By Julia Y. Ljubimova, MD, PhD
- Laminin-8 is dramatically increased in glial brain tumors
- Laminin-8 facilitates tumors' spread
- Brain tumors with laminin-8 overexpression recur faster after standard treatment and patients have shorter survival time
Attempts have been made to establish and characterize a number of glioma markers, but such research has not altered existing therapeutic approaches, treatment success rates or disease outcome prediction [1, 2]. Researchers then sought to identify novel glioma markers using powerful gene array technology [3-6].
In an earlier study using gene array technology, published in Cancer Research, we found that two genes were consistently up-regulated in all high-grade and low-grade gliomas and in tissues adjacent to GBMs, the most aggressive gliomas. One of the genes was already known to be over-expressed in gliomas. The other was the alpha-4 chain of laminin, a novel gene that had not been known to be overexpressed in any type of tumor. Laminins are heterogenic proteins, each of which are composed of three chains, alpha, beta and gamma, and about 15 laminins are currently known. Laminins are the major component of a thin "basement membrane" that lies beneath the surface layer of blood vessels. We found that during the tumor progression, laminin 9, which is expressed weakly in normal brain and low-grade glial tumors, switched to laminin 8. The data were validated by gene array analysis, semi-quantitative RT-PCR for RNA expression and immunohistochemistry for protein expression [4].
To examine the involvement of laminin-8 in glioma invasion, we needed reliable in vitro systems where it was possible to quantify invasion rates and to optimize the dosage of antisense laminin oligos. We used a cell culture system to meet these important needs. To better mimic the in vivo situation in glial tumors where the major cell types are glial (astrocytes) and endothelial cells, [12] (Figure 1)* we needed to combine glioma cells with brain endothelium in a co-culture [30]. In such a situation, endothelial cells can develop capillary-like structures, and this process is faster when endothelial cells are cultured with tumor astrocytes than with normal embryonic brain astrocytes [31]. We hypothesized that in glioma-endothelium co-cultures there would be more laminin-8 produced, and that this laminin might increase glioma invasion in a Matrigel assay. Research into these issues could facilitate GBM diagnosis and prognosis, help to find additional methods of treatment, and increase survival of brain cancer patients.
We found that during progression of human gliomas, the expression of capillary Basement Membrane (BM) laminins containing a4 chain switches from the predominant laminin-9 (a4B2y1) to laminin-8 (a4B1y1) (Figure 2) [4]. Laminin-8 and its receptors, integrins a3b1 and a6b1, appear to be important to the functioning of endothelial cell BMs, which play a role in the maintenance of the blood-brain barrier [10, 11]. Recently, the association of laminin a4 chain with angiogenesis has been demonstrated in vivo and in vitro [12]. Some cultured glioma cell lines can also produce a4-containing laminins. Laminin-8 is thought to play a role in cell migration during development, wound healing, and angiogenesis [7, 8, 10].
To probe the role of laminin-8 in glioma invasion, we attempted to use antisense oligos to block its expression. The potential of antisense is widely recognized, but it remained largely unfulfilled since, until recently, the available oligos suffered from poor specificity, instability, and undesirable non-antisense effects [22, 23]. These problems have been largely solved by the new generation of antisense oligos that offer the promise of safe and effective therapeutics for various diseases including cancer [23, 24]. New-generation antisense oligos are being used in studies to find effective medications and treatments for many disorders, including viruses and cancers. By blocking a gene's effects in a laboratory setting, they enable researchers to study the gene, its control, and the interactions between gene products. Antisense technology is being refined not only for drug validation and diagnostic purposes but also for the development of future treatments for patients.
The most promising types of oligos are Morpholino and peptide nucleic acid (PNA; they have nucleobases attached to a neutral "peptide-like" backbone) oligos [22, 24]. The new study used short strands of genetic code (Morpholino antisense oligonucleotides) to block the messenger RNA (mRNA) carrying the gene's "instructions." As a result, the gene's "protein product," laminin-8, was not produced and the invasiveness of glioma cells was significantly reduced. They work well in the presence or absence of serum, are totally resistant to nucleases, and remain intact in culture medium and in cells indefinitely. Morpholino oligos have a high affinity for RNA and efficiently invade even quite stable secondary structures in mRNAs. They have the highest sequence specificity of all antisense types over a very broad concentration range and appear to be free of non-antisense effects [24, 25]. They have high activity in a cell-free translation system and can block target protein production in cultured cells [26]. Morpholino are also effective in vivo [27]. Given these properties, Morpholino oligos have been chosen here to inhibit the expression of laminin-8 chains in culture. Special experiments have shown that Morpholino treatment did not affect the viability of any cell line used.
