Therapeutic Targeting of the GSK3ß-CUGBP1 Pathway in Myotonic Dystrophy.

Myotonic Dystrophy type 1 (DM1) is a neuromuscular disease associated with toxic RNA containing expanded CUG repeats. The developing therapeutic approaches to DM1 target mutant RNA or correct early toxic events downstream of the mutant RNA. We have previously described the benefits of the correction of the GSK3ß-CUGBP1 pathway in DM1 mice (HSALR model) expressing 250 CUG repeats using the GSK3 inhibitor tideglusib (TG). Here, we show that TG treatments corrected the expression of ~17% of genes misregulated in DM1 mice, including genes involved in cell transport, development and differentiation. The expression of chloride channel 1 (Clcn1), the key trigger of myotonia in DM1, was also corrected by TG. We found that correction of the GSK3ß-CUGBP1 pathway in mice expressing long CUG repeats (DMSXL model) is beneficial not only at the prenatal and postnatal stages, but also during adulthood. Using a mouse model with dysregulated CUGBP1, which mimics alterations in DM1, we showed that the dysregulated CUGBP1 contributes to the toxicity of expanded CUG repeats by changing gene expression and causing CNS abnormalities. These data show the critical role of the GSK3ß-CUGBP1 pathway in DM1 muscle and in CNS pathologies, suggesting the benefits of GSK3 inhibitors in patients with different forms of DM1.

Myotonic Dystrophy: From Molecular Pathogenesis to Therapeutics.

Current studies concerning myotonic dystrophy type 1 (DM1) are in the process of transitioning from molecular investigations to preclinical and clinical trials [...].

Development of Therapeutic Approaches for Myotonic Dystrophies Type 1 and Type 2.

Myotonic Dystrophies type 1 (DM1) and type 2 (DM2) are complex multisystem diseases without disease-based therapies. These disorders are caused by the expansions of unstable CTG (DM1) and CCTG (DM2) repeats outside of the coding regions of the disease genes: DMPK in DM1 and CNBP in DM2. Multiple clinical and molecular studies provided a consensus for DM1 pathogenesis, showing that the molecular pathophysiology of DM1 is associated with the toxicity of RNA CUG repeats, which cause multiple disturbances in RNA metabolism in patients' cells. As a result, splicing, translation, RNA stability and transcription of multiple genes are misregulated in DM1 cells. While mutant CCUG repeats are the main cause of DM2, additional factors might play a role in DM2 pathogenesis. This review describes current progress in the translation of mechanistic knowledge in DM1 and DM2 to clinical trials, with a focus on the development of disease-specific therapies for patients with adult forms of DM1 and congenital DM1 (CDM1).

Correction of GSK3ß at young age prevents muscle pathology in mice with myotonic dystrophy type 1.

Myotonic dystrophy type 1 (DM1) is a progressive neuromuscular disease caused by expanded CUG repeats, which misregulate RNA metabolism through several RNA-binding proteins, including CUG-binding protein/CUGBP1 elav-like factor 1 (CUGBP1/CELF1) and muscleblind 1 protein. Mutant CUG repeats elevate CUGBP1 and alter CUGBP1 activity via a glycogen synthase kinase 3ß (GSK3ß)-cyclin D3-cyclin D-dependent kinase 4 (CDK4) signaling pathway. Inhibition of GSK3ß corrects abnormal activity of CUGBP1 in DM1 mice [human skeletal actin mRNA, containing long repeats ( HSALR) model]. Here, we show that the inhibition of GSK3ß in young HSALR mice prevents development of DM1 muscle pathology. Skeletal muscle in 1-yr-old HSALR mice, treated at 1.5 mo for 6 wk with the inhibitors of GSK3, exhibits high fiber density, corrected atrophy, normal fiber size, with reduced central nuclei and normalized grip strength. Because CUG-GSK3ß-cyclin D3-CDK4 converts the active form of CUGBP1 into a form of translational repressor, we examined the contribution of CUGBP1 in myogenesis using Celf1 knockout mice. We found that a loss of CUGBP1 disrupts myogenesis, affecting genes that regulate differentiation and the extracellular matrix. Proteins of those pathways are also misregulated in young HSALR mice and in muscle biopsies of patients with congenital DM1. These findings suggest that the correction of GSK3ß-CUGBP1 pathway in young HSALR mice might have a positive effect on the myogenesis over time.-Wei, C., Stock, L., Valanejad, L., Zalewski, Z. A., Karns, R., Puymirat, J., Nelson, D., Witte, D., Woodgett, J., Timchenko, N. A., Timchenko, L. Correction of GSK3ß at young age prevents muscle pathology in mice with myotonic dystrophy type 1.

