Treatment of breast cancer tumor growth and metastasis with an anti-MMP-9 DNAzyme

描述（由申请人提供）：尽管在诊断、手术技术和化疗方面不断改进，但乳腺癌患者仍被癌症转移所克服。乳腺癌转移到脑、肺、骨、肝和局部淋巴结。早期发现和研究的进步提高了生存率和生活质量。然而，仍然需要改进。已知肿瘤细胞增殖、侵袭和转移至少部分地通过肿瘤和基质细胞产生的中性金属蛋白酶（MMP）降解基底膜来介导。有证据表明，MMP-9在乳腺肿瘤细胞的侵袭和转移中起重要作用。我们已经表明，针对MMP-9 mRNA产生的DNAzyme能够在体外降低该酶的表达，并且其瘤内注射使在MMTV-PyMT转基因小鼠模型中产生的乳腺肿瘤的大小减小50%。DNA酶是催化性DNA分子，其特异性结合并切割靶蛋白的mRNA，导致蛋白质表达降低。鉴于其全身给药和靶向的潜力，抗MMP-9 DNA酶（AM 9D）可能被证明是有用的治疗乳腺癌的侵袭。我们推测，AM 9D下调MMP-9的表达，将导致乳腺癌动物模型中肿瘤生长的抑制，肺转移的减少，实验转移的阻断和存活率的增加。为了实现这一目标，我们提出（1）确定在PyMT MTEC移植小鼠模型中显著降低肿瘤负荷而不引起显著局部毒性所需的AM 9D的最小有效剂量。将通过组织切片的组织病理学分析确定毒性。(2)评估AM 9D对继发性肿瘤发展的影响。将表达荧光素酶的PyMT MTEC移植到雌性小鼠的乳腺中，并将肿瘤用最佳浓度的AM 9D、对照DNAzyme或PBS处理4周。然后切除肿瘤，并将动物再饲养8周以形成肺继发性肿瘤。此外，将通过将PyMT MTEC与或不与AM 9D或对照DNazyme注射到FVB/Nj小鼠的尾静脉中来进行实验转移。肺转移的形成和发展随后将通过体内Xenogen生物成像系统进行。(3)将评估DNAzyme治疗对携带乳腺癌的动物的存活率的影响。因此，在本提案中，我们通过研究AM 9D治疗对肿瘤生长和转移的影响（发病率和死亡率的主要机制），采取了一种治疗乳腺癌的创新方法。

DESCRIPTION (provided by applicant): Despite continued improvements in diagnosis, surgical techniques, and chemotherapy, breast cancer patients are still being overcome by cancer metastasis. Breast cancer metastasizes to brain, lung, bone, liver and regional lymph nodes. Early detection and advancements in research have increased survival and enhanced quality of life. However, there Is still a need for improvement. 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 stroma cells. Evidence suggests that MMP-9 plays a significant role in breast tumor cell invasion and metastasis. We have shown that DNAzyme generated against MMP-9 mRNA was able to reduce the expression of the enzyme in vitro and its intratumoral injection reduced the size of mammary tumor, generated in a MMTV- PyMT transgenic mouse model, by 50%. DNAzymes are catalytic DNA molecules that specifically bind and cleave mRNA of the targeted protein, resulting in decreased protein expression. Given their potential for systemic administration and targeting, anti-MMP-9 DNAzyme (AM9D) could prove useful as a therapy against breast carcinoma invasion. We hypothesize the down regulation of MMP-9, by AM9D, will lead to the inhibition of tumor growth, decrease in lung metastasis, block experimental metastasis and increase survival in breast carcinoma animal model. Towards the achievement of this goal we propose to (1) determine the minimum effective dose of AM9D needed to significantly decrease tumor load in the PyMT MTEC transplant mouse model without causing significant local toxicity. Toxicity will be determined by histopathological analysis of tissue slices. (2) assess the affect of AM9D on development of secondary tumor. Luciferase expressing PyMT MTECs will be transplanted into female mice mammary glands of female mice and tumors will be treated with an optimum concentration of AM9D, control DNAzyme or PBS for 4 weeks. Tumors will then be removed and animal will be maintained for an additional 8 weeks to develop lung secondary tumors. In addition, experimental metastasis will be performed by injecting PyMT MTEC with or without AM9D or control DNazyme into tail vein of FVB/Nj mice. Formation and development of lung metastasis will then be followed by in vivo Xenogen bioimager system. (3) The effect of DNAzyme treatment on the survival rate of animals bearing breast carcinomas will be assessed. Thus, in this proposal we are taking an innovative approach to the treatment of breast carcinomas by studying the effect of the AM9D therapy on tumor growth and metastasis, the major mechanism of morbidity and mortality.