Functional characterization of novel or yet uncharacterized ATP7B missense variants detected in patients with clinical Wilson's disease.

Wilson's disease (WD, MIM#277900) is an autosomal recessive disorder resulting in copper excess caused by biallelic variants in the ATP7B gene (MIM#606882) encoding a copper transporting P-type ATPase. ATP7B variants of unknown significance (VUS) are detected frequently, sometimes impeding a clear diagnosis. Functional analyses can help to classify these variants as benign or pathogenic. Additionally, variants already classified as (likely) pathogenic benefit from functional analyses to understand their pathomechanism, thus contribute to the development of personalized treatment approaches in the future. We described clinical features of six WD patients and functionally characterized five ATP7B missense variants (two VUS, three yet uncharacterized likely pathogenic variants), detected in these patients. We determined the protein level, copper export capacity, and cellular localization in an in vitro model and potential structural consequences using an ATP7B protein model based on AlphaFold. Our analyses give insight into the pathomechanism and allowed reclassification for the two VUS to likely pathogenic and for two of the three likely pathogenic variants to pathogenic.

MiR-129-5p exerts Wnt signaling-dependent tumor-suppressive functions in hepatocellular carcinoma by directly targeting hepatoma-derived growth factor HDGF.

BACKGROUND: In hepatocellular carcinoma (HCC), histone deacetylases (HDACs) are frequently overexpressed. This results in chromatin compaction and silencing of tumor-relevant genes and microRNAs. Modulation of microRNA expression is a potential treatment option for HCC. Therefore, we aimed to characterize the epigenetically regulated miR-129-5p regarding its functional effects and target genes to understand its relevance for HCC tumorigenesis. METHODS: Global miRNA expression of HCC cell lines (HLE, HLF, Huh7, HepG2, Hep3B) and normal liver cell lines (THLE-2, THLE-3) was analyzed after HDAC inhibition by miRNA sequencing. An in vivo xenograft mouse model and in vitro assays were used to investigate tumor-relevant functional effects following miR-129-5p transfection of HCC cells. To validate hepatoma-derived growth factor (HDGF) as a direct target gene of miR-129-5p, luciferase reporter assays were performed. Survival data and HDGF expression were analyzed in public HCC datasets. After siRNA-mediated knockdown of HDGF, its cancer-related functions were examined. RESULTS: HDAC inhibition induced the expression of miR-129-5p. Transfection of miR-129-5p increased the apoptosis of HCC cells, decreased proliferation, migration and ERK signaling in vitro and inhibited tumor growth in vivo. Direct binding of miR-129-5p to the 3'UTR of HDGF via a noncanonical binding site was validated by luciferase reporter assays. HDGF knockdown reduced cell viability and migration and increased apoptosis in Wnt-inactive HCC cells. These in vitro results were in line with the analysis of public HCC datasets showing that HDGF overexpression correlated with a worse survival prognosis, primarily in Wnt-inactive HCCs. CONCLUSIONS: This study provides detailed insights into the regulatory network of the tumor-suppressive, epigenetically regulated miR-129-5p in HCC. Our results reveal for the first time that the therapeutic application of mir-129-5p may have significant implications for the personalized treatment of patients with Wnt-inactive, advanced HCC by directly regulating HDGF. Therefore, miR-129-5p is a promising candidate for a microRNA replacement therapy to prevent HCC progression and tumor metastasis.

MicroRNA-449a Inhibits Triple Negative Breast Cancer by Disturbing DNA Repair and Chromatid Separation.

Chromosomal instability (CIN) can be a driver of tumorigenesis but is also a promising therapeutic target for cancer associated with poor prognosis such as triple negative breast cancer (TNBC). The treatment of TNBC cells with defects in DNA repair genes with poly(ADP-ribose) polymerase inhibitor (PARPi) massively increases CIN, resulting in apoptosis. Here, we identified a previously unknown role of microRNA-449a in CIN. The transfection of TNBC cell lines HCC38, HCC1937 and HCC1395 with microRNA-449a mimics led to induced apoptosis, reduced cell proliferation, and reduced expression of genes in homology directed repair (HDR) in microarray analyses. EME1 was identified as a new target gene by immunoprecipitation and luciferase assays. The reduced expression of EME1 led to an increased frequency of ultrafine bridges, 53BP1 foci, and micronuclei. The induced expression of microRNA-449a elevated CIN beyond tolerable levels and induced apoptosis in TNBC cell lines by two different mechanisms: (I) promoting chromatid mis-segregation by targeting endonuclease EME1 and (II) inhibiting HDR by downregulating key players of the HDR network such as E2F3, BIRC5, BRCA2 and RAD51. The ectopic expression of microRNA-449a enhanced the toxic effect of PARPi in cells with pathogenic germline BRCA1 variants. The newly identified role makes microRNA-449a an interesting therapeutic target for TNBC.