Correction of RNA-Binding Protein CUGBP1 and GSK3ß Signaling as Therapeutic Approach for Congenital and Adult Myotonic Dystrophy Type 1.

Myotonic dystrophy type 1 (DM1) is a complex genetic disease affecting many tissues. DM1 is caused by an expansion of CTG repeats in the 3'-UTR of the DMPK gene. The mechanistic studies of DM1 suggested that DMPK mRNA, containing expanded CUG repeats, is a major therapeutic target in DM1. Therefore, the removal of the toxic RNA became a primary focus of the therapeutic development in DM1 during the last decade. However, a cure for this devastating disease has not been found. Whereas the degradation of toxic RNA remains a preferential approach for the reduction of DM1 pathology, other approaches targeting early toxic events downstream of the mutant RNA could be also considered. In this review, we discuss the beneficial role of the restoring of the RNA-binding protein, CUGBP1/CELF1, in the correction of DM1 pathology. It has been recently found that the normalization of CUGBP1 activity with the inhibitors of GSK3 has a positive effect on the reduction of skeletal muscle and CNS pathologies in DM1 mouse models. Surprisingly, the inhibitor of GSK3, tideglusib also reduced the toxic CUG-containing RNA. Thus, the development of the therapeutics, based on the correction of the GSK3ß-CUGBP1 pathway, is a promising option for this complex disease.

Reduction of toxic RNAs in myotonic dystrophies type 1 and type 2 by the RNA helicase p68/DDX5.

Myotonic dystrophies type 1 (DM1) and type 2 (DM2) are neuromuscular diseases, caused by accumulation of CUG and CCUG RNAs in toxic aggregates. Here we report that the increased stability of the mutant RNAs in both types of DM is caused by deficiency of RNA helicase p68. We have identified p68 by studying CCUG-binding proteins associated with degradation of the mutant CCUG repeats. Protein levels of p68 are reduced in DM1 and DM2 biopsied skeletal muscle. Delivery of p68 in DM1/2 cells causes degradation of the mutant RNAs, whereas delivery of p68 in skeletal muscle of DM1 mouse model reduces skeletal muscle myopathy and atrophy. Our study shows that correction of p68 may reduce toxicity of the mutant RNAs in DM1 and in DM2.

FXR-Gankyrin axis is involved in development of pediatric liver cancer.

The development of hepatoblastoma (HBL) is associated with failure of hepatic stem cells (HSC) to differentiate into hepatocytes. Despite intensive investigations, mechanisms of the failure of HSC to differentiate are not known. We found that oncogene Gankyrin (Gank) is involved in the inhibition of differentiation of HSC via triggering degradation of tumor suppressor proteins (TSPs) Rb, p53, C/EBPα and HNF4α. Our data show that the activation of a repressor of Gank, farnesoid X receptor, FXR, after initiation of liver cancer by Diethylnitrosamine (DEN) prevents the development of liver cancer by inhibiting Gank and rescuing tumor suppressor proteins. We next analyzed FXR-Gank-Tumor suppressor pathways in a large cohort of HBL patients which include 6 controls and 53 HBL samples. Systemic analysis of these samples and RNA-Seq approach revealed that the FXR-Gank axis is activated; markers of hepatic stem cells are dramatically elevated and hepatocyte markers are reduced in HBL samples. In the course of these studies, we found that RNA binding protein CUGBP1 is a new tumor suppressor protein which is reduced in all HBL samples. Therefore, we generated CUGBP1 KO mice and examined HBL signatures in the liver of these mice. Micro-array studies revealed that the HBL-specific molecular signature is developed in livers of CUGBP1 KO mice at very early ages. Thus, we conclude that FXR-Gank-TSPs-Stem cells pathway is a key determinant of liver cancer in animal models and in pediatric liver cancer. Our data provide a strong basis for development of FXR-Gank-based therapy for treatment of patients with hepatoblastoma.

Cooperation of C/EBP family proteins and chromatin remodeling proteins is essential for termination of liver regeneration.

