Antisense inhibition of laminin-8 expression to inhibit human gliomas

#This application is the National Phase of International Application PCT/US04/29956, filed Sep. 13, 2004, which designated the U.S. and that International Application was published under PCT Article 21(2) in English. This application also includes a claim of priority under 35 U.S.C. §119(e) to U.S. provisional patent application No. 60/502,729, filed Sep. 12, 2003.

#Glial tumors are the leading cause of cancer death in children [1]. Overall, they account for 1.4% of all cancers and 2.4% of all cancer deaths. Average survival time for low-grade astrocytoma or oligodendroglioma patients is 6 to 8 years. It decreases to 3 years for patients with anaplastic astrocytoma and drops to 12-18 months for glioblastoma multiforme (GBM). Currently, these tumors are treated by surgical removal, radiation therapy, chemotherapy or combinations of these treatments. The majority of GBMs is highly invasive and rapidly develops recurrences at the primary site. Tumor prognoses and responses to therapy can vary greatly even with the same histological diagnosis [2]. It is generally recognized that the improvement of prognosis, prediction of response to treatment, and development of novel effective therapeutic approaches for glial tumors may largely depend upon the introduction into clinical practice of novel specific markers involved in the development of different gliomas and their subsequent recurrences.

#Attempts have been made to establish and characterize a number of glioma markers, such as glial fibrillary acidic protein, vimentin, synaptophysin, and nestin. Determination of differential expression of these markers (immunophenotyping) in gliomas, however, has thus far not altered existing therapeutic approaches, treatment success rates, or disease outcome prediction [2, 3]. Researchers next sought to identify novel glioma markers using powerful gene array technology [4-7]. Recently, our group described a new molecular marker of glial tumors, laminin-8, that was differentially expressed in malignant tumors compared to benign tumors and normal brain tissues [5].

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#FIG. 1 shows uptake of Ac-LDL by various cultures and co-cultures; endothelial cells (HBMVEC) are positive (green fluorescence) but glioma cells (M59K) and normal astrocytes (HAST 040) are negative; in co-cultures HBMVEC+M059K and HBMVEC+HAST040, endothelial cells are positive, whereas other cells are negative (DAPI was used to counterstain cell nuclei (blue fluorescence)).

#FIG. 2 shows Laminin α4, β1, and β2 chain expression in cells and conditioned media of pure cultures:

#Since laminin-8 appears to be associated with GBM recurrence in vivo, we hypothesized that it might play a role in tumor invasion. Because of the complexity of in vivo experiments, we first explored this possibility in vitro using single cultures and co-cultures of brain microvascular endothelial cells, normal fetal brain astrocytes and several GBMs. We sought to analyze whether the patterns of laminin chain expression in cell culture would be similar to those seen in normal brain and in gliomas, and whether inhibition of laminin-8 expression by an antisense approach would alter glioma invasiveness through a reconstituted BM (Matrigel).

#Antisense oligonucleotides (oligos) that bind and inactivate specific RNA sequences may be the best tools for studying gene function, regulation of gene expression, and interactions between gene products. Highly specific antisense oligos that mimic the DNA template for RNA production are used to bind to the complementary RNA and to prevent protein translation [17,18]. Antisense oligos are the fastest, simplest and most cost effective tools for testing new therapeutic targets for drug development. The antisense approach was used in our present study to inhibit the expression of laminin-8 in cell culture.

#Our results show that normal cultured astrocytes and endothelial cells mostly express laminin-9 as seen in normal brain tissue. Glioma cells predominantly express laminin-8, again similar to the in vivo situation. Most importantly, antisense blocking of laminin-8 chain expression resulted in the inhibition of glioma invasion through Matrigel. These data show that laminin-8 is an important for glioma invasion and an effective target for antitumor therapy. That is, the differences in laminin-8 chain expression are not merely indicators of the malignant cells but are actually linked to invasiveness of the malignancy.