Helen L. Fillmore

The role of matrix metalloproteinase genes in glioma invasion: co-dependent and interactive proteolysis

Matrix metalloproteinases (MMPs) are cation-dependent endopeptidases which have been implicated in the malignancy of gliomas. It is thought that the MMPs play a critical role in both metastasis and angiogenesis, and that interference with proteases might therefore deter local tumor dissemination and neovascularization. However, the attempt to control tumor-associated proteolysis will rely on better definition of the normal tissue function of MMPs, an area of study still in its infancy in the central nervous system (CNS). Understanding the role of MMP-mediated proteolysis in the brain relies heavily on advances in other areas of molecular neuroscience, most notably an understanding of extracellular matrix (ECM) composition and the function of cell adhesion molecules such as integrins, which communicate knowledge of ECM composition intracellularly. Recently, protease expression and function has been shown to be strongly influenced by the functional state and signaling properties of integrins. Here we review MMP function and expression in gliomas and present examples of MMP profiling studies in glioma tissues and cell lines by RT-PCR and Western blotting. Co-expression of MMPs and certain integrins substantiates the gathering evidence of a functional intersection between the two, and inhibition studies using recombinant TIMP-1 and integrin antisera demonstrate significant inhibition of glioma invasion in vitro. Use of promising new therapeutic compounds with anti-MMP and anti-invasion effects are discussed. These data underline the importance of functional interaction of MMPs with accessory proteins such as integrins during invasion, and the need for further studies to elucidate the molecular underpinnings of this process.

Stem cell biology in traumatic brain injury: effects of injury and strategies for repair.

Approximately 350,000 individuals in the US are affected annually by severe and moderate traumatic brain injuries (TBI) that may result in long-term disability. This rate of injury has produced approximately 3.3 million disabled survivors in the US alone. There is currently no specific treatment available for TBI other than supportive care, but aggressive prehospital resuscitation, rapid triage, and intensive care have reduced mortality rates. With the recent demonstration that neurogenesis occurs in all mammals (including man) throughout adult life, albeit at a low rate, the concept of replacing neurons lost after TBI is now becoming a reality. Experimental rodent models have shown that neurogenesis is accelerated after TBI, especially in juveniles. Two approaches have been followed in these rodent models to test possible therapeutic approaches that could enhance neuronal replacement in humans after TBI. The first has been to define and quantify the phenomenon of de novo hippocampal and cortical neurogenesis after TBI and find ways to enhance this (for example by exogenous trophic factor administration). A second approach has been the transplantation of different types of neural progenitor cells after TBI. In this review the authors discuss some of the processes that follow after acute TBI including the changes in the brain microenvironment and the role of trophic factor dynamics with regard to the effects on endogenous neurogenesis and gliagenesis. The authors also discuss strategies to clinically harness the factors influencing these processes and repair strategies using exogenous neural progenitor cell transplantation. Each strategy is discussed with an emphasis on highlighting the progress and limiting factors relevant to the development of clinical trials of cellular replacement therapy for severe TBI in humans.

Encapsulation of a Radiolabeled Cluster Inside a Fullerene Cage, 177LuxLu(3−x)N@C80: An Interleukin-13-Conjugated Radiolabeled Metallofullerene Platform

In this communication, we describe the successful encapsulation of (177)Lu into the endohedral metallofullerene (177)Lu(x)Lu(3-x)N@C(80) (x = 1-3) starting with (177)LuCl(3) in a modified quartz Kraschmer-Huffman electric generator. We demonstrate that the (177)Lu (beta-emitter) in this fullerene cage is not significantly released for a period of up to at least one-half-life (6.7 days). We also demonstrate that this agent can be conjugated with an interleukin-13 peptide that is designed to target an overexpressed receptor in glioblastoma multiforme tumors. This nanoparticle delivery platform provides flexibility for a wide range of radiotherapeutic and radiodiagnostic multimodal applications.

Conjugation of functionalized gadolinium metallofullerenes with IL-13 peptides for targeting and imaging glial tumors.

BACKGROUND Glioblastoma multiforme is the most common and most lethal primary brain tumor in humans, with median survival of approximately 1 year. Owing to the ability of glioma cells to aggressively infiltrate normal brain tissue and survive exposure to current adjuvant therapies, there is a great need for specific targeted nanoplatforms capable of delivering both therapeutic and imaging agents directly to invasive tumor cells. METHOD Gadolinium-containing endohedral fullerenes, highly efficient contrast agents for MRI, were functionalized and conjugated with a tumor-specific peptide and assessed for their ability to bind to glioma cells in vitro. RESULTS We report the successful conjugation of the carboxyl functionalized metallofullerene Gd(3)N@C(80)(OH)(-26)(CH(2)CH(2)COOH)(-16) to IL-13 peptides and the successful targeting ability towards brain tumor cells that overexpress the IL-13 receptor (IL-13Rα2). CONCLUSION These studies demonstrate that IL-13 peptide-conjugated gadolinium metallofullerenes could serve as a platform to deliver imaging and therapeutic agents to tumor cells.