UNLABELLED: Liver cancer is the fifth most common cancer. A highly invasive surgical resection of the liver tumor is the main approach used to eliminate the tumor. Mechanisms that terminate liver regeneration when the liver reaches the original size are not known. The aims of this work were to generate an animal model that fails to stop liver regeneration after surgical resections and elucidate mechanisms that are involved in termination of liver regeneration. Because epigenetic control of liver function has been previously implicated in the regulation of liver proliferation, we generated C/EBPα-S193A knockin mice, which have alterations in formation of complexes of C/EBP family proteins with chromatin remodeling proteins. The C/EBPα-S193A mice have altered liver morphology and altered liver function leading to changes of glucose metabolism and blood parameters. Examination of the proliferative capacity of C/EBPα-S193A livers showed that livers of S193A mice have a higher rate of proliferation after birth, but stop proliferation at the age of 2 months. These animals have increased liver proliferation in response to liver surgery as well as carbon tetrachloride (CCl4 )-mediated injury. Importantly, livers of C/EBPα-S193A mice fail to stop liver regeneration after surgery when livers reach the original, preresection, size. The failure of S193A livers to stop regeneration correlates with the epigenetic repression of key regulators of liver proliferation C/EBPα, p53, FXR, SIRT1, PGC1α, and TERT by C/EBPß-HDAC1 complexes. The C/EBPß-HDAC1 complexes also repress promoters of enzymes of glucose synthesis PEPCK and G6Pase. CONCLUSION: Proper cooperation of C/EBP and chromatin remodeling proteins is essential for the termination of liver regeneration after surgery and for maintenance of liver functions.

Smaug/SAMD4A restores translational activity of CUGBP1 and suppresses CUG-induced myopathy.

We report the identification and characterization of a previously unknown suppressor of myopathy caused by expansion of CUG repeats, the mutation that triggers Myotonic Dystrophy Type 1 (DM1). We screened a collection of genes encoding RNA-binding proteins as candidates to modify DM1 pathogenesis using a well established Drosophila model of the disease. The screen revealed smaug as a powerful modulator of CUG-induced toxicity. Increasing smaug levels prevents muscle wasting and restores muscle function, while reducing its function exacerbates CUG-induced phenotypes. Using human myoblasts, we show physical interactions between human Smaug (SMAUG1/SMAD4A) and CUGBP1. Increased levels of SMAUG1 correct the abnormally high nuclear accumulation of CUGBP1 in myoblasts from DM1 patients. In addition, augmenting SMAUG1 levels leads to a reduction of inactive CUGBP1-eIF2α translational complexes and to a correction of translation of MRG15, a downstream target of CUGBP1. Therefore, Smaug suppresses CUG-mediated muscle wasting at least in part via restoration of translational activity of CUGBP1.

Age-associated change of C/EBP family proteins causes severe liver injury and acceleration of liver proliferation after CCl4 treatments.

The aged liver is more sensitive to the drug treatments and has a high probability of developing liver disorders such as fibrosis, cirrhosis, and cancer. Here we present mechanisms underlying age-associated severe liver injury and acceleration of liver proliferation after CCl4 treatments. We have examined liver response to CCl4 treatments using old WT mice and young C/EBPα-S193D knockin mice, which express an aged-like isoform of C/EBPα. Both animal models have altered chromatin structure as well as increased liver injury and proliferation after acute CCl4 treatments. We found that these age-related changes are associated with the repression of key regulators of liver biology: C/EBPα, Farnesoid X Receptor (FXR) and telomere reverse transcriptase (TERT). In quiescent livers of old WT and young S193D mice, the inhibition of TERT is mediated by HDAC1-C/EBPα complexes. After CCl4 treatments, TERT, C/EBPα and FXR are repressed by different mechanisms. These mechanisms include the increase of a dominant negative isoform, C/EBPß-LIP, and subsequent repression of C/EBPα, FXR, and TERT promoters. C/EBPß-LIP also disrupts Rb-E2F1 complexes in C/EBPα-S193D mice after CCl4 treatments. To examine if these alterations are involved in drug-mediated liver diseases, we performed chronic treatments of mice with CCl4. We found that C/EBPα-S193D mice developed fibrosis much more rapidly than WT mice. Thus, our data show that the age-associated alterations of C/EBP proteins create favorable conditions for the increased liver proliferation after CCl4 treatments and for development of drug-mediated liver diseases.

Transcriptional and translational regulation of C/EBPß-HDAC1 protein complexes controls different levels of p53, SIRT1, and PGC1α proteins at the early and late stages of liver cancer.

