Inhibition of specific matrix metalloproteinases (MMP) is an attractive noncytotoxic approach to cancer therapy.

Extracellular proteases have been shown to cooperatively influence matrix degradation and tumor cell invasion through proteolytic cascades, with individual proteases having distinct roles in tumor growth, invasion, migration and angiogenesis. Matrix metalloproteases (MMP)-9 and cathepsin B have been shown to participate in the processes of tumor growth, vascularization and invasion of gliomas. In the present study, we used a cytomegalovirus promoter-driven DNA template approach to induce hairpin RNA (hpRNA)-triggered RNA interference (RNAi) to block MMP-9 and cathepsin B gene expression with a single construct. Transfection of a plasmid vectorexpressing double-stranded RNA (dsRNA) for MMP-9 and cathepsin B significantly inhibited MMP-9 and cathepsin B expression and reduced the invasive behavior of SNB19, glioblastoma cell line in Matrigel and spheroid invasion models. Downregulation of MMP-9 and cathepsin B using RNAi in SNB19 cells reduced cell-cell interaction of human microvascular endothelial cells, resulting in the disruption of capillary network formation in both in vitro and in vivo models. Direct intratumoral injections of plasmid DNA expressing hpRNA for MMP-9 and cathepsin B significantly inhibited established glioma tumor growth and invasion in intracranial tumors in vivo. Further intraperitoneal (ip) injections of plasmid DNA expressing hpRNA for MMP-9 and cathepsin B completely regressed pre-established tumors for a long time (4 months) without any indication of these tumor cells. For the first time, these observations demonstrate that the simultaneous RNAi-mediated targeting of MMP-9 and cathepsin B has potential application for the treatment of human gliomas.

Mouse syngeneic tumor models are widely used tools to demonstrate activity of novel anti-cancer immunotherapies. Despite their widespread use, a comprehensive view of their tumor-immune compositions and their relevance to human tumors has only begun to emerge. We propose each model possesses a unique tumor-immune infiltrate profile that can be probed with immunotherapies to inform on anti-tumor mechanisms and treatment strategies in human tumors with similar profiles. In support of this endeavor, we characterized the tumor microenvironment of four commonly used models and demonstrate they encompass a range of immunogenicities, from highly immune infiltrated RENCA tumors to poorly infiltrated B16F10 tumors. Tumor cell lines for each model exhibit different intrinsic factors in vitro that likely influence immune infiltration upon subcutaneous implantation. Similarly, solid tumors in vivo for each model are unique, each enriched in distinct features ranging from pathogen response elements to antigen presentation machinery. As RENCA tumors progress in size, all major T cell populations diminish while myeloid-derived suppressor cells become more enriched, possibly driving immune suppression and tumor progression. In CT26 tumors, CD8 T cells paradoxically increase in density yet are restrained as tumor volume increases. Finally, immunotherapy treatment across these different tumor-immune landscapes segregate into responders and non-responders based on features partially dependent on pre-existing immune infiltrates. Overall, these studies provide an important resource to enhance our translation of syngeneic models to human tumors. Future mechanistic studies paired with this resource will help identify responsive patient populations and improve strategies where immunotherapies are predicted to be ineffective.

Growth/differentiation factor-5 (GDF5), also known as cartilage-derived morphogenetic protein-1 (CDMP-1), is a secreted signaling molecule that participates in skeletal morphogenesis. Heterozygous mutations in GDF5, which maps to human chromosome 20, occur in individuals with autosomal dominant brachydactyly type C (BDC). Here we show that BDC is locus homogeneous by reporting a GDF5 frameshift mutation segregating with the phenotype in a family whose trait was initially thought to map to human chromosome 12. We also describe heterozygous mutations in nine additional probands/families with BDC and show nonpenetrance in a mutation carrier. Finally, we show that mutant GDF5 polypeptides containing missense mutations in their active domains do not efficiently form disulfide-linked dimers when expressed in vitro. These data support the hypothesis that BDC results from functional haploinsufficiency for GDF5.

