Glial fibrillary acidic protein is pathologically modified in Alexander disease.

Here, we describe pathological events potentially involved in the disease pathogenesis of Alexander disease (AxD). This is a primary genetic disorder of astrocyte caused by dominant gain-of-function mutations in the gene coding for an intermediate filament protein glial fibrillary acidic protein (GFAP). Pathologically, this disease is characterized by the upregulation of GFAP and its accumulation as Rosenthal fibers. Although the genetic basis linking GFAP mutations with Alexander disease has been firmly established, the initiating events that promote GFAP accumulation and the role of Rosenthal fibers (RFs) in the disease process remain unknown. Here, we investigate the hypothesis that disease-associated mutations promote GFAP aggregation through aberrant posttranslational modifications. We found high molecular weight GFAP species in the RFs of AxD brains, indicating abnormal GFAP crosslinking as a prominent pathological feature of this disease. In vitro and cell-based studies demonstrate that cystine-generating mutations promote GFAP crosslinking by cysteine-dependent oxidation, resulting in defective GFAP assembly and decreased filament solubility. Moreover, we found GFAP was ubiquitinated in RFs of AxD patients and rodent models, supporting this modification as a critical factor linked to GFAP aggregation. Finally, we found that arginine could increase the solubility of aggregation-prone mutant GFAP by decreasing its ubiquitination and aggregation. Our study suggests a series of pathogenic events leading to AxD, involving interplay between GFAP aggregation and abnormal modifications by GFAP ubiquitination and oxidation. More important, our findings provide a basis for investigating new strategies to treat AxD by targeting abnormal GFAP modifications.

Stability dynamics of neurofilament and GFAP networks and protein fragments.

Neurofilaments (NFs) and GFAP are cytoskeletal intermediate filaments (IFs) that support cellular processes unfolding within the uniquely complex environments of neurons and astrocytes, respectively. This review highlights emerging concepts on the transitions between stable and destabilized IF networks in the nervous system. While self-association between transiently structured low-complexity IF domains promotes filament assembly, the opposing destabilizing actions of phosphorylation-mediated filament severing facilitate faster intracellular transport. Cellular proteases, including caspases and calpains, produce a variety of IF fragments, which may interact with N-degron and C-degron pathways of the protein degradation machinery. The rapid adoption of NF and GFAP-based clinical biomarker tests is contrasted with the lagging understanding of the dynamics between the native IF proteins and their fragments.

Intermediate filament dysregulation in astrocytes in the human disease model of KLHL16 mutation in giant axonal neuropathy (GAN).

Giant Axonal Neuropathy (GAN) is a pediatric neurodegenerative disease caused by KLHL16 mutations. KLHL16 encodes gigaxonin, which regulates intermediate filament (IF) turnover. Previous neuropathological studies and examination of postmortem brain tissue in the current study revealed involvement of astrocytes in GAN. To develop a clinically-relevant model, we reprogrammed skin fibroblasts from seven GAN patients to pluripotent stem cells (iPSCs), which were used to generate neural progenitor cells (NPCs), astrocytes, and brain organoids. Multiple isogenic control clones were derived via CRISPR/Cas9 gene editing of one patient line carrying the G332R gigaxonin mutation. All GAN iPSCs were deficient for gigaxonin and displayed patient-specific increased vimentin expression. GAN NPCs had lower nestin expression and fewer nestin-positive cells compared to isogenic controls, but nestin morphology was unaffected. GAN brain organoids were marked by the presence of neurofilament and GFAP aggregates. GAN iPSC-astrocytes displayed striking dense perinuclear vimentin and GFAP accumulations and abnormal nuclear morphology. In over-expression systems, GFAP oligomerization and perinuclear aggregation were augmented in the presence of vimentin. GAN patient cells with large perinuclear vimentin aggregates accumulated significantly more nuclear KLHL16 mRNA compared to cells without vimentin aggregates. As an early effector of KLHL16 mutations, vimentin may be a potential target in GAN.

