LGI1, a putative tumor metastasis suppressor gene, controls in vitro invasiveness and expression of matrix metalloproteinases in glioma cells through the ERK1/2 pathway.

Gliomas take a number of different genetic routes in the progression to glioblastoma multiforme, a highly invasive variant that is mostly unresponsive to current therapies. Gliomas express elevated levels of matrix metalloproteinases (MMPs), which have been implicated in the control of proliferation and invasion as well as neovascularization. Progressive loss of LGI1 expression has been associated with the development of high grade gliomas. We have shown previously that the forced re-expression of LGI1 in different glioma cells inhibits proliferation, invasiveness, and anchorage-independent growth in cells null for its expression. Here, using Affymetrix gene chip analysis, we show that reexpression of LGI1 in T98G cells results in the down-regulation of several MMP genes, in particular MMP1 and MMP3. LGI1 expression also results in the inhibition of ERK1/2 phosphorylation but not p38 phosphorylation. Inhibition of the MAPK pathway using the pharmacological inhibitors PD98059, U0126, and SB203580 in T98G LGI1-null cells inhibits MMP1 and MMP3 production in an ERK1/2-dependent manner. Treatment of LGI1-expressing cells with phorbol myristate acetate prevents the inhibition of MMP1/3 and restores invasiveness and ERK1/2 phosphorylation, suggesting that LGI1 acts through the ERK/MAPK pathway. Furthermore, LGI1 expression promotes phosphorylation of AKT, which leads to phosphorylation of Raf1(Ser-259), an event shown previously to negatively regulate ERK1/2 signaling. These data suggest that LGI1 plays a major role in suppressing the production of MMP1/3 through the phosphatidylinositol 3-kinase/ERK pathway. Loss of LGI1 expression, therefore, may be an important event in the progression of gliomas that leads to a more invasive phenotype in these cells.

Glioblastoma multiforme is the most common malignant tumor of the adult central nervous system and has a median survival time of less than 12 months (1-2). The highly lethal nature of this tumor results from the acquisition of an invasive phenotype that allows the tumor cells to infiltrate surrounding brain tissue. Despite considerable heterogeneity in the genetic abnormalities detected in the various etiologies and histopathologies of these tumors (3), 90% of glioblastoma multi-formes share losses of regions on chromosome 10 (4 -8). Using a positional cloning strategy, Chernova et al. (9) identified the LGI1 gene associated with an apparently reciprocal t (10,19)(q24;q13) chromosome translocation in the T98G glioma cell line.
LGI1 is expressed in low grade tumors but not in most of the high grade gliomas or permanent cell lines tested (9 -10). The coincident loss of LGI1 expression with loss of chromosome 10 suggested that it might be important in the malignant progression of gliomas.
LGI1 carries a leucine-rich repeat (LRR) 1 motif (9) that places it in the F20 family of LRR genes. Members of this family are predominantly involved in either receptor functions or attachment to the extracellular matrix (11). The strongest homology of the LGI1 LRR is with the slit genes (9), which are involved in the control of axonal movement in Drosophila. The loss of LGI1 function in glial cells at the transition from low grade to high grade tumors also suggested a possible role for this gene in controlling the invasive/migratory potential of these cells. To investigate this possible function in glioma cells, we recently expressed an exogenous copy of the wild type LGI1 gene in glioma cells that do not express detectable levels of LGI1 endogenously (12). Re-expression of LGI1 resulted in a significant reduction in growth potential but, significantly, almost completely suppressed the ability of these cells to invade an extracellular matrix or to form colonies in soft agar. Both of these in vitro phenotypes are indicative of a loss of malignant potential. We suggest, therefore (12), that LGI1 may be a member of the emerging family of genes referred to as "tumor metastasis suppressors" (13).