The Regulation and Function of Lactate Dehydrogenase A: Therapeutic Potential in Brain Tumor

There are over 120 types of brain tumor and approximately 45% of primary brain tumors are gliomas, of which glioblastoma multiforme (GBM) is the most common and aggressive with a median survival rate of 14 months. Despite progress in our knowledge, current therapies are unable to effectively combat primary brain tumors and patient survival remains poor. Tumor metabolism is important to consider in therapeutic approaches and is the focus of numerous research investigations. Lactate dehydrogenase A (LDHA) is a cytosolic enzyme, predominantly involved in anaerobic and aerobic glycolysis (the Warburg effect); however, it has multiple additional functions in non‐neoplastic and neoplastic tissues, which are not commonly known or discussed. This review summarizes what is currently known about the function of LDHA and identifies areas that would benefit from further exploration. The current knowledge of the role of LDHA in the brain and its potential as a therapeutic target for brain tumors will also be highlighted. The Warburg effect appears to be universal in tumors, including primary brain tumors, and LDHA (because of its involvement with this process) has been identified as a potential therapeutic target. Currently, there are, however, no suitable LDHA inhibitors available for tumor therapies in the clinic.

Microarray analysis of MRI-defined tissue samples in glioblastoma reveals differences in regional expression of therapeutic targets

Microarray technologies have come into prominence for the assessment of molecular diagnostic profiles in cancer tissue biopsies. To better understand the effect of sampling bias, we paired image-guided stereotactic biopsy and microarray technology to study regional intratumoral differences in tumor periphery and core regions of untreated glioblastoma. RNA was extracted from serial frozen sections using an integral histopathologic scoring approach. Gene expression analysis was performed using high-density oligonucleotide microarrays (22,283 probe sets). A consensus list of 643 genes (784 probe sets) with greater than 2-fold difference between intratumoral periphery and core samples was obtained using Microarray Suite 5.0, model-based expression indexes, and robust multiarray analysis algorithms. Results were validated using quantitative polymerase chain reaction and Western blotting analyses. Reproducible profiles emerged, in which multiple therapeutic targets significant to glioblastoma [matrix metalloproteinases, AKT1 (v-akt murine thymoma viral oncogene homolog 1), epidermal growth factor receptor, vascular endothelial growth factor] showed significant differences in regional expression that may affect treatment response. This study suggests important intratumoral regional differences in the molecular phenotype of glioblastoma.

Induction of membrane-type-1 matrix metalloproteinase by epidermal growth factor-mediated signaling in gliomas

Increased expression of membrane-type matrix metalloproteinases (MT-MMPs) has previously been reported to correlate with increasing grade of malignancy in gliomas, a relationship shared with alterations in epidermal growth factor receptor (EGFR) signaling. To investigate the possibility of a causative role for EGFR signaling in increasing MT-MMP expression and subsequent peritumoral proteolysis, we characterized glioma cell lines for expression of MT1-MMP, MT2-MMP, MT3-MMP, and MT5-MMP by Western blotting and by quantitative real-time polymerase chain reaction analysis, and for MMP-2 activity following epidermal growth factor (EGF) stimulation. EGF stimulation of glioma cell lines resulted in a 2- to 4-fold increase in MT1-MMP mRNA levels. Although there were slight differences in MT2-, MT3-, and MT5-MMP mRNA expression following EGF stimulation, none of these demonstrated an increase similar to that of MT1-MMP expression. Treatment of high-grade glioma cell lines U251MG and IPSB-18 with EGF for 24 h resulted in a several-fold increase in MT1-MMP protein (2.5- and 5.1-fold, respectively) and in cyclin D1 (2.9-fold), as compared to untreated controls. No significant increase was detected in other MT-MMPs at the protein level. Although there was no detectable increase in proMMP-2 protein, there was an increase in MMP-2 activity. Furthermore, the MT1-MMP induction by EGF was prevented by pretreatment with the EGFR-specific tyrphostin inhibitor AG1478. Similarly, treatment with the phosphatidylinositol 3-kinase inhibitor LY294002 prevented the induction of MT1-MMP protein by EGF stimulation. These compounds additionally inhibited EGF-stimulated invasion in Matrigel Transwell assays. Our results indicate that one mechanism of EGFR-mediated invasiveness in gliomas may involve the induction of MT1-MMP.

Association of a single nucleotide polymorphism in the matrix metalloproteinase-1 promoter with glioblastoma.