LINC00152 Drives a Competing Endogenous RNA Network in Human Hepatocellular Carcinoma.

Genomic and epigenomic studies revealed dysregulation of long non-coding RNAs in many cancer entities, including liver cancer. We identified an epigenetic mechanism leading to upregulation of the long intergenic non-coding RNA 152 (LINC00152) expression in human hepatocellular carcinoma (HCC). Here, we aimed to characterize a potential competing endogenous RNA (ceRNA) network, in which LINC00152 exerts oncogenic functions by sponging miRNAs, thereby affecting their target gene expression. Database and gene expression data of human HCC were integrated to develop a potential LINC00152-driven ceRNA in silico. RNA immunoprecipitation and luciferase assay were used to identify miRNA binding to LINC00152 in human HCC cells. Functionally active players in the ceRNA network were analyzed using gene editing, siRNA or miRNA mimic transfection, and expression vectors in vitro. RNA expression in human HCC in vivo was validated by RNA in situ hybridization. Let-7c-5p, miR-23a-3p, miR-125a-5p, miR-125b-5p, miR-143a-3p, miR-193-3p, and miR-195-5p were detected as new components of the potential LINC00152 ceRNA network in human HCC. LINC00152 was confirmed to sponge miR143a-3p in human HCC cell lines, thereby limiting its binding to their respective target genes, like KLC2. KLC2 was identified as a central mediator promoting pro-tumorigenic effects of LINC00152 overexpression in HCC cells. Furthermore, co-expression of LINC00152 and KLC2 was observed in human HCC cohorts and high KLC2 expression was associated with shorter patient survival. Functional assays demonstrated that KLC2 promoted cell proliferation, clonogenicity and migration in vitro. The LINC00152-miR-143a-3p-KLC2 axis may represent a therapeutic target in human HCC.

Ago-RIP Sequencing Identifies New MicroRNA-449a-5p Target Genes Increasing Sorafenib Efficacy in Hepatocellular Carcinoma.

BACKGROUND: Patients with hepatocellular carcinoma (HCC) have very limited treatment options. For the last fourteen years, the multi-tyrosine kinase inhibitor sorafenib has been used as standard-of-care therapeutic agent in advanced HCC. Unfortunately, drug resistance develops in many cases. Therefore, we aimed to find a way to mitigate drug resistance and to improve the sorafenib efficacy in HCC cells. MicroRNAs play a significant role in targeting genes involved in tumor control suggesting microRNA/sorafenib combination therapy as a promising treatment option in advanced HCC. METHODS: MiR-449a-5p target genes were identified by Ago-RIP sequencing and validated by luciferase reporter assays and expression analyses. Target gene expression and survival data were analyzed in public HCC datasets. Tumor-relevant functional effects of miR-449a-5p and its target genes as well as their impact on the effects of sorafenib were analyzed using in vitro assays. An indirect transwell co-culture system was used to survey anti-angiogenic effects of miR-449a-5p. RESULTS: PEA15, PPP1CA and TUFT1 were identified as direct target genes of miR-449a-5p. Overexpression of these genes correlated with a poor outcome of HCC patients. Transfection with miR-449a-5p and repression of miR-449a-5p target genes inhibited cell proliferation and angiogenesis, induced apoptosis and reduced AKT and ERK signaling in HLE and Huh7 cells. Importantly, miR-449a-5p potentiated the efficacy of sorafenib in HCC cells via downregulation of PEA15, PPP1CA and TUFT1. CONCLUSIONS: This study provides detailed insights into the targetome and regulatory network of miR-449a-5p. Our results demonstrate for the first time that targeting PEA15, PPP1CA and TUFT1 via miR-449a overexpression could have significant implications in counteracting sorafenib resistance suggesting miR-449a-5p as a promising candidate for a microRNA/sorafenib combination therapy.

