Targeting epigenetic regulators using bacterial macromolecule delivery

描述(由申请人提供)：表观遗传学提供了许多可能彻底改变癌症治疗的靶点。由于对再生组织的有害影响，表观遗传抑制物的全身输送是不可行的。通过将抑制剂与癌症特异性细菌一起提供，可以通过专门针对肿瘤和转移瘤的治疗来克服这一关键问题。到目前为止，还没有人培育出调节表观遗传靶标的细菌。细菌递送系统将通过将多肽和shRNA运送到癌细胞来发挥作用。抑制关键靶点EZH2、NIPP1和PP1将扰乱基本的癌细胞过程，并消除癌症干细胞。这将阻止肿瘤扩散，防止转移形成。这项拟议的研究有四个具体目标，结合了表观遗传学和细菌癌症靶向领域的最新发展。AIM 1将创建细菌大分子递送系统；AIM 2将创建针对NIPP1和EZH2的细菌shRNA递送系统；AIM 3将创建破坏NIPP1：PP1复合体的细菌肽递送系统；AIM 4将创建故障保护电路，以清除治疗后的细菌。基因敲除和复合干扰的效果将通过靶蛋白测量、免疫沉淀和磷酸酶磷酸化酶检测来测试。细胞活力和癌症干细胞数量将在芯片上肿瘤、小鼠皮下肿瘤和自发转移的设备中进行量化。本研究的基础工作已建立在以下方面：1)创建一种无毒的细菌大分子递送系统，2)展示其关键成分(细胞内触发的裂解和细胞质定位)及其传递蛋白质和DNA的能力；3)验证NIPP1和EZH2在癌细胞和肿瘤中的击倒作用；以及4)证明细菌释放NIPP1：PP1解离肽可诱导癌细胞死亡。这项拟议的研究将建立在尼尔·福布斯(美国马萨诸塞州阿默斯特大学化学工程)与埃利德·范·恩德和马蒂厄·博伦(比利时鲁汶大学细胞和分子医学专家)之间的合作基础上，他们是细菌癌症治疗和表观遗传调节方面的专家。这项研究将为控制基因和蛋白质进入癌细胞建立一个新的治疗平台。通过细菌瞬时传递基因和蛋白质将能够直接靶向蛋白质和功能，特别是在癌细胞中。这项研究将创造一种表观遗传疗法，有可能根除转移并防止其形成，这是两个紧迫的临床问题。拟议的动物实验将首次证明以蛋白质-磷酸酶为导向的疗法的好处，这种疗法可以破坏全酶中的蛋白质-蛋白质相互作用。该项目的最终目标是开发一种治疗原发癌症和转移性疾病的方法。

DESCRIPTION (provided by applicant): Epigenetics offers many targets that could revolutionize cancer therapy. Systemic delivery of epigenetic inhibitors is unfeasible due to deleterious effects on regenerating tissue. Delivering inhibitors with cancer- specific bacteria would conquer this critical problem by specifically focusing treatment to tumors and metastases. To date, no one has produced bacteria that modulate epigenetic targets. Bacterial delivery systems would function by transporting peptides and shRNA into cancer cells. Inhibiting the key targets EZH2, NIPP1 and PP1 would disrupt essential cancer cell processes and eliminate cancer stems cells. This will stop tumor spreading and prevent metastasis formation. The proposed research has four Specific Aims that combine the newest developments in the fields of epigenetics and bacterial cancer targeting. Aim 1 will create a bacterial macromolecule delivery system; Aim 2 will create a bacterial shRNA delivery system that targets NIPP1 and EZH2; Aim 3 will create a bacterial peptide delivery system that disrupts NIPP1:PP1 complexes; and Aim 4 will create failsafe circuitry to clear bacteria after treatment. The effect of gene knockdown and complex disruption will be tested by target-protein measurement, immunoprecipitation, and phosphatase phosphorylase assay. Cell viability and the cancer stem cell populations will be quantified in a tumor-on-a-chip device, subcutaneous tumors and spontaneous metastases in mice. The groundwork for this study has been established by 1) creating a non-toxic bacterial macromolecule delivery system, 2) demonstrating its key components (intracellularly triggered lysis and cytoplasmic localization) and its ability to delive proteins and DNA; 3) validating NIPP1 and EZH2 knockdowns in cancer cells and tumors; and 4) showing that bacterial release of NIPP1:PP1 dissociative peptides induces cancer cell death. The proposed research will build upon a collaboration between Neil Forbes (Chemical Engineering, UMass Amherst, USA), and Aleyde Van Eynde and Mathieu Bollen (Cellular and Molecular Medicine, KU Leuven, Belgium), experts in bacterial cancer treatment and epigenetic regulators. This research will establish a new therapeutic platform for controlled gene and protein delivery into cancer cells. Transient delivery of genes and proteins with bacteria will enable direct targeting of proteins and functions specifically in cancer cells. This study will create an epigenetic therapy with the potential to eradicate metastases and prevent their formation, two urgent clinical problems. Proposed animal experiments will be the first to demonstrate the benefit of a protein-phosphatase-directed therapy that disrupts protein-protein interactions in holoenzymes. The ultimate goal of this project is development of a treatment modality for primary cancers and metastatic disease.