Cancer changes biological processes in the liver by altering gene expression at the levels of transcription, translation, and protein modification. The RNA binding protein CUGBP1 is a key regulator of translation of CCAAT enhancer binding protein ß and histone deacetylase 1 (HDAC1). These proteins form complexes that are involved in the regulation of liver biology. Here we show a critical role of the translational activation of CCAAT/enhancer binding protein ß-HDAC1 complexes in the development of liver cancer mediated by diethylnitrosamine. We found that diethylnitrosamine increases the levels of CUGBP1 and activates CUGBP1 by phosphorylation, leading to the formation of the CUGBP1-eIF2 complex, which is an activator of translation of CUGBP1-dependent mRNAs. The elevation of the CUGBP1-eIF2 complex increases translation of C/EBPß and HDAC1, resulting in an increase of C/EBPß-HDAC1 complexes at later stages of liver cancer. We found that C/EBPß-HDAC1 complexes repress promoters of three key regulators of liver functions: p53, SIRT1, and PGC1α. As the result of this suppression, the p53-, SIRT1-, and PGC1α-dependent downstream pathways are reduced, leading to increased liver proliferation. We also found that the proper regulation of C/EBPß-HDAC1 complexes is required for the maintenance of biological levels of p53, SIRT1, and PGC1α in quiescent livers and at early stages of liver cancer. Taken together, these studies showed that the development of liver cancer includes a tight regulation of levels of C/EBPß-HDAC1 complexes on the levels of transcription, translation, and posttranslational modifications.

Inhibition of Histone Deacetylase Activity Increases Cisplatin Efficacy to Eliminate Metastatic Cells in Pediatric Liver Cancers.

The pediatric liver cancers, hepatoblastoma and hepatocellular carcinoma, are dangerous cancers which often spread to the lungs. Although treatments with cisplatin significantly improve outcomes, cisplatin may not eliminate metastasis-initiating cells. Our group has recently shown that the metastatic microenvironments of hepatoblastoma contain Cancer Associated Fibroblasts (CAFs) and neuron-like cells, which initiate cancer spread from liver to lungs. In this study, we found that these cells express high levels of HDAC1; therefore, we examined if histone deacetylase inhibition improves cisplatin anti-proliferative effects and reduces the formation of tumor clusters in pediatric liver cancer metastatic microenvironments. METHODS: New cell lines were generated from primary hepatoblastoma liver tumors (hbl) and lung metastases (LM) of HBL patients. In addition, cell lines were generated from hepatocellular neoplasm, not otherwise specified (HCN-NOS) tumor samples, and hcc cell lines. Hbl, LM and hcc cells were treated with cisplatin, SAHA or in combination. The effect of these drugs on the number of cells, formation of tumor clusters and HDAC1-Sp5-p21 axis were examined. RESULTS: Both HBL and HCC tissue specimens have increased HDAC1-Sp5 pathway activation, recapitulated in cell lines generated from the tumors. HDAC inhibition with vorinostat (SAHA) increases cisplatin efficacy to eliminate CAFs in hbl and in hcc cell lines. Although the neuron-like cells survive the combined treatments, proliferation was inhibited. Notably, combining SAHA with cisplatin overcame cisplatin resistance in an LM cell line from an aggressive case with multiple metastases. Underlying mechanisms of this enhanced inhibition include suppression of the HDAC1-Sp5 pathway and elevation of an inhibitor of proliferation p21. Similar findings were found with gemcitabine treatments suggesting that elimination of proliferative CAFs cells is a key event in the inhibition of mitotic microenvironment. CONCLUSIONS: Our studies demonstrate the synergistic benefits of HDAC inhibition and cisplatin to eliminate metastasis-initiating cells in pediatric liver cancers.

Cellular origin and molecular mechanisms of lung metastases in patients with aggressive hepatoblastoma.

