PurposeThere are conflicting reports regarding the function of EFEMP1 in different cancer types. In this study, we sought to evaluate the role of EFEMP1 in malignant glioma biology.Experimental DesignReal-time qRT-PCR was used to quantify EFEMP1 expression in 95 glioblastoma multiforme (GBM). Human high-grade glioma cell lines and primary cultures were engineered to express ectopic EFEMP1, a small hairpin RNA of EFEMP1, or treated with exogenous recombinant EFEMP1 protein. Following treatment, growth was assayed both in vitro and in vivo (subcutaneous (s.c.) and intracranial (i.c.) xenograft model systems).ResultsCox regression revealed that EFEMP1 is a favorable prognostic marker for patients with GBM. Over-expression of EFEMP1 eliminated tumor development and suppressed angiogenesis, cell proliferation, and VEGFA expression, while the converse was true with knock-down of endogenous EFEMP1 expression. The EFEMP1 suppression of tumor onset time was nearly restored by ectopic VEGFA expression; however, overall tumor growth rate remained suppressed. This suggested that inhibition of angiogenesis was only partly responsible for EFEMP1's impact on glioma development. In glioma cells that were treated by exogenous EFEMP1 protein or over-expressed endogenous EFEMP1, the EGFR level was reduced and AKT signaling activity attenuated. Mixing of EFEMP1 protein with cells prior to s.c. and i.c. implantations or injection of the protein around the established s.c. xenografts, both significantly suppressed tumorigenicity.ConclusionsOverall, our data reveals that EEFEMP1 suppresses glioma growth in vivo, both by modulating the tumor extracellular microenvironment and by altering critical intracellular oncogenic signaling pathways.

Glioblastoma multiforme (GBM) is the most invasive brain tumor. We have previously reported that the transcription factor PAX6 suppresses the tumorigenecity of GBM cells. By an in vitro Matrigel invasion assay on two GBM cell lines stably transfected with wild-type and/or two mutant forms of PAX6, this study displays the first evidence that PAX6 inhibits the invasiveness of GBM cells and that the DNA-binding domain of PAX6 is required for this function. Using real-time quantitative reverse transcription-PCR (RT-PCR), gelatin zymography, and immunohistochemistry assays, the expression of the gene encoding matrix metalloproteinase-2 (MMP2) in GBM cell lines grown in vitro or in intracranial xenografts in nude mice was shown to be repressed by either stable or adenoviralmediated overexpression of PAX6. Luciferase promoter assays revealed PAX6-mediated suppression of MMP2 promoter activity. Electrophoretic mobility shift assays showed direct binding of PAX6 to the MMP2 promoter. A significant reverse correlation (P < 0.05) occurred between PAX6 and MMP2 expression quantified by real-time quantitative RT-PCR in 41 GBMs, 43 anaplastic astrocytomas, and 7 adjacent normal tissues. Interestingly, the degree and significance of the reverse correlation increased after excluding astrocytomas, whereas it became insignificant after excluding GBMs. In GBM cells stably transfected with a dominant negative mutant PAX6 showing increased MMP2 expression and invasiveness, knockdown of MMP2 revealed that MMP2 is one of the PAX6 target genes mediating its suppression of invasion. Overall data delineated a mechanism for the suppressive function of PAX6 in GBM: suppression of cell invasion by repressing the expression of proinvasive genes such as MMP2. (Cancer Res 2006; 66(20): 9809-17)

Aneuploidy with chromosome instability is a cancer hallmark. We studied chromosome 7 (Chr7) copy number variation (CNV) in gliomas and in primary cultures derived from them. We found tumor heterogeneity with cells having Chr7-CNV commonly occurs in gliomas, with a higher percentage of cells in high-grade gliomas carrying more than 2 copies of Chr7, as compared to low-grade gliomas. Interestingly, all Chr7-aneuploid cell types in the parental culture of established glioma cell lines reappeared in single-cell-derived subcultures. We then characterized the biology of three syngeneic glioma cultures dominated by different Chr7-aneuploid cell types. We found phenotypic divergence for cells following Chr7 mis-segregation, which benefited overall tumor growth in vitro and in vivo. Mathematical modeling suggested the involvement of chromosome instability and interactions among cell subpopulations in restoring the optimal equilibrium of tumor cell types. Both our experimental data and mathematical modeling demonstrated that the complexity of tumor heterogeneity could be enhanced by the existence of chromosomes with structural abnormality, in addition to their mis-segregations. Overall, our findings show, for the first time, the involvement of chromosome instability in maintaining tumor heterogeneity, which underlies the enhanced growth, persistence and treatment resistance of cancers.

Granzyme B expression is essential for eliciting NK cell cytotoxicity and T cell function. However, its transcriptional regulatory mechanism is not well understood. In this report, we demonstrate in human NK cells and T cells that the NF-κB-signaling pathway is involved in such control. Furthermore, a novel downstream human granzyme B gene sequence (GGAGATTCCC) was identified for NF-κB binding. EMSA, luciferase, and chromatin immunoprecipitation assays in vitro and in vivo indicated that this NF-κB binding site is functional in an NK cell line and its primary counterpart. Our data also demonstrate that this binding site is functional in Jurkat T cells. Taken together, we identified a novel NF-κB binding site, which plays a pivotal role in controlling human granzyme B gene transcription.

Human hepatocellular carcinoma (HCC) is one of the most common tumors worldwide, in which the genetic mechanisms of oncogenesis are still unclear. To investigate whether the genomic DNA copy number alterations may contribute to primary HCC, the cDNA microarray-based comparative genomic hybridization (CGH) analysis was here performed in 41 primary HCC infected by hepatitis B virus and 12 HCC cell lines. The resulting data showed that, on average, 7.25% of genomewide DNA copy numbers was significantly altered in those samples (4.61 ± 2.49% gained and 2.64 ± 1.78% lost). Gains involving 1q, 6p, 8q and 9p were frequently observed in these cases; and whilst, losses involving Ip, 16q and 19p occurred in most patients. To address the correlation between the alteration of genomic DNA copy numbers and transcriptional expression, the same cDNA microarray was further applied in 20 HCC specimens and all available cell lines to figure out the gene expression profiles of those samples. Interestingly, the genomic DNA copy number alterations of most genes appeared not to be in generally parallel with the corresponding transcriptional expression. However, the transcriptional deregulation of a few genes, such as osteopontin (SPP1), transgelin 2 (TAGLN2) and PEG10, could be ascribed partially to their genomic aberrations, although the many alternative mechanisms could be involved in the deregulation of these genes. In general, this work would provide new insights into the genetic mechanisms in hepatocarcinogenesis associated with hepatitis B virus through the comprehensive survey on correlation between genomic DNA copy number alterations and transcriptional expression.