Reexpression of LGI1 in glioma cells results in dysregulation of genes implicated in the canonical axon guidance pathway.

The LGI1 gene suppresses invasion in glioma cells and predisposes to epilepsy. In a gene expression array comparison between parental cells and T98G cell clones forced to express LGI1, we demonstrate that the canonical axon guidance pathway is the most significantly affected. In particular, aspects of axon guidance that involve reorganization of the actin cytoskeleton, which is also involved in cell movement and invasion, were affected. Analysis of actin fiber organization using fluorescence microscopy demonstrated that different T98G cell clones expressing the exogenous LGI1 gene show high levels of stress fibers compared with controls. Since stress fiber formation is associated with loss of cell mobility, we used scratch wound assays to demonstrate that LGI1-expressing clones show a significant reduction in cell mobility. LGI1 reexpression also resulted in loss of the PDGFRA and EGFR proteins, suggesting a rapid turnover of these receptors despite increased mRNA levels for PDGFRA. LGI1 suppression of invasion is associated with loss of ERK/MAPK1 activation. LGI1 is a secreted protein, and when the culture supernatant from cells expressing FLAG- and GFP-tagged proteins were applied to parental T98G cells, ERK/MAPK1 phosphorylation and cell mobility was suppressed, demonstrating that the LGI1 protein acts as a suppressive agent for cell movement in this assay. These observations support a previous suggestion that LGI1 can reduce cellular invasion in in vitro assays and, as a secreted agent, may be developed as a means of treating metastatic cancer. In addition, this observation provides a mechanistic link for LGI1's common role in metastasis and epilepsy development.

Inactivation of LGI1 expression accompanies early stage hyperplasia of prostate epithelium in the TRAMP murine model of prostate cancer.

The LGI1 gene has been implicated in tumor cell invasion through regulation of the ERK pathway. To determine whether human prostate cancer cells (PC3, 22RV, Du145) are similarly affected by exposure to LGI1, we conducted scratch wound assays and demonstrated that the secreted LGI1 protein can reduce cell motility, an essential component of invasion and metastasis. These studies have now been extended to an in vivo mouse model of prostate cancer. Using a BAC transgenic mouse expressing a GFP reporter gene under the control of cis regulatory elements, we demonstrated that LGI1 is highly expressed in the normal prostate epithelium. To determine whether loss of LGI1 expression is associated with development and progression of murine prostate cancer, we bred the GFP reporter BAC transgenic mice with TRAMP mice which undergo early hyperplasia and progressive stages of prostate cancer. In the F1 animals, although the surrounding normal prostate epithelium expressed high levels of LGI1 in the double transgenic mice, the LGI1 gene had been inactivated even at the earliest stages of hyperplasia. This observation supports the suggestion that inactivation of LGI1 in certain cell types is related to tumor progression. Taken together these results suggest that LGI1 may be an important molecule for the arrest of prostate cancer cell invasion and possibly as a biomarker for early detection of prostate hyperplasia.

Arsenic trioxide affects signal transducer and activator of transcription proteins through alteration of protein tyrosine kinase phosphorylation.

PURPOSE: Arsenic trioxide decreases proliferation of acute myeloid leukemia (AML) cells, but its precise mechanism of action is unknown. EXPERIMENTAL DESIGN: We studied the effect of arsenic trioxide on patient samples and the AML cell line HEL, which, like leukemic blasts from 50% of AML cases, has constitutively activated signal transducer and activator of transcription (STAT) proteins. RESULTS: Arsenic trioxide induced mitotic arrest starting at 24 hours and significant cell death at 48 hours. These events were preceded by an arsenic trioxide dose-dependent down-regulation of activated STAT proteins starting at 6 hours. We hypothesized that arsenic trioxide inhibits protein tyrosine kinases (PTK), which, among others, phosphorylate and activate STATs. We therefore studied arsenic trioxide effects on Janus kinases and on three oncogenic PTKs that are known to activate STATs [FLT3, ZNF198/fibroblast growth factor receptor 1 (FGFR1), and BCR/ABL]. Arsenic trioxide reduced STAT3 activation by Janus kinases, altered phosphorylation and electrophoretic mobility of ZNF198/fibroblast growth factor receptor 1, reduced kinase protein level, and decreased STAT3 protein phosphorylation. Arsenic trioxide also reduced the phosphorylation of BCR/ABL and FLT3 with corresponding decreased STAT5 phosphorylation. CONCLUSIONS: These results suggest a selective activity of arsenic trioxide on PTKs and will assist in developing clinical trials in AML.

