Milota Kaluzova

EGFRvIII Antibody–Conjugated Iron Oxide Nanoparticles for Magnetic Resonance Imaging–Guided Convection-Enhanced Delivery and Targeted Therapy of Glioblastoma

The magnetic nanoparticle has emerged as a potential multifunctional clinical tool that can provide cancer cell detection by magnetic resonance imaging (MRI) contrast enhancement as well as targeted cancer cell therapy. A major barrier in the use of nanotechnology for brain tumor applications is the difficulty in delivering nanoparticles to intracranial tumors. Iron oxide nanoparticles (IONP; 10 nm in core size) conjugated to a purified antibody that selectively binds to the epidermal growth factor receptor (EGFR) deletion mutant (EGFRvIII) present on human glioblastoma multiforme (GBM) cells were used for therapeutic targeting and MRI contrast enhancement of experimental glioblastoma, both in vitro and in vivo, after convection-enhanced delivery (CED). A significant decrease in glioblastoma cell survival was observed after nanoparticle treatment and no toxicity was observed with treatment of human astrocytes (P < 0.001). Lower EGFR phosphorylation was found in glioblastoma cells after EGFRvIIIAb-IONP treatment. Apoptosis was determined to be the mode of cell death after treatment of GBM cells and glioblastoma stem cell-containing neurospheres with EGFRvIIIAb-IONPs. MRI-guided CED of EGFRvIIIAb-IONPs allowed for the initial distribution of magnetic nanoparticles within or adjacent to intracranial human xenograft tumors and continued dispersion days later. A significant increase in animal survival was found after CED of magnetic nanoparticles (P < 0.01) in mice implanted with highly tumorigenic glioblastoma xenografts (U87DeltaEGFRvIII). IONPs conjugated to an antibody specific to the EGFRvIII deletion mutant constitutively expressed by human glioblastoma tumors can provide selective MRI contrast enhancement of tumor cells and targeted therapy of infiltrative glioblastoma cells after CED.

Proteasomal Inhibition Attenuates Transcriptional Activity of Hypoxia-Inducible Factor 1 (HIF-1) via Specific Effect on the HIF-1α C-Terminal Activation Domain

ABSTRACT The ubiquitin-proteasome pathway (UPP) is involved in regulation of multiple cellular processes. Hypoxia-inducible factor 1α (HIF-1α) is a prototypic target of the UPP and, as such, is stabilized under conditions of proteasomal inhibition. Using carbonic anhydrase IX (CAIX) and vascular endothelial growth factor (VEGF) expression as paradigmatic markers of HIF-1 activity, we found that proteasomal inhibitors (PI) abrogated hypoxia-induced CAIX expression in all cell lines tested and VEGF expression in two out of three. Mapping of the inhibitory effect identified the C-terminal activation domain (CAD) of HIF-1α as the primary target of PI. PI specifically inhibited the HIF-1α CAD despite activating the HIF-1α coactivator p300 and another p300 cysteine/histidine-rich domain 1-dependent transcription factor, STAT-2. Coimmunoprecipitation and glutathione S-transferase pull downs indicated that PI does not disrupt interactions between HIF-1α and p300. Mutational analysis failed to confirm involvement of sites of known or putative posttranslational modifications in regulation of HIF-1α CAD function by PI. Our data provide evidence for the counterintuitive hypothesis that inhibition of HIF-1 function could be responsible for at least some of the antitumor effects of proteasomal inhibition. Further studies of the mechanism of the PI-induced attenuation of HIF-1α will provide important, potentially novel insight into regulation of HIF-1 activity and possibly identify new targets for HIF-directed therapy.