BACKGROUND AND AIMS: Lung metastases are the most threatening signs for patients with aggressive hepatoblastoma (HBL). Despite intensive studies, the cellular origin and molecular mechanisms of lung metastases in patients with aggressive HBL are not known. The aims of these studies were to identify metastasis-initiating cells in primary liver tumors and to determine if these cells are secreted in the blood, reach the lung, and form lung metastases. APPROACH: We have examined mechanisms of activation of key oncogenes in primary liver tumors and lung metastases and the role of these mechanisms in the appearance of metastasis-initiating cells in patients with aggressive HBL by RNA-Seq, immunostaining, chromatin immunoprecipitation, Real-Time Quantitative Reverse Transcription PCR and western blot approaches. Using a protocol that mimics the exit of metastasis-initiating cells from tumors, we generated 16 cell lines from liver tumors and 2 lines from lung metastases of patients with HBL. RESULTS: We found that primary HBL liver tumors have a dramatic elevation of neuron-like cells and cancer-associated fibroblasts and that these cells are released into the bloodstream of patients with HBL and found in lung metastases. In the primary liver tumors, the ph-S675-ß-catenin pathway activates the expression of markers of cancer-associated fibroblasts; while the ZBTB3-SRCAP pathway activates the expression of markers of neurons via cancer-enhancing genomic regions/aggressive liver cancer domains leading to a dramatic increase of cancer-associated fibroblasts and neuron-like cells. Studies of generated metastasis-initiating cells showed that these cells proliferate rapidly, engage in intense cell-cell interactions, and form tumor clusters. The inhibition of ß-catenin in HBL/lung metastases-released cells suppresses the formation of tumor clusters. CONCLUSIONS: The inhibition of the ß-catenin-cancer-enhancing genomic regions/aggressive liver cancer domains axis could be considered as a therapeutic approach to treat/prevent lung metastases in patients with HBL.

Intracellular signaling and hepatocellular carcinoma.

Liver cancer is the fifth most common cancer and the third most common cause of cancer related death in the world. The recent development of new techniques for the investigations of global change in the gene expression, signaling pathways and wide genome binding has provided novel information for the mechanisms underlying liver cancer progression. Although these studies identified gene expression signatures in hepatocellular carcinoma, the early steps of the development of hepatocellular carcinomas (HCC) are not well understood. The development of HCC is a multistep process which includes the progressive alterations of gene expression leading to the increased proliferation and to liver cancer. This review summarizes recent progress in the identification of the key steps of the development of HCC with the focus on early events of carcinogenesis and on the role of translational and epigenetic alterations in the development of HCC. Quiescent stage of the liver is supported by several tumor suppressor proteins including p53, Rb and C/EBPα. Studies with chemical models of liver carcinogenesis and with human HCC have shown that the elevation of gankyrin is responsible for the elimination of these three proteins at early steps of carcinogenesis. Later stages of progression of the liver cancer are associated with alterations in many signaling pathways including translation which leads to epigenetic silencing/activation of many genes. Particularly, recent reports suggest a critical role of histone deacetylase 1, HDAC1, in the development of HCC through the interactions with transcription factors such as C/EBP family proteins.

RNA Foci, CUGBP1, and ZNF9 are the primary targets of the mutant CUG and CCUG repeats expanded in myotonic dystrophies type 1 and type 2.

Expansions of noncoding CUG and CCUG repeats in myotonic dystrophies type 1 (DM1) and DM2 cause complex molecular pathology, the features of which include accumulation of RNA aggregates and misregulation of the RNA-binding proteins muscleblind-like 1 (MBNL1) and CUG-binding protein 1 (CUGBP1). CCUG repeats also decrease amounts of the nucleic acid binding protein ZNF9. Using tetracycline (Tet)-regulated monoclonal cell models that express CUG and CCUG repeats, we found that low levels of long CUG and CCUG repeats result in nuclear and cytoplasmic RNA aggregation with a simultaneous increase of CUGBP1 and a reduction of ZNF9. Elevation of CUGBP1 and reduction of ZNF9 were also observed before strong aggregation of the mutant CUG/CCUG repeats. Degradation of CUG and CCUG repeats normalizes ZNF9 and CUGBP1 levels. Comparison of short and long CUG and CCUG RNAs showed that great expression of short repeats form foci and alter CUGBP1 and ZNF9; however, long CUG/CCUG repeats misregulate CUGBP1 and ZNF9 much faster than high levels of the short repeats. These data suggest that correction of DM1 and DM2 might be achieved by complete and efficient degradation of CUG and CCUG repeats or by a simultaneous disruption of CUG/CCUG foci and correction of CUGBP1 and ZNF9.

Phosphorylation-Mediated Activation of ß-Catenin-TCF4-CEGRs/ALCDs Pathway Is an Essential Event in Development of Aggressive Hepatoblastoma.

