NOTCH localizes to mitochondria through the TBC1D15-FIS1 interaction and is stabilized via blockade of E3 ligase and CDK8 recruitment to reprogram tumor-initiating cells.

The P53-destabilizing TBC1D15-NOTCH protein interaction promotes self-renewal of tumor-initiating stem-like cells (TICs); however, the mechanisms governing the regulation of this pathway have not been fully elucidated. Here, we show that TBC1D15 stabilizes NOTCH and c-JUN through blockade of E3 ligase and CDK8 recruitment to phosphodegron sequences. Chromatin immunoprecipitation (ChIP-seq) analysis was performed to determine whether TBC1D15-dependent NOTCH1 binding occurs in TICs or non-TICs. The TIC population was isolated to evaluate TBC1D15-dependent NOTCH1 stabilization mechanisms. The tumor incidence in hepatocyte-specific triple knockout (Alb::CreERT2;Tbc1d15Flox/Flox;Notch1Flox/Flox;Notch2Flox/Flox;HCV-NS5A) Transgenic (Tg) mice and wild-type mice was compared after being fed an alcohol-containing Western diet (WD) for 12 months. The NOTCH1-TBC1D15-FIS1 interaction resulted in recruitment of mitochondria to the perinuclear region. TBC1D15 bound to full-length NUMB and to NUMB isoform 5, which lacks three Ser phosphorylation sites, and relocalized NUMB5 to mitochondria. TBC1D15 binding to NOTCH1 blocked CDK8- and CDK19-mediated phosphorylation of the NOTCH1 PEST phosphodegron to block FBW7 recruitment to Thr-2512 of NOTCH1. ChIP-seq analysis revealed that TBC1D15 and NOTCH1 regulated the expression of genes involved in mitochondrial metabolism-related pathways required for the maintenance of TICs. TBC1D15 inhibited CDK8-mediated phosphorylation to stabilize NOTCH1 and protect it from degradation The NUMB-binding oncoprotein TBC1D15 rescued NOTCH1 from NUMB-mediated ubiquitin-dependent degradation and recruited NOTCH1 to the mitochondrial outer membrane for the generation and expansion of liver TICs. A NOTCH-TBC1D15 inhibitor was found to inhibit NOTCH-dependent pathways and exhibited potent therapeutic effects in PDX mouse models. This unique targeting of the NOTCH-TBC1D15 interaction not only normalized the perinuclear localization of mitochondria but also promoted potent cytotoxic effects against TICs to eradicate patient-derived xenografts through NOTCH-dependent pathways.

Protocol for generation of humanized HCC mouse model and cancer-driver mutations using CRISPR-Cas9.

We detail procedures for generating a humanized mouse model of hepatocellular carcinoma (HCC) recapitulating genetic mutations associated with metabolic liver diseases (MLD). We humanized liver parenchymal, non-parenchymal, and hematopoietic cells. We employed CRISPR-Cas9-based ARID1A knockout and constitutively active CTNNB1 knockin combined with an alcohol Western diet to generate cancer-driver mutations commonly found in MLD-HCC patients. This HCC model facilitates the study of tumor-promoting gene-environment interactions. For complete details on the use and execution of this protocol, please refer to Yeh et al.1.

ß-CATENIN stabilizes HIF2 through lncRNA and inhibits intravenous immunoglobulin immunotherapy.