KIF12 Variants and Disturbed Hepatocyte Polarity in Children with a Phenotypic Spectrum of Cholestatic Liver Disease.

KIF12 has been identified as a cholestasis-associated candidate gene. We describe 6 cases from 4 unrelated families with diverse cholestatic phenotypes carrying 2 different homozygous KIF12 truncating variants. Immunofluorescence investigations of paraffin-embedded liver sections suggest that KIF12-associated impaired functional cell polarity may be the underlying cause.

MicroRNA-192-5p inhibits migration of triple negative breast cancer cells and directly regulates Rho GTPase activating protein 19.

Among the different breast cancer subtypes, triple-negative breast cancer (TNBC) is associated with a poor prognosis, low survival rates, and high expression of histone deacetylases. Treatment with histone deacetylase inhibitor trichostatin A (TSA) leads to an increased expression of potential tumor-suppressive miRNAs. Characterization of these miRNAs can help to find new molecular targets for treatment of TNBC. We identified differentially expressed miRNAs by microarray analyses after treatment with TSA in the TNBC cell lines HCC38, HCC1395, and HCC1935. The gene locus of hsa-miRNA-192-5p (miR-192) and hsa-miR-194-2 (miR-194-2) with its host gene, long noncoding RNA miR-194-2HG, has been linked to inhibition of migration in different tumor types. Therefore, we examined tumor-relevant functional effects using WST-1-based proliferation, capsase-3/7-based apoptosis, and trans-well migration assays after transfection with miRNA mimics or specific siRNAs. We demonstrated the tumor-suppressive capacity of miR-192 in TNBC cells, which was exerted through inhibition of proliferation, induction of apoptosis, and reduction of migration. Gene expression and bioinformatics analyses of TNBC cell lines transfected with miR-192 mimics, identified a number of genes involved in migration including the Rho GTPase Activating Protein ARHGAP19. Through RNA immunoprecipitation we demonstrated the direct binding of miR-192 and ARHGAP19. Downregulation of ARHGAP19 expression by either miR-192 or siRNA inhibited migration of TNBC cells significantly. Our findings demonstrate that overexpression of epigenetically deregulated miR-192 decreases proliferation, promotes apoptosis, and inhibits migration of TNBC cell lines.

Wnt status-dependent oncogenic role of BCL9 and BCL9L in hepatocellular carcinoma.

BACKGROUND: Activation of Wnt/ß-catenin pathway is a frequent event in hepatocellular carcinoma and is associated with enhanced cell survival and proliferation. Therefore, targeting this signaling pathway is discussed as an attractive therapeutic approach for HCC treatment. BCL9 and BCL9L, two homologous coactivators of the ß-catenin transcription factor complex, have not yet been comprehensively characterized in HCC. We aimed to elucidate the roles of BCL9 and BCL9L, especially regarding Wnt/ß-catenin signaling and their prognostic value in HCC. METHODS: Expression of BCL9/BCL9L was determined in HCC cell lines (HLE, HLF, Huh7, HepG2, Hep3B, and Huh6) and normal liver cell lines (THLE-2 and THLE-3). To analyze proliferation and apoptosis, BCL9 and/or BCL9L were knocked down in Wnt-inactive HLE and Wnt-active HepG2 and Huh6 cells using siRNA. Subsequently, Wnt reporter assays were performed in HepG2 and Huh6 cells. BCL9 and BCL9L expression, clinicopathological and survival data of public HCC datasets were analyzed, taking the Wnt signaling status into account. RESULTS: Knockdown of BCL9L, but not of BCL9, reduced Wnt signaling activity. Knockdown of BCL9 and/or BCL9L reduced cell viability and increased apoptosis of Wnt-inactive HCC cells, but had no effect in Wnt-active cells. Expression of BCL9 and BCL9L was upregulated in human HCC and increased with progressing dedifferentiation. For BCL9L, higher expression was observed in tumors of larger size. Overexpression of BCL9 and BCL9L correlated with poor overall survival, especially in HCC without activated Wnt signaling. CONCLUSION: Oncogenic BCL9 proteins represent promising targets for cancer therapy and inhibiting them may be particularly beneficial in Wnt-inactive HCCs.

MTF1 binds to metal-responsive element e within the ATP7B promoter and is a strong candidate in regulating the ATP7B expression.