To determine whether LGI1 exerts its effect by regulating specific signaling pathways, we used oligonucleotide microarray analysis to compare the gene expression profiles from T98G cells that do not express LGI1 with those that are forced to re-express it. The re-expression of LGI1 induced the suppression of a number of extracellular matrix genes, including members of the matrix metalloproteinase (MMP) family, in particular MMP1 and MMP3. High levels of the expression of MMPs have been reported in high grade gliomas, where it is proposed that their action serves to facilitate invasion and metastasis (14,15). Recently, Mercapide et al. (16) showed that MMP3 is responsible for the invasive phenotype in astrocytomas. Matrix metalloproteinases are a family of structurally related zinc-dependent neutral peptidases collectively capable of degrading essentially all components of the extracellular matrix (17).
These genes have been shown to play an important role in controlled tissue remodeling in physiological situations, including developmental morphogenesis, tissue repair, and angiogenesis (18 -19). The increased expression of various MMPs by peripheral tumors is strongly associated with invasive phenotypes. We now demonstrate that the re-expression of LGI1 in glioma cells suppresses ERK-dependent expression of MMP1 and MMP3, which correlates with the severe loss of in vitro invasiveness. Several pathways have been reported that lead to activation of MMPs. Using a variety of pathway-specific inhibitors, we now demonstrate that LGI1 affects MMP production predominantly by inhibiting the MAPK/ERK pathway.
Cell Culture and Treatments-The T98G cell line was maintained in DMEM with 10% FBS under 10% CO 2 . The T98G LGI1 stable transfectants were maintained in DMEM plus 10% FBS and 500 g/ml G418 sulfate. For all inhibition assays, inhibitors were added to the cells, which were then kept overnight in serum-free medium pending supernatant collection. For PMA treatment, cells were incubated with the appropriate concentration of the inhibitor for 30 min before the addition of PMA for 10 min.
Gene Expression Analysis-Total RNA was extracted from three independently isolated T98G clones that showed exogenous expression of LGI1 and was used to prepare cRNA for hybridization to the Affymetrix U133A oligonucleotide arrays as described previously (20). The gene expression profile from these cells was compared with that obtained from T98G clones that had been stably transfected with the empty pcDNA3 vector. To compare the vector-only clones with the LGI1-expressing clones, the base line-corrected data were imported into the Affymetrix Data Mining Tool (DMT 4.0) by using the publishing tool MicroDataBase (MDB 3.0). The genes were then sorted using a count and percent tool, and only those genes showing altered expression in at least two of both the vector-only clones and LGI1ϩ clones (two crosscomparisons) were used to define the final list. A cutoff of an average 2-fold or greater change was selected. The second analysis approach employed the dChip program developed at Harvard for analysis of GeneChip data and was available for downloading from www.dchip.org. This approach is based on the Li and Wong statistical model (21) that analyzes oligonucleotide array data at the individual probe level. dChip performs a multi-chip analysis to calculate a probe sensitivity index that captures the response characteristics of a specific probe pair and produces model-based expression index values for probe sets. The Affymetrix Microarray Suite (5.0) generated ".CEL" files were converted into ".DCP" files using dChip. The .DCP files were then normalized, and gene expression data were generated using the dChip system of modelbased analysis. The vector-only clones were designated as the baseline (B), and the T98G LGI1 samples were designated as experimental (E). Genes that displayed 2-fold up-or down-regulation in the T98G-LGI1 clones, as compared with clones carrying the vector, were identified by defining the appropriate filtering criteria in the dChip software (mean E mean B Ͼ 2; mean E Ͻ 2 mean B ϭ 100; p ϭ 0.01; t test).
Genes that were found by both data analysis tools to be significantly changed were considered to be consistent between experiments. All functional annotation and chromosomal locations were obtained using NetAffx (22).
