Victoria Petkova

In vivo imaging of siRNA delivery and silencing in tumors

With the increased potential of RNA interference (RNAi) as a therapeutic strategy, new noninvasive methods for detection of siRNA delivery and silencing are urgently needed. Here we describe the development of dual-purpose probes for in vivo transfer of siRNA and the simultaneous imaging of its accumulation in tumors by high-resolution magnetic resonance imaging (MRI) and near-infrared in vivo optical imaging (NIRF). These probes consisted of magnetic nanoparticles labeled with a near-infrared dye and covalently linked to siRNA molecules specific for model or therapeutic targets. Additionally, these nanoparticles were modified with a membrane translocation peptide for intracellular delivery. We show the feasibility of in vivo tracking of tumor uptake of these probes by MRI and optical imaging in two separate tumor models. We also used proof-of-principle optical imaging to corroborate the efficiency of the silencing process. These studies represent the first step toward the advancement of siRNA delivery and imaging strategies, essential for cancer therapeutic product development and optimization.

Pin1 is overexpressed in breast cancer and cooperates with Ras signaling in increasing the transcriptional activity of c-Jun towards cyclin D1.

Phosphorylation on serines or threonines preceding proline (Ser/Thr‐Pro) is a major signaling mechanism. The conformation of a subset of phosphorylated Ser/Thr‐Pro motifs is regulated by the prolyl isomerase Pin1. Inhibition of Pin1 induces apoptosis and may also contribute to neuronal death in Alzheimers disease. However, little is known about the role of Pin1 in cancer or in modulating transcription factor activity. Here we report that Pin1 is strikingly overexpressed in human breast cancers, and that its levels correlate with cyclin D1 levels in tumors. Overexpression of Pin1 increases cellular cyclin D1 protein and activates its promoter. Furthermore, Pin1 binds c‐Jun that is phosphorylated on Ser63/73‐Pro motifs by activated JNK or oncogenic Ras. Moreover, Pin1 cooperates with either activated Ras or JNK to increase transcriptional activity of c‐Jun towards the cyclin D1 promoter. Thus, Pin1 is up‐regulated in human tumors and cooperates with Ras signaling in increasing c‐Jun transcriptional activity towards cyclin D1. Given the crucial roles of Ras signaling and cyclin D1 overexpression in oncogenesis, our results suggest that overexpression of Pin1 may promote tumor growth.

PD-1 alters T-cell metabolic reprogramming by inhibiting glycolysis and promoting lipolysis and fatty acid oxidation

During activation, T cells undergo metabolic reprogramming, which imprints distinct functional fates. We determined that on PD-1 ligation, activated T cells are unable to engage in glycolysis or amino acid metabolism but have an increased rate of fatty acid β-oxidation (FAO). PD-1 promotes FAO of endogenous lipids by increasing expression of CPT1A, and inducing lipolysis as indicated by elevation of the lipase ATGL, the lipolysis marker glycerol and release of fatty acids. Conversely, CTLA-4 inhibits glycolysis without augmenting FAO, suggesting that CTLA-4 sustains the metabolic profile of non-activated cells. Because T cells utilize glycolysis during differentiation to effectors, our findings reveal a metabolic mechanism responsible for PD-1-mediated blockade of T-effector cell differentiation. The enhancement of FAO provides a mechanistic explanation for the longevity of T cells receiving PD-1 signals in patients with chronic infections and cancer, and for their capacity to be reinvigorated by PD-1 blockade.

Selective Effects of PD-1 on Akt and Ras Pathways Regulate Molecular Components of the Cell Cycle and Inhibit T Cell Proliferation