Matrigel invasion assay was developed for quantitative measurement of the invasiveness of tumor cells through a BM matrix. Most tested cells characterized as invasive and metastatic in vivo are able in vitro to invade Matrigel, which is a BM-like material from the mouse Engelbreth-Holm-Swarm tumor [13, 14].
When glioma-endothelial co-cultures were treated by antisense to block laminin 8 production, the inhibition of invasiveness on Matrigel was 62% and 53%, respectively, for two co-cultures of glioma cell lines U87MG and M059K, and normal and endothelial cells. In our experiments, a4 and B1 expression was inhibited more efficiently with a lower concentration of antisense oligos (0.25 + 0.25 mM) than with a higher concentration (0.5 + 0.5 mM), although no apparent toxicity was noticed at either concentration (Figure 3).
The use of antisense technology in vivo may offer an effective future tumor treatment because of its efficiency, specificity and ease of delivery to tumor cells [28, 29]. This technology is being continuously developed and refined not only for the drug validation and diagnostic purposes but also for the development of future treatments. The present data emphasize the feasibility of antisense approach using laminin-8 as a target for treatment of brain gliomas. Reduction of tumor invasion by antisense to laminin-8 may slow the growth and spread of aggressive GBMs (Figure 4). In combination with other treatment methods or with blocking of other targets as well (EGFR, MMPs) it may prolong disease-free periods and increase survival of glioma patients. Future developments of laminin-8 blocking for therapeutic purposes may also include the use of specific monoclonal antibodies and/or small interfering RNA (siRNA) that is an emerging very promising approach for gene silencing.
It remains to be established how laminin-8 promotes glioma invasiveness. One possible mechanism may be stimulation of cell migration. It was previously shown that at least one form of laminin-8 containing a4. A splice variant rather weakly supported cell adhesion and spreading compared to laminin-5 or laminin 10/11 [11, 32]. At the same time, laminin-8 stimulated cell migration better than several other laminin isoforms [11]. Increased expression of laminin-8 in both glioma cells and glioma-adjacent capillary endothelial cells [4, 11, this report] may reduce glial cell adhesion and enhance migration, which is necessary for local tumor invasiveness.
We recently studied a significantly larger number of glial tumors of different grades from 60 patients in an attempt to correlate laminin-8 expression with recurrent development and patient survival. Immunohistochemistry and Western blotting were used to detect laminin isoforms of interest. Both methods significantly detected expression of laminin-8 chains in three-quarters of GBMs and their adjacent tissues, whereas astrocytomas of lower grades mostly expressed a different isoform, laminin-9, also found in low amounts in normal brain and benign meningiomas. Overexpression of laminin-8 in GBM was significantly associated with shorter time to tumor recurrence (p<0.0001) and decreased patient survival time (p<0.05). Laminin-8 appears to facilitate tumor invasion and may serve as a target for glioma therapy.
In summary, we developed a glioma-endothelial co-culture model suitable for studying laminin-8 expression and its inhibition in vitro by antisense oligos. Morpholino proved to be efficient inhibitors of laminin-8 expression in co-cultures. Antisense oligos to laminin-8 chains also significantly inhibited invasion of two different glioma cell lines in vitro. The results suggest that laminin-8 may play an important role in glioma invasion. Morpholino oligos may provide an efficient method to block laminin-8 expression for future therapeutic purposes.
In combination with several new well-characterized proteins associated with glioma progression, such as tenascin-C, MMP-2 and MMP-9, [4, 9, 15-19], laminin-8 may be an important tool for potential diagnosis or treatment of gliomas. Previously, only laminin-5 was shown to play a role in melanoma invasion [20]. Our present data suggest that "vascular" laminin-8 also plays a significant role in glioma cell invasiveness. Since matrix-degrading proteinases are also important for glioma invasion [21], future research should explore whether proteolysis of laminin is required for glioma invasion. Our recent data suggest that laminin-8, which may facilitate tumor invasion, contributes to tumor regrowth after therapy. Laminin-8 may be used as a predictor of tumor recurrence, patient survival and as a potential molecular target for glioma therapy.