Magnetic nanoparticles: an emerging technology for malignant brain tumor imaging and therapy

Magnetic nanoparticles (MNPs) represent a promising nanomaterial for the targeted therapy and imaging of malignant brain tumors. Conjugation of peptides or antibodies to the surface of MNPs allows direct targeting of the tumor cell surface and potential disruption of active signaling pathways present in tumor cells. Delivery of nanoparticles to malignant brain tumors represents a formidable challenge due to the presence of the blood–brain barrier and infiltrating cancer cells in the normal brain. Newer strategies permit better delivery of MNPs systemically and by direct convection-enhanced delivery to the brain. Completion of a human clinical trial involving direct injection of MNPs into recurrent malignant brain tumors for thermotherapy has established their feasibility, safety and efficacy in patients. Future translational studies are in progress to understand the promising impact of MNPs in the treatment of malignant brain tumors.

DNA Damage Is a Prerequisite for p53-Mediated Proteasomal Degradation of HIF-1α in Hypoxic Cells and Downregulation of the Hypoxia Marker Carbonic Anhydrase IX

ABSTRACT We investigated the relationship between the tumor suppressor p53 and the hypoxia-inducible factor-1 (HIF-1)-dependent expression of the hypoxia marker, carbonic anhydrase IX (CAIX). MCF-7 (wt p53) and Saos-2 (p53-null) cells displayed similar induction of CAIX expression and CA9 promoter activity under hypoxic conditions. Activation of p53 by the DNA damaging agent mitomycin C (MC) was accompanied by a potent repression of CAIX expression and the CA9 promoter in MCF-7 but not in Saos-2 cells. The activated p53 mediated increased proteasomal degradation of HIF-1α protein, resulting in considerably lower steady-state levels of HIF-1α protein in hypoxic MCF-7 cells but not in Saos-2 cells. Overexpression of HIF-1α relieved the MC-induced repression in MCF-7 cells, confirming regulation at the HIF-1α level. Similarly, CA9 promoter activity was downregulated by MC in HCT 116 p53+/+ but not the isogenic p53−/− cells. Activated p53 decreased HIF-1α protein levels by accelerated proteasome-dependent degradation without affecting significantly HIF-1α transcription. In summary, our results demonstrate that the presence of wtp53 under hypoxic conditions has an insignificant effect on the stabilization of HIF-1α protein and HIF-1-dependent expression of CAIX. However, upon activation by DNA damage, wt p53 mediates an accelerated degradation of HIF-1α protein, resulting in reduced activation of CA9 transcription and, correspondingly, decreased levels of CAIX protein. A model outlining the quantitative relationship between p53, HIF-1α, and CAIX is presented.

Nanotechnology Applications for Glioblastoma

Glioblastoma remains one of the most difficult cancers to treat and represents the most common primary malignancy of the brain. Although conventional treatments have found modest success in reducing the initial tumor burden, infiltrating cancer cells beyond the main mass are responsible for tumor recurrence and ultimate patient demise. Targeting residual infiltrating cancer cells requires the development of new treatment strategies. The emerging field of cancer nanotechnology holds promise in the use of multifunctional nanoparticles for imaging and targeted therapy of glioblastoma. This article examines the current state of nanotechnology in the treatment of glioblastoma and directions of further study.

Intraoperative Spectroscopy with Ultrahigh Sensitivity for Image-Guided Surgery of Malignant Brain Tumors

Intraoperative cancer imaging and fluorescence-guided surgery have attracted considerable interest because fluorescence signals can provide real-time guidance to assist a surgeon in differentiating cancerous and normal tissues. Recent advances have led to the clinical use of a natural fluorophore called protoporphyrin IX (PpIX) for image-guided surgical resection of high-grade brain tumors (glioblastomas). However, traditional fluorescence imaging methods have only limited detection sensitivity and identification accuracy and are unable to detect low-grade or diffuse infiltrating gliomas (DIGs). Here we report a low-cost hand-held spectroscopic device that is capable of ultrasensitive detection of protoporphyrin IX fluorescence in vivo, together with intraoperative spectroscopic data obtained from both animal xenografts and human brain tumor specimens. The results indicate that intraoperative spectroscopy is at least 3 orders of magnitude more sensitive than the current surgical microscopes, allowing ultrasensitive detection of as few as 1000 tumor cells. For detection specificity, intraoperative spectroscopy allows the differentiation of brain tumor cells from normal brain cells with a contrast signal ratio over 100. In vivo animal studies reveal that protoporphyrin IX fluorescence is strongly correlated with both MRI and histological staining, confirming that the fluorescence signals are highly specific to tumor cells. Furthermore, ex vivo spectroscopic studies of excised brain tissues demonstrate that the hand-held spectroscopic device is capable of detecting diffuse tumor margins with low fluorescence contrast that are not detectable with current systems in the operating room. These results open new opportunities for intraoperative detection and fluorescence-guided resection of microscopic and low-grade glioma brain tumors with invasive or diffusive margins.

