Yingmiao Liu

In vivo selection of tumor-targeting RNA motifs

In an effort to target the in vivo context of tumor-specific moieties, a large library of nuclease-resistant RNA oligonucleotides was screened in tumor-bearing mice to identify candidate molecules with the ability to localize to hepatic colon cancer metastases. One of the selected molecules is an RNA aptamer that binds to protein p68, an RNA helicase that has been shown to be upregulated in colorectal cancer.

RNA Aptamer-targeted Inhibition of NF-κB Suppresses Non-small Cell Lung Cancer Resistance to Doxorubicin

Due to the prevalence of tumor chemoresistance, the clinical response of advanced non-small cell lung cancer (NSCLC) to chemotherapy is poor. We suppressed tumor resistance to doxorubicin (Dox) in A549 cells, a human NSCLC cell line, both in vitro and in vivo in a lung tumor xenograft model, using a novel adenoviral expression system to deliver an RNA aptamer (A-p50) that specifically inhibits nuclear factor-κB (NF-κB) activation. By achieving selective, targeted, and early inhibition of NF-κB activity, we demonstrate that NF-κB plays a critical role in Dox-induced chemoresistance by regulating genes involved in proliferation (Ki-67), response to DNA damage (GADD153), antiapoptosis (Bcl-XL), and pH regulation (CA9). This Dox-induced NF-κB activation and subsequent chemoresistance is dependent on expression of p53. We also demonstrate that NF-κB promotes angiogenesis in the presence of Dox via the hypoxia-inducible factor-1α/vascular endothelial growth factor (HIF-1α/VEGF) pathway, revealing a previously unknown mechanism of NSCLC resistance to Dox. These studies provide important insights into the mechanisms of Dox-induced chemoresistance, and they demonstrate a novel, effective, and clinically practical strategy for interfering with these processes.

Aptamers selected against the unglycosylated EGFRvIII ectodomain and delivered intracellularly reduce membrane-bound EGFRvIII and induce apoptosis.

Abstract Epidermal growth factor receptor variant III (EGFRvIII) is a glycoprotein uniquely expressed in glioblastoma, but not in normal brain tissues. To develop targeted therapies for brain tumors, we selected RNA aptamers against the histidine-tagged EGFRvIII ectodomain, using an Escherichia coli system for protein expression and purification. Representative aptamer E21 has a dissociation constant (Kd) of 33×10-9 m, and exhibits high affinity and specificity for EGFRvIII in ELISA and surface plasmon resonance assays. However, selected aptamers cannot bind the same protein expressed from eukaryotic cells because glycosylation, a post-translational modification present only in eukaryotic systems, significantly alters the structure of the target protein. By transfecting EGFRvIII aptamers into cells, we find that membrane-bound, glycosylated EGFRvIII is reduced and the percentage of cells undergoing apoptosis is increased. We postulate that transfected aptamers can interact with newly synthesized EGFRvIII, disrupt proper glycosylation, and reduce the amount of mature EGFRvIII reaching the cell surface. Our work establishes the feasibility of disrupting protein post-translational modifications in situ with aptamers. This finding is useful for elucidating the function of proteins of interest with various modifications, as well as dissecting signal transduction pathways.

H1 RNA polymerase III promoter-driven expression of an RNA aptamer leads to high-level inhibition of intracellular protein activity

Aptamers offer advantages over other oligonucleotide-based approaches that artificially interfere with target gene function due to their ability to bind protein products of these genes with high affinity and specificity. However, RNA aptamers are limited in their ability to target intracellular proteins since even nuclease-resistant aptamers do not efficiently enter the intracellular compartments. Moreover, attempts at expressing RNA aptamers within mammalian cells through vector-based approaches have been hampered by the presence of additional flanking sequences in expressed RNA aptamers, which may alter their functional conformation. In this report, we successfully expressed a ‘pure’ RNA aptamer specific for NF-κB p50 protein (A-p50) utilizing an adenoviral vector employing the H1 RNA polymerase III promoter. Binding of the expressed aptamer to its target and subsequent inhibition of NF-κB mediated intracellular events were demonstrated in human lung adenocarcinoma cells (A549), murine mammary carcinoma cells (4T1) as well as a human tumor xenograft model. This success highlights the promise of RNA aptamers to effectively target intracellular proteins for in vitro discovery and in vivo applications.