Western Blot Analysis-Protein levels of MMP1 and MMP3 were examined in the serum-free supernatants from the cell lines stably transfected with either the pcDNA3 vector or LGI1 by immunoblotting. For all other experiments, total cell extracts were prepared in radioimmune precipitation assay buffer (50 mM Tris containing 150 mM NaCl, 1% sodium deoxycholate, 0.1% SDS, and 1% Triton X-100) supplemented with 0.2% protease-and phosphatase-inhibitor mixtures (Sigma). The extracts were kept on ice for 10 min, after which the supernatants were recovered by centrifugation at 14000 ϫ g for 20 min. Protein concentrations were determined using the Bio-Rad protein assay method. Cell extracts containing 40 g of protein were size-fractionated in 12% SDS-PAGE gels (Bio-Rad) and electroblotted onto nitrocellulose membranes (Bio-Rad). The membranes were blocked using 3% bovine serum albumin in PBS-Tween 20 (0.05%) and then probed overnight at 4°C with specific primary antibodies diluted in 3% bovine serum albumin. The blots were then washed three times with PBS-Tween 20 for 10 min each wash, and then anti-rabbit or anti-mouse antibodies (Jackson Laboratories, Bar Harbor, ME) were added as secondary antibodies to the respective membranes at a 1:7500 dilution in 5% nonfat dry milk. After incubation for 1 h at room temperature, the membranes were washed three times with PBS-Tween 20 for 10 min each wash. Immunoreactive bands were visualized using the Western Lightning chemiluminescence kit (PerkinElmer Life Sciences), followed by exposure to Kodak X-Omat AR films.
Matrigel Chamber Invasion Assay-Matrigel (BD Biosciences) chambers with 8 m membrane pores, stored at Ϫ20°C, were kept at room temperature for 1 h, after which 750 l of DMEM was added to the empty wells. The inserts were then transferred to the medium-containing wells. 500 l of DMEM was added to the inserts and kept at 37°C in an incubator with 5% CO 2 for equilibration. After 2 h, the medium in the inserts was aspirated, and the inserts were placed into the wells containing DMEM and 5% FBS. FBS in this case acts as a chemoattractant for the cells to migrate. 50,000 cells in 500 l of DMEM were added to the inserts. The plates were incubated for 22 h in a CO 2 incubator at 37°C. The chamber inserts were then stained using the Diff-Quick staining kit (Dade-Behring, Newark, DE) according to the manufacturer's instructions. Finally, the membranes were separated with a sterile scalpel and observed using a light microscope. The number of cells that had passed through the membranes were counted as a measure of their migration potential. These assays were performed in triplicate.
Casein Zymography-Cells were incubated in serum-free medium overnight, and the supernatants were collected and mixed with SDS-PAGE sample buffer in the absence of reducing agents and electrophoresed through 4 -16% gradient polyacrylamide gels containing 0.1% (w/v) blue casein (Novex® zymogram gels from Invitrogen). Electrophoresis was carried out at 125 V. After electrophoresis, the gel was equilibrated by incubating in 1ϫ zymogram renaturing buffer (Invitrogen) for 30 min at room temperature with gentle agitation to remove the SDS from the gel. 1ϫ zymogram-developing buffer was added to the gel, which was incubated for 48 h at 37°C to identify the proteolytic activity of the MMP enzymes. The protease activity was then visualized as a negative staining area (clear bands) on the blue casein gel, which otherwise has a dark blue background.

RESULTS
The predicted location of the LGI1 protein at or in the cell membrane (9), together with the membership of LGI1 in the family of F20 LRR genes, suggests that it may have a signaling function, as do many of these F20 family members (11). To test this possibility, we compared the gene expression profile of LGI1-null glioma cells carrying the pcDNA3 vector with clones expressing exogenous LGI1. The full-length LGI1 open reading frame described by Chernova et al. (9), fused to a C-terminal FLAG epitope by PCR, was cloned into pcDNA3 and then stably transfected into T98G cells (12).