Circulating Ectonucleotidases Signal Impaired Myocardial Perfusion at Rest and Stress.

Background Ectonucleotidases maintain vascular homeostasis by metabolizing extracellular nucleotides, modulating inflammation and thrombosis, and potentially, myocardial flow through adenosine generation. Evidence implicates dysfunction or deficiency of ectonucleotidases CD39 or CD73 in human disease; the utility of measuring levels of circulating ectonucleotidases as plasma biomarkers of coronary artery dysfunction or disease has not been previously reported. Methods and Results A total of 529 individuals undergoing clinically indicated positron emission tomography stress testing between 2015 and 2019 were enrolled in this single-center retrospective analysis. Baseline demographics, clinical data, nuclear stress test, and coronary artery calcium score variables were collected, as well as a blood sample. CD39 and CD73 levels were assessed as binary (detectable, undetectable) or continuous variables using ELISAs. Plasma CD39 was detectable in 24% of White and 8% of Black study participants (P=0.02). Of the clinical history variables examined, ectonucleotidase levels were most strongly associated with underlying liver disease and not other traditional coronary artery disease risk factors. Intriguingly, detection of circulating ectonucleotidase was inversely associated with stress myocardial blood flow (2.3±0.8 mL/min per g versus 2.7 mL/min per g±1.1 for detectable versus undetectable CD39 levels, P<0.001) and global myocardial flow reserve (Pearson correlation between myocardial flow reserve and log(CD73) -0.19, P<0.001). A subanalysis showed these differences held true independent of liver disease. Conclusions Vasodilatory adenosine is the expected product of local ectonucleotidase activity, yet these data support an inverse relationship between plasma ectonucleotidases, stress myocardial blood flow (CD39), and myocardial flow reserve (CD73). These findings support the conclusion that plasma levels of ectonucleotidases, which may be shed from the endothelial surface, contribute to reduced stress myocardial blood flow and myocardial flow reserve.

Intermediate filament dysregulation and astrocytopathy in the human disease model of KLHL16 mutation in giant axonal neuropathy (GAN).

Giant Axonal Neuropathy (GAN) is a pediatric neurodegenerative disease caused by KLHL16 mutations. KLHL16 encodes gigaxonin, a regulator of intermediate filament (IF) protein turnover. Previous neuropathological studies and our own examination of postmortem GAN brain tissue in the current study revealed astrocyte involvement in GAN. To study the underlying mechanisms, we reprogrammed skin fibroblasts from seven GAN patients carrying different KLHL16 mutations to iPSCs. Isogenic controls with restored IF phenotypes were derived via CRISPR/Cas9 editing of one patient carrying a homozygous missense mutation (G332R). Neural progenitor cells (NPCs), astrocytes, and brain organoids were generated through directed differentiation. All GAN iPSC lines were deficient for gigaxonin, which was restored in the isogenic control. GAN iPSCs displayed patient-specific increased vimentin expression, while GAN NPCs had decreased nestin expression compared to isogenic control. The most striking phenotypes were observed in GAN iPSC-astrocytes and brain organoids, which exhibited dense perinuclear IF accumulations and abnormal nuclear morphology. GAN patient cells with large perinuclear vimentin aggregates accumulated nuclear KLHL16 mRNA. In over-expression studies, GFAP oligomerization and perinuclear aggregation were potentiated in the presence of vimentin. As an early effector of KLHL16 mutations, vimentin may serve as a potential therapeutic target in GAN.

Assay of Endocannabinoid Oxidation by Cytochrome P450.

Cytochrome P450 enzymes are a large family of heme-containing proteins that have important functions in the biotransformation of xenobiotics, including pharmacologic and environmental agents, as well as endogenously produced chemicals with broad structural and functional diversity. Anandamide and 2-arachidonoylglycerol (2-AG) are substrates for P450s expressed in multiple tissues, leading to the production of a diverse set of mono- and di-oxygenated metabolites. This chapter describes tools and methods that have been used to identify major endocannabinoid metabolizing P450s and their corresponding products using subcellular tissue fractions, cultured cells, and purified recombinant enzymes in a reconstituted system.

