Ryo Miyahara

Melanoma differentiation-associated gene-7 (mda-7 )/interleukin (IL)-24 induces anticancer immunity in a syngeneic murine model

Previous studies have shown that the human melanoma differentiation-associated gene-7 (mda-7)/interleukin-24 (IL-24) has tumor-suppressor activity in vitro and in vivo. Additionally, in vitro studies using human peripheral blood mononuclear cells indicate that mda-7/IL-24 has TH1 cytokine-like activity. However, the individual properties of mda-7/IL-24 have been previously examined separately. Thus, there is not a single study that has examined both, antitumor and proimmune properties of mda-7/IL-24. Furthermore, the tumor suppressive activity and the cytokine activity of mda-7/IL-24 have not been previously tested in an immunocompetent setting. We therefore in the present study evaluated the antitumor and immune properties of mda-7/IL-24 in a murine syngeneic tumor model. In vitro, adenovirus-mediated mda-7 gene (Ad-mda7) transfer to murine fibrosarcoma (UV2237m; MCA16) and normal (10T1/2) cells significantly inhibited growth (P=0.001) and induced apoptosis in tumor cells but not in normal cells. In vivo, intratumoral administration of Ad-mda7 resulted in significant inhibition of tumor growth (P<0.05), with a subset of mice showing complete tumor regression. We next evaluated the immune potentiation activity of Ad-mda7 in a cancer vaccine model. UV2237m cells transfected with Ad-mda7 and injected into syngeneic immunocompetent C3H mice were unable to grow; however, they did grow in immunocompromised nude mice. These tumor-free C3H mice, when challenged with parental tumor cells experienced no tumor growth, suggesting induction of systemic immunity. Moreover, splenocytes prepared from vaccinated C3H mice demonstrated higher proliferative activity and produced elevated levels of TH1 cytokines compared with those from control mice. An in vitro subset analysis of splenocytes from vaccinated mice demonstrated a significant increase in the CD3+CD8+ but not the CD3+CD4+ cell population (P=0.019). Thus Ad-mda7 treatment of syngeneic tumors induces tumor cell death and promotes immune activation, leading to anticancer immunity.

Selective induction of cell cycle arrest and apoptosis in human prostate cancer cells through adenoviral transfer of the melanoma differentiation-associated -7 (mda-7)/interleukin-24 (IL-24) gene.

We have previously reported that overexpression of the melanoma differentiation-associated gene -7 (mda-7) using a replication-defective adenovirus (Ad-mda7), results in tumor-specific growth suppression and induction of apoptosis in wide variety of cancer cells. In the present study, we investigated the antitumor activity of Ad-mda7 and the underlying mechanism in human prostate cancer cells and normal prostate epithelial cells. Overexpression of MDA-7 induced significant (P=.001) suppression of cell growth and apoptosis in prostate cancer cells (DU 145, LNCaP, and PC-3). In normal prostate epithelial cells (PrEC) some degree of growth inhibition but not apoptosis was observed. However, the inhibitory effects in normal cells were less compared to tumor cells. Growth inhibitory effects were mediated by the intracellular and not by extracellular MDA-7 protein. Molecular effectors that are involved in Ad-mda7-mediated tumor killing included activation of the caspase cascade, and the induction of G2 phase cell cycle arrest through the inhibition of Cdc25C pathway. These results demonstrate the mechanisms by which Ad-mda7 exerts its antitumor activity in human prostate cancer cells. The antitumor activity combined with previously reported antiangiogenic and proimmune properties of Ad-mda7 can serve as a potential therapeutic agent for treatment of primary and disseminated prostate cancer.We have previously reported that overexpression of the melanoma differentiation-associated gene -7 (mda-7) using a replication-defective adenovirus (Ad-mda7), results in tumor-specific growth suppression and induction of apoptosis in wide variety of cancer cells. In the present study, we investigated the antitumor activity of Ad-mda7 and the underlying mechanism in human prostate cancer cells and normal prostate epithelial cells. Overexpression of MDA-7 induced significant (P=.001) suppression of cell growth and apoptosis in prostate cancer cells (DU 145, LNCaP, and PC-3). In normal prostate epithelial cells (PrEC) some degree of growth inhibition but not apoptosis was observed. However, the inhibitory effects in normal cells were less compared to tumor cells. Growth inhibitory effects were mediated by the intracellular and not by extracellular MDA-7 protein. Molecular effectors that are involved in Ad-mda7-mediated tumor killing included activation of the caspase cascade, and the induction of G2 phase cell cycle arrest through the inhibition of Cdc25C pathway. These results demonstrate the mechanisms by which Ad-mda7 exerts its antitumor activity in human prostate cancer cells. The antitumor activity combined with previously reported antiangiogenic and proimmune properties of Ad-mda7 can serve as a potential therapeutic agent for treatment of primary and disseminated prostate cancer.

