Targeting Loss of Tumor Suppressor Function and Oncogene Overexpression

tumor suppressor genes, including Rb, and APC, have been identified by their association with hereditary cancers. Many sporadic tumors harbor inactivating or recessive mutations in one or more tumor suppressor genes. Gene transfer techniques can b plied to introduce wild-type copies of tumor suppressor genes into malignant cells, thus potentially reversing the neoplastic pheno-type. The p53 tumor suppressor gene has been of interest because p53 mutation occurs commonly in a variety of human cancers, including breast, lung, colon, prostate, bladder, and cervix. The use of adenoviral vectors to deliver the p53 transgene to human tumors is now under evaluation in several clinical trials (145).The overexpression of Fas ligand caused by adenovirus-mediated wild-type p53 gene transfer induces neutrophil infiltration into human colorectal tumors, which may play a critical role in the bystander effect of p53 gene therapy (146).

Besides the p53 gene, other tumor suppressor genes that regulate the cell cycle have been used in cancer gene therapy. Among them are Rb, BRCA1, PTEN, pl6, E2F, and fragile his-tidine triad (FHIT) genes. Clinical trials with BRCA1 and Rb have been initiated (147).

Protooncogenes, in contrast to tumor suppressor genes, gain dominant mutation resulting in excessive expression of their protein products, which lead to development of the malignant phenotype. Three members of the Ras family of oncogenes (H-ras, K-ras, and N-ras) are among the most commonly activated oncogenes in human cancers. Several strategies have been designed to combat K-ras mutations, including antisense nucleotide, ri-bozyrnes (148-150), and intracellular single-chain antibodies (151). cDNA encoding antisense RNA can be delivered using the viral vector system approach. In vivo gene therapy with K-ras, c-fos, and c-myc antisense nucleo-tides is currently being applied in clinical trials.

5.6.4 Angiogenesis Control. Gene therapy offers a new strategy for the delivery of angiogenesis inhibitors. By engineering and delivering vectors that carry the coding sequence for an antiangiogenic protein, it is possible to produce high levels of antiangiogenic factors in the tumor location or to systemically prevent the growth of distant metastasis. Several inhibitors, such as angiostatin (152), endostatin (153), plasminogen activator inhibitor type 1 (154), and truncated VEGF receptor (155), have been tested using this ap proach. These studies have demonstrated that retroviral and adenoviral vectors could be used to inhibit endothelial cell growth in vitro and angiogenesis in vivo. The inhibition of tumor-associated angiogenesis results in increased apoptotic tumor cell death, leading to inhibition of tumor growth.

5.6.5 Matrix Metalloproteinase. As mentioned earlier in the chapter, MMPs are capable of proteolytic degradation of stromal ECM, which is essential in cancer cell migration and invasion, as well as in tumor-induced angio-genesis. The activity of MMPs in vivo is inhibited by TIMPs, small secreted proteins with molecular weight of between 20 and 30 kDa. TIMPs inhibit MMPs by binding to both the latent and active forms of MMPs. The following properties of TIMPs such as secretion, diffusion (TIMP-1, -2 and -4), induction of apo-ptosis (TIMP-3), and inhibition of multiple MMPs make them very attractive tools for gene therapy application.

Inhibition of cancer cell invasion after overexpression of TIMPs using different gene delivery vectors has been shown in vitro in gastric cancer cells and mammary carcinoma cells (156,157). Overexpression in vitro of TIMP-2, which was delivered by a recombinant ade;io-virus (AdTIMP-2), inhibited the invasion of both tumor and endothelial cells in models without affecting cell proliferation (158). Its in vivo efficiency has been evaluated in the LLC murine lung cancer model, the colon cancer C51 model, as well as in MDA-human breast cancer in athymic mice. Preinfection of tumor cells by AdTIMP-2 resulted in an inhibition of tumor establishment in more than 50% of mice in LLC and C51 models and in 100% of mice in the MDA-MB231 model. A single local injection of AdTIMP-2 into preestablished tumors of these three tumor types reduced tumor growth rates by 60-80%, and the tumor-associated angiogenesis index by 25-75%. Lung metastasis of LLC tumors was inhibited by >90%. In addition, AdTIMP-2-treated mice showed a significantly prolonged survival in all the cancer models tested. These data demonstrate the potential of adenovirus-mediated TIMP-2 therapy in cancer treatment.

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