Wilson's disease is an autosomal recessive disorder resulting from copper excess. Some patients with clinical Wilson's disease symptoms exhibit no or only heterozygous pathogenic variants in the coding region of the disease-causing ATP7B gene. Therefore, the ATP7B promoter region is of special interest. Metal-responsive elements (MREs) located in the ATP7B promoter are promising motifs in modulating the ATP7B expression. We studied protein interaction of MREe, MREc, and MREd by electrophoretic mobility shift assays and revealed specific interactions for all MREs. We further narrowed down the specific binding site. Proteins potentially binding to the three MREs were identified by MatInspector analyses. Metal regulatory transcription factor 1 (MTF1) could be validated to bind to MREe by electrophoretic mobility shift assays. ATP7B promoter-driven reporter gene expression was significantly increased because of this interaction. MTF1 is a strong candidate in regulating the ATP7B expression through MREe binding.

Homozygous frame shift variant in ATP7B exon 1 leads to bypass of nonsense-mediated mRNA decay and to a protein capable of copper export.

Wilson disease (WD) is an autosomal recessive disease of copper excess due to pathogenic variants in the ATP7B gene coding for a copper-transporting ATPase. We present a 5-year-old girl with the homozygous frame shift variant NM_000053.3: c.19_20del in exon 1 of ATP7B (consecutive exon numbering with c.1 as first nucleotide of exon 1), detected by whole-exome sequencing as a secondary finding. The variant leads to a premature termination codon in exon 2. The girl exhibited no WD symptoms and no abnormalities in liver biopsy. ATP7B liver mRNA expression was comparable to healthy controls suggesting that nonsense-mediated mRNA decay (NMD) could be bypassed by the mechanism of translation reinitiation. To verify this hypothesis, a CMV-driven ATP7B minigene (pcDNA3) was equipped with the authentic ATP7B 5' untranslated region  and a truncated intron 2. We introduced c.19_20del by site-directed mutagenesis and overexpressed the constructs in HEK293T cells. We analyzed ATP7B expression by qRT-PCR, northern and western blot, and examined protein function by copper export capacity assays. Northern blot, qRT-PCR, and western blot revealed that c.19_20del ATP7B mRNA and protein is expressed in size and amount comparable to wild-type. Copper export capacity was also comparable to wild-type. Our results indicate that c.19_20del in ATP7B is able to bypass NMD by translation reinitiation, demonstrating that the classification of truncating variants as pathogenic without additional investigations should be done carefully.

Effect of TP53 contact and conformational mutations on cell survival and erythropoiesis of human hematopoietic stem cells in a long term culture model.

TP53 deficiencies characterize myeloid malignancies with a dismal prognosis. To unravel the pathomechanism of TP53 mutations in the development of myeloid malignancies, we analyzed the functional properties of TP53 conformational and contact mutations and TP53 loss in human CD34+ cells. We show for the first time that the analyzed conformational mutations lead to higher cell viability in human hematopoietic stem progenitor cells. In contrast to these conformational mutations, contact mutations interfered with efficient erythropoiesis. These findings show that not only the detection of a TP53 mutation is important, but also the specific mutation may play a role in malignant transformation and progression.

hsa-mir-183 is frequently methylated and related to poor survival in human hepatocellular carcinoma.

AIM: To screen clinically relevant microRNAs (miRNAs) silenced by DNA methylation in human hepatocellular carcinoma (HCC). METHODS: Knockdown of DNA methyltransferases (DNMTs) using siRNAs and miRNA profiling in HCC cell lines were performed to identify DNA hypermethylation-mediated miRNA downregulation. Confirmation using individual quantitative real-time PCR (qRT-PCR) assays was then performed followed by DNA methylation quantification at the promoter of the miRNA genes. Quantification of DNA methylation and miRNA expression was then performed in primary HCC tumor samples and related with clinicopathological variables. RESULTS: miRNA profiling after DNMT knockdown in HCC cell lines revealed upregulation of miR-23, miR-25 and miR-183. After qRT-PCR confirmation and CpG island methylation quantification of these miRNAs in cell lines, further analysis in primary HCC specimens showed that hsa-miR-183 is hypermethylated in 30% of HCC (n = 40). Expression of mature miR-183 showed an inverse correlation with DNA methylation levels. In HCC cells, DNMT knockdown and 5-aza-2'-deoxycytidine treatment reduced methylation and stimulated expression of miR-183. In HCC patients, hypermethylation at hsa-miR-183 promoter significantly correlates with poor survival (log-rank test P = 0.03). DNA methylation analysis in healthy liver, benign liver tumors (hepatocellular adenoma and focal nodular hyperplasia) and their corresponding adjacent tissues showed absence of hypermethylation supporting the notion that aberrant methylation at hsa-miR-183 is specific for the malignant transformation of hepatocytes. CONCLUSION: Our data indicate that hypermethylation of hsa-miR-183 is a frequent event in HCC and potentially useful as a novel surrogate diagnostic and prognostic marker.