BACKGROUND AND AIMS: Hepatoblastoma (HBL), a deadly malignancy in children, is the most common type of pediatric liver cancer. We recently demonstrated that ß-catenin, phosphorylated at S675 (ph-S675-ß-catenin), causes pathological alterations in fibrolamellar hepatocellular carcinoma (FLC), by activating oncogenes and fibrotic genes via human genomic regions, known as cancer-enhancing genomic regions or aggressive liver cancer domains (CEGRs/ALCDs). The aim of this study was to determine the role of the ph-S675-ß-catenin-TCF4-CEGRs/ALCDs pathway in HBL. METHODS: The ph-S675-ß-catenin-TCF4-CEGRs/ALCDs pathway was examined in a large cohort of HBL specimens, in HBL cell lines HepG2 and Huh6, and in patient-derived xenografts (PDXs). RESULTS: ß-catenin is phosphorylated at S675 in a large portion of tested HBL patients. In these patients, ph-S675-ß-catenin forms complexes with TCF4 and opens CEGRs/ALCDs-dependent oncogenes for transcription, leading to a massive overexpression of the oncogenes. The inhibition of the ß-catenin-TCF4-CEGRs/ALCDs axis inhibits the proliferation of cancer cells and tumor growth in HBL cell lines and HBL-PDXs. The ph-S675-ß-catenin is abundant in mitotic cells. We found that markers of HBL Glypican 3 (GPC3) and Alpha Fetoprotein (AFP) are increased in HBL patients by ß-catenin-TCF4-p300 complexes. CONCLUSIONS: The phosphorylation-mediated activation of the ß-catenin-TCF4-p300-CEGRs/ALCDs pathway increases oncogene expression in patients with aggressive liver cancer and promotes the development of hepatoblastoma.

ß-catenin cancer-enhancing genomic regions axis is involved in the development of fibrolamellar hepatocellular carcinoma.

Fibrolamellar hepatocellular carcinoma (FLC) is a disease that occurs in children and young adults. The development of FLC is associated with creation of a fusion oncoprotein DNAJB1-PKAc kinase, which activates multiple cancer-associated pathways. The aim of this study was to examine the role of human genomic regions, called cancer-enhancing genomic regions or aggressive liver cancer domains (CEGRs/ALCDs), in the development of FLC. Previous studies revealed that CEGRs/ALCDs are located in multiple oncogenes and cancer-associated genes, regularly silenced in normal tissues. Using the regulatory element locus intersection (RELI) algorithm, we searched a large compendium of chromatin immunoprecipitation-sequencing (ChIP) data sets and found that CEGRs/ALCDs contain regulatory elements in several human cancers outside of pediatric hepatic neoplasms. The RELI algorithm further identified components of the ß-catenin-TCF7L2/TCF4 pathway, which interacts with CEGRs/ALCDs in several human cancers. Particularly, the RELI algorithm found interactions of transcription factors and chromatin remodelers with many genes that are activated in patients with FLC. We found that these FLC-specific genes contain CEGRs/ALCDs, and that the driver of FLC, fusion oncoprotein DNAJB1-PKAc, phosphorylates ß-catenin at Ser675, resulting in an increase of ß-catenin-TCF7L2/TCF4 complexes. These complexes increase a large family of CEGR/ALCD-dependent collagens and oncogenes. The DNAJB1-PKAc-ß-catenin-CEGR/ALCD pathway is preserved in lung metastasis. The inhibition of ß-catenin in FLC organoids inhibited the expression of CEGRs/ALCDs-dependent collagens and oncogenes, preventing the formation of the organoid's structure. Conclusion: This study provides a rationale for the development of ß-catenin-based therapy for patients with FLC.

Expansion of CUG RNA repeats causes stress and inhibition of translation in myotonic dystrophy 1 (DM1) cells.

The purpose of this study was to investigate the role of the mutant CUGn RNA in the induction of stress in type 1 myotonic dystrophy (DM1) cells and in the stress-mediated inhibition of protein translation in DM1. To achieve our goals, we performed HPLC-based purification of stress granules (SGs), immunoanalysis of SGs with stress markers TIA-1, CUGBP1, and ph-eIF2, site-specific mutagenesis, and examinations of RNA-protein and protein-protein interactions in myoblasts from control and DM1 patients. The cause-and-effect relationships were addressed in stable cells expressing mutant CUG repeats. We found that the mutant CUGn RNA induces formation of SGs through the increase of the double-stranded RNA-dependent protein kinase (PKR) and following inactivation of eIF2α, one of the substrates of PKR. We show that SGs trap mRNA coding for the DNA repair and remodeling factor MRG15 (MORF4L1), translation of which is regulated by CUGBP1. As the result of the trapping, the levels of MRG15 are reduced in DM1 cells and in CUG-expressing cells. These data show that CUG repeats cause stress in DM1 through the PKR-ph-eIF2α pathway inhibiting translation of certain mRNAs, such as MRG15 mRNA. The repression of protein translation by stress might contribute to the progressive muscle loss in DM1.

