Date of Award

5-2011

Document Type

Dissertation

Degree Name

Doctor of Philosophy (PhD)

Program

Biomedical Sciences

Track

Cancer and Developmental Biology

Research Advisor

Tayebeh Pourmotabbed, Ph.D.

Committee

Yi Lu, Ph.D. Radhakrishna Rao, Ph.D. Tiffany N. Seagroves, Ph.D. Trevor W. Sweatman, Ph.D.

Abstract

BACKGROUND: Tumor cell proliferation, invasion and metastasis are known to be mediated, at least in part, through degradation of basement membrane by neutral metalloproteinases (MMP) produced by tumor and stromal cells. MMP-9 is known to play a significant role in breast tumor cell invasion and metastasis via degradation of ECM components and activation of cytokines and chemokines. MMP-9 is known to cleave type IV collagen, one of the most abundant collagens of the extracellular matrix. Our lab has found that the fully processed (68 kDa) form of MMP-9 generated in tumor and stromal cells is also capable of cleaving type I collagen molecule with similar characteristics of MMP‑1. This observation raises questions regarding the significance of each MMP in tumor cell invasion and metastases. Thus, in combating tumor growth and invasion, understanding the mechanism of substrate specificity and design of a novel therapeutic compound that is capable of specifically down regulating MMP-9 expression are of interest.

METHOD: DNAzymes are catalytic oligonucleotides that bind to and cleave specific mRNA, resulting in a decreased protein expression. The safety and efficacy of anti-MMP-9 DNAzyme (AM9D) in vivo was determined by injecting 35S‑labeled AM9D (35S-AM9D) into the tail vein of healthy and MMTV-PyMT transgenic mice and the amount of 35S-AM9D accumulated in different tissues was measured as a function of time. The effect of AM9D treatment on breast tumor progression was tested by four once‑weekly intratumoral injections of two concentrations of AM9D into mammary tumors of MMTV-PyMT transgenic mice at early stages of tumor development. The tumor sizes were measured at the end of the experiments. The effect of AM9D treatment on early stage experimental metastasis and survival was evaluated by injecting FVB female mice with 1x106 luciferase labeled mammary tumor epithelial cells (luc‑MTEC) isolated from MMTV‑PyMT mice and treating the animals with AM9D, control DNAzyme, or PBS for 3 weeks or up to 10 weeks, respectively.

The mechanism of substrate specificity of 68 kDa MMP‑9 was determined by generating a series of truncated and site directed mutant forms of MMP‑9. Specific amino acid residue in sequence 444PRPEPEPRPPTTT456 in the hinge region responsible for substrate selectivity of the enzyme was then identified by alanine‑scanning site directed mutagenesis. The charged amino acid residues (Arg445, Glu447, Glu449, and Arg451) were individually replaced by Ala and the proteolytic activity of the mutant enzymes toward Mca‑PLGL(Dpa)AR‑NH2 , gelatin, and type I collagen was determined.

RESULTS: Treatment of MDA‑MB‑231 breast cancer cell line with AM9D in vitro resulted in decreased invasion potential of the cells and intratumoral treatment of MMTV‑PyMT mice in vivo resulted in delayed rate of tumor growth and retarded final tumor volume by up to 51%. This decrease in tumor growth was correlated with decreased MMP-9 protein production within the treated tumor tissues. Tumors treated with AM9D were also less vascular compared to control and untreated tumors. Furthermore, DNAzyme administered IV is distributed to major organs including lung, without showing any organ toxicity. Intravenous administration of AM9D and control DNAzyme in animals bearing luc‑MTECs were able to decrease the number of gross lung macro metastasis. In addition, AM9D treatment increased progression‑free survival but did not have an effect on overall survival of animals inoculated with luc‑MTECs compared to control DNAzyme and PBS treatment. These data indicate that AM9D can be used individually or as an adjuvant to current chemotherapy for breast cancer.

To further illuminate the role of MMP-9 in tumor growth and metastases the mechanism of substrate specificity of the enzyme was studied. Site directed and deletion mutagenesis revealed that interaction of Glu415 in the active site with Glu447 and/or Arg451 in the hinge region makes the active site rigid, preventing full length MMP-9 from cleaving type I collagen. Disruption of this interaction, on the other hand, provides the flexibility necessary for the enzyme's active site to change conformation and be able to bind and cleave type I collagen substrate.

CONCLUSION: Given that MMP-9 is capable of cleaving type I collagen and its down regulation hinders the development of breast cancer in an animal model, AM9D could prove useful as an adjuvant therapy against breast carcinoma cell invasion.

DOI

10.21007/etd.cghs.2011.0124

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