

Propofol (2,6-diisopropylphenol) is one of the most commonly used intravenous anesthetics and possesses a number of non-anesthetic effects, including antitumor function. The aim of the present study was to elucidate the antitumor molecular mechanism of propofol on A549 and H1299 cells. A549 and H1299 cells were treated in the presence or absence of different concentrations (0, 60 or 120 µmol) of propofol for different durations (0, 24, 48 or 72 h), and proliferation was detected by MTT and colony formation assays; the protein levels of optineurin (OPTN) ubiquitination, HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1), methyl-CpG binding domain protein 3 (MBD3) and Microtubule-associated protein 1A/1B-light chain 3 were detected by immunoblotting or quantitative (q)PCR; the methylation state of the HACE1 gene promoter was detected by bisulfite DNA sequencing; and binding of MBD3 on HACE1 gene promoter was detected by chromatin immunoprecipitation-qPCR. Propofol inhibited proliferation of A549 and H1299 cells and promoted HACE1-OPTN axis-mediated selective autophagy activity by increasing the protein expression levels of HACE1 via demethylating its promoter region. Furthermore, propofol promoted expression levels of MBD3 and binding to the −1,000 to −1 bp (transcription start site) region of HACE1 gene promoter. MBD3-knockdown experiments indicated that propofol inhibited proliferation of A549 cells in a MBD3-dependent manner. Thus, the findings of the present study provided a potential antitumor molecular mechanism mediated by propofol.

Demethylation of the HACE1 gene promoter inhibits the proliferation of human liver cancer cells

HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1) is frequently downregulated or lost in numerous types of cancer, including liver cancer. The aim of the present study was to examine whether demethylation of the HACE1 gene could inhibit tumour progression. The expression of HACE1 was detected in liver cancer cell lines. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated (Cas)-based demethylation single guide RNAs for the HACE1 gene promoter were designed and transfected into liver cancer cells. Subsequently, proliferation was detected by MTT and colony formation assays, and optineurin (OPTN) ubiquitination and microtubule-associated proteins 1A/1B light chain 3B protein levels were detected by immunoblotting. The levels of HACE1 were significantly reduced in liver cancer cell lines compared with in a normal liver cell line. Demethylation of the HACE1 gene promoter increased HACE1 expression, inhibited the proliferation of liver cancer cells, and promoted OPTN ubiquitination and autophagy activity in liver cancer cells. In conclusion, activation of HACE1 expression by promoter demethylation may provide a suitable approach for anticancer therapy.

HACE1 is a potential tumor suppressor in osteosarcoma

Osteosarcoma is a malignant bone sarcoma characterized by extensive genomic disruption and a propensity for metastatic spread. Osteoid production suggests a close relationship with normal osteoblasts, and the latter are the presumptive cell of origin of this disease. The HACE1 gene, localized to human chromosome 6q21, encodes the HACE1 HECT E3 ligase, a tumor suppressor in diverse tumors that acts in part by targeting the activated form of RAC1 GTPase for proteasomal degradation. Disruption or loss of 6q21 is relatively common in osteosarcomas, and Hace1−/−/Tp53+/− mice frequently develop osteosarcomas, in contrast to Tp53+/− mice, which do not. This suggests an unexplored link between HACE1 loss and osteosarcoma. Here we compared HACE1 expression in normal osteoblasts and osteosarcoma cell lines in vitro by western blotting and quantitative RT-PCR, and in human osteosarcoma specimens by immunohistochemistry. Both HACE1 transcript and protein levels were reduced in osteosarcoma compared to osteoblasts in vitro. Reduced HACE1 expression in osteosarcoma tumors was observed in 76% of cases and associated with high-grade lesions. Further, clonally derived pairs of high and low metastatic osteosarcoma cell lines showed significant downregulation in the high compared to corresponding low metastatic cells. Ectopic expression of HACE1 markedly inhibited anchorage-independent growth and cell motility of HACE1 osteosarcoma cell lines, and was associated with reduced RAC1 activation and decreased reactive oxygen species (ROS). Finally, HACE1 overexpression blocked osteosarcoma xenograft growth and dramatically reduced pulmonary metastases. These findings point to a potential tumor suppressor function for HACE1 in osteosarcoma.