Calpain-mediated proteolysis of vimentin filaments is augmented in giant axonal neuropathy fibroblasts exposed to hypotonic stress.

Giant Axonal Neuropathy (GAN) is a pediatric neurodegenerative disease caused by loss-of-function mutations in the E3 ubiquitin ligase adaptor gigaxonin, which is encoded by the KLHL16 gene. Gigaxonin regulates the degradation of multiple intermediate filament (IF) proteins, including neurofilaments, GFAP, and vimentin, which aggregate in GAN patient cells. Understanding how IFs and their aggregates are processed under stress can reveal new GAN disease mechanisms and potential targets for therapy. Here we tested the hypothesis that hypotonic stress-induced vimentin proteolysis is impaired in GAN. In both GAN and control fibroblasts exposed to hypotonic stress, we observed time-dependent vimentin cleavage that resulted in two prominent ∼40-45 kDa fragments. However, vimentin proteolysis occurred more rapidly and extensively in GAN cells compared to unaffected controls as both fragments were generated earlier and at 4-6-fold higher levels. To test enzymatic involvement, we determined the expression levels and localization of the calcium-sensitive calpain proteases-1 and -2 and their endogenous inhibitor calpastatin. While the latter was not affected, the expression of both calpains was 2-fold higher in GAN cells compared to control cells. Moreover, pharmacologic inhibition of calpains with MDL-28170 or MG-132 attenuated vimentin cleavage. Imaging analysis revealed striking colocalization between large perinuclear vimentin aggregates and calpain-2 in GAN fibroblasts. This colocalization was dramatically altered by hypotonic stress, where selective breakdown of filaments over aggregates occurred rapidly in GAN cells and coincided with calpain-2 cytoplasmic redistribution. Finally, mass spectrometry-based proteomics revealed that phosphorylation at Ser-412, located at the junction between the central "rod" domain and C-terminal "tail" domain on vimentin, is involved in this stress response. Over-expression studies using phospho-deficient and phospho-mimic mutants revealed that Ser-412 is important for filament organization, solubility dynamics, and vimentin cleavage upon hypotonic stress exposure. Collectively, our work reveals that osmotic stress induces calpain- and proteasome-mediated vimentin degradation and IF network breakdown. These effects are significantly augmented in the presence of disease-causing KLHL16 mutations that alter intermediate filament organization. While the specific roles of calpain-generated vimentin IF fragments in GAN cells remain to be defined, this proteolytic pathway is translationally-relevant to GAN because maintaining osmotic homeostasis is critical for nervous system function.

Effects of Alexander disease-associated mutations on the assembly and organization of GFAP intermediate filaments.

Alexander disease is a primary genetic disorder of astrocytes caused by dominant mutations in the gene encoding glial fibrillary acidic protein (GFAP). How single-amino-acid changes can lead to cytoskeletal catastrophe and brain degeneration remains poorly understood. In this study, we have analyzed 14 missense mutations located in the GFAP rod domain to investigate how these mutations affect in vitro filament assembly. Whereas the internal rod mutants assembled into filaments that were shorter than those of wild type, the rod end mutants formed structures with one or more of several atypical characteristics, including short filament length, irregular width, roughness of filament surface, and filament aggregation. When transduced into primary astrocytes, GFAP mutants with in vitro assembly defects usually formed cytoplasmic aggregates, which were more resistant to biochemical extraction. The resistance of GFAP to solubilization was also observed in brain tissues of patients with Alexander disease, in which a significant proportion of insoluble GFAP were accumulated in Rosenthal fiber fractions. These findings provide clinically relevant evidence that link GFAP assembly defects to disease pathology at the tissue level and suggest that altered filament assembly and properties as a result of GFAP mutation are critical initiating factors for the pathogenesis of Alexander disease.