Suppression of the cell proliferation and invasion phenotypes in glioma cells by the LGI1 gene.

The leucine-rich, glioma-inactivated (LGI1) gene, located in 10q24, was originally identified because it was interrupted and inactivated by a reciprocal chromosome translocation in the T98G glioma cell line. Loss of LGI1 expression in high-grade brain tumors is correlated with the frequent loss of chromosome 10 during progression of gliomas. To investigate whether this gene can suppress the malignant phenotype in glioma cells, we introduced the LGI1 gene into cells that do (U87) and do not (T98G and A172) express LGI1 endogenously. A172 and T98G cells showed a significant reduction in cell proliferation potential as a result of re-expression of LGI1, whereas U87 cells did not. Using BD matrigel matrix chamber assays we were also able to show that the migration ability of the reconstituted A172 and T98G cells was also reduced considerably. Finally, these reconstituted T98G and A172 cells showed a significant reduction in the ability to form colonies in soft agar compared with the parental cells. This analysis clearly demonstrates that re-expression of the LGI1 gene in glioma cells that were null for its activity can greatly reduce their malignant potential. These observations provide the opportunity to investigate the role of LGI1 in gliomagenesis and, since LGI1 is predicted to be a membrane-bound protein, potentially provides the opportunity to develop novel treatment strategies for malignant gliomas.

ZNF198 protein, involved in rearrangement in myeloproliferative disease, forms complexes with the DNA repair-associated HHR6A/6B and RAD18 proteins.

A highly specific t(8;13)(p11;q12) translocation has been consistently identified in bone marrow cells from patients with an atypical myeloproliferative disease that is associated with peripheral blood eosinophila and T- or B-cell leukemias. In all patients analysed to date, the translocation event results in a chimeric gene in which the atypical zinc-finger domain of ZNF198 is fused to the N-terminal end of the catalytic domain of the FGFR1 receptor tyrosine kinase. To understand more about the consequences of this rearrangement we have investigated the normal function of the ZNF198 gene. Using yeast two-hybrid analysis we identified HHR6 as a protein binding partner and confirmed this using immunoprecipitation studies. The ZNF198/FGFR1 fusion protein also binds to HHR6. We demonstrate here that the human RAD18 is also present in the ZNF198/HHR6 protein complex, although it does not coimmunoprecipitate with the fusion kinase. Cells expressing the fusion kinase gene show a marked increased sensitivity to UVB irradiation, suggesting that it acts in a dominant-negative way to affect DNA repair. These observations support the idea that ZNF198, through its interaction with HHR6 and RAD18, may be involved in the DNA repair process.

Mass spectrometry identifies LGI1-interacting proteins that are involved in synaptic vesicle function in the human brain.

The LGI1 gene has been shown to predispose to epilepsy and influence cell invasion in glioma cells. To identify proteins that interact with LGI1 and gain a better understanding of its function, we have used co-immunoprecipitation (co-IP) of a secreted green fluorescent protein-tagged LGI1 protein combined with mass spectrometry to identify interacting partners from lysates prepared from human subcortical white matter. Proteins were recovered from polyacrylamide gels and analyzed using liquid chromatography coupled to tandem mass spectrometry. This analysis identified a range of proteins, but in particular synaptotagmin, synaptophysin, and syntaxin 1A. Each of these proteins is found associated with synaptic vesicles. These interactions were confirmed independently by co-IP and Western blotting and implicate LGI1 in synapse biology in neurons. Other vesicle-related proteins that were recovered by co-IP include clathrin heavy chain 1, syntaxin binding protein 1, and a disintegrin and metalloprotease 23. These observations support a role for LGI1 in synapse vesicle function in neurons.

