Astrocyte elevated gene-1 (AEG-1) was initially identified as an HIV-1-and tumor necrosis factor A (TNF-A)-inducible transcript in primary human fetal astrocytes by a rapid subtraction hybridization approach. Interestingly, AEG-1 expression is elevated in subsets of breast cancer, glioblastoma multiforme and melanoma cells and AEG-1 cooperates with Ha-ras to promote transformation of immortalized melanocytes. Activation of the transcription factor nuclear factor KB (NF-KB), a TNF-A downstream signaling component, is associated with several human illnesses, including cancer, and NF-KB controls the expression of multiple genes involved in tumor progression and metastasis. We now document that AEG-1 is a significant positive regulator of NF-KB. Enhanced expression of AEG-1 via a replication-incompetent adenovirus (Ad.AEG-1) in HeLa cells markedly increased binding of the transcriptional activator p50/p65 complex of NF-KB. The NF-KB activation induced by AEG-1 corresponded with degradation of IKBA and nuclear translocation of p65 that resulted in the induction of NF-KB downstream genes. Infection with an adenovirus expressing the mt32IKBA superrepressor (Ad.IKBA-mt32), which prevents p65 nuclear translocation, inhibited AEG-1-induced enhanced agar cloning efficiency and increased matrigel invasion of HeLa cells. We also document that TNF-A treatment resulted in nuclear translocation of both AEG-1 and p65 wherein these two proteins physically interacted, suggesting a potential mechanism by which AEG-1 could activate NF-KB. Our findings suggest that activation of NF-KB by AEG-1 could represent a key molecular mechanism by which AEG-1 promotes anchorage-independent growth and invasion, two central features of the neoplastic phenotype.

The International Council on Harmonization (ICH) S7B and E14 regulatory guidelines are sensitive but not specific for predicting which drugs are pro-arrhythmic. In response, the Comprehensive In Vitro Proarrhythmia Assay (CiPA) was proposed that integrates multi-ion channel pharmacology data in vitro into a human cardiomyocyte model in silico for proarrhythmia risk assessment. Previously, we reported the model optimization and proarrhythmia metric selection based on CiPA training drugs. In this study, we report the application of the prespecified model and metric to independent CiPA validation drugs. Over two validation datasets, the CiPA model performance meets all pre-specified measures for ranking and classifying validation drugs, and outperforms alternatives, despite some in vitro data differences between the two datasets due to different experimental conditions and quality control procedures. This suggests that the current CiPA model/metric may be fit for regulatory use, and standardization of experimental protocols and quality control criteria could increase the model prediction accuracy even further.

Metastasis is a significant event in cancer progression and continues to pose the greatest challenge for a cancer cure. Defining genes that control metastasis in vivo may provide new targets for intervening in this process with profound therapeutic implications. Melanoma differentiation associated gene-9 (mda-9) was initially identified by subtraction hybridization as a novel gene displaying biphasic expression during terminal differentiation in human melanoma cells. Mda-9, also known as syntenin, is a PDZ-domain protein overexpressed in many types of human cancers, where it is believed to function in tumor progression. However, a functional role of mda-9/ syntenin in tumor growth and metastasis and the signaling pathways involved in mediating these biological activities remain to be defined. Evidence is now provided, using weakly and highly metastatic isogenic melanoma variants, that mda-9/ syntenin regulates metastasis. Expression of mda-9/syntenin correlates with advanced stages of melanoma progression. Regulating mda-9/syntenin expression using a replicationincompetent adenovirus expressing either sense or antisense mda-9/syntenin modifies the transformed phenotype and alters metastatic ability in immortal human melanocytes and metastatic melanoma cells in vitro and in vivo in newborn rats. A direct relationship is observed between mda-9/syntenin expression and increased phosphorylation of focal adhesion kinase, c-Jun-NH 2 -kinase, and p38. This study provides the first direct link between mda-9/syntenin expression and tumor cell dissemination in vivo and indicates that mda-9/syntenin expression activates specific signal transduction pathways, which may regulate melanoma tumor progression. Based on its ability to directly alter metastasis, mda-9/syntenin provides a promising new focus for melanoma cancer research with potential therapeutic applications for metastatic diseases.

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Melanoma differentiation-associated gene-7/interleukin-24 (mda-7/IL-24) is a unique member of the IL-10 gene family that induces cancer-selective growth suppression and apoptosis in a wide spectrum of human cancers in cell culture and animal models. Additionally, recent clinical trials confirm safety and document significant clinical activity of mda-7/ IL-24 in patients with diverse solid cancers and melanomas. Despite intensive study the molecular basis of tumor-cell selectivity of mda-7/IL-24 is not well characterized. Using deletion analysis, a specific mutant of MDA-7/IL-24, M4, consisting of amino acids 104 to 206, is described that retains the cancer-specific growth-suppressive and apoptosis-inducing properties of the full-length protein. Employing rationally designed mutational analysis, we show that MDA-7/IL-24 and M4 physically interact with BiP/GRP78 through their C and F helices, localize in the endoplasmic reticulum, and activate p38 MAPK and GADD gene expression, culminating in cancerselective apoptosis. These studies provide novel mechanistic insights into the discriminating antitumor activity of MDA-7/ IL-24 by elucidating BiP/GRP78 as a defined intracellular target of action and present an unparalleled opportunity to develop improved therapeutic versions of this cancer-specific apoptosis-inducing cytokine. (Cancer Res 2006; 66(16): 8182-91)

Melanoma differentiation associated gene-7/interleukin-24 (Mda-7/IL-24), a novel member of the IL-10 family of cytokines, uniquely displays cancer-specific apoptosis-inducing activity. Positive results in ongoing phase I/II clinical trials have strengthened the possibility of its utilization as a cancer gene therapeutic. Previous studies document that signaling events leading to Ad.mda-7-induced transformed cell apoptosis are tyrosine kinase-independent. These results suggest that mda-7/IL-24 cancer cell-specific activity could occur through mechanisms independent of binding to its currently recognized cognate receptors and might even occur independent of receptor function. An adenovirus vector expressing a nonsecreted version of MDA-7/IL-24 protein was generated via deletion of its signal peptide. This nonsecreted protein was as effective as wild-type secreted MDA-7/IL-24 in inducing apoptosis in prostate carcinoma cell lines and displayed transformed cell specificity and localization of MDA-7/IL-24 in the Golgi/endoplasmic reticulum compartments. Our results indicate that mda-7/IL-24-mediated apoptosis can be triggered through a combination of intracellular as well as secretory mechanisms and can occur efficiently in the absence of protein secretion.

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