Introduction: Tumor-initiating cells (TICs) are rare, stem-like, and highly malignant. Although intravenous hepatitis B and C immunoglobulins have been used for HBV and HCV neutralization in patients, their tumor-inhibitory effects have not yet been examined. Hepatitis B immunoglobulin (HBIG) therapy is employed to reduce hepatocellular carcinoma (HCC) recurrence in patients after living donor liver transplantations (LDLT). Hypothesis: We hypothesized that patient-derived intravenous immunoglobulin (IVIG) binding to HCC associated TICs will reduce self-renewal and cell viability driven by ß-CATENIN-downstream pathways. ß-CATENIN activity protected TICs from IVIG effects. Methods: The effects of HBIG and HCIG binding to TICs were evaluated for cell viability and self-renewal. Results: Inhibition of ß-CATENIN pathway(s) augmented TIC susceptibility to HBIG- and HCIG-immunotherapy. HBV X protein (HBx) upregulates both ß-CATENIN and NANOG expression. The co-expression of constitutively active ß-CATENIN with NANOG promotes self-renewal ability and tumor-initiating ability of hepatoblasts. HBIG bound to HBV+ cells led to growth inhibition in a TIC subset that expressed hepatitis B surface antigen. The HBx protein transformed cells through ß-CATENIN-inducible lncRNAs EGLN3-AS1 and lnc-ß-CatM. Co-expression of constitutively active ß-CATENIN with NANOG promoted self-renewal ability of TICs through EGLN3 induction. ß-CATENIN-induced lncRNAs stabilized HIF2 to maintain self-renewal of TICs. Targeting of EGLN3-AS1 resulted in destabilization of EZH2-dependent ß-CATENIN activity and synergized cell-killing of TICs by HBIG or HCIG immunotherapy. Discussion: Taken together, WNT and stemness pathways induced HIF2 of TICs via cooperating lncRNAs resulting in resistance to cancer immunotherapy. Therefore, therapeutic use of IVIG may suppress tumor recurrence through inhibition of TICs.

Polycomb repressive complex 2 binds and stabilizes NANOG to suppress differentiation-related genes to promote self-renewal.

The synergistic effect of alcohol and HCV mediated through TLR4 signaling transactivates NANOG, a pluripotency transcription factor important for the stemness of tumor-initiating stem-like cells (TICs). NANOG together with the PRC2 complex suppresses expression of oxidative phosphorylation (OXPHOS) genes to generate TICs. The phosphodegron sequence PEST domain of NANOG binds EED to stabilize NANOG protein by blocking E3 ligase recruitment and proteasome-dependent degradation, while the tryptophan-rich domain of NANOG binds EZH2 and SUZ12. Human ARID1A gene loss results in the resistance to combined FAO and PRC2 inhibition therapies due to reduction of mitochondrial ROS levels. CRISPR-Cas9-mediated ARID1A knockout and/or constitutively active CTNNB1 driver mutations promoted tumor development in humanized FRG HCC mouse models, in which use of an interface inhibitor antagonizing PRC2-NANOG binding and/or FAO inhibitor blocked tumor growth. Together, the PRC2-NANOG interaction becomes a new drug target for HCC via inducing differentiation-related genes, destabilizing NANOG protein, and suppressing NANOG activity.

MSI2 promotes translation of multiple IRES-containing oncogenes and virus to induce self-renewal of tumor initiating stem-like cells.

RNA-binding protein Musashi 2 (MSI2) is elevated in several cancers and is linked to poor prognosis. Here, we tested if MSI2 promotes MYC and viral mRNA translation to induce self-renewal via an internal ribosome entry sequence (IRES). We performed RIP-seq using anti-MSI2 antibody in tumor-initiating stem-like cells (TICs). MSI2 binds the internal ribosome entry site (IRES)-containing oncogene mRNAs including MYC, JUN and VEGFA as well as HCV IRES to increase their synthesis and promote self-renewal and tumor-initiation at the post-transcriptional level. MSI2 binds a lncRNA to interfere with processing of a miRNA that reduced MYC translation in basal conditions. Deregulation of this integrated MSI2-lncRNA-MYC regulatory loop drives self-renewal and tumorigenesis through increased IRES-dependent translation of MYC mRNA. Overexpression of MSI2 in TICs promoted their self-renewal and tumor-initiation properties. Inhibition of MSI2-RNA binding reduced HCV IRES activity, viral replication and liver hyperplasia in humanized mice predisposed by virus infection and alcohol high-cholesterol high-fat diet. Together MSI2, integrating the MYC oncogenic pathway, can be employed as a therapeutic target in the treatment of HCC patients. A hypothetical model shows that MSI2 binds and activates cap-independent translation of MYC, c-JUN mRNA and HCV through MSI2-binding to Internal Ribosome Entry Sites (IRES) resulting in upregulated MYC, c-JUN and viral protein synthesis and subsequent liver oncogenesis. Inhibitor of the interaction between MYC IRES and MSI2 reduces liver hyperplasia, viral mRNA translation and tumor formation.