Comparative Gene Expression Analysis-RNA isolated from three independently derived T98G clones that showed exogenous expression of LGI1 (12) was used to prepare cRNA for hybridization to the Affymetrix U133A arrays. The gene expression profiles from these cells were compared with those produced from T98G clones transfected with the empty pcDNA3 vector. The data from the three clones was then filtered to display only those genes that were at least 2-fold up-regulated or 2-fold down-regulated in all three independent clones compared with the control. Of a total of 22,214 genes and expressed sequence tags analyzed, 614 genes were up-regulated and 475 were down-regulated using the Affymetrix Data Mining Tool analysis alone. However, combining this analysis with dChip, only 189 genes were up-regulated and 15 were down-regulated. Within this series of genes it was striking that a significant number of them were associated with the extracellular matrix (Table I). In addition, several members of the MMP gene family also showed down-regulation. This gene family has been associated with invasion characteristics in brain tumors (23,24). MMP1 and MMP3 were the most consistently down-regulated in all of the clones in our analysis. Only one clone showed down-regulation of MMP11 and MMP12. The up-regulation of TIMP3, a known antagonist of MMP action, was also seen in all of the clones expressing LGI1.
Effect of LGI1 on MMP1 and MMP3 Expression-Because MMP1 and MMP3 were the most consistently and severely down-regulated MMP genes in the T98G/LGI1 cells, we concentrated our subsequent analysis on these two family members (see discussion). Using reverse transcription PCR, we demonstrated that MMP1 and MMP3 are expressed endogenously in the T98G clones that carried the empty vector (Fig.  1A). In contrast, MMP1 and MMP3 transcripts were below detectable levels in cells forced to re-express LGI1.
The MMPs are structurally and functionally related zinc-dependent endopeptidases belonging to four different subfamilies (17), namely collagenases, gelatinases, stromelysins, and membrane-type MMPs (MT-MMPs). These proteolytic enzymes are secreted as inactive proenzymes (pro-MMPs), which are subsequently activated extracellularly by proteolytic cleavage. To determine whether MMP1 and MMP3 proteins were secreted from the T98G cells, supernatants were harvested from cultures grown overnight in the absence of serum and then analyzed by Western blotting. Analysis using anti-MMP1 and anti-MMP3 antibodies demonstrated high levels of these proteins in the supernatant of the cells carrying the empty vector (Fig. 1B) but not in any of the T98G cell clones expressing LGI1, confirming the LGI1-induced suppression of MMP1/3 mRNA.
To determine that the MMPs seen in the parental T98G cells were active, 10 and 20 l of the supernatants from T98G cells carrying the empty vector were size fractionated on Novex zymogram gels (Fig. 1) and compared with supernatants from T98G clones carrying the exogenous LGI1 gene. MMP activity was clearly seen in cells that did not express LGI1 but was absent in clones that expressed it. These results demonstrate that the presence of LGI1 suppresses MMP activity.
Analysis of the ERK and p38 MAPK Pathways-Earlier reports have shown that expression and activation of the MMP1 and MMP3 gene products are coordinately controlled by the activation of various extracellular, signal-related protein kinases, including ERK1/2 and p38. Several reports also indicate that the enhancement of human MMP1 and MMP3 transcription involves the activation of the AP-1 elements in the 5Јflanking regulatory regions of these genes through distinct MAPK pathways (25,26). It has also been shown that activation of ERK1/2 plays a major role in MMP1/3 expression, whereas the activation of p38 apparently has little effect on the transcriptional activity of the MMP1 gene (25).
To determine whether activation of these signaling pathways controls MMP1 and MMP3 production in the LGI1-expressing T98G cells, we analyzed the ERK proteins in these cells using Western blotting. T98G/vector clones show high levels of phosphorylated ERK1/2 (Fig. 2). In contrast, T98G cells expressing LGI1 contained low levels of phosphorylated ERK1/2 proteins. Total levels of ERK proteins were unaffected by the re-expression of LGI1 (Fig. 2). In contrast, neither p38 phosphorylation nor total p38 protein levels were affected by LGI1 re-expression (Fig. 2). These results suggest that LGI1 may be controlling the expression of MMP1/3 transcription through the ERK1/2 pathway.
To further examine the roles of ERK1/2 in the regulation of MMP1 and MMP3 production by LGI1 (Fig. 3A), we treated the different T98G cell clones with the pharmacological MEK1 inhibitors PD98059 (50 M) and U0126 (20 M). Both inhibitors severely diminished relative ERK1/2 phosphorylation levels in the T98G/vector cells (data not shown). In these cells, PD98056 causes complete suppression of MMP1/3 production (Fig. 3A), and U0126 causes significant down-regulation of protein production (Fig. 3A), which is consistent with observations in other cell systems.