A novel loss-of-function mutation in HACE1 is linked to a genetic disorder in a patient from India

A large number of congenital disorders are very rare and localized to rural areas in India, a country that practices both endogamy and consanguinity. Recent advances in genomics can aid in the identification of causative genomic elements when exploring therapeutic interventions and developing neonatal screening to assign novel functions. Here, we report a novel loss-of-function mutation (p.Trp370*) in the HACE1 gene that is associated with a rare congenital neurodevelopmental disorder in a boy from a remote village in southern India.

Bayesian Cox Proportional Hazards Model in Survival Analysis of HACE1 Gene with Age at Onset of Alzheimer's Disease.

Alzheimer's disease (AD), the most common form of dementia, is a chronic neurodegenerative disease. The HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1 (HACE1) gene is expressed in human brain and may play a role in the pathogenesis of neurodegenerative disorders. Till now, no previous study has reported the association of the HACE1 gene with the risk and age at onset (AAO) of AD; while few studies have checked the proportional hazards assumption in the survival analysis of AAO of AD using Cox proportional hazards model. In this study, we examined the associations of 14 single nucleotide polymorphisms (SNPs) in the HACE1 gene with the risk and the AAO of AD using 791 AD patients and 782 controls. Multiple logistic regression model identified one SNP (rs9499937 with p = 1.8×10-3) to be associated with the risk of AD. For survival analysis of AAO, both classic Cox regression model and Bayesian survival analysis using the Cox proportional hazards model were applied to examine the association of each SNP with the AAO. The hazards ratio (HR) with its 95% confidence interval (CI) was estimated. Survival analysis using the classic Cox regression model showed that 4 SNPs were significantly associated with the AAO (top SNP rs9499937 with HR=1.33, 95%CI=1.13-1.57, p=5.0×10-4). Bayesian Cox regression model showed similar but a slightly stronger associations (top SNP rs9499937 with HR=1.34, 95%CI=1.11-1.55) compared with the classic Cox regression model. Using an independent family-based sample, one SNP rs9486018 was associated with the risk of AD (p=0.0323) and the T-T-G haplotype from rs9786015, rs9486018 and rs4079063 showed associations with both the risk and AAO of AD (p=2.27×10-3 and 0.0487, respectively). The findings of this study provide first evidence that several genetic variants in the HACE1 gene were associated with the risk and AAO of AD.

Association Between HACE1 Gene Polymorphisms and Wilms' Tumor Risk in a Chinese Population

ABSTRACTWilms' tumor is one of the most common solid tumors of childhood; however, the genetic basis underlying the majority of cases remains largely unknown. HACE1 is a putative Wilms' tumor susceptibility gene. We investigated the association between five HACE1 gene polymorphisms and Wilms' tumor susceptibility in a Chinese population consisting of 145 patients and 531 controls. We found a significant association between HACE1 rs9404576 polymorphism and decreased Wilms' tumor risk. No significant association was detected for other polymorphisms in the overall analysis. Our results indicated that HACE1 rs9404576 polymorphism may be associated with Wilms' tumor susceptibility in the Chinese population.

Heterogeneous epigenetic regulation of HACE1 in Burkitt- Lymphoma-derived cells

We examined the consequences of 3-deazaneplanocin A (DZNep) on HACE1 expression in human Burkitt- Lymphoma-derived cells to investigate fundamental molecular mechanisms that control its expression. We treated the human Burkitt- Lymphoma-derived cells lines Ramos and Raji with DZNep and examined HACE1 mRNA expression by RT-PCR. We also studied the effect of DZNep on the methylation of lysine 9 and 27 of histone 3 (H3K27me3 and H3K9me2) associated with the CpG88 and CpG177 islands of the HACE1 promoters by chromatin immunoprecipitation and quantitative PCR. CpG88 (hypomethylated) of the HACE1 promoter was enriched for histone marks H3K27me3 and H3K9me2 whereas CpG177 (hypermethylated) was only enriched for H3K9me2. DZNep treatment increased HACE1 gene expression which was further increased by the addition of trichostatine A (TSA), a promising therapeutic compound for the treatment of human B-Lymphoma. Histone methylation (both H3K9me2 and H3K27me3) of the HACE1 promoter concomitantly decreased. Our experiments suggest that HACE1 can be downregulated by methylation of its promoter region chromatin (H3K27me3 and H3K9me2), making HACE1 a potential target for DZNep combined with TSA. These results highlight the heterogeneity of HACE1 regulation in B-lymphoma and suggest that successful drug-induced restoration of epigenetically silenced tumor suppressor genes will require accurate characterization of cell type- and locus-specific gene silencing mechanisms.