Dr. Julia Y. Ljubimova is Director of Molecular Oncology at the Maxine Dunitz Neurosurgical Institute at Cedars-Sinai Medical Center in Los Angeles.
1. Shapiro WR, Shapiro JR. Biology and treatment of malignant glioma. Oncology. 12: 233-240, 1998.
2. Kaye AH, Laws ER. (eds). Brain Tumors. pp. 990. Churchill Livingstone, 1997.
3. Ljubimova JY, Khazenzon NM, Chen Z, Neyman YI, Turner L, Riedinger MS, Black KL. Gene array analysis of differentially expressed genes in human glial tumors. Int J Oncol. 18: 287-295, 2001.
4. Ljubimova JY, Lakhter AJ, Loksh A,Yong WH, Riedinger MS, Miner JH, Sorokin ML, Ljubimov AV, Black KL. Overexpression of a4 chain-containing laminins in human glial tumors identified by gene microarray analysis. Cancer Res. 61: 5601-5610, 2001.
5. Sehgal A. Molecular changes during the genesis of human gliomas. Seminars Surg Oncol. 14: 3-12, 1998.
6. Lal A, Lash AE, Altschul SF, Velculescu V, Zhang L, McLendon RE, Marra MA, Prange C, Morin PJ, Polyak K, Papadopoulos N, Vogelstein B, Kinzler KW, Strausberg RL, Riggins GJ. A public database for gene expression in human cancers. Cancer Res. 59: 5403-5407, 1999.
7. Miner JH, Patton BL, Lentz SI, Gilbert DJ, Snider WD, Jenkins NA, Copeland NG, Sanes JR. The laminin alpha chains: expression, developmental transitions, and chromosomal locations of a1-5, identification of heterotrimeric laminins 8-11, and cloning of a novel a3 isoform. J Cell Biol. 137: 685-701, 1997.
8. Patarroyo M, Tryggvason K, Virtanen I. Laminin isoforms in tumor invasion, angiogenesis and metastasis. Semin Cancer Biol. 12: 197-207, 2002.
9. Kulla A, Liigant A, Piirsoo A, Rippin G, Asser T. Tenascin expression patterns and cells of monocyte lineage: relationship in human gliomas. Mod Pathol. 13: 56-67, 2000.
10. Thyboll J, Kortesmaa J, Cao R, Soininen R, Wang L, Iivanainen A, Sorokin L, Risling M, Cao Y, Tryggvason K. Deletion of the laminin a4 chain leads to impaired microvessel maturation. Mol Cell Biol. 22:1194-1202, 2002.
11. Fujiwara H, Kikkawa Y, Sanzen N, Sekiguchi K. Purification and characterization of human laminin-8. Laminin-8 stimulates cell adhesion and migration through a3b1 and a6b1 integrins. J Biol Chem. 276: 17550-17558, 2001.
12. Gonzalez AM, Gonzales M, Herron GS, Nagavarapu U, Hopkinson SB, Tsuruta D, Jones JC. Complex interactions between the laminin a4 subunit and integrins regulate endothelial cell behavior in vitro and angiogenesis in vivo. Proc Natl Acad Sci USA. 99: 16075-16080, 2002.
13. Albini A, Iwamoto Y, Aaronson SA, Kozlowski JM, McEwan RN. A rapid in vitro assay for quantitating the invasive potential of tumor cells. Cancer Res. 47: 3239-3245, 1987.
14. Kleinman HK, McGarvey ML, Hassell JR, Star VL, Gannon FB, Laurie GW, Martin GR. Basement membrane complexes with biological activity. Biochemistry. 25: 312-318, 1986.
15. Herold-Mende C, Mueller MM, Bonsanto MM, Schmitt HP, Kunze S, Steiner HH. Clinical impact and functional aspects of tenascin-C expression during glioma progression. Int J Cancer. 98: 362-369, 2002.