The elegant complexity of mammalian ecto-5'-nucleotidase (CD73).

Purinergic signaling is a fundamental mechanism used by all cells to control their internal activities and interact with the environment. A key component of the purinergic system, the enzyme ecto-5'-nucleotidase (CD73) catalyzes the last step in the extracellular metabolism of ATP to form adenosine. Efforts to harness the therapeutic potential of endogenous adenosine in cancer have culminated in the ongoing clinical development of multiple CD73-targeting antibodies and small-molecule inhibitors. However, recent studies are painting an increasingly complex picture of CD73 mRNA and protein regulation and function in cellular homeostasis, physiological adaptation, and disease development. This review discusses the latest conceptual and methodological advances that are helping to unravel the complexity of this important enzyme that was identified nearly 90 years ago.

CD73 Maintains Hepatocyte Metabolic Integrity and Mouse Liver Homeostasis in a Sex-Dependent Manner.

BACKGROUND & AIMS: Metabolic imbalance and inflammation are common features of chronic liver diseases. Molecular factors controlling these mechanisms represent potential therapeutic targets. CD73 is the major enzyme that dephosphorylates extracellular adenosine monophosphate (AMP) to form the anti-inflammatory adenosine. CD73 is expressed on pericentral hepatocytes, which are important for long-term liver homeostasis. We aimed to determine if CD73 has nonredundant hepatoprotective functions. METHODS: Liver-specific CD73 knockout (CD73-LKO) mice were generated by targeting the Nt5e gene in hepatocytes. The CD73-LKO mice and hepatocytes were characterized using multiple approaches. RESULTS: Deletion of hepatocyte Nt5e resulted in an approximately 70% reduction in total liver CD73 protein (P < .0001). Male and female CD73-LKO mice developed normally during the first 21 weeks without significant liver phenotypes. Between 21 and 42 weeks, the CD73-LKO mice developed spontaneous-onset liver disease, with significant severity in male mice. Middle-aged male CD73-LKO mice showed hepatocyte swelling and ballooning (P < .05), inflammation (P < .01), and variable steatosis. Female CD73-LKO mice had lower serum albumin levels (P < .05) and increased inflammatory genes (P < .01), but did not show the spectrum of histopathologic changes in male mice, potentially owing to compensatory induction of adenosine receptors. Serum analysis and proteomic profiling of hepatocytes from male CD73-LKO mice showed significant metabolic imbalance, with increased blood urea nitrogen (P < .0001) and impairments in major metabolic pathways, including oxidative phosphorylation and AMP-activated protein kinase (AMPK) signaling. There was significant hypophosphorylation of AMPK substrates in CD73-LKO livers (P < .0001), while in isolated hepatocytes treated with AMP, soluble CD73 induced AMPK activation (P < .001). CONCLUSIONS: Hepatocyte CD73 supports long-term metabolic liver homeostasis through AMPK in a sex-dependent manner. These findings have implications for human liver diseases marked by CD73 dysregulation.

Integrated Multiomics Reveals Glucose Use Reprogramming and Identifies a Novel Hexokinase in Alcoholic Hepatitis.

