About MeTumor-targeted cancer gene therapy, viral oncolysis, and mechanisms of viral transformation.
Although much progress has been made toward the prevention and therapy of cancer, more Canadians die each year from cancer or cancer-related causes than from any other disease. This has prompted investigators to examine new anti-cancer
agents and new approaches to optimize administration
of these agents. Many of these agents can have significant side effects when active in normal tissues.
One area of research in my laboratory focuses on the development of gene therapy vectors that minimize these side effects without compromising anticancer activity. Currently
we are investigating adenovirus vectors carrying modified capsid proteins and/or tissue-specific regulatory elements to target expression of cytotoxic genes specifically to the tumor
. As an alternative to transfer of therapeutic genes to tumor
cells, we are investigating the ability of replicating
viruses (both adenovirus and poxviruses) to specifically eliminate tumor
cells in vitro and in tumor
models. A third
line of study involves the investigation of mechanisms of viral transformation as a model for early stages of tumorigenesis.
My laboratory primarily uses the adenovirus (Ad) vector system to transduce cancer cells with potentially therapeutic genes. The advantages of Ad are its high efficiency of transduction, ease of propagation and purification, stability, and the wide range of cell types, both proliferating and quiescent, that can be infected by the virus. Unfortunately, many tumor
cells display reduced levels of the receptors required for Ad infection, resulting in greater gene transfer to normal cells than tumor
cells under normal conditions. This problem can be circumvented by modifying the virus capsid so that it recognizes alternate receptors on the tumor
cell surface. In collaboration with Dr.
Frank Graham at McMaster University, we have recently generated a vector that binds to ErbB3 and ErbB4 receptors that are frequently over-expressed on tumor
cells. This vector modification enhances in vitro transduction of cancer cells but not normal cells (MacLeod et al., 2012). Whether this modification results in reduced toxicity to normal tissues in vivo is currently under investigation in my lab. Transcriptional targeting.
Another approach for reducing toxicity to normal cells is to target expression of the therapeutic gene using tissue-specific or tumor-specific promoters. In particular, we have focused on regulatory sequences derived from the mammaglobin gene which is expressed at high levels in mammary carcinoma cells, to a lesser extent in normal mammary cells, and is undetectably
expressed in nearly all other normal cells (Shi et al. 2005, 2006). We have recently isolated a 340 bp minimal promoter sequence immediately 5' to the mammaglobin open reading frame that appears to be responsible for the high degree of specificity of expression. In addition, we have identified an upstream enhancer element that boosts expression levels by at least 10-fold in breast cancer cells. Therapeutic genes.
The utility of Ad vectors for the transfer of anti-cancer genes directly to tumors
has been investigated by numerous labs over the past 20 years. Some of these vectors, including those encoding the cytokines IL-2, IL-12 and the costimulatory factor B7-1 (CD80) developed by myself and colleagues at McMaster University and University of Toronto, have been used in clinical trials for cancer therapy. Results of both pre-clinical and clinical trials demonstrate the potential of Ad vectors, but also indicate that the genes being transferred are not potent enough in tumor
cell killing. Extremely potent death-inducing vectors can be difficult to rescue and amplify due
premature killing of 'packaging' cell lines. We are currently developing systems that will attenuate expression of toxic
genes in the cell lines used to rescue and propagate vectors, as well as in normal cells. One way to modulate expression exploits the RNA interference pathway. We are investigating whether insertion of sequences targeted by naturally-occurring or introduced microRNAs can facilitate silencing of virus-encoded targeted genes. We hope to use these new systems to produce vectors encoding pro-apoptotic proteins for tumor-targeted cancer gene therapy. Viral oncolysis.
Viral oncolysis is the selective killing of tumor
cells by replicating viruses. One mechanism for such selective killing is to exploit cellular processes that are altered in both infected cells and in tumor
cells. Such processes include evasion of innate host immune responses (such as the interferon response), stimulation of DNA synthesis and cell cycling, activation of signaling
pathways, and inactivation of tumor
suppressors such as p53 and pRb
. Viruses can be designed or selected that are unable to replicate in normal cells due to host control of these pathways, yet can replicate efficiently in tumor
cells which have lost regulation of these pathways. This category of oncolytic viruses includes adenoviruses from which the E1B and/or VA-RNA genes have been deleted. One E1B protein, E1B-55K binds p53 and sequesters it into an aggresome to prevent its activity. Another E1B product is involved in blocking apoptosis. VA RNAs are known to play an important role in counteracting the interferon response in normal cells, a function which is redundant in many tumor
cells. Characterization of VA-RNA deleted viruses is underway (Sharon et al., 2013).
In addition, in collaboration with Dr.
David Evans (Medical Microbiology and Immunology)
we are investigating poxviruses (myxoma virus, Shope fibroma virus and vaccinia) for their oncolytic potential. Leporipoxviruses exhibit a very narrow host range limited to rabbits and hares. However
the host range can be expanded to encompass transformed human cells as a result of the constitutive activation of the protein kinase B/Akt signaling
pathways in some tumor
cells. This forms the basis for using myxoma virus as an oncolytic agent. We are currently testing the efficacy of these viruses in vitro and in mouse tumor
models (Irwin et al., in press). Recent experiments suggest that vaccinia virus can be modified to improve its safety as an anticancer agent. This modified vaccinia is under investigation in breast cancer and bladder cancer (Potts et al., 2012) tumor
models to test its activity and specificity for tumor
cells. Viral transformation.
Many of the cellular processes that are subverted at early stages of virus infection are also subverted at early stages of tumorigenesis. In collaboration with Dr.
Andy Shaw (Experimental Oncology), we are investigating a novel cellular protein that may play an important role in intracellular trafficking of regulatory molecules during adenoviral replication and transformation.
bladder cancer, breast cancer, cancer, epithelial-mesenchymal transition, immune checkpoint inhibitors, small molecule drugs, invasion and metastasis, nitrofen, oncolytic adenovirus, oncolytic virus, reovirus, oncolytic virus, mouse tumor models, vaccinia, vaccinia, cancer, mouse models