Correlation of angiogenic biomarker signatures with clinical outcomes in metastatic colorectal cancer patients receiving capecitabine, oxaliplatin, and bevacizumab

A novel combination of capecitabine, oxaliplatin, and bevacizumab was evaluated in colorectal cancer patients enrolled in a phase II clinical trial. In this retrospective analysis, plasma samples from patients receiving capecitabine, oxaliplatin, and bevacizumab were analyzed to investigate biomarkers of clinical benefit. Forty‐one protein biomarkers were tested in 38 patients at baseline and after two cycles of drug administration. Correlations among analytes were evaluated by Spearman analysis. Analyte levels at baseline and changes on‐treatment were correlated with progression‐free survival (PFS) and overall survival (OS) by univariate analysis. Multivariate analyses were determined using the Cox proportional hazard model. Time to event analyses were evaluated by Kaplan–Meier analysis and compared by log‐rank test. Baseline levels of vWF and Ang‐2 significantly correlated with PFS, while levels of VCAM‐1, vWF, TSP‐2, IL‐8, MMP‐2, and Ang‐2 correlated with OS (P < 0.05). The fold change of IGF‐1 levels from baseline to the end of cycle 2 was correlated with PFS, while fold changes of Ang‐2, TSP‐2, and TGF‐β2 correlated with OS. A baseline signature of Ang‐2, IGFBP‐3, IL‐6, and VCAM‐1 identified a low‐risk and high‐risk group of patients (OS: 33.9 months vs. 18.1 months, respectively, P = 0.016). For treatment‐related changes, a signature consisting of Ang‐2, E‐Cadherin, IL‐6, MCP‐1, OPN, and TGF‐β1 was able to stratify patients into high‐ and low‐risk groups (PFS: 7.7 months vs. 15.5 months, P = 0.004). Multiplex analysis of patient plasma in this trial identified several baseline‐ and treatment‐related biomarkers associated with clinical outcome. These findings merit further exploration in larger, controlled clinical trials.

Neural plasticity and addiction: integrin-linked kinase and cocaine behavioral sensitization.

Behavioral sensitization of psychostimulants was accompanied by alterations in a variety of biochemical molecules in different brain regions. However, which change is actually related to drug‐produced sensitization lacks of accurate clarification. In this study, we investigated the role of integrin‐linked kinase (ILK) in both the induction and expression of cocaine sensitization. Conditional inhibition of ILK expression was established in the nucleus accumbens (NAc) core by microinjecting recombinant adeno‐associated virus‐carrying, tetracycline‐on‐regulated small interfering RNA which reversed the chronic cocaine‐induced psychomotor sensitization, as well as the changes in protein kinase B Ser473 phosphorylation, dendritic density, and dendritic spine numbers locally. Importantly, the reversed psychomotor sensitization did not recover after cessation of the silencing for 8 days. We also demonstrated that inhibition of ILK expression pre‐ and during‐chronic cocaine treatments blocked the induction of cocaine psychomotor sensitization and abolished the stimulant effect of cocaine on ILK expression. In contrast, inhibition of ILK expression in the NAc core has no significant effect on cocaine‐induced stereotypical behaviors. This concludes that ILK is involved in cocaine sensitization with the earlier induction and later expression functioning as a kinase to regulate protein kinase B Ser473 phosphorylation and a scaffolding protein to regulate the reorganization of the NAc spine morphology.

Effects of the combination of TRC105 and bevacizumab on endothelial cell biology

SummaryEndoglin, or CD105, is a cell membrane glycoprotein that is overexpressed on proliferating endothelial cells (EC), including those found in malignancies and choroidal neovascularization. Endoglin mediates the transition from quiescent endothelium, characterized by the relatively dominant state of Smad 2/3 phosphorylation, to active angiogenesis by preferentially phosphorylating Smad 1/5/8. The monoclonal antibody TRC105 binds endoglin with high avidity and is currently being tested in phase 1b and phase 2 clinical trials. In this report, we evaluated the effects of TRC105 on primary human umbilical vascular endothelial cells (HUVEC) as a single agent and in combination with bevacizumab. As single agents, both TRC105 and bevacizumab efficiently blocked HUVEC tube formation, and the combination of both agents achieved even greater levels of inhibition. We further assessed the effects of each drug on various aspects of HUVEC function. While bevacizumab was observed to inhibit HUVEC viability in nutrient-limited medium, TRC105 had little effect on HUVEC viability, either alone or in combination with bevacizumab. Additionally, both drugs inhibited HUVEC migration and induced apoptosis. At the molecular level, TRC105 treatment of HUVEC lead to decreased Smad 1/5/8 phosphorylation in response to BMP-9, a primary ligand for endoglin. Together, these results indicate that TRC105 acts as an effective anti-angiogenic agent alone and in combination with bevacizumab.

Modulation of circulating protein biomarkers following TRC105 (anti-endoglin antibody) treatment in patients with advanced cancer