HSPA1A is an important regulator of the stability and function of ZNF198 and its oncogenic derivative, ZNF198-FGFR1.

Mass spectroscopy analysis demonstrated that the HSPA1A protein is found in complex with the ZNF198 protein which is involved in a chromosome rearrangement with the FGFR1 gene in an atypical myeloproliferative disease. HSPA1A is a member of the HSP70 family of genes which has been shown to be inducible in a variety of circumstances. Exogenous expression of the ZNF198-FGFR1 fusion kinase gene as well as ZNF198 in a model cell system results in a large (>650-fold) increase in HSP70 mRNA levels. Using KNK437, a specific inhibitor of HSP70 transcription, we have demonstrated that an important function of HSPA1A is to stabilize the ZNF198 and ZNF198-FGFR1 proteins. In the absence of HSPA1A, specific functions of ZNF198-FGFR1 such as STAT3 phosphorylation is also lost. Treatment of cells with KNK437 in the presence of MG132, an inhibitor of proteasomal degradation of proteins, suggested that only the ZNF198-FGFR1 protein is subject to the proteasomal degradation pathway, while ZNF198 is not. These observations suggest an important role for HSPA1A in ZNF198 and ZNF198-FGFR1 mediated cellular function.

Induction of the plasminogen activator inhibitor-2 in cells expressing the ZNF198/FGFR1 fusion kinase that is involved in atypical myeloproliferative disease.

The ZNF198/FGFR1 fusion kinase associated with an atypical myeloproliferative disease is constitutively activated and regulates several STAT transcription factors. We used oligonucleotide microarrays to compare the gene-expression profiles between HEK-293 cells that stably express either the ZNF198/FGFR1 chimeric protein or the wild-type ZNF198 gene. Expression of the plasminogen activator inhibitor-2 (PAI-2/SERPINB2) was highly increased in cells expressing the fusion gene. Western blot analysis demonstrated that HEK-293 cells do not express PAI-2 endogenously, but in ZNF198/FGFR1-expressing cells 2 molecular forms of PAI-2, which were 47 kDa and 32 kDa, were expressed intracellularly, and a 60-kDa form was secreted. Similarly, expression of ZNF198/FGFR1 in BaF/3 mouse hematopoietic cells also induced the expression of the PAI-2 protein. Immunoprecipitation analysis revealed that both intracellular forms of PAI-2 bind to the ZNF198/FGFR1 kinase. Treatment of HEK-293 and BaF/3 cells with TNF-alpha in the presence of cycloheximide, induced apoptosis in both cases. In contrast, HEK-293 and BaF/3 cells expressing ZNF198/FGFR1 were resistant to TNF-alpha-induced apoptosis. These observations suggest that expression of the ZNF198/FGFR1 fusion gene is associated with specific PAI-2-mediated resistance to apoptosis which may contribute to the highly malignant nature of leukemic cells carrying this fusion kinase gene.

ZNF198, a zinc finger protein rearranged in myeloproliferative disease, localizes to the PML nuclear bodies and interacts with SUMO-1 and PML.