The microRNA-449 family inhibits TGF-ß-mediated liver cancer cell migration by targeting SOX4.

BACKGROUND & AIMS: Modulation of microRNA expression is a potential treatment for hepatocellular carcinoma (HCC). Therefore, the epigenetically regulated microRNA-449 family (miR-449a, miR-449b, miR-449c) was characterized with regards to its functional effects and target genes in HCC. METHODS: After transfection of miR-449a, miR-449b, and/or miR-449c, tumor-relevant functional effects were analyzed using in vitro assays and a xenograft mouse model. Binding specificities, target genes, and regulated pathways of each miRNA were identified by microarray analyses. Target genes were validated by luciferase reporter assays and expression analyses in vitro. Furthermore, target gene expression was analyzed in 61 primary human HCCs compared to normal liver tissue. RESULTS: Tumor suppressive effects, binding specificities, target genes, and regulated pathways of miR-449a and miR-449b differed from those of miR-449c. Transfection of miR-449a, miR-449b, and/or miR-449c inhibited cell proliferation and migration, induced apoptosis, and reduced tumor growth to different extents. Importantly, miR-449a, miR-449b, and, to a lesser degree, miR-449c directly targeted SOX4, which codes for a transcription factor involved in epithelial-mesenchymal transition and HCC metastasis, and thereby inhibited TGF-ß-mediated cell migration. CONCLUSIONS: This study provides detailed insights into the regulatory network of the epigenetically regulated miRNA-449 family and, for the first time, describes distinct tumor suppressive effects and target specificities of miR-449a, miR-449b, and miR-449c. Our results indicate that particularly miR-449a and miR-449b may be considered for miRNA replacement therapy to prevent HCC progression and metastasis. LAY SUMMARY: In this study, we demonstrated that the microRNA-449 family acts as a tumor suppressor in liver cancer by causing cell death and inhibiting cell migration. These effects are caused by downregulation of the oncogene SOX4, which is frequently overexpressed in liver cancer. We conclude that the microRNA-449 family may be a target for liver cancer therapy.

HDAC inhibition activates the apoptosome via Apaf1 upregulation in hepatocellular carcinoma.

BACKGROUND: Histone deacetylation, a common hallmark in malignant tumors, strongly alters the transcription of genes involved in the control of proliferation, cell survival, differentiation and genetic stability. We have previously shown that HDAC1, HDAC2, and HDAC3 (HDAC1-3) genes encoding histone deacetylases 1-3 are upregulated in primary human hepatocellular carcinoma (HCC). The aim of this study was to characterize the functional effects of HDAC1-3 downregulation and to identify functionally important target genes of histone deacetylation in HCC. METHODS: Therefore, HCC cell lines were treated with the histone deacetylase inhibitor (HDACi) trichostatin A and by siRNA-knockdown of HDAC1-3. Differentially expressed mRNAs were identified after siRNA-knockdown of HDAC1-3 using mRNA expression profiling. Findings were validated after siRNA-mediated silencing of HDAC1-3 using qRTPCR and Western blotting assays. RESULTS: mRNA profiling identified apoptotic protease-activating factor 1 (Apaf1) to be significantly upregulated after HDAC inhibition (HLE siRNA#1/siRNA#2 p < 0.05, HLF siRNA#1/siRNA#2 p < 0.05). As a component of the apoptosome, a caspase-activating complex, Apaf1 plays a central role in the mitochondrial caspase activation pathway of apoptosis. Using annexin V, a significant increase in apoptosis could also be shown in HLE (siRNA #1 p = 0.0034) and HLF after siRNA against HDAC1-3 (Fig. 3a, b). In parallel, caspase-9 activity was increased after siRNA-knockdown of HDAC1-3 leading to enhanced apoptosis after HDAC inhibition (Fig. 3c, d). CONCLUSIONS: The present data show that siRNA-knockdown of HDAC1-3 plays a major role in mediating apoptotic response to HDAC inhibitors through regulation of Apaf1.