16. Zagzag D, Capo V. Angiogenesis in the central nervous system: a role for vascular endothelial growth factor/vascular permeability factor and tenascin-C. Common molecular effectors in cerebral neoplastic and non-neoplastic "angiogenic diseases". Histol Histopathol. 17: 301-321, 2002.
17. Qin H, Sun Y, Benveniste EN. The transcription factors Sp1, Sp3, and AP-2 are required for constitutive matrix metalloproteinase-2 gene expression in astroglioma cells. J Biol Chem. 274: 29130-29137, 1999.
18. Kachra Z, Beaulieu E, Delbecchi L, Mousseau N, Berthelet F, Moumdjian R, Del Maestro R, Beliveau R. Expression of matrix metalloproteinases and their inhibitors in human brain tumors. Clin Exp Metastasis. 17: 555-566, 1999.
19. MacDonald TJ, DeClerck YA, Laug WE. Urokinase induces receptor mediated brain tumor cell migration and invasion. J Neurooncol. 40: 215-226, 1998.
20. Tsuj T, Kawada Y, Kai-Murozono M, Komatsu S, Han SA, Takeuchi K, Mizushima H, Miyazaki K, Irimura T. Regulation of melanoma cell migration and invasion by laminin-5 and a3b1 integrin (VLA-3). Clin Exp Metastasis. 19: 127-134, 2002.
21. Kondraganti S, Mohanam S, Chintala SK, Kin Y, Jasti SL, Nirmala C, Lakka SS, Adachi Y, Kyritsis AP, Ali-Osman F, Sawaya R, Fuller GN, Rao JS. Selective suppression of matrix metalloproteinase-9 in human glioblastoma cells by antisense gene transfer impairs glioblastoma cell invasion. Cancer Res. 60: 6851-6855, 2000.
22. Nielsen PE. Peptide nucleic acid targeting of double-stranded DNA. Methods Enzymol. 340: 329-340, 2001.
23. Dias N, Stein CA. Antisense oligonucleotides: basic concepts and mechanisms. Mol Cancer Ther. 1: 347-355, 2002.
24. Summerton J, Weller D. Morpholino antisense oligomers: Design, preparation and properties. Antisense Nucleic Acid Drug Dev. 7: 187-195, 1997.
25. Lacerra G, Sierakowska H, Carestia C, Fucharoen S, Summerton J, Weller D, Kole R. Restoration of hemoglobin A synthesis in erythroid cells from peripheral blood of thalassemic patients. Proc Natl Acad Sci USA. 97 :9591-9596, 2000.
26. Taylor MF, Paulauskis JD, Weller DD, Kobzik L. Comparison of efficacy of antisense oligomers directed toward TNF-a in helper T and macrophage cell lines. Cytokine. 9: 672-681, 1997.
27. Arora V, Knapp DC, Smith BL, Statdfield ML, Stein DA, Reddy MT, Weller DD, Iversen PL. c-Myc antisense limits rat liver regeneration and indicates role for c-myc in regulating cytochrome P-450 3A activity. J Pharmacol Exp Ther. 292: 921-928, 2000.
28. Jansen B, Wacheck V, Heere-Ress E, Schlagbauer-Wadl H, Hoeller C, Lucas T, Hoermann M, Hollenstein U, Wolff K, Pehamberger H. Chemosensitisation of malignant melanoma by BCL2 antisense therapy. Lancet. 356: 1728-1733, 2000.
29. Shi N, Boado RJ, Pardridge WM. Antisense imaging of gene expression in the brain in vivo. Proc Natl Acad Sci USA. 97: 14709-14714, 2000.
30. Boado RJ, Kazantsev A, Apostol BL, Thompson LM, Pardridge WM. Antisense-mediated down-regulation of the human huntingtin gene. J Pharmacol Exp Ther. 295: 239-243, 2000.
31. Minakawa T, Bready J, Berliner J, Fisher M, Cancilla PA. In vitro interaction of astrocytes and pericytes with capillary-like structures of brain microvessel endothelium. Lab Invest. 65: 32-40, 1991.
32. Knott JC, Mahesparan R, Garcia-Cabrera I, Bolge Tysnes B, Edvardsen K, Ness GO, Mork S, Lund-Johansen M, Bjerkvig R. Stimulation of extracellular matrix components in the normal brain by invading glioma cells. Int J Cancer. 75: 864-872, 1998.
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