NKX6.3 Regulates Reactive Oxygen Species Production by Suppressing NF-kB and DNMT1 Activities in Gastric Epithelial Cells

NKX6.3 plays an important role in gastric epithelial differentiation and also acts as a gastric tumor suppressor. The specific aim of this study was to determine whether NKX6.3 contributes to gastric mucosal barrier function by regulating reactive oxygen species (ROS) production. NKX6.3 reduced ROS production and regulated expression of anti-oxidant genes, including Hace1. In addition, NKX6.3 reduced DNMT1 expression and activity by down-regulating NF-kB family gene transcription. Silencing of Hace1 recovered ROS production, whereas knock-down of DNMT1 and NF-kB reduced ROS production and induced Hace1 expression by hypomethylating its promoter region. In addition, NKX6.3 inhibited CagA effects on cell growth, ROS production, and NF-kB and DNMT1 activity. In gastric mucosae and cancers, NKX6.3 and Hace1 expression was significantly reduced. The NKX6.3 expression was positively correlated with Hace1 and Nrf2 genes, but negatively correlated with DNMT1. Hypermethylation of Hace1 gene was observed only in gastric mucosae with H. pylori, atrophy and intestinal metaplasia. Thus, these results suggest that NKX6.3 inhibits ROS production by inducing the expression of Hace1 via down-regulating NF-kB and DNMT1 activity in gastric epithelial cells.

Common variations within HACE1 gene and neuroblastoma susceptibility in a Southern Chinese population.

Neuroblastoma is a common fatal pediatric cancer of the developing sympathetic nervous system, which accounts for ~10% of all pediatric cancer deaths. To investigate genetic risk factors related to neuroblastoma, many genome-wide association studies have been performed, and single nucleotide polymorphisms (SNPs) within HACE1 gene have been identified to associate with neuroblastoma risk. However, the association of the HACE1 SNPs with neuroblastoma needs to be validated in Southern Chinese children. We genotyped five SNPs located in the HACE1 gene (rs4336470 C>T, rs9404576 T>G, rs4079063 A>G, rs2499663 T>C, and rs2499667 A>G) in 256 Southern Chinese patients in comparison with 531 ethnically matched healthy controls. Single locus analysis showed no significant association between any of HACE1 SNPs and neuroblastoma risk in Southern Chinese children. However, when all the risk genotypes were combined, we found a borderline significant trend toward an increased neuroblastoma risk with 4–5 risk genotypes (adjusted odds ratio =1.36, 95% confidence interval =0.98–1.89, P=0.065). Moreover, stratified analysis found that carriers of 4–5 risk genotypes tended to develop neuroblastoma in the retroperitoneal region and have more aggressive tumors, progressing to advanced clinical stages III/IV, when compared with those of 0–3 risk genotypes. In conclusion, HACE1 gene may have weak effect on neuroblastoma risk in Southern Chinese children. Large well-designed studies are needed to strengthen our findings.