Inhibition of nuclear factor-κB augments antitumor activity of adenovirus-mediated melanoma differentiation-associated gene-7 against lung cancer cells via mitogen-activated protein kinase kinase kinase 1 activation

Nuclear factor-κB (NF-κB) activation promotes cell survival and growth. Reports show that chemotherapeutic agents and cytokines that are used for cancer therapy activate NF-κB expression in tumor cells and its suppression enhanced the antitumor activity. We hypothesized that adenovirus-mediated overexpression of melanoma differentiation-associated gene-7/interleukin-24 (Ad-mda7/IL-24) induces NF-κB expression and that inhibition of this expression results in enhanced tumor cell killing. Treatment of human lung tumor (H1299 and A549) cells with Ad-mda7 resulted in NF-κB activation in a dose- and time-dependent manner before activation of cell death pathways. To establish that inhibition of Ad-mda7–mediated NF-κB activation results in enhanced tumor cell killing, H1299 cells that overexpress the dominant-negative IκBα (dnIκBα) were treated with Ad-mda7 in vitro. An enhanced growth arrest and apoptosis was observed in Ad-mda7–treated H1299-dnIκBα compared with H1299-Neo cells. This Ad-mda7–mediated enhanced killing of H1299-dnIκBα cells involved cleavage of mitogen-activated protein kinase kinase kinase 1 (MEKK1) and caspase-3 in a feedback loop mechanism. The inhibition of MEKK1 or caspase-3 cleavage in H1299-dnIκBα cells resulted in reduced Ad-mda7–mediated cell killing. In vivo, the treatment of H1299-dnIκBα s.c. tumors with Ad-mda7 resulted in increased drug sensitivity and delayed the tumor growth rate compared with Ad-mda7–treated H1299-Neo tumors. Molecular analysis of Ad-mda7–treated H1299-dnIκBα tumors showed increased MEKK1 cleavage and activation of caspase-3 compared with Ad-mda7–treated H1299-Neo tumors. Our findings thus showed that the NF-κB activation induced by Ad-mda7 treatment of lung cancer cells is an intrinsic survival mechanism and that the inhibition of this NF-κB expression results in enhanced tumor cell killing. [Mol Cancer Ther 2007;6(4):1440–9]

MDA-7/IL-24 as a Multi-Modality Therapy for Cancer

The protein encoded by the melanoma differentiation-association gene 7 (MDA-7/IL-24) is a novel interleukin (IL)-10 family cytokine with unique tumor-specific apoptotic and antiangiogenic properties that make it especially attractive for use in cancer gene therapy applications. Mda-7 gene transfer with a replication incompetent adenoviral vector (Ad-mda7) induces apoptosis in a tumor specific manner, an effect that is independent of the status of other tumor suppressor genes, such as p53, Rb, or pl6INK4. In addition to its direct cytotoxic effects, Ad-mda7 transduction causes secretion of a processed, glycosylated form of MDA-7 protein. MDA-7 is a novel interleukin (IL-24) with unique apoptotic functions. Studies on the secreted MDA-7/IL-24 protein have shown that it can act as a pro-Thl cytokine, and induces secretion of interferon-gamma, tumor necrosis factor-α, IL-6, IL-12, and granulocyte macrophage colony-stimulating factor in human peripheral blood mononuclear cells. Additional studies in syngeneic mice indicate that MDA-7 can function as an immune adjuvant and enhance immune reactivity against tumors. More recently, our group demonstrated that MDA-7 protein functions as a potent antiangiogenic factor in vitro and in vivo that is 50-fold more active than angiostatin or endostatin. In phase I clinical trials intratumoral delivery of Ad-mda7 showed tumor regression in patients with advanced carcinomas who failed conventional therapies. This chapter provides a comprehensive perspective on MDA-7/IL-24 research, highlighting its proapoptotic, antimetastatic, and antiangiogenic properties. The combination of these potent effector mechanisms makes mda-7/IL-24 a promising and novel approach for the treatment of cancer.

500. Local and systemic inhibition of lung tumor growth after liposome mediated mda-7/IL-24 gene delivery

The melanoma differentiation associated gene-7 (mda-7), also known as interleukin-24 (IL-24), is a novel gene with tumor suppressor, anti-angiogenic and cytokine properties. In vitro adenovirus-mediated gene transfer of the mda-7/IL-24 gene (Ad-mda7) results in ubiquitous growth suppression of human cancer cells with minimal toxicity to normal cells. Intratumoral administration of Ad-mda7 to lung tumor xenografts results in growth suppression via induction of apoptosis and anti-angiogenic mechanisms. Although these results are encouraging, one limitation of this approach is its locoregional clinical application-systemic delivery of adenoviruses for treatment of disseminated cancer is not feasible at the present time. An alternative approach that is suitable for systemic application is liposomal gene delivery. We recently demonstrated that DOTAP:cholesterol (DOTAP:Chol) liposomes effectively deliver tumor suppressor genes to primary and disseminated lung tumors. In the present study, therefore, we evaluated liposome-mediated delivery of the mda-7/IL-24 gene to primary and disseminated lung tumors in vivo. We demonstrate that DOTAP:Chol efficiently delivers the mda-7/IL-24 gene to human lung tumor xenografts resulting in suppression of tumor growth. Growth-inhibitory effects were observed in both primary (P = 0.001) and metastatic lung tumors (P = 0.02). Furthermore, tumor vascularization was reduced in mda-7/IL-24-treated tumors. Finally, growth was also inhibited in murine syngenic tumors treated with DOTAP:Chol-mda-7 complex (P = 0.01). This is the first report demonstrating (1) systemic therapeutic effects of mda-7/IL-24 in lung cancer and (2) anti-tumor effects of mda-7 in syngeneic cancer models. Our findings are important for the development of mda-7/IL-24 treatments for primary and disseminated cancers.