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

描述(申请人提供)：尽管在诊断、手术技术和化疗方面不断改进，乳腺癌患者仍然被癌症转移所困扰。乳腺癌转移到脑、肺、骨、肝和区域淋巴结。早期发现和研究的进步提高了存活率和生活质量。然而，仍有改进的必要。肿瘤细胞的增殖、侵袭和转移至少部分是通过肿瘤和间质细胞产生的中性金属蛋白酶(MMPs)降解基底膜来实现的。有证据表明，基质金属蛋白酶-9在乳腺肿瘤细胞的侵袭和转移中起重要作用。我们已经证明，针对MMP-9mRNA产生的DNAzyme能够在体外减少该酶的表达，其瘤内注射能够将在MMTV-PyMT转基因小鼠模型中产生的乳腺肿瘤的大小减少50%。DNAzyme是一种催化的DNA分子，它能特异性地结合和切割目标蛋白质的mRNA，导致蛋白质表达减少。鉴于其全身给药和靶向的潜力，抗基质金属蛋白酶-9脱氧核酶(AM9D)可能被证明是一种有效的治疗乳腺癌侵袭的方法。我们推测AM9D通过下调基质金属蛋白酶-9的表达，抑制肿瘤生长，减少肺转移，阻断实验性转移，提高乳腺癌动物模型的生存率。为了实现这一目标，我们建议(1)确定在不引起明显局部毒性的情况下，在PYMT MTEC移植小鼠模型中显著降低肿瘤负荷所需的AM9D的最小有效剂量。毒性将通过组织切片的组织病理学分析来确定。(2)评价AM9D在继发性肿瘤发生中的作用。将表达荧光素酶的pYMT mTECs移植到雌性小鼠乳腺内，以AM9D、对照DNAzyme或PBS的最佳浓度处理肿瘤4周。然后，肿瘤将被切除，动物将再维持8周，以形成肺部继发性肿瘤。FVB/NJ小鼠尾静脉注射含或不含AM9D或对照DNAzyme的PYMT MTEC，进行实验性转移实验。肺转移的形成和发展将通过体内异种生物成像系统进行跟踪。(3)评估DNAzyme治疗对荷瘤动物存活率的影响。因此，在这项建议中，我们通过研究AM9D治疗对肿瘤生长和转移的影响，这是发病率和死亡率的主要机制，来采取一种创新的方法来治疗乳腺癌。

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.