Volume 10, Issue 6, Pages 1085-1095 (December 2004) Bystander activity of Ad-mda7: Human MDA-7 protein kills melanoma cells via an IL-20 receptor-dependent but STAT3-independent mechanism  Sunil Chada, Abner M. Mhashilkar, Rajagopal Ramesh, John B. Mumm, R. Bryan Sutton, Dora Bocangel, Mingzhong Zheng, Elizabeth A. Grimm, Suhendan Ekmekcioglu  Molecular Therapy  Volume 10, Issue 6, Pages 1085-1095 (December 2004) DOI: 10.1016/j.ymthe.2004.08.020 Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 1 Ad-mda7 transduction of melanoma cells induces death independent of the PKR pathway. (A) Melanoma cell lines (MeWo, WM35, and A375-S2) were untreated (−) or transduced with Ad-luc (L) or Ad-mda7 (M) for 48 h and the lysate or supernatant was immunoblotted using anti-MDA-7 antibody. Intracellular MDA-7 runs as a 30/23-kDa doublet, whereas secreted MDA-7 is approx 40 kDa. (B) Melanoma cells (MeWo, WM35, and A375-S2) and keratinocytes (HaCaT) were transduced with Ad-mda7 or Ad-luc (3000 vp/cell) and were analyzed for apoptosis 4 days later by annexin V staining. (C) To determine activation of the PKR pathway, melanoma lines were treated with medium (−), Ad-luc (L), or Ad-mda7 (M) as above and lysates analyzed by immunoblot using specific antisera to PKR and apoptosis pathway proteins. Molecular Therapy 2004 10, 1085-1095DOI: (10.1016/j.ymthe.2004.08.020) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 2 STAT3 activation by MDA-7. (A) HaCaT keratinocytes and MeWo melanoma and A549 lung tumor cells were treated with 20 ng/ml MDA-7 protein for 4 h, and pSTAT3 was analyzed using immunofluorescence microscopy. Note nuclear localization of pSTAT3 in HaCaT and MeWo cells. (B) HaCaT cells were treated with 20 ng/ml MDA-7 protein for the indicated times and samples analyzed by immunofluorescence for STAT3 activation. (C) Melanoma (MeWo) and HaCaT cells were treated with 20 ng/ml MDA-7 for the indicated times and the kinetics of pSTAT3 activation was analyzed. Data are plotted as means + SD. Molecular Therapy 2004 10, 1085-1095DOI: (10.1016/j.ymthe.2004.08.020) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 3 MDA-7 activates STAT3 via engagement of both type 1 and type 2 IL-20R. (A) HaCaT cells were treated with increasing doses of MDA-7 protein (M) or 100 ng/ml antibodies against MDA-7, IL-20R1, or IL-22R1 or 50 ng/ml MDA-7 plus antibodies at 100 ng/ml. MDA-7 antibodies blocked pSTAT3 activation by >90%, whereas anti-receptor antibodies partially blocked pSTAT3. Treatment with 500 ng/ml STAT3 inhibitor blocked pSTAT3 activation by >90%. (B) Melanoma (MeWo) cells were treated with increasing doses of MDA-7 protein (M) or 50 ng/ml MDA-7 plus indicated antibodies (at 100 ng/ml) or STAT3 inhibitor. Anti-MDA7 blocks STAT3 activation by >85%; anti-IL-20R1 and anti-IL-22R1 partially block STAT3, whereas STAT3 inhibitor blocks STAT3 activation by >90%. Data are plotted as means + SD. Molecular Therapy 2004 10, 1085-1095DOI: (10.1016/j.ymthe.2004.08.020) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 4 Ligand-, dose-, and receptor-dependent analysis of MDA7-induced cell death in melanoma cells. MeWo (A, C) and WM35 (B) melanoma cells were treated with increasing doses of MDA-7 protein for 4 days (A and B) or 5 days (C) and cytotoxicity was assessed. Cells were also treated with antibodies (100 ng/ml) to MDA-7 (Pab, polyclonal antibody; Mab, monoclonal antibody), control antibody (IgG), IL-20R1 antibody, or IL-22R1 antibody or treated with STAT3 inhibitor. MDA-7 protein (M, 20 ng/ml) was added to antibodies or inhibitors and cell killing assessed. MDA-7 kills melanoma cells via ligand binding to type 1 IL-20R. Blocking IL-22R1 or STAT3 does not inhibit killing. Data are plotted as means + SD. *P < 0.001 compared to control; #P > 0.2. Molecular Therapy 2004 10, 1085-1095DOI: (10.1016/j.ymthe.2004.08.020) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 5 MDA-7/IL-24 is the only IL-10 family member to kill melanoma cells. (A) IL-19, IL-20, IL-22, and IL-24 (MDA-7) activate STAT3 in melanoma cells. Cells were treated with 50 ng/ml cytokines and pSTAT3 activation was assessed. Cells were also treated with antibodies against MDA-7, control IgG, and antibodies against IL-20R1 and IL-22R1 in the presence of cytokines and STAT3 activation was quantitated. *Not done. (B) MDA-7/IL-24 is the only IL-10 family member that kills melanoma cells directly. Cells were treated with the indicated doses of cytokines (50 or 100 ng/ml) or bacterially produced MDA-7 (bMDA-7) for 4 days and cytotoxicity was assessed. Data are shown as means + SD. Molecular Therapy 2004 10, 1085-1095DOI: (10.1016/j.ymthe.2004.08.020) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 6 MDA-7 induces apoptosis and cell cycle arrest in melanoma cells. (A) MeWo melanoma cells were treated with MDA-7 at indicated doses or treated with MDA-7 (60 ng/ml) in the presence of antibodies against MDA-7 (Pab, polyclonal; Mab, monoclonal) or control IgG (100 ng/ml) and apoptosis was assessed using annexin V assay. Parallel cultures were treated with Ad-luc or Ad-mda7 at 1000 vp/cell. (B) Western analysis of lysates from MeWo cells treated with increasing doses of MDA-7 for 48 h and immunoblotted for BAX and p21. (C) MeWo cells were treated with MDA-7 protein (0-20 ng/ml) or Ad-mda7 or Ad-luc (2000 vp/cell) and cell cycle analysis was performed. MDA-7 protein causes G1 block in melanoma cells, whereas Ad-mda7 induces G2/M block. Molecular Therapy 2004 10, 1085-1095DOI: (10.1016/j.ymthe.2004.08.020) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions

FIG. 7 MDA-7 killing of tumor cells correlates with receptor expression. (A) RNA was isolated from the indicated cell lines and subjected to RT-PCR with primers specific for (lane 1) GAPDH, (lane 2, top) IL-20R1, (lane 3, top) IL-20R2, and (lane 2, bottom) IL-22R1. Amplified PCR products were visualized by agarose gel electrophoresis. Only melanoma and HaCaT cells express receptors for MDA-7. (B) MDA-7 kills receptor-positive tumor cells. Trypan blue cytotoxicity assay was performed on MeWo (receptor positive–tumor), A549 NSCLC (receptor negative–tumor), and HaCaT cells (receptor positive–normal). Cells were treated with increasing doses of MDA-7 protein and cell death was quantitated. Data are shown as means + SD. Molecular Therapy 2004 10, 1085-1095DOI: (10.1016/j.ymthe.2004.08.020) Copyright © 2004 The American Society of Gene Therapy Terms and Conditions