Profiling of Circulating Tumor Cells for Screening of Selective Inhibitors of Tumor-Initiating Stem-Like Cells.

A critical barrier to effective cancer therapy is the improvement of drug selectivity, toxicity, and reduced recurrence of tumors expanded from tumor-initiating stem-like cells (TICs). The aim is to identify circulating tumor cell (CTC)-biomarkers and to identify an effective combination of TIC-specific, repurposed federal drug administration (FDA)-approved drugs. Three different types of high-throughput screens targeting the TIC population are employed: these include a CD133 (+) cell viability screen, a NANOG expression screen, and a drug combination screen. When combined in a refined secondary screening approach that targets Nanog expression with the same FDA-approved drug library, histone deacetylase (HDAC) inhibitor(s) combined with all-trans retinoic acid (ATRA) demonstrate the highest efficacy for inhibition of TIC growth in vitro and in vivo. Addition of immune checkpoint inhibitor further decreases recurrence and extends PDX mouse survival. RNA-seq analysis of TICs reveals that combined drug treatment reduces many Toll-like receptors (TLR) and stemness genes through repression of the lncRNA MIR22HG. This downregulation induces PTEN and TET2, leading to loss of the self-renewal property of TICs. Thus, CTC biomarker analysis would predict the prognosis and therapy response to this drug combination. In general, biomarker-guided stratification of HCC patients and TIC-targeted therapy should eradicate TICs to extend HCC patient survival.

Intranasal Delivery of miR133b in a NEO100-Based Formulation Induces a Healing Response in Spinal Cord-Injured Mice.

Despite important advances in the pre-clinical animal studies investigating the neuroinhibitory microenvironment at the injury site, traumatic injury to the spinal cord remains a major problem with no concrete response. Here, we examined whether (1) intranasal (IN) administration of miR133b/Ago2 can reach the injury site and achieve a therapeutic effect and (2) NEO100-based formulation of miR133b/Ago2 can improve effectiveness. 24 h after a cervical contusion, C57BL6 female mice received IN delivery of miR133b/Ago2 or miR133b/Ago2/NEO100 for 3 days, one dose per day. The pharmacokinetics of miR133b in the spinal cord lesion was determined by RT-qPCR. The role of IN delivery of miR133b on motor function was assessed by the grip strength meter (GSM) and hanging tasks. The activity of miR133b at the lesion site was established by immunostaining of fibronectin 1 (FN1), a miR133b target. We found that IN delivery of miR133b/Ago2 (1) reaches the lesion scar and co-administration of miR133b with NEO100 facilitated the cellular uptake; (2) enhanced the motor function and addition of NEO100 potentiated this effect and (3) targeted FN1 expression at the lesion scar. Our results suggest a high efficacy of IN delivery of miR133b/Ago2 to the injured spinal cord that translates to improved healing with NEO100 further potentiating this effect.

LIN28 and histone H3K4 methylase induce TLR4 to generate tumor-initiating stem-like cells.

Chemoresistance and plasticity of tumor-initiating stem-like cells (TICs) promote tumor recurrence and metastasis. The gut-originating endotoxin-TLR4-NANOG oncogenic axis is responsible for the genesis of TICs. This study investigated mechanisms as to how TICs arise through transcriptional, epigenetic, and post-transcriptional activation of oncogenic TLR4 pathways. Here, we expressed constitutively active TLR4 (caTLR4) in mice carrying pLAP-tTA or pAlb-tTA, under a tetracycline withdrawal-inducible system. Liver progenitor cell induction accelerated liver tumor development in caTLR4-expressing mice. Lentiviral shRNA library screening identified histone H3K4 methylase SETD7 as central to activation of TLR4. SETD7 combined with hypoxia induced TLR4 through HIF2 and NOTCH. LIN28 post-transcriptionally stabilized TLR4 mRNA via de-repression of let-7 microRNA. These results supported a LIN28-TLR4 pathway for the development of HCCs in a hypoxic microenvironment. These findings not only advance our understanding of molecular mechanisms responsible for TIC generation in HCC, but also represent new therapeutic targets for the treatment of HCC.