Interferon-Mediated Cytokine Induction Determines Sustained Virus Control in Chronic Hepatitis C Virus Infection.

Hepatitis C virus (HCV) infection is a major cause of chronic liver disease and associated complications such as liver cirrhosis and hepatocellular carcinoma. Interferons (IFNs) are crucial for HCV clearance and a sustained virological response (SVR), but a significant proportion of patients do not respond to IFNα. The underlying mechanisms of an insufficient IFN response remain largely unknown. In this study, we found that patients responding to IFNα with viral clearance had significantly higher serum levels of TNF-related apoptosis inducing ligand (TRAIL), compared with patients who failed to control HCV. In addition, upon direct IFNα exposure, peripheral blood mononuclear cells (PBMCs) from patients with SVR upregulated TRAIL, as well as IFN-γ and the chemokines CXCL9 and CXCL10, much more strongly than cells from patients with antiviral treatment failure. As a possible mechanism of the stronger IFNα-induced cytokine response, we identified higher levels of expression and phosphorylation of the transcription factor STAT1 in PBMCs from patients with SVR. Increased TRAIL expression additionally involved the NF-κB and JNK signaling pathways. Thus, SVR in chronic HCV infection is associated with a strong IFNα-induced cytokine response, which might allow for the early prediction of treatment efficacy in HCV infection.

The heteromeric transcription factor GABP activates the ITGAM/CD11b promoter and induces myeloid differentiation.

The heteromeric transcription factor GA-binding protein (GABP) consists of two subunits, the alpha subunit (GABPA) carrying the DNA-binding ETS domain, and the beta subunit (GABPB1) harbouring the transcriptional activation domain. GABP is involved in haematopoietic stem cell maintenance and differentiation of myeloid and lymphoid lineages in mice. To elucidate the molecular function of GABP in human haematopoiesis, the present study addressed effects of ectopic overexpression of GABP focussing on the myeloid compartment. Combined overexpression of GABPA and GABPB1 caused a proliferation block in cell lines and drastically reduced the colony-forming capacity of murine lineage-negative cells. Impaired proliferation resulted from perturbed cellular cycling and induction of myeloid differentiation shown by surface markers and myelomonocytic morphology of U937 cells. Depending on the dosage and functional integrity of GABP, ITGAM expression was induced. ITGAM encodes CD11b, the alpha subunit of integrin Mac-1, whose beta subunit, ITGB2/CD18, was already described to be regulated by GABP. Finally, Shield1-dependent proteotuning, luciferase reporter assays and chromatin immunoprecipitation showed that GABP activates the ITGAM/CD11b promoter via three binding sites close to the translational start site. In conclusion, the present study supports the crucial role of GABP in myeloid cell differentiation and identified ITGAM/CD11b as a novel GABP target gene.

Insulin-like growth factor 2 mRNA-binding protein 1 (IGF2BP1) is an important protumorigenic factor in hepatocellular carcinoma.

UNLABELLED: Hepatocarcinogenesis is a stepwise process. It involves several genetic and epigenetic alterations, e.g., loss of tumor suppressor gene expression (TP53, PTEN, RB) as well as activation of oncogenes (c-MYC, MET, BRAF, RAS). However, the role of RNA-binding proteins (RBPs), which regulate tumor suppressor and oncogene expression at the posttranscriptional level, are not well understood in hepatocellular carcinoma (HCC). Here we analyzed RBPs induced in human liver cancer, revealing 116 RBPs with a significant and more than 2-fold higher expression in HCC compared to normal liver tissue. We focused our subsequent analyses on the Insulin-like growth factor 2 messenger RNA (mRNA)-binding protein 1 (IGF2BP1) representing the most strongly up-regulated RBP in HCC in our cohort. Depletion of IGF2BP1 from multiple liver cancer cell lines inhibits proliferation and induces apoptosis in vitro. Accordingly, murine xenograft assays after stable depletion of IGF2BP1 reveal that tumor growth, but not tumor initiation, strongly depends on IGF2BP1 in vivo. At the molecular level, IGF2BP1 binds to and stabilizes the c-MYC and MKI67 mRNAs and increases c-Myc and Ki-67 protein expression, two potent regulators of cell proliferation and apoptosis. These substrates likely mediate the impact of IGF2BP1 in human liver cancer, but certainly additional target genes contribute to its function. CONCLUSION: The RNA-binding protein IGF2BP1 is an important protumorigenic factor in liver carcinogenesis. Hence, therapeutic targeting of IGF2BP1 may offer options for intervention in human HCC.

