Denise M. Gibo

EphA2 as a Novel Molecular Marker and Target in Glioblastoma Multiforme

We investigated the presence of EphA2, and its ligand, ephrinA1, in glioblastoma multiforme (GBM), a malignant neoplasm of glial cells, and normal brain. We also initially examined the functional importance of the interaction between EphA2 and ephrinA1 in glioma cells. Expression and localization of EphA2 and ephrinA1 in human GBM and normal brain were examined using Western blotting, immunofluorescence, and immunohistochemistry. A functional role for EphA2 was investigated by assessing the activation status of the receptor and the effect of ephrinA1 on the anchorage-independent growth and invasiveness of GBM cells. We found EphA2 to be elevated in ∼90% of GBM specimens and cell lines but not in normal brain, whereas ephrinA1 was present at consistently low levels in both GBM and normal brain. EphA2 was activated and phosphorylated by ephrinA1 in GBM cells. Furthermore, ephrinA1 induced a prominent, dose-dependent inhibitory effect on the anchorage-independent growth and invasiveness of GBM cells highly overexpressing EphA2, which was not seen in cells expressing low levels of the receptor. Thus, EphA2 is both specifically overexpressed in GBM and expressed differentially with respect to its ligand, ephrinA1, which may reflect on the oncogenic processes of malignant glioma cells. EphA2 seems to be functionally important in GBM cells and thus may play an important role in GBM pathogenesis. Hence, EphA2 represents a new marker and novel target for the development of molecular therapeutics against GBM.

Novel anti–brain tumor cytotoxins specific for cancer cells

The vast majority of brain cancers (gliomas) express a receptor (R) for interleukin 13 (IL13). In order to achieve specific targeting of the IL13R in gliomas, we have mutagenized human (h) IL13. The mutation was made to alter IL13 interaction with the shared functional IL13/4 normal tissue receptor, but not with the glioma-associated receptor. We have thus produced MIL13.E13K (glutamic acid at position 13 changed to lysine) and fused it to derivatives of Pseudomonas exotoxin A. The hlL13.E13K-based cytotoxins are less active on normal cells and thus less toxic, and are better antitumor agents compared with the cytotoxins containing nonmutagenized hlL13.

A novel, potent, and specific ephrinA1-based cytotoxin against EphA2 receptor–expressing tumor cells

We have previously shown that the EphA2 receptor tyrosine kinase is overexpressed in glioblastoma multiforme (GBM) and represents a novel, attractive therapeutic target for the treatment of brain tumors. Here, we have developed an EphA2-targeted agent, ephrinA1-PE38QQR, a novel cytotoxin composed of ephrinA1, a ligand for EphA2, and PE38QQR, a mutated form of Pseudomonas aeruginosa exotoxin A. EphrinA1-PE38QQR showed potent and dose-dependent killing of GBM cells overexpressing the EphA2 receptor in cell viability and clonogenic survival assays, with an average IC50 of ∼10−11 mol/L. The conjugate was also highly effective in killing breast and prostate cancer cells overexpressing EphA2. The cytotoxic effect of ephrinA1-PE38QQR was specific, as it was neutralized by an excess of EphA2 ligands. Moreover, normal human endothelial cells and breast cancer cells that do not overexpress EphA2, as well as GBM cells that have down-regulated EphA2, were not susceptible to the cytotoxin. EphrinA1-PE38QQR–mediated cytotoxicity induced caspase-dependent apoptosis, which was, however, not responsible for cell death in response to the conjugate. In addition, the conjugate elicited no changes in the activity of survival pathways such as phosphoinositide 3-kinase, measured by AKT phosphorylation. This is the first attempt to create a cytotoxic therapy using any of the ephrin ligands of either class (A or B) conjugated to a bacterial toxin. EphrinA1-PE38QQR is very potent and specific, produces cell death that is caspase independent, and forms the basis for the further development of clinically applicable EphA2-targeted cytotoxins. [Mol Cancer Ther 2007;6(12):3208–18]