Gut-derived Endotoxin-TLR4 Signaling Drives MYC-Ig Translocation to Promote Lymphoproliferation through c-JUN and STAT3 Activation.

Synergism between obesity and virus infection promotes the development of B-cell lymphoma. In this study, we tested whether obesity-associated endotoxin release induced activation-induced cytidine deaminase (AID). TLR4 activation in turn caused c-JUN-dependent and STAT3-dependent translocations of MYC loci to suppress transactivation of CD95/FAS. We used viral nucleocapside Core transgenic (Tg) mice fed alcohol Western diet to determine whether oncogenesis arising from obesity and chronic virus infection occurred through TLR4-c-JUN-STAT3 pathways. Our results showed B cell-specific, c-Jun and/or Stat3 disruption reduced the incidence of splenomegaly in these mice. AID-dependent t(8;14) translocation was observed between the Ig promoter and MYC loci. Comparison with human B cells showed MYC-immunoglobulin (Ig) translocations after virus infection with lipopolysaccharide stimulation. Accordingly, human patients with lymphoma with virus infections and obesity showed a 40% incidence of MYC-Ig translocations. Thus, obesity and virus infection promote AID-mediated translocation between the Ig promoter and MYC through the TLR4-c-JUN axis, resulting in lymphoproliferation. Taken together, preventative treatment targeting either c-JUN and/or STAT3 may be effective strategies to prevent tumor development. IMPLICATIONS: Obesity increases gut-derived endotoxin which induces Toll-like receptor-mediated MYC-Ig translocation via c-JUN-STAT3, leading to lymphoproliferation.

c-JUN inhibits mTORC2 and glucose uptake to promote self-renewal and obesity.

Metabolic syndrome is associated with obesity, insulin resistance, and the risk of cancer. We tested whether oncogenic transcription factor c-JUN metabolically reprogrammed cells to induce obesity and cancer by reduction of glucose uptake, with promotion of the stemness phenotype leading to malignant transformation. Liquid alcohol, high-cholesterol, fat diet (HCFD), and isocaloric dextrin were fed to wild-type or experimental mice for 12 months to promote hepatocellular carcinoma (HCC). We demonstrated 40% of mice developed liver tumors after chronic HCFD feeding. Disruption of liver-specific c-Jun reduced tumor incidence 4-fold and improved insulin sensitivity. Overexpression of c-JUN downregulated RICTOR transcription, leading to inhibition of the mTORC2/AKT and glycolysis pathways. c-JUN inhibited GLUT1, 2, and 3 transactivation to suppress glucose uptake. Silencing of RICTOR or c-JUN overexpression promoted self-renewal ability. Taken together, c-JUN inhibited mTORC2 via RICTOR downregulation and inhibited glucose uptake via downregulation of glucose intake, leading to self-renewal and obesity.

Immunotherapy and Microbiota for Targeting of Liver Tumor-Initiating Stem-like Cells.

Cancer contains tumor-initiating stem-like cells (TICs) that are resistant to therapies. Hepatocellular carcinoma (HCC) incidence has increased twice over the past few decades, while the incidence of other cancer types has trended downward globally. Therefore, an understanding of HCC development and therapy resistance mechanisms is needed for this incurable malignancy. This review article describes links between immunotherapies and microbiota in tumor-initiating stem-like cells (TICs), which have stem cell characteristics with self-renewal ability and express pluripotency transcription factors such as NANOG, SOX2, and OCT4. This review discusses (1) how immunotherapies fail and (2) how gut dysbiosis inhibits immunotherapy efficacy. Gut dysbiosis promotes resistance to immunotherapies by breaking gut immune tolerance and activating suppressor immune cells. Unfortunately, this leads to incurable recurrence/metastasis development. Personalized medicine approaches targeting these mechanisms of TIC/metastasis-initiating cells are emerging targets for HCC immunotherapy and microbiota modulation therapy.

p53 destabilizing protein skews asymmetric division and enhances NOTCH activation to direct self-renewal of TICs.

