Kyung-Sun Hwang

E2-EPF UCP targets pVHL for degradation and associates with tumor growth and metastasis

The von Hippel-Lindau tumor suppressor, pVHL, forms part of an E3 ubiquitin ligase complex that targets specific substrates for degradation, including hypoxia-inducible factor-1α (HIF-1α), which is involved in tumor progression and angiogenesis. It remains unclear, however, how pVHL is destabilized. Here we show that E2-EPF ubiquitin carrier protein (UCP) associates with and targets pVHL for ubiquitin-mediated proteolysis in cells, thereby stabilizing HIF-1α. UCP is detected coincidently with HIF-1α in human primary liver, colon and breast tumors, and metastatic cholangiocarcinoma and colon cancer cells. UCP level correlates inversely with pVHL level in most tumor cell lines. In vitro and in vivo, forced expression of UCP boosts tumor-cell proliferation, invasion and metastasis through effects on the pVHL-HIF pathway. Our results suggest that UCP helps stabilize HIF-1α and may be a new molecular target for therapeutic intervention in human cancers.

Adenovirus-mediated interleukin-18 mutant in vivo gene transfer inhibits tumor growth through the induction of T cell immunity and activation of natural killer cell cytotoxicity

We report here that gene transfer using recombinant adenoviruses encoding interleukin (IL)-18 mutants induces potent antitumor activity in vivo. The precursor form of IL-18 (ProIL-18) is processed by caspase-1 to produce bioactive IL-18, but its cleavage by caspase-3 (CPP32) produces an inactive form. To prepare IL-18 molecules with an effective antitumor activity, a murine IL-18 mutant with the signal sequence of murine granulocyte-macrophage (GM)- colony stimulating factor (CSF) at the 5′-end of mature IL-18 cDNA (GMmIL-18) and human IL-18 mutant with the prepro leader sequence of trypsin (PPT), which is not cleaved by caspase-3 (PPThIL-18CPP32−), respectively, were constructed. Adenovirus vectors carrying GMmIL-18 or PPThIL-18CPP32− produced bioactive IL-18. Ad.GMmIL-18 had a more potent antitumor effect than Ad.mProIL-18 encoding immature IL-18 in renal cell adenocarcinoma (Renca) tumor-bearing mice. Tumor-specific cytotoxic T lymphocytes, the induction of Th1 cytokines, and an augmented natural killer (NK) cell activity were detected in Renca tumor-bearing mice treated with Ad.GMmIL-18. An immunohistological analysis revealed that CD4+ and CD8+ T cells abundantly infiltrated into tumors of mice treated with Ad.GMmIL-18. Huh-7 human hepatoma tumor growth in nude mice with a defect of T cell function was significantly inhibited by Ad.PPThIL-18CPP32− compared with Ad.hProIL-18 encoding immature IL-18. Nude mice treated with Ad.PPThIL-18CPP32− contained NK cells with increased cytotoxicity. The results suggest that the release of mature IL-18 in tumors is required for achieving an antitumor effect including tumor-specific cellular immunity and augmented NK cell-mediated cytotoxicity. These optimally designed IL-18 mutants could be useful for improving the antitumor effectiveness of wild-type IL-18.

Adeno-associated virus-mediated gene transfer of a secreted form of TRAIL inhibits tumor growth and occurrence in an experimental tumor model

Tumor necrosis factor‐related apoptosis‐inducing ligand (TRAIL) induces cell death in various tumor cells, but relatively spares normal cells. Recombinant adeno‐associated virus (rAAV) vectors have a number of advantages including in vivo long‐term gene expression. Here, we assessed the biological activity of a novel, secreted form of TRAIL (sTRAIL) for cancer gene therapy using a rAAV2 vector.

Adenovirus-mediated interleukin-12 gene transfer combined with cytosine deaminase followed by 5-fluorocytosine treatment exerts potent antitumor activity in Renca tumor-bearing mice

BackgroundTherapeutic gene transfer affords a clinically feasible and safe approach to cancer treatment but a more effective modality is needed to improve clinical outcomes. Combined transfer of therapeutic genes with different modes of actions may be a means to this end. Interleukin-12 (IL-12), a heterodimeric immunoregulatory cytokine composed of covalently linked p35 and p40 subunits, has antitumor activity in animal models. The enzyme/prodrug strategy using cytosine deaminase (CD) and 5-fluorocytosine (5-FC) has been used for cancer gene therapy. We have evaluated the antitumor effect of combining IL-12 with CD gene transfer in mice bearing renal cell carcinoma (Renca) tumors.MethodsAdenoviral vectors were constructed encoding one or both subunits of murine IL-12 (Ad.p35, Ad.p40 and Ad.IL-12) or cytosine deaminase (Ad.CD). The functionality of the IL-12 or CD gene products expressed from these vectors was validated by splenic interferon (IFN)-γ production or viability assays in cultured cells. Ad.p35 plus Ad.p40, or Ad.IL-12, with or without Ad.CD, were administered (single-dose) intratumorally to Renca tumor-bearing mice. The animals injected with Ad.CD also received 5-FC intraperitoneally. The antitumor effects were then evaluated by measuring tumor regression, mean animal survival time, splenic natural killer (NK) cell activity and IFN-γ production.ResultsThe inhibition of tumor growth in mice treated with Ad.p35 plus Ad.p40 and Ad.CD, followed by injection of 5-FC, was significantly greater than that in mice treated with Ad.CD/5-FC, a mixture of Ad.p35 plus Ad.p40, or Ad.GFP (control). The combined gene transfer increased splenic NK cell activity and IFN-γ production by splenocytes. Ad.CD/5-FC treatment significantly increased the antitumor effect of Ad.IL-12 in terms of tumor growth inhibition and mean animal survival time.ConclusionThe results suggest that adenovirus-mediated IL-12 gene transfer combined with Ad.CD followed by 5-FC treatment may be useful for treating cancers.

951. Antitumor Activity of AAV-2 Vector Encoding Modified TRAIL Gene

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) induces apoptosis in a variety of cancer cell lines, but is relatively nontoxic to normal cells. Adeno-associated virus (AAV) vectors have a number of advantages including the potential sustained expression of transgenes. Here, we investigated potential usefulness of the TRAIL gene for systemic cancer therapy using AAV-2 vector. We constructed a plasmid encoding modified human TRAIL protein composed of signal peptide, an isoleucine zipper sequence, and active domain of TRAIL (pTR-PLF-ITRAD), which allows the TRAIL gene product to be released from the cells. TRAIL-sensitive cells were killed by transfection with pTR-PLF-ITRAD or a plasmid encoding full-length TRAIL (pTR-TRAIL), indicating that the plasmids produce bioactive TRAIL proteins. The culture media from TRAIL-resistant cells transfected with pTR-PLF-ITRAD, but not pTR-TRAIL induce apoptosis in a number of cancer cell lines, indicating that the modified TRAIL gene product, but not full-length TRAIL is released into the culture media. AAV-2 vector producing the modified TRAIL protein (AAV-2/PLF-ITRAD) inhibited tumor growth in A549 lung tumor-bearing nude mice. Our results suggest that AAV-2/PLF-ITRAD may be useful for systemic cancer gene therapy, provided that TRAIL is not toxic to normal cells.