Expression of a Restrictive Receptor for Interleukin 13 is Associated with Glial Transformation

We have previously documented that the vast majority of high-grade gliomas over-express binding sites for interleukin 13 (IL13) in situ. We now extend this analysis to evaluate the distribution of the binding of IL13 among other brain tumors. Tumor specimens from patients with low-grade gliomas, oligodendrogliomas, ependymomas, pilocytic astrocytomas, gliosarcomas, medulloblastomas, meningiomas, and metastases to the brain were analyzed and compared to a new series of gioblastoma multiforme (GBM) samples. Serial tumor tissue sections were incubated with 125I-labeled (i) IL13, (ii) antibody against transferrin (Tf) receptor, and (iii) epidermal growth factor (EGF). Most (17/18) GBMs stained specifically for IL13 binding sites while sections from 3/11 low-grade gliomas, 5/5 high-grade gliomas (grade III), 3/5 oligodendrogliomas (all three were anaplastic), and 1/2 gliosarcomas also showed specific binding for IL13. We did not detect IL13 binding sites in medulloblastomas (0/4) and found them only in 2/20 meningiomas. Metastases to the brain (4/12, i.e., lung adenocarcinomas and renal cell carcinoma) showed some binding of 125I-IL13. The presence of receptors for Tf was ubiquitous among all studied tumors while EGF receptor expression was much more variable. Since it appears that primarily the least differentiated forms of gliomas possess IL13 binding sites in abundance, it is plausible that IL13 receptor expressed in low-grade gliomas might be a prognostically significant marker associated with their progression to high-grade gliomas. Finally, we demonstrate that the glioma-associated IL13 receptor is truly more restrictive in nature also due to its selective representation among brain tumors of glial origin.

Interleukin-13 receptor alpha 2, EphA2, and Fos-related antigen 1 as molecular denominators of high-grade astrocytomas and specific targets for combinatorial therapy.

Purpose: We investigated the expression of interleukin-13 receptor α2 (IL-13Rα2), EphA2, and Fos-related antigen 1 (Fra-1) in astrocytomas and normal brain. We sought to document whether the expression of the three factors changed with progression to higher grade malignancy and whether two or three targets in combination might be sufficient to target all patients with high-grade astrocytomas. Experimental Design: Immunohistochemistry was done for IL-13Rα2, EphA2, and Fra-1 using human brain tumor tissue microarrays containing 30 specimens of WHO grades II and III astrocytomas, 46 glioblastoma multiformes (GBM), and 9 normal brain samples. Sections were scored based on frequency and intensity of expression. Western blotting was done for all three markers using GBM tumor specimens and xenograft cell lines. Two cytotoxins, IL-13.E13K.PE38QQR and ephrinA1-PE38QQR, which target IL-13Rα2 or EphA2, respectively, were tested for cytotoxicity against human GBM primary explant cells and established cells. Results: Expression of all three proteins was significantly higher in GBM compared with normal brain, low-grade, and anaplastic astrocytomas. Greater than 95% of GBM overexpressed at least two of the three markers. Importantly, every GBM overexpressed at least one marker. Human GBM primary explant cells and cell lines were potently killed by IL-13.E13K.PE38QQR and ephrinA1-PE38QQR, in accordance with their level of expression of IL-13Rα2 and EphA2, respectively. Conclusions: IL-13Rα2, EphA2, and Fra-1 are attractive therapeutic targets representing molecular denominators of high-grade astrocytomas. One hundred percent of GBM tumors overexpress at least one of these proteins, providing the basis for rational combinatorial targeted therapies/diagnostics suitable for all patients with this disease.