To investigate the relative role of the p38 pathway in MMP1/3 production, T98G/vector cells were treated with SB203580, a specific inhibitor of the p38 MAPK pathway. In these experiments, expression of the MMP1/3 was suppressed (Fig. 3B). These data suggest that p38 MAPK is also involved in MMP1/3 production in these cells. Despite the fact that ERK1/2 and p38 MAPK seem to be involved in MMP1/3 production in T98G/vector cells, because the phosphorylation status of p38 in T98G/LGI1 clones showed only a marginal decrease, it is apparently not the pathway through which LGI1 exerts its biological effect.
Effect of PMA on ERK1/2 Activation and MMP Production-In some cell systems, activation of protein kinase C (PKC) activates the RAF1-MEK-ERK signaling pathway (27). It has been shown that PKC also activates MMP1 production in malignant glioma cells (28). To determine whether PKC activation affects MMP production in T98G/vector and T98G/LGI1 cells that express exogenous LGI1, we treated these cells with PMA, a pharmacological activator of PKC. This treatment resulted in a further increase in the normal steady state expression levels of MMP1 and MMP3 in T98G/vector cells (Fig. 3). Interestingly, the inhibitory effects of LGI1 on MMP expression was abolished in T98G/LGI1 cells (Fig. 3), demonstrating that the LGI1-mediated inhibition of MMPs is sensitive to the activation of PKC. The PMA-induced reactivation of MMP1/3 in T98G/LGI1 was accompanied by phosphorylation of ERK1/2 (Fig. 4) This result confirms the earlier suggestion (Fig. 2) that MMP1/3 production is regulated through the ERK pathway. To assess the migration potential of the T98G cells following treatment with PMA, we performed Matrigel matrix invasion assays (Fig. 5). T98G cells transfected with pcDNA3 migrate LGI1 Controls the MMP Production freely through these substrates, whereas T98G cells expressing exogenous LGI1 do not (Fig. 5). However, when T98G/LGI1 cells were treated with PMA, they were able to migrate through the Matrigel matrix membrane (Fig. 5). In contrast, when these cells were treated with PD98059 alone and in combination with PMA, they were no longer able to pass through the matrix, thus behaving in a similar matter to cells forced to re-express LGI1 (Fig. 5). These data demonstrate that LGI1-mediated inhibition of invasiveness can be reversed as a result of activating PKC. When T98G/vector and T98G/LGI1 cells were co-treated with the either U0126 or PD98059, inhibitors of the ERK1/2 pathway, together with PMA, the expression of MMPs induced by PMA was suppressed (Fig. 3). These experiments demonstrate that LGI1 may affect the ERK1/2 pathway upstream of the point where PKC acts. On the other hand, cells treated with the p38 MAPK inhibitor SB203580 showed reversal of MMP1/3 inhibition following treatment with PMA in both T98G/vector and T98G/LGI1 cells.
Effect of AKT Phosphorylation on the MAPK Pathway-In some circumstances, control of cell migration has been shown to operate through the PI3K/AKT signaling pathway (29). To investigate whether LGI1 has an effect on signaling through this pathway, we analyzed the phosphorylation status of Akt Ser-473 in the presence and absence of LGI1. In T98G/vector cells, the level of Akt phosphorylation was barely detectable (Fig. 6A). In contrast, in cells expressing exogenous LGI1, higher levels of phosphorylated Akt were seen (Fig. 6A).