A key feature in the malignant behavior of glioblastoma is the tendency to invade host brain tissue surrounding the primary tumor site. Several members of the matrix metalloproteinase family are thought to contribute to this invasive capacity. A single nucleotide polymorphism has been described in the matrix metalloproteinase‐1 (MMP‐1) promoter that consists of either the presence or absence of a guanine nucleotide at position −1607. The presence of the guanine base creates a functional binding site for members of the ETS family of transcription factors and has been shown to increase MMP‐1 transcription. The purpose of our study was to characterize this polymorphism in human glioblastoma. Promoter genotyping was performed on brain tumor tissue obtained from 81 patients and compared to 57 healthy individuals. The 2G/2G genotype is more prevalent in glioblastoma tissue compared to healthy individuals (p = 0.01). mRNA and protein expression were measured in a subset of brain tumor and normal brain tissue samples. MMP‐1 protein levels are significantly higher in glioblastoma tissue compared to normal brain (p = 0.001). Electromobility shift assays and promoter assays were performed to assess binding capability and transcriptional activity, respectively. Proteins present in glioma cell lines can specifically bind the 2G promoter probe. MMP‐1 transcription is significantly higher in cells transfected with the 2G promoter when compared to cells transfected with the 1G promoter (p<0.02). This polymorphism may provide a mechanism for increased expression of MMP‐1 in malignant gliomas via elevation of MMP‐1 mRNA transcription and may underlie the invasive phenotype.

Antiproliferative effect of c-myc antisense phosphorothioate oligodeoxynucleotides in malignant glioma cells

OBJECTIVE To improve the prognosis for primary malignant tumors of the central nervous system, new therapeutic strategies are needed. Antisense oligodeoxynucleotides (ODNs) offer the potential to block the expression of specific genes within cells. The proto-oncogene c-myc has long been implicated in the control of normal cell growth and its deregulation in the development of neoplasia. We therefore reasoned that a strategy using ODNs complementary to c-myc messenger ribonucleic acid would be a potent inhibitor of glioma cell proliferation. METHODS A variety of antisense, sense, and scrambled (15-mer) phosphorothioate ODNs targeted to rat and human c-myc messenger ribonucleic acid were synthesized and added to the media of cultured RT-2 cells (a rat glioblastoma cell line). Cell growth was assessed by 3-[4,5-dimethylthiazol-2yl]-2,5-diphenyltetrazolium bromide dye assay 1 to 5 days after adding the ODNs. c-Myc protein expression was analyzed by Western blot analysis. The stability of the ODNs was confirmed by gel electrophoresis. RESULTS Compared with cultures containing standard media, two of three antisense ODNs significantly inhibited the growth of glioma cells, whereas sense and scrambled sequence ODNs did not significantly affect cell growth at the concentrations tested. A human c-myc antisense sequence, which differed from the rat sequence by one base substitution, also had an inhibitory effect on RT-2 cells. Western blot analysis demonstrated that expression of immunoreactive c-Myc protein was also greatly reduced in the rat antisense ODN-treated cells (and not in sense-, scrambled-, or control-treated cells). The degree of reduction of c-Myc protein expression correlated well with the decrease in cell growth observed with several antisense ODNs. Phosphorothioate ODNs were stable in cell culture media for at least 5 days. CONCLUSION These results suggest that c-Myc plays a critical role in glioma cell proliferation and demonstrate that antisense ODNs can suppress proto-oncogene expression and inhibit the proliferation of glioma cells. Our results indicate that the antiproliferative activity of these ODNs was mediated predominantly through sequence-specific antisense mechanisms, but that sequence-specific nonantisense effects may also contribute to the strongest effects demonstrated. These findings support a potential role for antisense strategies designed to inhibit c-myc expression in the treatment of malignant gliomas.

Epidermal growth factor induces matrix metalloproteinase-1 (MMP-1) expression and invasion in glioma cell lines via the MAPK pathway

Glioblastoma multiforme (GBM) is an aggressive cancer with a poor survival rate. A key component that contributes to the poor prognosis is the capacity of glioma cells to invade local brain tissue in a diffuse manner. Among various proteases that aid in the process of invasion, matrix metalloproteinase-1 (MMP-1) has been identified as an important contributory factor in various cancers. Apart from its traditional role in cleaving its primary extracellular matrix (ECM) substrates, and like other members of the matrix metalloproteinase family, MMP-1 can activate latent forms of bio-active molecules initiating downstream pro-invasive and pro-oncogenic signaling mechanisms. MMP-1 expression is regulated by several growth factors including epidermal growth factor (EGF). Due to the fact that the epidermal growth factor receptor (EGFR) is aberrantly overexpressed in GBM, we wanted to examine in greater detail the signaling mechanisms by which MMP-1 expression and invasion is driven by EGF in GBM cells. T98G cells treated with EGF resulted in an induction of MMP-1 expression following EGFR activation. Inhibition of EGFR by both pharmacologic and genetic approaches abrogated this induction. Repression of the mitogen activated protein kinase (MAPK) signaling led to the inhibition of EGF-induced MMP-1 whereas the PI3-kinase/AKT signaling was not associated with EGFR-mediated MMP-1 induction. Inhibition of EGFR signaling also led to a decrease in T98G invasion. These data suggest that EGFR mediated MMP-1 regulation is mainly via the MAPK pathway in T98G cells and inhibition of EGFR and MMP-1 results in a decrease in T98G cell invasion.