Survivin Promoter-Driven DFF40 Gene Expression Sensitizes Melanoma Cancer Cells to Chemotherapy

Downregulation of the apoptotic protein DNA fragmentation factor 40 (DFF40) is correlated with poor overall survival in some malignancies, including melanoma. In this study, DFF40 gene expression driven by survivin promoter, a tumor-specific promoter, was used to selectively induce cytotoxicity in melanoma cells. The activity and strength of survivin promoter were examined in B16F10 murine melanoma, and L929 murine normal fibroblast cell lines using enhanced green fluorescent protein reporter assay and reverse transcription polymerase chain reaction. The effect of expression of DFF40 under the control of cytomegalovirus (CMV) or survivin promoter on viability of cancerous and normal cells was determined by MTT [3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide] assay. Apoptosis induction by expression of DFF40 was evaluated using Annexin-V/propidium iodide staining. Our findings showed high activity of survivin promoter comparable to the control promoter (ie, CMV) in melanoma cells, while survivin activity in normal cells was negligible. Survivin promoter-derived DFF40 gene expression led to selective inhibition of cell viability and induction of apoptosis in cancerous cells. Low and sublethal concentrations of a chemotherapeutic drug, dacarbazine, significantly enhanced the growth inhibitory effect of DFF40 gene therapy. Combination of survivin-driven gene therapy and chemotherapy could be considered as a potential therapeutic treatment for melanoma and possibly other malignancies with similar features.

Combined oncolytic adenovirus carrying MnSOD and mK5 genes both regulated by survivin promoter has synergistic inhibitory effect on gastric cancer

Gastric cancer (GC) is one of the major causes of cancer related mortality. The use of oncolytic virus for cancer gene-virotherapy is a new approach for the treatment of human cancers. In this study, a novel Survivin promoter driven recombinant oncolytic adenovirus carrying mK5 or MnSOD gene was constructed, which was modified after deletion of E1B gene. Human plasminogen Kringle 5 mutant ( mK5 ) and manganese superoxide dismutase ( MnSOD ) are both potential tumor suppressor genes. To construct Ad-Surp- mK5 and Ad-Surp- MnSOD oncolytic adenovirus, we hypothesized that the combination of the two viruses would enhance the therapeutic efficacy of GC as compared to the virus alone. The results of the in vitro experiments revealed that the combination of adenovirus carrying mK5 and MnSOD gene exhibited stronger cytotoxicity to GC cell lines as compared to the virus alone, Additionally, the virus could selectively kill cancer cells and human somatic cells. Cell staining, flow cytometry and western blot analysis showed that the combination of two adenovirus containing therapeutic genes could promote the apoptosis of cancer cells. In vivo experiments further verified that Ad-Surp- mK5 in combination with Ad-Surp- MnSOD exhibited significant inhibitory effect on the growth of GC tumor xenograft as compared to the virus alone, and no significant difference was observed in the body weight of treatment and the normal mice. In conclusion, the combination of our two newly constructed recombinant oncolytic adenovirus containing mK5 or MnSOD therapeutic genes could significantly inhibit gastric cancer growth by inducing apoptosis, suggestive of its potential for GC therapy.

JCPyV T-Antigen Activation of the Anti-Apoptotic Survivin Promoter—Its Role in the Development of Progressive Multifocal Leukoencephalopathy

Progressive Multifocal Leukoencephalopathy (PML) is a fatal demyelinating disease of the CNS, resulting from the lytic infection of oligodendrocytes by the human neurotropic polyomavirus JC (JCPyV), typically associated with severe immunocompromised states and, in recent years, with the use of immunotherapies. Apoptosis is a homeostatic mechanism to dispose of senescent or damaged cells, including virally infected cells, triggered in the vast majority of viral infections of the brain. Previously, we showed upregulation of the normally dormant anti-apoptotic protein Survivin in cases of PML, which—in vitro—resulted in protection from apoptosis in JCPyV-infected primary cultures of astrocytes and oligodendrocytes. In the present study, we first demonstrate the absence of apoptotic DNA fragmentation and the lack of caspase activity in 16 cases of PML. We also identified the viral protein large T-Antigen as being responsible for the activation of the Survivin promoter. Chromatin Immunoprecipitation assay shows a direct binding between T-Antigen and the Survivin promoter DNA. Finally, we have identified the specific region of T-Antigen, spanning from amino acids 266 and 688, which binds to Survivin and translocates it to the nucleus, providing evidence of a mechanism that results in the efficient replication of JCPyV and a potential target for novel therapies.

EXTH-41. A NANOPARTICLE-BASED CANCER-SELECTIVE GENE TRANSFER STRATEGY FOR LOCALIZED THERAPY OF MALIGNANT HIGH GRADE GLIOMAS

Treatment for malignant high grade gliomas entails surgery, followed by chemotherapy and/or radiation. Despite the intensive care, there is a ~90% recurrence rate. This is attributed to suboptimal efficacy of treatments in tackling widespread cancer cells. We have previously developed nanoparticles that provide widespread therapeutic distribution both in healthy and tumor tissues, but are incapable of differentiating between them. This limitation applies to the current standard-of-care and state-of-the-art gene therapies. Here, we evaluate the efficacy of DNA nanoparticles carrying promoters that drive transgene expression specifically in tumor cells to achieve widespread yet cancer-selective gene transfer in high grade gliomas. To identify tumor-specific promoters, we used ELISA to confirm elevated expression of proteins previously reported to be upregulated in tumor tissue. We observed that expression of survivin in cancer cells was significantly greater than that of other cancer-rich proteins, exhibiting two orders of magnitude greater levels in rodent and human cancer cells compared to their respective healthy cells. Furthermore, the CMV promoter mediated similarly high expression in healthy cells, whereas the level achieved by the survivin promoter was significantly lower, if not negligible, suggesting its tumor specificity. Likewise, CMV-driven plasmids delivered into the brain by the nanoparticles mediated virtually identical volumetric distribution of transgene expression in both normal and tumor tissues in vivo. In contrast, nanoparticles carrying survivin-driven plasmids provided widespread transgene expression only in an orthotopically established tumor, but not healthy brain tissue. Additionally, we demonstrate therapeutic efficacy in an established brain tumor model using the DNA nanoparticles carrying survivin promoter-driven plasmids expressing a therapeutic protein. We identified survivin promoter as a lead TSP and confirmed its ability to mediate highly efficient and widespread but cancer-selective transgene expression with the aid of our nanoparticles uniquely designed to penetrate in healthy and tumor tissues.