The inhibitory receptor PD-1 blocks T cell proliferation by preventing cells from leaving the G1 phase of the cell cycle. PD-1 Inhibits the Cell Cycle Machinery Effector T cells are stimulated through the T cell receptor (TCR) to become activated and proliferate. However, inhibitory receptors, such as PD-1, counteract these stimulatory signals and block T cell proliferation. Patsoukis et al. found that PD-1–mediated inhibition of Akt and Ras signaling pathways affected a number of components of the cell cycle machinery to “lock” T cells in the G1 phase and prevent their proliferation. These mechanistic details may help in the development of therapeutics designed to block PD-1 function and enable T cell activation in the context of antiviral or antitumor responses. The receptor programmed death 1 (PD-1) inhibits T cell proliferation and plays a critical role in suppressing self-reactive T cells, and it also compromises antiviral and antitumor responses. To determine how PD-1 signaling inhibits T cell proliferation, we used human CD4+ T cells to examine the effects of PD-1 signaling on the molecular control of the cell cycle. The ubiquitin ligase SCFSkp2 degrades p27kip1, an inhibitor of cyclin-dependent kinases (Cdks), and PD-1 blocked cell cycle progression through the G1 phase by suppressing transcription of SKP2, which encodes a component of this ubiquitin ligase. Thus, in T cells stimulated through PD-1, Cdks were not activated, and two critical Cdk substrates were not phosphorylated. Activation of PD-1 inhibited phosphorylation of the retinoblastoma gene product, which suppressed expression of E2F target genes. PD-1 also inhibited phosphorylation of the transcription factor Smad3, which increased its activity. These events induced additional inhibitory checkpoints in the cell cycle by increasing the abundance of the G1 phase inhibitor p15INK4 and repressing the Cdk-activating phosphatase Cdc25A. PD-1 suppressed SKP2 transcription by inhibiting phosphoinositide 3-kinase–Akt and Ras–mitogen-activated and extracellular signal–regulated kinase kinase (MEK)–extracellular signal–regulated kinase (ERK) signaling. Exposure of cells to the proliferation-promoting cytokine interleukin-2 restored activation of MEK-ERK signaling, but not Akt signaling, and only partially restored SKP2 expression. Thus, PD-1 blocks cell cycle progression and proliferation of T lymphocytes by affecting multiple regulators of the cell cycle.

PU.1 expression is modulated by the balance of functional sense and antisense RNAs regulated by a shared cis-regulatory element

The transcription factor PU.1 is an important regulator of hematopoiesis; precise expression levels are critical for normal hematopoietic development and suppression of leukemia. We show here that noncoding antisense RNAs are important modulators of proper dosages of PU.1. Antisense and sense RNAs are regulated by shared evolutionarily conserved cis-regulatory elements, and we can show that antisense RNAs inhibit PU.1 expression by modulating mRNA translation. We propose that such antisense RNAs will likely be important in the regulation of many genes and may be the reason for the large number of overlapping complementary transcripts with so far unknown function.

Dexamethasone and corticosterone induce similar, but not identical, muscle wasting responses in cultured L6 and C2C12 myotubes

Dexamethasone‐treated L6 (a rat cell line) and C2C12 (a mouse cell line) myotubes are frequently used as in vitro models of muscle wasting. We compared the effects of different concentrations of dexamethasone and corticosterone (the naturally occurring glucocorticoid in rodents) on protein breakdown rates, myotube size, and atrogin‐1 and MuRF1 mRNA levels in the two cell lines. In addition, the expression of the glucocorticoid receptor (GR) and its role in glucocorticoid‐induced metabolic changes were determined. Treatment with dexamethasone or corticosterone resulted in dose‐dependent increases in protein degradation rates in both L6 and C2C12 myotubes accompanied by 25–30% reduction of myotube diameter. The same treatments increased atrogin‐1 mRNA levels in L6 and C2C12 myotubes but, surprisingly, upregulated the expression of MuRF1 in L6 myotubes only. Both cell types expressed the GR and treatment with dexamethasone or corticosterone downregulated total cellular GR levels while increasing nuclear translocation of the GR in both L6 and C2C12 myotubes. The GR antagonist RU38486 inhibited the dexamethasone‐ and corticosterone‐induced increases in atrogin‐1 and MuRF1 expression in L6 myotubes but not in C2C12 myotubes. Interestingly, RU38486 exerted agonist effects in the C2C12, but not in the L6 myotubes. The present results suggest that muscle wasting‐related responses to dexamethasone and corticosterone are similar, but not identical, in L6 and C2C12 myotubes. Most notably, the regulation by glucocorticoids of MuRF1 and the role of the GR may be different in the two cell lines. These differences need to be taken into account when cultured myotubes are used in future studies to further explore mechanisms of muscle wasting. J. Cell. Biochem. 105: 353–364, 2008.

PD-1 Increases PTEN Phosphatase Activity While Decreasing PTEN Protein Stability by Inhibiting Casein Kinase 2