1,25-Dihydroxyvitamin D decreases HTRA1 promoter activity in the rhesus monkey--a plausible explanation for the influence of vitamin D on age-related macular degeneration?

Age-related macular degeneration is the major cause of blindness in the elderly worldwide and the risk is influenced by both environmental and genetic risk factors. One important disease-associated region in humans is located on 10q26 and includes the two candidate genes ARMS2 and HTRA1. However, determination of the causative gene has not yet been possible and examining the situation in the rhesus monkey may help understand the situation in humans. In a recent paper, we characterized the rhesus monkey 10q26-orthologue region on chromosome 9 in detail and identified the drusen-associated HTRA1 promoter SNP rs196357513 as a putative risk factor. In this study, we predicted 9 binding sites for the vitamin D-dependent transcription factor vitamin D receptor in the rhesus HTRA1 promoter, one of which is destroyed by the rs196357513-risk allele. As patients with vitamin D deficit are at increased risk for age-related macular degeneration, a luciferase assay in transiently transfected ARPE19-cells was performed to evaluate the influence of the SNP rs196357513 and of 1,25-dihydroxyvitamin D on the rhesus monkey HTRA1 promoter activity. This revealed that the luciferase activity of the promoter construct containing the rs196357513 wild type allele was significantly reduced after vitamin D stimulation. An in silico analysis and literature search imply that this regulation could also play a role in human HTRA1 expression. Moreover, HTRA1 promoter activity of the construct containing the rs196357513 risk allele appeared diminished in comparison to the construct with the wild type allele, albeit this difference was not significant. The lower promoter activity due to the rhesus monkey rs196357513 risk allele apparently contradicts the common hypothesis for the human HTRA1 promoter risk allele of SNP rs11200638, for which a higher promoter activity has been observed. Our data point to a yet unexpected effect of decreased HTRA1 expression on drusen pathogenesis. Thus not only a higher HTRA1 expression, but an imbalance of HTRA1 might be disease-relevant. Both findings require closer analysis, but if relevance for humans proves true, it would impact current age-related macular degeneration research and treatment.

Posttranscriptional destabilization of the liver-specific long noncoding RNA HULC by the IGF2 mRNA-binding protein 1 (IGF2BP1).

UNLABELLED: Selected long noncoding RNAs (lncRNAs) have been shown to play important roles in carcinogenesis. Although the cellular functions of these transcripts can be diverse, many lncRNAs regulate gene expression. In contrast, factors that control the expression of lncRNAs remain largely unknown. Here we investigated the impact of RNA binding proteins on the expression of the liver cancer-associated lncRNA HULC (highly up-regulated in liver cancer). First, we validated the strong up-regulation of HULC in human hepatocellular carcinoma. To elucidate posttranscriptional regulatory mechanisms governing HULC expression, we applied an RNA affinity purification approach to identify specific protein interaction partners and potential regulators. This method identified the family of IGF2BPs (IGF2 mRNA-binding proteins) as specific binding partners of HULC. Depletion of IGF2BP1, also known as IMP1, but not of IGF2BP2 or IGF2BP3, led to an increased HULC half-life and higher steady-state expression levels, indicating a posttranscriptional regulatory mechanism. Importantly, HULC represents the first IGF2BP substrate that is destabilized. To elucidate the mechanism by which IGF2BP1 destabilizes HULC, the CNOT1 protein was identified as a novel interaction partner of IGF2BP1. CNOT1 is the scaffold of the human CCR4-NOT deadenylase complex, a major component of the cytoplasmic RNA decay machinery. Indeed, depletion of CNOT1 increased HULC half-life and expression. Thus, IGF2BP1 acts as an adaptor protein that recruits the CCR4-NOT complex and thereby initiates the degradation of the lncRNA HULC. CONCLUSION: Our findings provide important insights into the regulation of lncRNA expression and identify a novel function for IGF2BP1 in RNA metabolism.