Fos-Related Antigen 1 Modulates Malignant Features of Glioma Cells

Malignant gliomas, and high-grade gliomas (HGG) in particular, are nonmetastasizing but locally infiltrating, hypervascularized brain tumors of poor prognosis. We found previously that a c-fos-inducible vascular endothelial growth factor D is ubiquitously up-regulated in HGG grade IV, glioblastoma multiforme, and that glioblastoma multiforme overexpress Fos-related antigen 1 (Fra-1) rather than the c-Fos. We have thus become interested in the role Fra-1 may play in malignant glioma progression/maintenance, because Fra-1 has the capacity to modulate transcription of a variety of target genes. In this work, we have analyzed the biological effects of ectopic Fra-1 expression or Fra-1 knockdown in malignant glioma cells. Ectopic Fra-1 induced prominent phenotypic changes in all three malignant glioma cell lines examined: H4, U-87 MG, and A-172 MG. These changes were reflected in cells becoming more elongated with larger number of cellular processes. Furthermore, Fra-1 transgene caused H4 cells, which do not form tumor xenografts, to regain tumorigenic capacity. The genotype of these cells changed too, because 50 of 1,056 genes examined became either up-regulated or down-regulated. Conversely, Fra-1 knockdown altered prominently the morphology, anchorage-independent growth, tumorigenic potential, and Fra-1 effector expression, such as vascular endothelial growth factor D, in HGG cells. For example, cells transfected with antisense fra-1 showed shorter cellular processes than the control cells that did not grow in agar, and their tumorigenic potential was significantly diminished. Thus, Fra-1 may likely play an important role in the maintenance/progression of malignant gliomas and potentially represents a new target for therapeutic interventions.

Molecular targeting with recombinant cytotoxins of interleukin-13 receptor α2-expressing glioma

SummaryA restricted receptor for interleukin 13 (IL-13Rα2) is over-expressed in high-grade astrocytoma (HGA), but not in normal organs. In order to design and examine new anti-HGA therapies, which are molecularly directed against IL-13Rα2, we established an IL-13Rα2-expressing syngeneic immunocompetent murine model of HGA. The model was obtained by transfecting G-26 murine glioma cells with IL-13Rα2. G-26-IL-13Rα2(+) cells, but not mock-transfected cells, became susceptible to IL-13 mutant-based cytotoxic proteins that kill human HGA cells. G-26-IL-13Rα2(+) cells maintained their tumorigenicity in immunocompetent C57BL/J6 mice and preserved their expression of IL-13Rα2in vivo. These characteristics of the G-26-IL-13Rα2(+) tumors allowed us to test molecularly defined anti-glioma passive immunotherapy. A targeted recombinant chimera cytotoxin composed of multiply mutated IL-13 (IL-13.E13Y/R66D/S69D) and a derivative ofPseudomonas exotoxin (PE), PE1E, IL-13.E13Y/R66D/S69D-PE1E, was used in anti-tumor experiments. G-26-IL-13Rα2(+) cells were killed by IL-13.E13Y/R66D/S69D-PE1E in an IL-4-independent fashion. To test the cytotoxinin vivo, G-26-IL-13Rα2(+) tumors were established in C57BL/J6 mice and when the tumors reached a size of at least 50 mm3, the mice were treated with IL-13.E13Y/R66D/S69D-PE1E. In the mice treated with the targeted fusion cytotoxin, the tumors regressed and 80% of the animals were cured. This study documents the establishment of an IL-13Rα2-positive model of HGA in immunocompetent rodents. Furthermore, the effectiveness and safety of the targeted IL-13-based cytotoxin against IL-13Rα2-expressing tumors in a more clinically relevantin vivo HGA model is promising with regard to the future clinical utility of the cytotoxin.