HACE1 is a putative tumor suppressor gene in B-cell lymphomagenesis and is down-regulated by both deletion and epigenetic alterations

HECT domain and ankyrin repeat containing E3 ubiquitin protein ligase 1, HACE1, located on chromosome 6q, encodes an E3 ubiquitin ligase and is downregulated in many human tumors. Here, we report HACE1 as a candidate tumor suppressor gene down-regulated by a combination of deletion and epigenetic mechanisms. HACE1 deletions were observed in 40% of B-cell lymphoma tumors. Hypermethylation of the HACE1 promoter CpG177 island was found in 60% (68/111) of cases and in all tested B-cell lymphoma lines. Using HDAC inhibitors, we observed predominantly inactive chromatin conformation (methylated H3 histones H3K9me2) in HACE1 gene promoter region. We demonstrated in Ramos and Raji cells that down-regulation of HACE1 expression was associated with a significant decrease in apoptosis and an accumulation of cells in the S and G2/M phases. Our experiments indicate that HACE1 can act as a haploinsufficient tumor suppressor gene in most B-cell lymphomas and can be downregulated by deacetylation of its promoter region chromatin, which makes HACE1 a potential target for HDAC inhibitors.

Abstract 4947: HACE1 is a putative tumor suppressor gene in B-cell lymphomagenesis down-regulated by both deletion and epigenetic mechanisms

Abstract HACE1, located on chromosome 6q, encodes an E3 ubiquitin ligase and is downregulated in human tumors such as neuroblastomas and natural killer (NK) lymphomas. HACE1 has been shown to ubiquitylate Rac1, a protein involved in cell proliferation and G2/M cell cycle progression. The function of HACE1 and the factors involved in its transcriptional regulation are largely unknown in the context of B-cell lymphomas. We show here, by RT-qPCR, that HACE1 gene is constitutively expressed in Normal lymph nodes and in normal B-cells isolated from peripheral blood, contrasting with a strong downregulation of its expression in more than 70% (77/111) of diffuse large B-cell lymphoma (DLBCL) cases and in four tested B-Lymphoma cell lines. HACE1 gene copy number was assessed by quantitative multiplex PCR of short fluorescent fragments (QMPSF) and array for comparative genomic hybridization (aCGH) in 91 DLBCL cases. A HACE1 heterozygous deletion was observed in 38.1% and an homozygous deletion in 2.4% of cases. These deletions were associated with a significant gene expression decrease. The molecular epigenetic mechanisms underlying HACE1 downregulation were also investigated. Using pyrosequencing assays, as compared to normal B-cells, we observed an hypermethylation of HACE1 promoter CpG177 island in 60% (68/111) of DLBCL cases and in all tested B-Lymphoma cell lines. However, no significant correlation between promoter methylation status and gene expression level was demonstrated. Furthermore, RT-qPCR assays revealed that the demethylating agent 5′azacytidine (5′AZA) did not induce a HACE1 gene expression increase in the different cell lines. By contrast, the histone deacetylase inhibitors (HDACi) trichostatin A (TSA) and LBH589 strongly reactivated the expression of HACE1 in Ramos, Raji and RL cells in which the CpG 177 island was fully methylated. We next performed ChIP experiments to determine whether HACE1 locus chromatin was in an active or inactive conformation in Ramos cell line, the most sensitive cell line to TSA effect. We found that the chromatin of HACE1 gene promoter region was predominantly in the inactive conformation (methylated H3 histones). TSA treatment was able to reverse this pattern, switching the conformation of HACE1 promoter chromatin to an active one predominantly associated with acetylated H3 histones. The putative role of HACE1 in B-cell lymphomagenesis was further investigated using lentiviral transduction (shHACE1). We demonstrated in Ramos and Raji cells that a down-regulation of HACE1 expression was associated with a significant decrease of apoptosis level and cell cycle arrest in G2/M phase. To conclude, our experiments indicate that HACE1 can act as a haploinsufficient tumor suppressor gene in most B-cell lymphomas and be downregulated by deacetylation and methylation of its promoter region chromatin constituting a potential target for HDAC inhibitors. Citation Format: Abdelilah Bouzelfen, Marion Alcantara, Hafid Kora, Philippe Bertrand, Sylvain Mareschal, Elodie Bohers, Catherine Maingonnat, Philippe Ruminy, Sahil Adriouch, Gaetan Riou, Martin Figeac, Thierry Fest, Christian Bastard, Hervé Tilly, Jean-Baptiste Latouche, Fabrice Jardin. HACE1 is a putative tumor suppressor gene in B-cell lymphomagenesis down-regulated by both deletion and epigenetic mechanisms. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4947. doi:10.1158/1538-7445.AM2015-4947

Common genetic variants in NEFL influence gene expression and neuroblastoma risk.