It has been reported (30) that hyperphosphorylation of Akt can down-regulate the ERK1/2 MAPK pathway by phosphorylation at the serine 259 position in Raf1. To determine whether the increased Akt phosphorylation seen in the presence of LGI1 expression operates in this way, we analyzed the phosphoryl- LGI1 exerts its effect upstream of PKC, which acts on the Raf-MEK pathway. When the cells that do and do not express LGI1 are stimulated with PMA and, at the same time, exposed to a potent synthetic inhibitor of ERK1/2 (PD98059), MMP1/3 production is again severely down-regulated. Treatment of T98G cells with U0126, another inhibitor of the ERK1/2 pathway, demonstrates the same inhibition of MMP production with or without the inclusion of PMA. Similarly, when the same cells are treated with SB203580 (an inhibitor for the p38 MAPK pathway), loss of MMP1 and MMP3 production was seen, showing that p38 contributes to the control of MMP expression. This inhibition by SB203580 is lost in the presence of PMA. ation status of Raf1 at the previously implicated serine 259 residue. Using an antibody that is specific for the phospho-Raf1 Ser-259 , we demonstrated that, in the presence of the exogenous expression of LGI1, Raf1 Ser-259 is distinctly phosphorylated. In T98G/vector cells, phospho-Akt was not identified and neither was phospho-Raf1 Ser-259 (Fig. 6B). These data demonstrate that re-expression of LGI1 in glioma cells can apparently influence the inactivation of the ERK/MAPK pathway through Akt phosphorylation (Fig. 6B). When these cells were treated with a PI3K inhibitor, LY294002, phosphorylation of both Akt and Raf1 Ser-259 was inhibited. On the other hand, even though treatment of cells with LY294002 inhibited Akt phosphorylation, ERK1/2 phosphorylation was partially restored (Fig. 6B). This suggests that LGI1 may also regulate the ERK/MAPK pathway through other mechanisms. DISCUSSION Brain tumors are the third most frequent cause of cancerrelated deaths in adults and the second most common cause of cancer-related deaths in children. Even though most primary brain tumors do not metastasize and rarely disseminate through the cerebrospinal fluid, they do invade the surrounding normal brain tissue. This characteristic local invasiveness of gliomas contributes substantially to the inability to achieve total resection by surgery and often results in a recurrence at the primary site and throughout the brain. Glial cell invasion is a multistep process that requires these cells to first attach to the barrier matrix and create a proteolytic defect in it. These cells then migrate through the defect. Several reports have shown that there is a strong association between expression of various proteases, such as serine proteases, metalloproteases, and the plasminogen activation/plasmin system, and the invasive behavior of gliomas (31)(32)(33)(34)(35). The MMPs in particular, because they are extracellular endopeptidases and thus responsible for degradation of the extracellular matrix, have been implicated in the invasion of the tumor cells. The complexity of the regulation of MMPs is only just being explored, and here we demonstrate that the LGI1 gene regulates the production of MMP1/3 through the MAPK pathway (Fig. 7). Previous studies had suggested that the proteolytic capacity of various cell types was regulated through ERK1/2 and p38 MAPK, which, in turn, regulate the activation of MMP1 and MMP3 as well as gelatinase B (MMP-9) and collagenase-3 (MMP13) (36 -40). The demonstration that the biological consequences of LGI1 expression functions by regulating the ERK pathway provides a link between this cell surface effector and MMP regulation in glioma cells. Although only MMP1 and MMP3 were analyzed in T98G cells, in an independent gene expression profiling study using A172 cells that were forced to re-express LGI1 (12), we found that LGI1 also inhibited other MMPs, the most signifi- cant of which were MMP1, MMP-2, and MMP-9 (data not shown). This observation strongly suggests that LGI1 may be a more general inhibitor of MMPs and that its inhibitory action depends largely on which MMPs are endogenously expressed by the individual tumor cells.