BACKGROUND & AIMS: We recently showed that alcoholic hepatitis (AH) is characterized by dedifferentiation of hepatocytes and loss of mature functions. Glucose metabolism is tightly regulated in healthy hepatocytes. We hypothesize that AH may lead to metabolic reprogramming of the liver, including dysregulation of glucose metabolism. METHODS: We performed integrated metabolomic and transcriptomic analyses of liver tissue from patients with AH or alcoholic cirrhosis or normal liver tissue from hepatic resection. Focused analyses of chromatin immunoprecipitation coupled to DNA sequencing was performed. Functional in vitro studies were performed in primary rat and human hepatocytes and HepG2 cells. RESULTS: Patients with AH exhibited specific changes in the levels of intermediates of glycolysis/gluconeogenesis, the tricarboxylic acid cycle, and monosaccharide and disaccharide metabolism. Integrated analysis of the transcriptome and metabolome showed the used of alternate energetic pathways, metabolite sinks and bottlenecks, and dysregulated glucose storage in patients with AH. Among genes involved in glucose metabolism, hexokinase domain containing 1 (HKDC1) was identified as the most up-regulated kinase in patients with AH. Histone active promoter and enhancer markers were increased in the HKDC1 genomic region. High HKDC1 levels were associated with the development of acute kidney injury and decreased survival. Increased HKDC1 activity contributed to the accumulation of glucose-6-P and glycogen in primary rat hepatocytes. CONCLUSIONS: Altered metabolite levels and messenger RNA expression of metabolic enzymes suggest the existence of extensive reprogramming of glucose metabolism in AH. Increased HKDC1 expression may contribute to dysregulated glucose metabolism and represents a novel biomarker and therapeutic target for AH.

Hepatic lipocalin 2 promotes liver fibrosis and portal hypertension.

Advanced fibrosis and portal hypertension influence short-term mortality. Lipocalin 2 (LCN2) regulates infection response and increases in liver injury. We explored the role of intrahepatic LCN2 in human alcoholic hepatitis (AH) with advanced fibrosis and portal hypertension and in experimental mouse fibrosis. We found hepatic LCN2 expression and serum LCN2 level markedly increased and correlated with disease severity and portal hypertension in patients with AH. In control human livers, LCN2 expressed exclusively in mononuclear cells, while its expression was markedly induced in AH livers, not only in mononuclear cells but also notably in hepatocytes. Lcn2-/- mice were protected from liver fibrosis caused by either ethanol or CCl4 exposure. Microarray analysis revealed downregulation of matrisome, cell cycle and immune related gene sets in Lcn2-/- mice exposed to CCl4, along with decrease in Timp1 and Edn1 expression. Hepatic expression of COL1A1, TIMP1 and key EDN1 system components were elevated in AH patients and correlated with hepatic LCN2 expression. In vitro, recombinant LCN2 induced COL1A1 expression. Overexpression of LCN2 increased HIF1A that in turn mediated EDN1 upregulation. LCN2 contributes to liver fibrosis and portal hypertension in AH and could represent a new therapeutic target.

Alternative Splicing in Hepatocellular Carcinoma.

Hepatocellular carcinoma (HCC) accounts for the majority of primary liver cancer cases, with more than 850,000 new diagnoses per year globally. Recent trends in the United States have shown that liver cancer mortality has continued to increase in both men and women, while 5-year survival remains below 20%. Understanding key mechanisms that drive chronic liver disease progression to HCC can reveal new therapeutic targets and biomarkers for early detection of HCC. In that regard, many studies have underscored the importance of alternative splicing as a source of novel HCC prognostic markers and disease targets. Alternative splicing of pre-mRNA provides functional diversity to the genome, and endows cells with the ability to rapidly remodel the proteome. Genes that control fundamental processes, such as metabolism, cell proliferation, and apoptosis, are altered globally in HCC by alternative splicing. This review highlights the major splicing factors, RNA binding proteins, transcriptional targets, and signaling pathways that are of key relevance to HCC. We highlight primary research from the past 3-5 years involving functional interrogation of alternative splicing in rodent and human liver, using both large-scale transcriptomic and focused mechanistic approaches. Because this is a rapidly advancing field, we anticipate that it will be transformative for the future of basic liver biology, as well as HCC diagnosis and management.

Site-specific phosphorylation and caspase cleavage of GFAP are new markers of Alexander disease severity.

