ABSTRACT
Metastatic involvement of the skeleton is a frequent consequence of advanced prostate cancer. These skeletal metastases cause a number of debilitating complications and are refractory to current treatments. New therapeutic options are being explored, including conditionally replicating adenoviruses (CRAds). CRAds are engineered to selectively replicate in and destroy tumor cells and can be 'armed' with exogenous transgenes for enhanced potency. We hypothesized that a CRAd armed with osteoprotegerin (OPG), an inhibitor of osteoclastogenesis, would inhibit the progression of prostate cancer bone metastases by directly lysing tumor cells and by reducing osteoclast activity. Although prostate cancer bone metastases are predominantly osteoblastic in nature, increased osteoclast activity is critical for the growth of these lesions. Ad5-Δ24-sOPG-Fc-RGD is a CRAd that carries a fusion of the ligand-binding domains of OPG and the Fc region of human IgG1 in place of the viral E3B genes. To circumvent low tumor cell expression of the native adenoviral receptor, an arginine-glycine-aspartic acid (RGD) peptide insertion within the viral fiber knob allows infection of cells expressing α(v) integrins. A 24-base pair deletion (Δ24) within viral E1A limits replication to cells with aberrant retinoblastoma cell cycle regulator/tumor suppressor expression. We have confirmed that Ad5-Δ24-sOPG-Fc-RGD replicates within and destroys prostate cancer cells and, in both murine and human coculture models, that infection of prostate cancer cells inhibits osteoclastogenesis in vitro. In a murine model, progression of advanced prostate cancer bone metastases was inhibited by treatment with Ad5-Δ24-sOPG-Fc-RGD but not by an unarmed control CRAd.
Subject(s)
Bone Neoplasms/secondary , Bone Neoplasms/therapy , Oncolytic Virotherapy/methods , Osteoprotegerin/pharmacology , Prostatic Neoplasms/pathology , Adenoviridae/genetics , Analysis of Variance , Animals , Cell Line, Tumor , Humans , Immunoglobulin G/genetics , Luciferases , Male , Mice , Oligopeptides/genetics , Oligopeptides/metabolism , Osteoclasts/drug effects , Osteoprotegerin/genetics , Osteoprotegerin/metabolism , Real-Time Polymerase Chain Reaction , X-Ray MicrotomographyABSTRACT
Ovarian cancer remains difficult to treat mainly due to presentation of the disease at an advanced stage. Conditionally-replicating adenoviruses (CRAds) are promising anti-cancer agents that selectively kill the tumor cells. The present study evaluated the efficacy of a novel CRAd (Ad5/3-CXCR4-TIMP2) containing the CXCR4 promoter for selective viral replication in cancer cells together with TIMP2 as a therapeutic transgene, targeting the matrix metalloproteases (MMPs) in a murine orthotopic model of disseminated ovarian cancer. An orthotopic model of ovarian cancer was established in athymic nude mice by intraperitonal injection of the human ovarian cancer cell line, SKOV3-Luc, expressing luciferase. Upon confirmation of peritoneal dissemination of the cells by non-invasive imaging, mice were randomly divided into four treatment groups: PBS, Ad-ΔE1-TIMP2, Ad5/3-CXCR4, and Ad5/3-CXCR4-TIMP2. All mice were imaged weekly to monitor tumor growth and were sacrificed upon reaching any of the predefined endpoints, including high tumor burden and significant weight loss along with clinical evidence of pain and distress. Survival analysis was performed using the Log-rank test. The median survival for the PBS cohort was 33 days; for Ad-ΔE1-TIMP2, 39 days; for Ad5/3-CXCR4, 52.5 days; and for Ad5/3-CXCR4-TIMP2, 63 days. The TIMP2-armed CRAd delayed tumor growth and significantly increased survival when compared to the unarmed CRAd. This therapeutic effect was confirmed to be mediated through inhibition of MMP9. Results of the in vivo study support the translational potential of Ad5/3-CXCR4-TIMP2 for treatment of human patients with advanced ovarian cancer.