The genetic etiology of sporadic neuroblastoma is still largely obscure. In a genome-wide association study, we identified single-nucleotide polymorphisms (SNP) associated with neuroblastoma at the CASC15, BARD1, LMO1, DUSP12, HSD17B12, HACE1, and LIN28B gene loci, but these explain only a small fraction of neuroblastoma heritability. Other neuroblastoma susceptibility genes are likely hidden among signals discarded by the multiple testing corrections. In this study, we evaluated eight additional genes selected as candidates for further study based on proven involvement in neuroblastoma differentiation. SNPs at these candidate genes were tested for association with disease susceptibility in 2,101 cases and 4,202 controls, with the associations found replicated in an independent cohort of 459 cases and 809 controls. Replicated associations were further studied for cis-effect using gene expression, transient overexpression, silencing, and cellular differentiation assays. The neurofilament gene NEFL harbored three SNPs associated with neuroblastoma (rs11994014: Pcombined = 0.0050; OR, 0.88; rs2979704: Pcombined = 0.0072; OR, 0.87; rs1059111: Pcombined = 0.0049; OR, 0.86). The protective allele of rs1059111 correlated with increased NEFL expression. Biologic investigations showed that ectopic overexpression of NEFL inhibited cell growth specifically in neuroblastoma cells carrying the protective allele. NEFL overexpression also enhanced differentiation and impaired the proliferation and anchorage-independent growth of cells with protective allele and basal NEFL expression, while impairing invasiveness and proliferation of cells homozygous for the risk genotype. Clinically, high levels of NEFL expression in primary neuroblastoma specimens were associated with better overall survival (P = 0.03; HR, 0.68). Our results show that common variants of NEFL influence neuroblastoma susceptibility and they establish that NEFL expression influences disease initiation and progression.

Methylation of the HACE1 gene is frequently detected in hepatocellular carcinoma.

Recently, it has been reported that HACE1, the E3 ubiquitin ligase, is epigenetically inactivated in human Wilms' tumors and HACE 1 expression was also down-regulated in colorectal and gastric carcinomas. In this study, methylation status of the HACE1 gene was examined in primary carcinomas and the corresponding normal tissues derived from 27 patients with HCC using quantitative methylation-specific PCR (qMSP). Methylation of the HACE1 gene was detected in 18 out of the 27 (67%) HCCs, suggesting that the methylation of HACE1 was frequently observed in HCC. The clinicopathological data were then correlated with these results. In the value of serum AFP (α-fetoprotein), a significant difference was observed (p=0.0025). All stages of HCCs presented HACE1 methylation, indicating that the HACE1 gene has been methylated from the early stages of HCCs.

The Hace1 E3 ligase: linking misfolded protein load and oncogenesis

Hace1 is a new member of the HECT family of E3 protein‐ubiquitin ligases. We previously showed that the Hace1 gene is a tumor suppressor that is inactivated in human Wilms' tumor, and that Hace1‐/‐ mice develop spontaneous tumors. Moreover, various stresses dramatically increases tumor incidence in these mice. The goal of this study is to characterize the role Hace1 is playing in cellular stress and more specifically in misfolded protein handling by the cell. We now show that Hace1 decreases the half‐life of GFP‐Huntingtin‐128Q and eliminates its aggregates in a proteasome independent manner. This indicates that Hace1 may mediate mutant Huntingtin degradation via other pathways such as autophagy. Indeed, Hace1‐/‐ cells show decreased autophagy induction. Fluorescence microscopy suggests that Hace1 is localized to a recently described cellular structure known as IPOD‐ Insoluble Protein Deposit. Using fluorescence recovery after photo‐bleaching we determined that GFP‐Hace1 interacts with IPOD in a highly dynamic manner suggesting that Hace1 plays a role in the function of IPOD rather than being transported to them as aggregates. Taken together, these data indicate that the tumor suppressor Hace1 reduces accumulation of misfolded proteins by promoting autophagy, suggesting that the deregulation of non‐proteasomal processes that manage misfolded protein load may promote oncogenesis in the cell.