Matrix metalloproteinase 9 (MMP-9) is known to play a major role in cell migration and invasion in both physiological and pathological processes. Our previous work has shown that increased MMP-9 levels are associated with human glioma tumor progression. In this study, we evaluated the ability of an adenovirus containing a 528 bp cDNA sequence in antisense orientation to the 5' end of the human MMP-9 gene (Ad-MMP-9AS) to inhibit the invasiveness and migratory capacity of the human glioblastoma cell line SBN19 in in vitro and in vivo models. Infection of glioma cells with Ad-MMP-9AS reduced MMP-9 enzyme activity by approximately 90% compared with mock-or Ad-CMV-infected cells. Migration and invasion of glioblastoma cells infected with Ad-MMP-9AS were significantly inhibited relative to Ad-CMV-infected controls in spheroid and Matrigel assays. Intracranial injections of SNB19 cells infected with Ad-MMP-9AS did not produce tumors in nude mice. However, injecting the Ad-MMP-9AS construct into subcutaneous U87MG tumors in nude mice caused regression of tumor growth. These results support the theory that adenoviral-mediated delivery of the MMP-9 gene in the antisense orientation has therapeutic potential for treating gliomas.

Urokinase-type plasminogen activator (uPA) and its receptor (uPAR) play an important role in the invasiveness of gliomas and other infiltrative tumors. In glioma cell lines and tumors, high grade correlates with increased expression of uPAR and uPA. We report here the downregulation of uPAR and uPA by delivery of antisense sequences of uPAR and uPA in a single adenoviral vector, Ad-uPAR-uPA (Ad, adenovirus). The bicistronic construct (Ad-uPAR-uPA) infected glioblastoma cell line had significantly reduced levels of uPAR, uPA enzymatic activity and immunoreactivity for these proteins when compared to controls. The Ad-uPAR-uPA infected cells showed a markedly lower level of invasion in the Matrigel invasion assays, and their spheroids failed to invade the fetal rat brain aggregates in the coculture system. Intracranial injection of SNB19 cells with the AduPAR-uPA antisense bicistronic construct showed inhibited invasiveness and tumorigenicity. Subcutaneous injections of bicistronic antisense constructs into established tumors (U87 MG) caused regression of those tumors. Our results support the therapeutic potential of targeting the individual components of the uPAR-uPA system by using a single adenovirus construct for the treatment of glioma and other invasive cancers.

We have shown previously that the tissue factor pathway inhibitor-2 (TFPI-2), a broad range proteinase inhibitor, is highly expressed in low-grade gliomas, but, minimally expressed or undetectable in glioblastomas, and that enforced expression of this gene reduces the invasive properties of brain tumor cells. Here, we examined the role of promoter methylation as a mechanism of TFPI-2 gene silencing. In SNB19 glioblastoma cells, which have no detectable TFPI-2 expression, 5-aza-2 0 -deoxycytidine (5aC), an inhibitor of DNA methyltransferase, induced TFPI-2 mRNA in a dose-dependent manner. Trichostatin A (TSA), the histone deacetylase (HDAC) inhibitor, by itself, was more efficient than 5aC in inducing TFPI-2 transcripts, and the 5aC þ TSA combination resulted in highly synergistic reactivation of the gene, both at the transcript and protein levels. In Hs683 glioma cells, which express the TFPI-2 gene at high levels, transfection of the in vitro methylated TFPI-2 promoter constructs resulted in a drastic decrease of promoter activity compared to the unmethylated promoter. Further, the methylation-specific PCR in SNB19 and Hs683 cells showed that TFPI-2 gene repression was closely linked with methylation of the CpG islands in the promoter. Finally, the chromatin immunoprecipitation assays in SNB19 cells showed that the methylated and repressed TFPI-2 promoter was associated with the methyl-CpG binding protein 2 (MeCP2), and that gene reactivation resulted in the loss of MeCP2 from this site. These studies establish that TFPI-2 is transcriptionally silenced through promoter methylation in SNB19 cells.

Increases in the abundance of cathepsin B transcript and protein with increased tumor grade and changes in subcellular localization and activity of this enzyme. We observed progressive reductions in levels of the protease inhibitor cystatin C, an inhibitor of cathepsin B with corresponding increases in the malignancy of glioma cell lines, implying an inverse correlation between cystatin C and tumor grade. To investigate the role of cystatin C in the invasion of brain tumor cells, we stably transfected SNB19 glioblastoma cells with either a 0.4-kb cDNA construct of human cystatin C in the sense orientation or an empty vector. Clones expressing sense-cystatin C cDNA had higher cystatin C mRNA and protein levels than did control cells. Sense-transfected cells were also markedly less invasive than control cells in a Matrigel invasion assay and in a coculture assay of SNB19 spheroids and fetal rat brain aggregates. Finally, the sense-transfected cells did not form tumors in nude mice upon intracerebral injection. These results strongly implicate cystatin C in the invasiveness of human glioblastoma cells and suggest that sense transcripts of cystatin C may prove useful in cancer therapy.