ABSTRACT Programmed death 1 (PD-1) is a potent inhibitor of T cell responses. PD-1 abrogates activation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, but the mechanism remains unclear. We determined that during T cell receptor (TCR)/CD3- and CD28-mediated stimulation, PTEN is phosphorylated by casein kinase 2 (CK2) in the Ser380-Thr382-Thr383 cluster within the C-terminal regulatory domain, which stabilizes PTEN, resulting in increased protein abundance but suppressed PTEN phosphatase activity. PD-1 inhibited the stabilizing phosphorylation of the Ser380-Thr382-Thr383 cluster within the C-terminal domain of PTEN, thereby resulting in ubiquitin-dependent degradation and diminished abundance of PTEN protein but increased PTEN phosphatase activity. These effects on PTEN were secondary to PD-1-mediated inhibition of CK2 and were recapitulated by pharmacologic inhibition of CK2 during TCR/CD3- and CD28-mediated stimulation without PD-1. Furthermore, PD-1-mediated diminished abundance of PTEN was reversed by inhibition of ubiquitin-dependent proteasomal degradation. Our results identify CK2 as a new target of PD-1 and reveal an unexpected mechanism by which PD-1 decreases PTEN protein expression while increasing PTEN activity, thereby inhibiting the PI3K/Akt signaling axis.

Expression and activity of C/EBPβ and δ are upregulated by dexamethasone in skeletal muscle

The influence of glucocorticoids on the expression and activity of the transcription factors CCAAT/enhancer binding protein (C/EBP)β and δ in skeletal muscle was examined by treating rats or cultured L6 myotubes with dexamethasone. Treatment of rats with 10 mg/kg of dexamethasone resulted in increased C/EBPβ and δ DNA binding activity in the extensor digitorum longus muscle as determined by electrophoretic mobility shift assay (EMSA) and supershift analysis. A similar response was noticed in dexamethasone‐treated myotubes. In other experiments, myocytes were transfected with a plasmid containing a promoter construct consisting of multiple C/EBP binding elements upstream of a luciferase reporter gene. Treatment of these cells with dexamethasone resulted in a fourfold increase in luciferase activity, suggesting that glucocorticoids increase C/EBP‐dependent gene activation in muscle cells. In addition, dexamethasone upregulated the protein and gene expression of C/EBPβ and δ in the myotubes in a time‐ and dose‐dependent fashion as determined by Western blotting and real‐time PCR, respectively. The results suggest that glucocorticoids increase C/EBPβ and δ activity and expression through a direct effect in skeletal muscle.

Context-dependent differences in miR-10b breast oncogenesis can be targeted for the prevention and arrest of lymph node metastasis.

Metastases, and not the primary tumor from which they originate, are the main reason for mortality from carcinoma. Although the molecular mechanisms behind metastasis are poorly understood, it is clear that epigenetic dysregulation at the level of microRNA expression is a key characteristic of the metastatic process that can be exploited for therapy. Here, we describe an miRNA-targeted therapeutic approach for the prevention and arrest of lymph node metastasis. Therapy relies on the inhibition of the pro-metastatic microRNA-10b. It is delivered to primary and lymph node metastatic tumor cells using an imaging-capable nanodrug that is designed to specifically home to these tissues. Treatment of invasive human breast tumor cells (MDA-MB-231) with the nanodrug in vitro downregulates miR-10b and abolishes the invasion and migration of the tumor cells. After intravenous delivery to mice bearing orthotopic MDA-MB-231-luc-D3H2LN tumors, the nanodrug accumulates in the primary tumor and lymph nodes. When treatment is initiated before metastasis to lymph nodes, metastasis is prevented. Treatment after the formation of lymph node metastases arrests the metastatic process without a concomitant effect on primary tumor growth raising the possibility of a context-dependent variation in miR-10b breast oncogenesis.

Dexamethasone upregulates the expression of the nuclear cofactor p300 and its interaction with C/EBPβ in cultured myotubes

Muscle wasting during sepsis and other catabolic conditions is, at least in part, mediated by glucocorticoids and is associated with upregulated transcription of multiple genes in the ubiquitin‐proteasome proteolytic pathway. In addition to transcription factors, nuclear cofactors, including p300, regulate gene transcription. We tested the hypothesis that glucocorticoids upregulate the expression of p300 in muscle cells. Treatment of cultured L6 myotubes, a rat skeletal muscle cell line, with dexamethasone resulted in a dose‐ and time‐dependent increase in p300 protein and mRNA levels. Surprisingly, the effect of dexamethasone on p300 levels was not inhibited by the glucocorticoid receptor (GR) antagonist RU38486 and RU38486 exerted an agonist effect on p300, increasing its expression. Co‐immunoprecipitation showed that treatment of the myotubes with dexamethasone resulted in protein–protein interaction between p300 and C/EBPβ, but not C/EBPδ. The present results suggest that glucocorticoids upregulate the expression of p300 and its interaction with C/EBPβ in skeletal muscle. Increased expression and activity of p300 may be involved in the regulation of gene transcription in glucocorticoid‐dependent muscle wasting. J. Cell. Biochem. 94: 1058–1067, 2005.