The ZNF198/FGFR1 fusion gene in atypical myeloproliferative disease produces a constitutively active cytoplasmic tyrosine kinase, unlike ZNF198 which is normally a nuclear protein. We have now shown that the ZNF198/FGFR1 fusion kinase interacts with the endogenous ZNF198 protein suggesting that the function of ZNF198 may be compromised in cells expressing it. Little is currently known about the endogenous function of ZNF198 and to investigate this further we performed a yeast two-hybrid analysis and identified SUMO-1 as a binding partner of ZNF198. These observations were confirmed using co-immunoprecipitation which demonstrated that ZNF198 is covalently modified by SUMO-1. Since many of the SUMO-1-modified proteins are targeted to the PML nuclear bodies we used confocal microscopy to show that SUMO-1, PML and ZNF198 colocalize to punctate structures, shown by immunocytochemistry to be PML bodies. Using co-immunoprecipitation we now show that PML and sumoylated ZNF198 can be found in a protein complex in the cell. Mutation of the SUMO-1 binding site in wild-type ZNF198 resulted in loss of distinct PML bodies, reduced PML levels and a more dispersed nuclear localization of the PML protein. In cells expressing ZNF198/FGFR1, which also lack the SUMO-1 binding site, SUMO-1 is preferentially localized in the cytoplasm, which is associated with loss of distinct PML bodies. Recently, arsenic trioxide (ATO) was proposed as an alternative therapy for APL that was resistant to traditional therapy. Treatment of cells expressing ZNF198/FGFR1 with ATO demonstrated reduced autophosphorylation of the ZNF198/FGFR1 protein and induced apoptosis, which is not seen in cells expressing wild-type ZNF198. Overall our results suggest that the sumoylation of ZNF198 is important for PML body formation and that the abrogation of sumoylation of ZNF198 in ZNF198/FGFR1 expressing cells may be an important mechanism in cellular transformation.

Mass spectroscopy identifies the splicing-associated proteins, PSF, hnRNP H3, hnRNP A2/B1, and TLS/FUS as interacting partners of the ZNF198 protein associated with rearrangement in myeloproliferative disease.

ZNF198 is fused with FGFR1 in an atypical myeloproliferative disease that results in constitutive activation of the kinase domain and mislocalization to the cytoplasm. We have used immunoprecipitation of a GFP-tagged ZNF198 combined with MALDI-TOF mass spectroscopy to identify interacting proteins. P splicing factor (PSF) was identified as one of the proteins and this interaction was confirmed by Western blotting. Other proteins identified were the spliceosomal components hnRNP A2/B1, hnRNP H3, and TLS/FUS. PSF is also known to interact with PTB, another member of the hnRNP family of proteins, and we further demonstrated that PTB interacts with ZNF198. The interaction between TLS/FUS and ZNF198 was confirmed using Western blot analysis. In 293 cells expressing the ZNF198/FGFR1 fusion protein, neither PSF nor PTB binds to the fusion protein, possibly because of their differential localization in the cell.

The oncogenic fusion protein-tyrosine kinase ZNF198/fibroblast growth factor receptor-1 has signaling function comparable with interleukin-6 cytokine receptors.

The reciprocal t(8;13) chromosome translocation results in a fusion gene (FUS) in which the N-terminal half of the zinc finger protein ZNF198 is combined with the cytoplasmic domain of the fibroblast growth factor receptor-1 (FGFR1). Expression of FUS is suggested to provide growth-promoting activity to myeloid cells similar to the activity of hematopoietic cytokine receptors. This study determined the specificity of FUS to activate signal transduction pathways. Because no tumor cell line expressing FUS was available, the mode of FUS action was identified in cells transiently and stably transfected with an expression vector for FUS. FUS acted as a constitutively active protein-tyrosine kinase and mediated phosphorylation of STAT1, 3, and 5 but not STAT4 and 6. The same specificity but lower activity was determined for normal FGFR1. STAT activation by FUS, similar to that by interleukin-6-type cytokines, promoted STAT-specific induction of genes. The functionality of FUS, as well as the relative recruitment of STAT isoforms, was determined by the dimerizing function of the zinc finger domain. Replacement of the ZNF198 portion by the Bcr portion as present in the t(8;22) translocation shifted the signaling toward a more prominent STAT5 activation. This study documents that both gene partners forming the fusion oncogene define the activity and the signaling specificity of the protein-tyrosine kinase of FGFR1.

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.