Breast Cancer Research 2013, 15:R12

Anti-matrix metalloproteinase-9 DNAzyme decreases tumor growth in the MMTV-PyMT mouse model of breast cancer

Miranda A Hallett, Bin Teng, Hisashi Hasegawa, Luciana P Schwab, Tiffany N Seagroves and Tayebeh Pourmotabbed*

 

Introduction

Despite continued improvements in diagnosis, surgical techniques, and chemotherapy, breast cancer patients are still overcome by cancer metastasis. Tumor cell proliferation, invasion and metastasis are mediated, at least in part, through degradation of basement membrane by neutral matrix metalloproteinases (MMP) produced by tumor and stromal cells. Evidence suggests that MMP-9 plays a significant role in breast tumor cell invasion and metastasis. DNAzymes or catalytic oligonucleotides are new classes of gene targeting molecules that bind and cleave a specific mRNA, resulting in decreased protein expression.

Methods

The application of anti-MMP-9 DNAzyme (AM9D) for the treatment of primary and metastatic breast cancer was evaluated in vitro and in vivo using MDA-MB-231 cells and the MMTV-PyMT transgenic breast cancer mouse model. Spontaneously developed mammary tumors in MMTV-PyMT transgenic mice were treated intratumorally with naked AM9D, once a week for 4 weeks. The stability of DNAzyme was determined in vitro and in vivo using fluorescently labeled DNAzyme.

Results

AM9D specifically inhibited expression of MMP-9 in MDA-MB-231 cells resulting in reduced invasive property of these cells by 43%. Weekly intratumoral treatment of spontaneously developed mammary tumors in MMTV-PyMT transgenic mice was sufficient to significantly reduce the rate of tumor growth and final tumor load in a dose dependent and statistically significant manner (P < 0.05). 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 vascularized and contained more apoptotic cells compared to control and untreated tumors.

Conclusions

These results show that targeting and down regulation of MMP-9 by AM9D could prove useful as a therapy against breast carcinoma tumor growth and invasion.

PMID: 23407024

 

Supplemental:

A role for Matrix Metalloproteinases in cancer growth and metastasis has been known for over a decade. In fact, a broad-spectrum inhibitor targeting these proteolytic molecules were tested in clinical trails. The lack of complete knowledge of these over 23 protein family members was the cause of failure, due to vast side effects. One family member, MMP-9, is important in tumor growth and metastasis. In the Pourmotabbed laboratory we developed and tested a MMP-9 specific DNA enzyme (AM9D) capable of cleaving MMP-9 mRNA and thereby decreasing protein expression. In our recent publication we show that this enzyme is specific to MMP-9, since it is not capable of cleaving other family members. Further, AM9D is stable when injected, without a carrier, intratumorally. Mice developing spontaneous mammary tumors were treated with AM9D, control DNAzyme, or PBS alone. Treatment with AM9D decreased the size of tumors, decreased angiogenesis and increased apoptotic areas within the tumors compared to control treatments (FIG). The stability of AM9D in vivo via intratumor or intravenous (Hallett, et al. Nucleic Acid Ther. 2013 Dec;23(6):379-88) injection, carrier-free delivery, and the MMP-9 selective targeting make this a promising therapeutic with decreased immunogenic response.

 TP pic1Down regulation of MMP-9 by AM9D and its effect on angiogenesis and apoptosis

 

Acknowledgement:  This work was partially supported by USPHS GRANT CA-107183 (TP) and USPHS F31CA144572 (MH).

 

Contact:

Tayebeh Pourmotabbed, Ph.D.

Professor

Department of Microbiology, Immunology and Biochemistry

858 Madison Ave.

University of Tennessee Health Science Center

Memphis, TN 38163

tpourmotabbe@uthsc.edu

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