Subject(s)
Adenoviridae/physiology , Ovarian Neoplasms/pathology , Tissue Inhibitor of Metalloproteinase-2/metabolism , Virus Replication/physiology , Animals , Cell Line, Tumor , Cell Proliferation , Disease Models, Animal , Female , Humans , Imaging, Three-Dimensional , Luminescent Measurements , Matrix Metalloproteinase 9/metabolism , Mice , Neovascularization, Pathologic/pathology , Ovarian Neoplasms/blood supply , Ovarian Neoplasms/enzymology , Platelet Endothelial Cell Adhesion Molecule-1/metabolism , Survival AnalysisABSTRACT
PURPOSE: Current treatments for ovarian cancer have limited therapeutic outcomes due to advanced stage of the disease at diagnosis. Among new therapies, conditionally replicating adenoviruses (CRAds), designed to selectively lyse cancer cells, hold promise. In clinical trials, CRAds exhibited limited efficacy thus far. Second-generation CRAds are being developed to express a therapeutic protein to enhance antitumor efficacy. One attractive target in the tumor microenvironment is the matrix metalloproteinases (MMPs) that degrade the extracellular matrix, and are upregulated in ovarian cancer. Tissue inhibitor of metalloproteinase 2 (TIMP2) is an endogenous inhibitor of MMPs. The present study developed and evaluated a novel CRAd (Ad5/3-CXCR4-TIMP2) for ovarian cancer therapy. EXPERIMENTAL DESIGN: A targeted CRAd, Ad5/3-CXCR4-TIMP2 was developed using the CXCR4 promoter for enhanced replication, and expressing the TIMP2 transgene. The efficacy of this armed CRAd was determined in both established human ovarian cancer cell lines and in primary ovarian tumor samples. RESULTS: Ad5/3-CXCR4-TIMP2 mediated expression of functional TIMP2, as demonstrated by the inhibition of MMP activity. In addition, arming with TIMP2 did not inhibit viral replication or oncolytic potency, as the TIMP2-armed viruses showed enhanced killing of cancer cells when compared to the unarmed viruses. We also examined viral replication in primary ovarian cancer tissues obtained from patients with stage III and IV ovarian cancer. In four of the five tumor samples, Ad5/3-CXCR4-TIMP2 revealed a 21- to 89-fold increase in replication when compared to the Ad5/3 virus. CONCLUSION: Results support the translational potential of Ad5/3-CXCR4-TIMP2 for treatment of patients with advanced ovarian cancer.
Subject(s)
Adenoviridae/genetics , DNA, Viral/metabolism , Oncolytic Virotherapy/methods , Tissue Inhibitor of Metalloproteinase-2/metabolism , Cell Line, Tumor , Female , Humans , Ovarian Neoplasms/therapy , Receptors, CXCR4/metabolism , Tissue Inhibitor of Metalloproteinase-2/genetics , Virus ReplicationABSTRACT
There are currently no effective therapies for metastatic melanoma and targeted immunotherapy results in the remission of only a very small percentage of tumors. In this study, we show that the noncanonical Wnt ligand, Wnt5A, can increase melanoma metastasis in vivo while down-regulating the expression of tumor-associated antigens important in eliciting CTL responses (e.g., MART-1, GP100, and tyrosinase). Melanosomal antigen expression is governed by MITF, PAX3, and SOX10 and is inhibited upon signal transducers and activators of transcription 3 (STAT3) activation, via decreases in PAX3 and subsequently MITF expression. Increasing Wnt5A in Wnt5A-low cells activated STAT3, and STAT3 was decreased upon Wnt5A knockdown. Downstream targets such as PAX3, MITF, and MART-1 were also affected by Wnt5A treatment or knockdown. Staining of a melanoma tissue array also highlighted the inverse relationship between MART-1 and Wnt5A expression. PKC activation by phorbol ester mimicked Wnt5A effects, and Wnt5A treatment in the presence of STAT3 or PKC inhibitors did not lower MART-1 levels. CTL activation studies showed that increases in Wnt5A correspond to decreased CTL activation and vice versa, suggesting that targeting Wnt5A before immunotherapy may lead to the enhancement of current targeted immunotherapy for patients with metastatic melanoma.