Tumor-initiating stem-like cells (TICs) are defective in maintaining asymmetric cell division and responsible for tumor recurrence. Cell-fate-determinant molecule NUMB-interacting protein (TBC1D15) is overexpressed and contributes to p53 degradation in TICs. Here we identify TBC1D15-mediated oncogenic mechanisms and tested the tumorigenic roles of TBC1D15 in vivo. We examined hepatocellular carcinoma (HCC) development in alcohol Western diet-fed hepatitis C virus NS5A Tg mice with hepatocyte-specific TBC1D15 deficiency or expression of non-phosphorylatable NUMB mutations. Liver-specific TBC1D15 deficiency or non-p-NUMB expression reduced TIC numbers and HCC development. TBC1D15-NuMA1 association impaired asymmetric division machinery by hijacking NuMA from LGN binding, thereby favoring TIC self-renewal. TBC1D15-NOTCH1 interaction activated and stabilized NOTCH1 which upregulated transcription of NANOG essential for TIC expansion. TBC1D15 activated three novel oncogenic pathways to promote self-renewal, p53 loss, and Nanog transcription in TICs. Thus, this central regulator could serve as a potential therapeutic target for treatment of HCC.

Intravenous delivery of microRNA-133b along with Argonaute-2 enhances spinal cord recovery following cervical contusion in mice.

BACKGROUND CONTEXT: Acute spinal cord injury (SCI) is a devastating condition for which spine decompression and stabilization of injury remains the only therapy available in the clinical setup. However, fibrous scar formation during the healing process significantly impairs full recovery. MicroRNAs (miRs) are small noncoding RNAs that regulate gene expression by binding to target mRNA(s) and initiating translational repression or mRNA degradation. It has been reported that microRNA-133b (miR133b) is highly expressed in regenerating neurons following a SCI in zebrafish, and lentiviral delivery of miR133b at the time of SCI in mice resulted in improved functional recovery. PURPOSE: The aim of this study was to investigate whether intravenous delivery of miR133b enhances spinal cord recovery when administered 24 hours following a cervical contusion injury in mice. STUDY DESIGN: This is an experimental animal study of acute SCI, investigating the effect of miR133b on spinal cord recovery by targeting scar lesion formation. The approach involved setting an acute SCI in mice, which was followed 24 hours later by intravenous co-delivery of miR133b and Argonaute 2 (Ago2), a protein involved in miRNA stabilization. Readouts of the impact of this intervention included analysis of RNA and protein expression at the lesion site, in particular with regard to markers of scar tissue formation, and determination of motor function recovery by the grip strength meter task. METHODS: C57BL6 female mice between 6 and 8 weeks of age were tested. The injury model employed was a unilateral moderate contusion at the cervical fifth level. Twenty-four hours following the injury, the authors co-delivered miR133b, or scrambled miRNA as negative control, along with Ago2 for 3 consecutive days, one dose per day via tail-vein injection. They first investigated the level of miR133b in the spinal cord and in spinal cord lesion after a single dose of injection. Next, they determined the efficacy of miR133b and/or Ago2 delivery in regulating gene and protein expression at the lesion site. Finally, they established the role of miR133b and/or Ago2 in enhancing forelimb gripping recovery as assessed by the grip strength meter task for 8 weeks post-SCI. RESULTS: Intravenous delivery of miR133b and/or Ago2 targeted the microenvironment at the lesion site and prevented the increased expression of certain extracellular matrix proteins (ECM), in particular collagen type 1 alpha 1 and tenascin N, which are known to have a key role in scar formation. It also reduced microglia and/or macrophage recruitment to the lesion site. Functional recovery in mice treated with miR133b and/or Ago2 started around 2 weeks postinjury and continued to improve over time, whereas mice in the control group displayed significantly poorer recovery. CONCLUSIONS: Our data indicate therapeutic activity of intravenous miR133b and/or Ago2 treatment, possibly via decreasing ECM protein expression and macrophage recruitment at the lesion site, thereby minimizing detrimental fibrous scar formation. CLINICAL SIGNIFICANCE: There is an urgent medical need for better treatments of SCIs. Based on our findings in a preclinical model, the miR133b and/or Ago2 system specifically targets fibrous scar formation, a barrier in neuronal regrowth, by remodeling ECM molecules at the injury site. Prevention of scar formation is critical to improved outcomes of treatment. Of note, delivery of miR133b and/or Ago2 was initiated 24 hours after traumatic impact, thus indicating a fairly long window of opportunity providing more time and flexibility for therapeutic intervention. Intravenous miR133b may become a beneficial therapeutic strategy to treat patients with acute SCI.