HDAC1-Dependent Repression of Markers of Hepatocytes and P21 Is Involved in Development of Pediatric Liver Cancer.

BACKGROUND & AIMS: Epigenetic regulation of gene expression plays a critical role in the development of liver cancer; however, the molecular mechanisms of epigenetic-driven liver cancers are not well understood. The aims of this study were to examine molecular mechanisms that cause the dedifferentiation of hepatocytes into cancer cells in aggressive hepatoblastoma and test if the inhibition of these mechanisms inhibits tumor growth. METHODS: We have analyzed CCAAT/Enhancer Binding Protein alpha (C/EBPα), Transcription factor Sp5, and histone deacetylase (HDAC)1 pathways from a large biobank of fresh hepatoblastoma (HBL) samples using high-pressure liquid chromatography-based examination of protein-protein complexes and have examined chromatin remodeling on the promoters of markers of hepatocytes and p21. The HDAC1 activity was inhibited in patient-derived xenograft models of HBL and in cultured hepatoblastoma cells and expression of HDAC1-dependent markers of hepatocytes was examined. RESULTS: Analyses of a biobank showed that a significant portion of HBL patients have increased levels of an oncogenic de-phosphorylated-S190-C/EBPα, Sp5, and HDAC1 compared with amounts of these proteins in adjacent regions. We found that the oncogenic de-phosphorylated-S190-C/EBPα is created in aggressive HBL by protein phosphatase 2A, which is increased within the nucleus and dephosphorylates C/EBPα at Ser190. C/EBPα-HDAC1 and Sp5-HDAC1 complexes are abundant in hepatocytes, which dedifferentiate into cancer cells. Studies in HBL cells have shown that C/EBPα-HDAC1 and Sp5-HDAC1 complexes reduce markers of hepatocytes and p21 via repression of their promoters. Pharmacologic inhibition of C/EBPα-HDAC1 and Sp5-HDAC1 complexes by Suberoylanilide hydroxamic acid (SAHA) and small interfering RNA-mediated inhibition of HDAC1 increase expression of hepatocyte markers, p21, and inhibit proliferation of cancer cells. CONCLUSIONS: HDAC1-mediated repression of markers of hepatocytes is an essential step for the development of HBL, providing background for generation of therapies for aggressive HBL by targeting HDAC1 activities.

Reduction of the rate of protein translation in patients with myotonic dystrophy 2.

Myotonic dystrophy 2 (DM2) is an autosomal dominant, multisystem disease, which primarily affects skeletal muscle. DM2 is caused by CCTGn expansion in the intron 1 of the ZNF9 gene. Expression of the mutant CCUGn RNA changes RNA processing in patients with DM2; however, the role of ZNF9 protein in DM2 pathology has been not elucidated. ZNF9 has been shown to regulate cap-dependent and cap-independent translation. We have examined a possible role of ZNF9 in the regulation of translation in DM2 patients. We found that ZNF9 interacts with the 5' UTRs of terminal oligopyrimidine (TOP) tract mRNAs encoding human ribosomal protein, RPS17, poly(A)-binding protein 1 (PABP1), and the elongation factors, eEF1A and eEF2. The binding activity of ZNF9 toward these TOP-containing 5' UTRs is reduced in DM2 muscle. Consistent with the reduction of this activity, the levels of RPS17, PABP, eEF1A, and eEF2 proteins are also diminished in DM2 muscle. The reduction of ZNF9 RNA-binding activity in DM2 correlates with a decrease of ZNF9 protein levels in cytoplasm of DM2 muscle cells. We found that the reduction of ZNF9 is caused by expression of the mutant CCUG repeats. This decrease of proteins of translational apparatus in DM2 correlates with a reduction of a rate of protein synthesis in myoblasts from DM2 patients. We found that the ectopic expression of ZNF9 in DM2 myoblasts corrects rate of protein synthesis, suggesting that the alterations in CCUG-ZNF9-TOP mRNAs pathway are responsible for the reduction of the rate of protein translation in DM2 muscle cells.