TRC105 is an endoglin‐targeting drug that possesses anti‐angiogenic and antitumor potential. Analysis of the initial phase I trial of TRC105 demonstrated good tolerability and efficacy in cancer patients. In this report, we analyzed multiple circulating biomarkers at baseline, cycle 2 day 1 (C2D1), and end of study (EOS) for each patient. The baseline level and the fold change from baseline to both C2D1 and EOS for each marker were statistically analyzed. At C2D1, seven markers were significantly downregulated (angiopoietin‐2 [Ang‐2], insulin‐like growth factor‐binding protein‐3 [IGFBP‐3], plasminogen activator inhibitor‐1 [PAI‐1] total, platelet‐derived growth factor [PDGF]‐AA, PDGF‐BB, thrombospondin‐1 [TSP‐1], and vascular endothelial growth factor [VEGF]‐D). Meanwhile, seven markers were upregulated by C2D1 (E‐Cadherin, soluble Endoglin [sEnd], E‐Selectin, interleukin‐6 [IL‐6], osteopontin [OPN], TSP‐2, and von Willebrand factor [vWF]). At EOS, seven markers were upregulated including Ang‐2, C‐reactive protein (CRP), intercellular adhesion molecule‐1 (ICAM‐1), IGFBP‐1, IL‐6, TSP‐2, and vascular cell adhesion molecule‐1 (VCAM‐1). A statistical trend was also seen for increases of VEGF‐A and placenta growth factor (PlGF) at EOS. Throughout treatment, sEnd levels significantly increased, an observation that was recapitulated in cultured endothelial cells. This is the first report of plasma‐based biomarkers in patients receiving TRC105. TRC105 treatment by C2D1 was associated with decreases in several angiogenic factors, including Ang‐2, PDGF isoforms, and VEGF isoforms, offering insight into the mechanisms underlying TRC105s anti‐angiogenic, antitumor function. Increases in sEnd were the most significant of all observed biomarker changes and may reflect direct drug effects. Additionally, biomarker changes in response to TRC105 are distinct from those seen in patients treated with VEGF‐targeting drugs, suggesting the possible utility of combining these two classes of angiogenesis inhibitors in patients.

Targeting inhibition of GluR1 Ser845 phosphorylation with an RNA aptamer that blocks AMPA receptor trafficking

Phosphorylation at glutamate receptor subunit 1(GluR1) Ser845 residue has been widely accepted to involve in GluR1‐containing α‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionic acid (AMPA) receptor trafficking, but the in vivo evidence has not yet been established. One of the main obstacles is the lack of effective methodologies to selectively target phosphorylation at single amino acid residue. In this study, the Escherichia coli‐expressed glutathione‐S‐transferase‐tagged intracellular carboxyl‐terminal domain of GluR1 (cGluR1) was phosphorylated by protein kinase A for in vitro selection. We have successfully selected aptamers which effectively bind to phospho‐Ser845 cGluR1 protein, but without binding to phospho‐Ser831 cGluR1 protein. Moreover, pre‐binding of the unphospho‐cGluR1 protein with these aptamers inhibits protein kinase A‐mediated phosphorylation at Ser845 residue. In contrast, the pre‐binding of aptamer A2 has no effect on protein kinase C‐mediated phosphorylation at Ser831 residue. Importantly, the representative aptamer A2 can effectively bind the mammalian GluR1 that inhibited GluR1/GluR1‐containing AMPA receptor trafficking to the cell surface and abrogated forskolin‐stimulated phosphorylation at GluR1 Ser845 in both green fluorescent protein–GluR1‐transfected human embryonic kidney cells and cultured rat cortical neurons. The strategy to use aptamer to modify single‐residue phosphorylation is expected to facilitate evaluation of the potential role of AMPA receptors in various forms of synaptic plasticity including that underlying psychostimulant abuse.

The role of angiogenesis in Group 3 medulloblastoma pathogenesis and survival

Background Of the 4 medulloblastoma subgroups, Group 3 is the most aggressive but the importance of angiogenesis is unknown. This study sought to determine the role of angiogenesis and identify clinically relevant biomarkers of tumor vascularity and survival in Group 3 medulloblastoma. Methods VEGFA mRNA expression and survival from several patient cohorts were analyzed. Group 3 xenografts were implanted intracranially in nude rats. Dynamic susceptibility weighted (DSC) MRI and susceptibility weighted imaging (SWI) were obtained. DSC MRI was used to calculate relative cerebral blood volume (rCBV) and flow (rCBF). Tumor vessel density and rat vascular endothelial growth factor alpha (VEGFA) expression were determined. Results Patient VEGFA mRNA levels were significantly elevated in Group 3 compared with the other subgroups (P < 0.001) and associated with survival. Xenografts D283, D341, and D425 were identified as Group 3 by RNA hierarchical clustering and MYC amplification. The D283 group had the lowest rCBV and rCBF, followed by D341 and D425 (P < 0.05). These values corresponded to histological vessel density (P < 0.05), rat VEGFA expression (P < 0.05), and survival (P = 0.002). Gene set enrichment analysis identified 5 putative genes with expression profiles corresponding with these findings: RNH1, SCG2, VEGFA, AGGF1, and PROK2. SWI identified 3 xenograft-independent categories of intratumoral vascular architecture with distinct survival (P = 0.004): organized, diffuse microvascular, and heterogeneous. Conclusions Angiogenesis plays an important role in Group 3 medulloblastoma pathogenesis and survival. DSC MRI and SWI are clinically relevant biomarkers for tumor vascularity and overall survival and can be used to direct the use of antivascular therapies for patients with Group 3 medulloblastoma.