Adult and developing human cerebella exhibit different profiles of opioid binding sites

The binding of [3H][D-Ala2,MePhe4,Gly-ol5]enkephalin (DAGO), [3H][D-Pen2,5]enkephalin (DPDPE), [3H]ethylketocyclazocine (EKC), and [3H][Met5]enkephalin (MET) was used to examine mu-, delta-, kappa-, and zeta-receptors, respectively, in the developing (birth to postnatal day 19) and adult human cerebellum. Specific and saturable binding of all ligands was recorded in developing brains, and of [3H]DAGO, [3H]DPDPE, and [3H]EKC in adult cerebellum; all data fit a single homogeneous binding site for each ligand. However, the ontogenic profile of opioid receptor subtypes differed. Delta- and kappa-receptor capacities were 7.8- and 3.6-fold, respectively, greater in infant cerebellum than in adults. The mu-receptor decreased over 7-fold in both binding affinity and capacity after day 2; by adulthood, the binding affinity was the same as in newborns but only one-half the binding capacity was recorded. The concentration of zeta-receptors was 20-fold greater in subjects 2-19 days of age than in newborns. These data demonstrate the presence, and distinct developmental profiles, of opioid receptors in human cerebellum. Although the function of mu-, delta-, and kappa-receptors in human cerebellum are unclear, the growth-related zeta-receptor is present at a time of cell replication and differentiation but is not detected in mature cerebellum.

An interleukin 13 receptor α 2–specific peptide homes to human Glioblastoma multiforme xenografts

Interleukin 13 receptor α 2 (IL-13Rα2) is a glioblastoma multiforme (GBM)-associated plasma membrane receptor, a brain tumor of dismal prognosis. Here, we isolated peptide ligands for IL-13Rα2 with use of a cyclic disulphide-constrained heptapeptide phages display library and 2 in vitro biopanning schemes with GBM cells that do (G26-H2 and SnB19-pcDNA cells) or do not (G26-V2 and SnB19-asIL-13Rα2 cells) over-express IL-13Rα2. We identified 3 peptide phages that bind to IL-13Rα2 in cellular and protein assays. One of the 3 peptide phages, termed Pep-1, bound to IL-13Rα2 with the highest specificity, surprisingly, also in a reducing environment. Pep-1 was thus synthesized and further analyzed in both linear and disulphide-constrained forms. The linear peptide bound to IL-13Rα2 more avidly than did the disulphide-constrained form and was efficiently internalized by IL-13Rα2-expressing GBM cells. The native ligand, IL-13, did not compete for the Pep-1 binding to the receptor and vice versa in any of the assays, indicating that the peptide might be binding to a site on the receptor different from the native ligand. Furthermore, we demonstrated by noninvasive near infrared fluorescence imaging in nude mice that Pep-1 binds and homes to both subcutaneous and orthotopic human GBM xenografts expressing IL-13Rα2 when injected by an intravenous route. Thus, we identified a linear heptapeptide specific for the IL-13Rα2 that is capable of crossing the blood-brain tumor barrier and homing to tumors. Pep-1 can be further developed for various applications in cancer and/or inflammatory diseases.

Novel way to increase targeting specificity to a human glioblastoma‐associated receptor for interleukin 13

Human brain cancers (gliomas) overexpress large numbers of a receptor for interleukin 13 (IL13), making this receptor an attractive target for anti‐glioma therapies. We have recently proposed that the glioma‐associated IL13 receptor is different from the one expressed on some hemopoietic and somatic cells. In an attempt to identify an even more glioma‐specific target, we have used an antagonist of a related cytokine, IL4, which neutralizes the physiological effects of both IL13 and IL4 on normal cells. Here we demonstrate that the IL4 antagonist also counteracts the action of cytotoxins targeted to the IL13 receptor on normal human cells. Importantly, the IL4 antagonist does not inhibit IL13‐based cytotoxins on glioma cells at all. Thus, the IL13 receptor on glioma cells can be categorized as tumor‐specific in the presence of an IL4 antagonist. We conclude that IL13 receptor‐directed cytotoxins can be delivered to glioma cells without being cytotoxic to normal cells. Int. J. Cancer 76:547–551, 1998.© 1998 Wiley‐Liss, Inc.