Comment on the role of FIH in the inhibitory effect of bortezomib on hypoxia-inducible factor-1

To the editor: Recently, Shin et al reported that the proteasomal inhibitor bortezomib inhibits the hypoxic response by stimulating the factor-inhibiting HIF-1 (FIH)–mediated repression of hypoxia-inducible factor-1 (HIF-1).[1][1] The essence of this study is that bortezomib enhances the FIH

5-Aminolevulinic Acid Guided Sampling of Glioblastoma Microenvironments Identifies Pro-Survival Signaling at Infiltrative Margins

Glioblastoma (GBM) contains diverse microenvironments with uneven distributions of oncogenic alterations and signaling networks. The diffusely infiltrative properties of GBM result in residual tumor at neurosurgical resection margins, representing the source of relapse in nearly all cases and suggesting that therapeutic efforts should be focused there. To identify signaling networks and potential druggable targets across tumor microenvironments (TMEs), we utilized 5-ALA fluorescence-guided neurosurgical resection and sampling, followed by proteomic analysis of specific TMEs. Reverse phase protein array (RPPA) was performed on 205 proteins isolated from the tumor margin, tumor bulk, and perinecrotic regions of 13 previously untreated, clinically-annotated and genetically-defined high grade gliomas. Differential protein and pathway signatures were established and then validated using western blotting, immunohistochemistry, and comparable TCGA RPPA datasets. We identified 37 proteins differentially expressed across high-grade glioma TMEs. We demonstrate that tumor margins were characterized by pro-survival and anti-apoptotic proteins, whereas perinecrotic regions were enriched for pro-coagulant and DNA damage response proteins. In both our patient cohort and TCGA cases, the data suggest that TMEs possess distinct protein expression profiles that are biologically and therapeutically relevant.

Abstract 415: Differential expression of therapeutic targets across tumor micro-environments and at infiltrative margins in glioblastoma

Glioblastoma (GBM; WHO grade IV) is the most common primary brain tumor and is associated with a poor prognosis. The Cancer Genome Atlas (TCGA) project has revealed discrete molecular classes of GBM, raising the possibility of targeted therapies. Molecular heterogeneity across tumor microenvironments presents a challenge to targeted therapy, since effective therapies will be directed at targets near resection margins rather than those expressed in bulk resected tumor. To this end, we initiated studies to delineate spatial and temporal variations of potential druggable targets in GBM, focusing on potential protein targets at the tumor margin. By utilizing 5-ALA (5-aminolevulinic acid, an oral compound that accumulates in malignant glioma cells and is metabolized to the fluorescent agent, protoporphyrin IX) fluorescence-guided surgery of primary and recurrent GBMs in a Phase II clinical trial, we were able to precisely define perinecrotic, bulk, and marginal regions within the glioma intraoperatively in order to sample tissues for further molecular characterization. Tissues harvested from these 3 tumor regions from 5 patients were analyzed using reverse phase protein array (RPPA) analysis with 218 antibodies (Array-Pro). Relative protein levels were determined and a heatmap was generated as a hierarchical cluster using Pearson Correlation and a center metric. Forty proteins had statistically significant altered expression at the tumor margin compared with bulk and perinecrotic tumor. Among these, STAT1, STAT2, STAT3, PDGFRA, PDGFRB, EGFR, JAK2, MET, PIK3R1, and ERBB2 were upregulated at the margins and are potential druggable targets worthy of further investigation. Also upregulated at the tumor margins were NFKBIA, JUN, ZAP70, GAPDH, and CD28, whereas STAT5A, EFNB2, NTRK1, KDR, FGFR1, PTPRA, IFNGR1, and EFNB1 were downregulated at margins. Our data demonstrate significant variation of druggable targets and signaling networks across tumor microenvironments in GBM. Marginal tumor differs from bulk and perinecrotic tumor by the relative overexpression of STAT proteins, growth factor receptors, and receptor kinases. Directing therapy at networks and targets present at the tumor margin of GBM may be an effective strategy. Citation Format: Myles R. McCrary, David Gutman, William Dunn, Milota Kaluzova, Alexandros Bouros, Merete Williams, Xialong Zhang, Lee AD Cooper, Constantinos G. Hadjipanayis, Daniel J. Brat. Differential expression of therapeutic targets across tumor micro-environments and at infiltrative margins in glioblastoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 415. doi:10.1158/1538-7445.AM2015-415