Placenta growth factor mediated gene regulation in sickle cell disease.

Sickle cell anemia (SCA) is an autosomal recessive disorder caused by mutation in the ß-globin gene. Pulmonary hypertension (PH), a complication of SCA, results in severe morbidity and mortality. PH is a multifactorial disease: systemic vasculopathy, pulmonary vasoconstriction, and endothelial dysfunction and remodeling. Placenta growth factor (PlGF), an angiogenic growth factor, elaborated from erythroid cells, has been shown to contribute to inflammation, pulmonary vasoconstriction and airway hyper-responsiveness (AH) in mouse models of sickle cell disease. In this review, we summarize the cell-signaling mechanism(s) by which PlGF regulates the expression of genes involved in inflammation, PH and AH in cell culture and corroborate these findings in mouse models of SCA and in individuals with SCA. The role of microRNAs (miRNAs) in the post-transcriptional regulation of these genes is presented and how these miRNAs located in their host genes are transcriptionally regulated. An understanding of the transcriptional regulation of these miRNAs provides a new therapeutic approach to ameliorate the clinical manifestations of SCA.

Hepatitis C virus has a genetically determined lymphotropism through co-receptor B7.2.

B-cell infection by hepatitis C virus (HCV) has been a controversial topic. To examine whether HCV has a genetically determined lymphotropism through a co-receptor specific for the infection by lymphotropic HCV, we established an infectious clone and chimeric virus of hepatotropic and lymphotropic HCV strains derived from an HCV-positive B-cell lymphoma. The viral envelope and 5'-UTR sequences of the lymphotropic HCV strain were responsible for the lymphotropism. Silencing of the virus sensor, RIGI, or overexpression of microRNA-122 promoted persistent viral replication in B cells. By cDNA library screening, we identified an immune cell-specific, co-stimulatory receptor B7.2 (CD86) as a co-receptor of lymphotropic HCV. Infection of B cells by HCV inhibited the recall reaction to antigen stimulation. Together, a co-receptor B7.2 enabled lymphotropic HCV to infect memory B cells, leading to inhibition of memory B-cell function and persistent HCV infection in HCV-infected hosts.

Activated Transcription Factor 3 in Association with Histone Deacetylase 6 Negatively Regulates MicroRNA 199a2 Transcription by Chromatin Remodeling and Reduces Endothelin-1 Expression.