Alexander disease (AxD) is a fatal neurodegenerative disorder caused by mutations in glial fibrillary acidic protein (GFAP), which supports the structural integrity of astrocytes. Over 70 GFAP missense mutations cause AxD, but the mechanism linking different mutations to disease-relevant phenotypes remains unknown. We used AxD patient brain tissue and induced pluripotent stem cell (iPSC)-derived astrocytes to investigate the hypothesis that AxD-causing mutations perturb key post-translational modifications (PTMs) on GFAP. Our findings reveal selective phosphorylation of GFAP-Ser13 in patients who died young, independently of the mutation they carried. AxD iPSC-astrocytes accumulated pSer13-GFAP in cytoplasmic aggregates within deep nuclear invaginations, resembling the hallmark Rosenthal fibers observed in vivo. Ser13 phosphorylation facilitated GFAP aggregation and was associated with increased GFAP proteolysis by caspase-6. Furthermore, caspase-6 was selectively expressed in young AxD patients, and correlated with the presence of cleaved GFAP. We reveal a novel PTM signature linking different GFAP mutations in infantile AxD.

Tumor-Selective Altered Glycosylation and Functional Attenuation of CD73 in Human Hepatocellular Carcinoma.

CD73, a cell-surface N-linked glycoprotein that produces extracellular adenosine, is a novel target for cancer immunotherapy. Although anti-CD73 antibodies have entered clinical development, CD73 has both protumor and antitumor functions, depending on the target cell and tumor type. The aim of this study was to characterize CD73 regulation in human hepatocellular carcinoma (HCC). We examined CD73 expression, localization, and activity using molecular, biochemical, and cellular analyses on primary HCC surgical specimens, coupled with mechanistic studies in HCC cells. We analyzed CD73 glycan signatures and global alterations in transcripts encoding other N-linked glycoproteins by using mass spectrometry glycomics and RNA sequencing (RNAseq), respectively. CD73 was expressed on tumor hepatocytes where it exhibited abnormal N-linked glycosylation, independent of HCC etiology, tumor stage, or fibrosis presence. Aberrant glycosylation of tumor-associated CD73 resulted in a 3-fold decrease in 5'-nucleotidase activity (P < 0.0001). Biochemically, tumor-associated CD73 was deficient in hybrid and complex glycans specifically on residues N311 and N333 located in the C-terminal catalytic domain. Blocking N311/N333 glycosylation by site-directed mutagenesis produced CD73 with significantly decreased 5'-nucleotidase activity in vitro, similar to the primary tumors. Glycosylation-deficient CD73 partially colocalized with the Golgi structural protein GM130, which was strongly induced in HCC tumors. RNAseq analysis further revealed that N-linked glycoprotein-encoding genes represented the largest category of differentially expressed genes between HCC tumor and adjacent tissue. Conclusion: We provide the first detailed characterization of CD73 glycosylation in normal and tumor tissue, revealing a novel mechanism that leads to the functional suppression of CD73 in human HCC tumor cells. The present findings have translational implications for therapeutic candidate antibodies targeting cell-surface CD73 in solid tumors and small-molecule adenosine receptor agonists that are in clinical development for HCC.

Cell type- and tissue-specific functions of ecto-5'-nucleotidase (CD73).

Ecto-5'-nucleotidase [cluster of differentiation 73 (CD73)] is a ubiquitously expressed glycosylphosphatidylinositol-anchored glycoprotein that converts extracellular adenosine 5'-monophosphate to adenosine. Anti-CD73 inhibitory antibodies are currently undergoing clinical testing for cancer immunotherapy. However, many protective physiological functions of CD73 need to be taken into account for new targeted therapies. This review examines CD73 functions in multiple organ systems and cell types, with a particular focus on novel findings from the last 5 years. Missense loss-of-function mutations in the CD73-encoding gene NT5E cause the rare disease "arterial calcifications due to deficiency of CD73." Aside from direct human disease involvement, cellular and animal model studies have revealed key functions of CD73 in tissue homeostasis and pathology across multiple organ systems. In the context of the central nervous system, CD73 is antinociceptive and protects against inflammatory damage, while also contributing to age-dependent decline in cortical plasticity. CD73 preserves barrier function in multiple tissues, a role that is most evident in the respiratory system, where it inhibits endothelial permeability in an adenosine-dependent manner. CD73 has important cardioprotective functions during myocardial infarction and heart failure. Under ischemia-reperfusion injury conditions, rapid and sustained induction of CD73 confers protection in the liver and kidney. In some cases, the mechanism by which CD73 mediates tissue injury is less clear. For example, CD73 has a promoting role in liver fibrosis but is protective in lung fibrosis. Future studies that integrate CD73 regulation and function at the cellular level with physiological responses will improve its utility as a disease target.

