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

Abstract

IntroductionDespite 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.MethodsThe 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.ResultsAM9D 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.ConclusionsThese 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.

The Distribution, Clearance, and Safety of an Anti-MMP-9 DNAzyme in Normal and MMTV-PyMT Transgenic Mice

“…This is consistent with our previous observation showing that AM9D is stable for at least 72 hours in MDA-MB-231 cells and for 10 days post single intratumoral injection in mammary tumors of MMTV-PyMT transgenic mice (Hallett et al, 2013). These results are also consistent with Cai et al's published data demonstrating that DNAzyme targeting c-jun ( 33 P-DZ13) is stable for up to 120 hours in serum at 37°C, and 33 P labeled Dz13 was retained in serum for at least 24 hours after a single intravenous administration in Sprague-Dawley rats (Cai et al, 2012).…”

“…Increasing evidence suggests that MMPs contribute to the formation of a microenvironment that promotes tumor growth during early stages of tumorigenesis. Our previous observations that AM9D treatment decreased MMP-9 production and reduced invasive behavior of human MDA-MB-231 (Hallett et al, 2013) and glioma cells (Batson et al, unpublished data) suggest that AM9D has a great potential as an antitumorigenic/antimetastatic agent. Thus, to utilize AM9D as a therapeutic agent in breast cancer preclinical and clinical studies, healthy and MMTV-PyMT transgenic mice bearing early to late stage tumors were used to test the distribution of AM9D to different organs and mammary tumors.…”

“…DZ13 inhibition of tumor growth, however, was partially due to the induction of tumor immunity (Cai et al, 2012). We have recently shown that down regulation of matrix metalloproteinase-9 (MMP-9) production with anti-MMP-9 DNAzyme (AM9D) in vivo leads to the inhibition of tumor growth and angiogenesis and induction of apoptosis in both animal models of glioma (Batson et al unpublished data) and in a MMTV-polyoma virus middle T (PyMT) transgenic mouse model of breast cancer (Hallett et al, 2013). AM9D selectively cleaves MMP-9 mRNA without affecting the expression of other related MMP members (Hallett et al, 2013).…”

“…We have recently shown that down regulation of matrix metalloproteinase-9 (MMP-9) production with anti-MMP-9 DNAzyme (AM9D) in vivo leads to the inhibition of tumor growth and angiogenesis and induction of apoptosis in both animal models of glioma (Batson et al unpublished data) and in a MMTV-polyoma virus middle T (PyMT) transgenic mouse model of breast cancer (Hallett et al, 2013). AM9D selectively cleaves MMP-9 mRNA without affecting the expression of other related MMP members (Hallett et al, 2013). MMPs are a family of enzymes that play a crucial role in both normal connective tissue remodeling and in the accelerated matrix breakdown and release of chemokines, cytokines, and other proteins associated with tumor cell invasion and metastasis (Egeblad and Werb, 2002).…”

“…DNAzymes are a class of nucleic acid-based gene therapy which bind to their complementary sequence in a target mRNA, cleaving the mRNA molecule, and subsequently causing inhibition of gene expression. Contrary to other small oligonucleotide-based gene therapy (short hairpin RNA, small interfering RNA, oligodeoxynucleotide, and ribozyme) techniques, DNAzymes have the potential to be highly effective anticancer drugs due to their specificity, catalytic activity, high stability in vitro and in vivo (Cai et al, 2012;Hallett et al, 2013), lack of side effects, in vivo safety and tolerability, ease of synthesis, and low development costs (Schubert and Kurreck, 2004). Most importantly, delivery of DNAzyme in vivo can be accomplished without a need for lipid-based compound or retroviral vectors (Hallett et al, 2013) that have been shown to provoke immediate and late inflammatory responses and continue to express native viral proteins (Morral et al, 1997).…”