Previous studies showed that high levels of placenta growth factor (PlGF) correlated with increased plasma levels of endothelin-1 (ET-1), a potent vasoconstrictor, in sickle cell disease (SCD). PlGF-mediated transcription of the ET-1 gene occurs by activation of hypoxia inducible factor 1α (HIF-1α) and posttranscriptionally by microRNA 199a2 (miR-199a2), which targets the 3' untranslated region (UTR) of HIF-1α mRNA. However, relatively less is known about how PlGF represses the expression of miR-199a2 located in the DNM3 opposite strand (DNM3os) transcription unit. Here, we show that PlGF induces the expression of activated transcription factor 3 (ATF3), which, in association with accessory proteins (c-Jun dimerization protein 2 [JDP2], ATF2, and histone deacetylase 6 [HDAC6]), as determined by proteomic analysis, binds to the DNM3os promoter. Furthermore, we show that association of HDAC6 with ATF3 at its binding site in this promoter was correlated with repression of miR-199a2 transcription, as shown by DNM3os transcription reporter and chromatin immunoprecipitation (ChIP) assays. Tubacin, an inhibitor of HDAC6, antagonized PlGF-mediated repression of DNM3os/pre-miR-199a2 transcription with a concomitant reduction in ET-1 levels in cultured endothelial cells. Analysis of lung tissues from Berkeley sickle (BK-SS) mice showed increased levels of ATF3 and increased expression of ET-1. Delivery of tubacin to BK-SS mice significantly attenuated plasma ET-1 and PlGF levels. Our studies demonstrated that ATF3 in conjunction with HDAC6 acts as a transcriptional repressor of the DNM3os/miR-199a2 locus.

NANOG Metabolically Reprograms Tumor-Initiating Stem-like Cells through Tumorigenic Changes in Oxidative Phosphorylation and Fatty Acid Metabolism.

Stem cell markers, including NANOG, have been implicated in various cancers; however, the functional contribution of NANOG to cancer pathogenesis has remained unclear. Here, we show that NANOG is induced by Toll-like receptor 4 (TLR4) signaling via phosphorylation of E2F1 and that downregulation of Nanog slows down hepatocellular carcinoma (HCC) progression induced by alcohol western diet and hepatitis C virus protein in mice. NANOG ChIP-seq analyses reveal that NANOG regulates the expression of genes involved in mitochondrial metabolic pathways required to maintain tumor-initiating stem-like cells (TICs). NANOG represses mitochondrial oxidative phosphorylation (OXPHOS) genes, as well as ROS generation, and activates fatty acid oxidation (FAO) to support TIC self-renewal and drug resistance. Restoration of OXPHOS activity and inhibition of FAO renders TICs susceptible to a standard care chemotherapy drug for HCC, sorafenib. This study provides insights into the mechanisms of NANOG-mediated generation of TICs, tumorigenesis, and chemoresistance through reprogramming of mitochondrial metabolism.

Peroxisome proliferator-activated receptor-α-mediated transcription of miR-301a and miR-454 and their host gene SKA2 regulates endothelin-1 and PAI-1 expression in sickle cell disease.

Endothelin-1 (ET-1) and plasminogen activator inhibitor-1 (PAI-1) play important roles in pulmonary hypertension (PH) in sickle cell disease (SCD). Our previous studies show higher levels of placenta growth factor (PlGF) in SCD correlate with increased plasma levels of ET-1, PAI-1, and other physiological markers of PH. PlGF-mediated ET-1 and PAI-1 expression occurs via activation of hypoxia-inducible factor-1α (HIF-1α). However, relatively little is understood regarding post-transcriptional regulation of PlGF-mediated expression of ET-1 and PAI-1. Herein, we show PlGF treatment of endothelial cells reduced levels of miR-301a and miR-454 from basal levels. In addition, both miRNAs targeted the 3'-UTRs of ET-1 and PAI-1 mRNAs. These results were corroborated in the mouse model of SCD [Berkeley sickle mice (BK-SS)] and in SCD subjects. Plasma levels of miR-454 in SCD subjects were significantly lower compared with unaffected controls, which correlated with higher plasma levels of both ET-1 and PAI-1. Moreover, lung tissues from BK-SS mice showed significantly reduced levels of pre-miR-301a and concomitantly higher levels of ET-1 and PAI-1. Furthermore, we show that miR-301a/miR-454 located in the spindle and kinetochore-associated protein-2 (SKA2) transcription unit was co-transcriptionally regulated by both HIF-1α and peroxisome proliferator-activated receptor-α (PPAR-α) as demonstrated by SKA2 promoter mutational analysis and ChIP. Finally we show that fenofibrate, a PPAR-α agonist, increased the expression of miR-301a/miR-454 and SKA2 in human microvascular endothelial cell line (HMEC) cells; the former were responsible for reduced expression of ET-1 and PAI-1. Our studies provide a potential therapeutic approach whereby fenofibrate-induced miR-301a/miR-454 expression can ameliorate PH and lung fibrosis by reduction in ET-1 and PAI-1 levels in SCD.