Abstract 3335: Identification of candidate proteins for the targeted imaging and therapy of glioma stem cells: Effect of antibody-conjugated magnetic nanoparticles and proteasomal inhibitor bortezomib on GBM-derived neurospheres and glioma stem cell marker CD133

Proceedings: AACR 101st Annual Meeting 2010‐‐ Apr 17‐21, 2010; Washington, DC Introduction: Glioblastoma multiforme (GBM) is the most common and lethal malignant primary brain tumor in adults. Glioma stem cells (GSCs) have properties of self renewal, pluripotency and high tumorigenicity. Magnetic iron oxide nanoparticles (IONPs) have emerged as a potential multifunctional clinical tool that can provide cancer cell detection by magnetic resonance imaging (MRI) contrast enhancement as well as therapy by cancer cell targeted delivery of agents. In this study, we have studied differential activation of signaling pathways in purified CD133-positive GSCs in an effort to identify candidate proteins for the targeted imaging and therapy of GSCs by magnetic nanoparticles. We have also studied the therapeutic effect of IONPs conjugated with an EGFRvIII antibody against GSCs and the regulation of the GSC marker, CD133, by the proteasomal inhibitor bortezomib. Methods: Glioblastoma tumor specimens were used to isolate neurospheres. Neurospheres of an EGFRvIII-negative and EGFRvIII-positive GBM tumors were further enriched for CD133-positive cells using Indirect CD133 MicroBeads kit or FACS analysis to ∼90% purity. Magnetic nanoparticles were conjugated with the EGFRvIII antibody using Ocean NanoTech kit. Neurospheres were treated with 200 nM bortezomib. Results: Using Western blot analysis of CD133-positive GSCs from different patients, we identified nestin, wt EGFR, EGFRvIII, PDGFRs, carbonic anhydrase IX (CAIX) podoplanin as well as CD133 marker as potential candidates for the targeted imaging and therapy of GSCs. Hypoxia (1% O2) greatly enhanced expression of CD133 antigen in EGFRvIII positive neurospheres in a HIF-2α-independent manner. Treatment with proteasomal inhibitor bortezomib decreased expression of CD133. EGFRvIII antibody conjugated to IONPs (EGFRvIIIAb-IONPs) promoted apoptosis of not only EGFRvIII-positive stem cells, but also CD133-negative cells. Conclusion: Candidate proteins found in CD133-positive GSCs include nestin, wt EGFR, EGFRvIII, PDGFRβ, CAIX, and podoplanin. Hypoxia activated expression of CD133 and CAIX, a well-established hypoxia marker. The proteasomal inhibitor bortezomib downregulated hypoxia-induced expression of CD133 and CAIX. Antibodies against the candidate proteins, conjugated to magnetic nanoparticles, may form the basis of targeted imaging and therapy of GSCs. IONPs conjugated to an antibody specific to the EGFRvIII deletion mutant can promote apoptosis in neurospheres, CD133-positive GSCs, and CD133-negative neurospheres. In conclusion, a combination therapy in which GSCs are targeted a) with antibody-conjugated IONPs b) with proteasomal inhibitors to downregulate GSCs markers may have a synergistic effect against GSCs. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 3335.