Vimentin on the move: new developments in cell migration.

The vimentin gene ( VIM) encodes one of the 71 human intermediate filament (IF) proteins, which are the building blocks of highly ordered, dynamic, and cell type-specific fiber networks. Vimentin is a multi-functional 466 amino acid protein with a high degree of evolutionary conservation among vertebrates. Vim -/- mice, though viable, exhibit systemic defects related to development and wound repair, which may have implications for understanding human disease pathogenesis. Vimentin IFs are required for the plasticity of mesenchymal cells under normal physiological conditions and for the migration of cancer cells that have undergone epithelial-mesenchymal transition. Although it was observed years ago that vimentin promotes cell migration, the molecular mechanisms were not completely understood. Recent advances in microscopic techniques, combined with computational image analysis, have helped illuminate vimentin dynamics and function in migrating cells on a precise scale. This review includes a brief historical account of early studies that unveiled vimentin as a unique component of the cell cytoskeleton followed by an overview of the physiological vimentin functions documented in studies on Vim -/- mice. The primary focus of the discussion is on novel mechanisms related to how vimentin coordinates cell migration. The current hypothesis is that vimentin promotes cell migration by integrating mechanical input from the environment and modulating the dynamics of microtubules and the actomyosin network. These new findings undoubtedly will open up multiple avenues to study the broader function of vimentin and other IF proteins in cell biology and will lead to critical insights into the relevance of different vimentin levels for the invasive behaviors of metastatic cancer cells.

The sweet side of vimentin.

A protein modification called O-linked glycosylation regulates the interactions between vimentin molecules under normal conditions, and the ability of Chlamydia bacteria to replicate after they infect cells.

An image-based small-molecule screen identifies vimentin as a pharmacologically relevant target of simvastatin in cancer cells.

Vimentin is a cytoskeletal intermediate filament protein that is expressed in mesenchymal cells and cancer cells during the epithelial-mesenchymal transition. The goal of this study was to identify vimentin-targeting small molecules by using the Tocriscreen library of 1120 biochemically active compounds. We monitored vimentin filament reorganization and bundling in adrenal carcinoma SW13 vimentin-positive (SW13-vim+) cells via indirect immunofluorescence. The screen identified 18 pharmacologically diverse hits that included 2 statins-simvastatin and mevastatin. Simvastatin induced vimentin reorganization within 15-30 min and significant perinuclear bundling within 60 min (IC50 = 6.7 nM). Early filament reorganization coincided with increased vimentin solubility. Mevastatin produced similar effects at >1 µM, whereas the structurally related pravastatin and lovastatin did not affect vimentin. In vitro vimentin filament assembly assays revealed a direct targeting mechanism, as determined biochemically and by electron microscopy. In SW13-vim+ cells, simvastatin, but not pravastatin, reduced total cell numbers (IC50 = 48.1 nM) and promoted apoptosis after 24 h. In contrast, SW13-vim- cell viability was unaffected by simvastatin, unless vimentin was ectopically expressed. Simvastatin similarly targeted vimentin filaments and induced cell death in MDA-MB-231 (vim+), but lacked effect in MCF7 (vim-) breast cancer cells. In conclusion, this study identified vimentin as a direct molecular target that mediates simvastatin-induced cell death in 2 different cancer cell lines.-Trogden, K. P., Battaglia, R. A., Kabiraj, P., Madden, V. J., Herrmann, H., Snider, N. T. An image-based small-molecule screen identifies vimentin as a pharmacologically relevant target of simvastatin in cancer cells.