Phosphorylation of ERK1/2 and p38 MAPK have been shown in other systems to be the mechanism through which these pathways promote the production of MMP1 and MMP3 (37). In our studies, inhibition of the ERK1/2 and p38 pathways in T98G cells prevents expression of MMP1 and MMP3. This observation demonstrates that both pathways are normally involved in the regulation of MMP production, but the fact that only the phosphorylation status of ERK1/2 is affected by LGI1 expression strongly suggests that this pathway (ERK1/2) is responsible for the biological effect of suppression of tumor cell invasiveness. MAPKs can affect MMP production directly by influencing transcription factors that regulate the expression of MMPs. In a recent report (36), it was suggested that ERK1/2 directly regulates the transcriptional activation of MMP1 and MMP3, whereas p38 MAPK helps stabilize these mRNAs. The data from the present study suggest that, although T98G cells depend on both the ERK1/2 and p38 MAPK pathways for the expression and, probably, mRNA stabilization of MMPs, the inhibition of the ERK1/2 pathway itself (through the re-introduction of LGI1) is sufficient to down-regulate the expression of MMP1 and MPP3. In support of this suggestion, Westermarck et al. (25) showed that the activation of ERK1/2 results in induction of the AP-1 transcription complex, which in turn results in the stimulation of MMP1 promoter activity, whereas that activation of p38 MAPK had only minimal effect. In light of these earlier observations and our present results, we suggest that the inhibition of ERK1/2 in the T98G glioma cell line, which results in inhibition of MMP1 and MMP3, is sufficient to inhibit in vitro invasiveness.
PKC has been shown to phosphorylate many cellular targets, including RAF1 and MEK1, both of which activate the RAF-MEK-ERK pathway, which is responsible for the activation of MMP1 and MMP3 transcription (36,41). The PMA-induced reversal of LGI1-mediated inhibition of MMP1 and MMP3 production, as well as ERK1/2 phosphorylation, clearly indicates that LGI1 acts upstream of Raf-1. Raf-1 activation has been shown to be a very important event in the activation of the MAPK pathway (42). Raf-1 is a serine/threonine kinase (43) that can be activated by a variety of extracellular stimuli and is strongly implicated in the induction of proliferation. Raf-1 activation itself is a complex process and involves the recruitment of various members of the 14-3-3 protein family to the membrane, which results in multisite phosphorylation of Raf-1, causing activation of the MEK/ERK pathway (44 -45). It was reported earlier that there is a cross-talk between the PI3K/Akt pathway and the Raf-MEK-ERK pathway. It was also shown that, following hyperphosphorylation, Akt Ser-473 phosphorylates Raf-1 on the serine 259 residue (30). This phosphorylation of Raf-1 has been shown to be an inhibitory signal that abolishes the activation of the MAPK pathway (30). The AKTmediated regulation of the MEK-ERK pathway was shown to be cell-and stage-specific (29). In our present study, we found that high levels of Akt phosphorylation resulted in high levels of Raf-1 Ser-259 phosphorylation and inhibition of the MAPK pathway. This signaling cascade was not seen in T98G/vector cells where, instead, high levels of phosphorylation of ERK1/2 were seen. These results show that the forced re-expression of LGI1 is responsible for inhibition of the ERK/MAPK pathway. Furthermore, the inhibition of PI3K using LY294002 completely inhibited the phosphorylation of Raf-1 Ser-259 and partially restored ERK1/2 phosphorylation, which indicates that phosphorylation of Akt may be playing a role in the inhibition of the ERK1/2-MAPK pathway. However, because a partial reversal of the ERK1/2 pathway occurs following LY 294002 treatment, LGI1 may be exerting its effect on ERK-MAPK signaling through other pathways.
Inhibition of MMP function has already been explored as a means to treat cancer, although most of the compounds that have been tested so far have failed (46). These approaches included the injection of TIMPs and various synthetic inhibitors targeting the active sites of MMPs. The use of TIMPs in cancer therapy, however, has been disappointing, because these protein-based treatments are difficult to administer and generally have poor pharmacokinetics. Likewise, synthetic MMP inhibitors produce indiscriminate inhibition of related proteases. It is possible, therefore, that a better understanding of the regulatory mechanisms that control MMP production may lead to more effective strategies. The demonstration by at least two groups that LGI1 is expressed in low grade tumors and is lost in invasive cells (12,13), together with the demonstration here that LGI1 controls MMP production, raises the possibility that the LGI1 signal transduction pathway may be one such potential target for the treatment of metastatic/invasive brain tumors.