Erythropoietin-mediated expression of placenta growth factor is regulated via activation of hypoxia-inducible factor-1α and post-transcriptionally by miR-214 in sickle cell disease.

Placental growth factor (PlGF) plays an important role in various pathological conditions and diseases such as inflammation, cancer, atherosclerosis and sickle cell disease (SCD). Abnormally high PlGF levels in SCD patients are associated with increased inflammation and pulmonary hypertension (PHT) and reactive airway disease; however, the transcriptional and post-transcriptional mechanisms regulating PlGF expression are not well defined. Herein, we show that treatment of human erythroid cells and colony forming units with erythropoietin (EPO) increased PlGF expression. Our studies showed EPO-mediated activation of HIF-1α led to subsequent binding of HIF-1α to hypoxia response elements (HREs) within the PlGF promoter, as demonstrated by luciferase transcription reporter assays and ChIP analysis of the endogenous gene. Additionally, we showed miR-214 post-transcriptionally regulated the expression of PlGF as demonstrated by luciferase reporter assays using wild-type (wt) and mutant PlGF-3'-UTR constructs. Furthermore, synthesis of miR-214, located in an intron of DNM3 (dynamin 3), was transcriptionally regulated by transcription factors, peroxisome proliferator-activated receptor-α (PPARα) and hypoxia-inducible factor-1α (HIF-1α). These results were corroborated in vivo wherein plasma from SCD patients and lung tissues from sickle mice showed an inverse correlation between PlGF and miR-214 levels. Finally, we observed that miR-214 expression could be induced by fenofibrate, a Food and Drug Administration (FDA) approved PPARα agonist, thus revealing a potential therapeutic approach for reduction in PlGF levels by increasing miR-214 transcription. This strategy has potential clinical implications for several pathological conditions including SCD.

MicroRNA 648 Targets ET-1 mRNA and is cotranscriptionally regulated with MICAL3 by PAX5.

Pulmonary hypertension (PHT) is associated with high mortality in sickle cell anemia (SCA). Previously, we showed that elevated levels of placenta growth factor (PlGF) in SCA patients correlate with increased levels of the potent vasoconstrictor endothelin-1 (ET-1) and PHT. Moreover, PlGF induced the expression of ET-1 via hypoxia-inducible factor 1α. Here, we show a novel example of ET-1 posttranscriptional regulation by PlGF via action of microRNA 648 (miR-648), which is subject to transcriptional coregulation with its host gene, MICAL3 (microtubule-associated monooxygenase, calponin, and LIM domain containing 3gene). PlGF repressed expression of miR-648 in endothelial cells. Luciferase reporter assays using wild-type and mutant ET-1 3' untranslated region (UTR) constructs, and transfection of miR-648 mimics showed that miR-648 targets the 3' UTR of ET-1 mRNA. Since miR-648 is located in a 5'-proximal intron of MICAL3, we examined which of three potential promoters was responsible for its expression. The MICAL3 distal promoter (P1) was the predominant promoter used for transcription of pre-miR-648, and it was under positive control by PAX5 (paired box protein 5) transcription factor, as demonstrated by the loss and gain of function of PAX5 activity, and chromatin immunoprecipitation analysis. These studies provide a novel link wherein PlGF-mediated downregulation of PAX5 attenuates miR-648 expression leading to increased ET-1 levels that are known to induce PHT in SCA.