Specific activation of tumor suppressors repressed by oncogenes to treat cancer

肝细胞癌是最常见的恶性肝癌，是世界上与癌症相关的死亡的第二大常见原因。目前，对肝细胞癌患者的治疗选择非常有限，导致肝细胞癌形成的潜在机制仅被很少地阐明。因此，迫切需要开发一种副作用很小或没有副作用的专门治疗方法。转录因子SAL样蛋白4(SALL4)是一个很有前景的基因靶点，在包括肝癌在内的大量实体癌中表达，而在健康的成人组织中不表达。SALL4与核小体重塑和脱乙酰酶(NuRD)复合体的视网膜母细胞瘤结合蛋白4(RBBp4)结合，从而抑制包括肿瘤抑制基因在内的各种基因，最终导致癌症。最近有研究表明，一种合成肽(FFW)可以阻断SALL4-RBBp4/NuRD之间的相互作用，从而减少小鼠移植瘤的生长，这表明它是肝癌患者非常有希望的靶点。然而，由于RBBp4还存在于其他核复合体中，因此阻断RBBp4是一种非选择性的治疗方法。为了克服这一障碍，拟议的研究项目旨在将SALL4-RBBp4/NuRD复合体专门针对特定抑制肿瘤抑制因子的启动子。在该项目的第一部分，将验证受SALL4-RBBp4/NuRD复合体影响的最有希望的肿瘤抑制基因。因此，它们对肝癌细胞的致瘤特性的影响，如增殖和迁移，将在培养中进行测试。在第二部分中，SALL4-RBBp4/NuRD相互作用将被新的CRISPR/dCas9技术在一种被证实具有强大抗肿瘤作用的肿瘤抑制因子的特定启动子上中断。通过设计特定的引导RNA并将dCas9蛋白与FFW肽融合，FFW将在预期的启动子上特异性和排他性地破坏SALL4-RBBp4/NuRD复合体，而不是在其他基因组位置上非特异性地破坏SALL4-RBBp4/NuRD复合体。因此，这种特殊的肿瘤抑制因子将重新激活。在第三部分中，将分析激活的抑癌基因在肝癌细胞中的抗肿瘤作用。最后，将在异种小鼠模型中研究dCas9-FFW构建体治疗肝脏肿瘤的治疗潜力。综上所述，拟议的研究项目将针对SALL4阳性肝癌中下调的相关肿瘤抑制因子，提供一种非常新颖和创新的技术来特异性地重新激活肝癌患者中被抑制的肿瘤抑制因子。这代表了个性化癌症治疗的一种非常有前途的策略。由于SALL4阳性癌症的高丰度，这一策略还可以转移到各种其他类型的癌症上。

Hepatocellular carcinoma (HCC) is the most common type of malignant liver cancer representing the second most common cause of cancer-related deaths in the world. Currently, the therapeutic options for HCC patients are highly limited and the underlying mechanisms driving HCC formation are only poorly elucidated. Therefore, there is an urgent need to develop a specialized treatment with little or no side effects. The transcription factor Sal-like protein 4 (SALL4) is a promising gene target that is expressed in a significant number of solid cancers, including HCC, and not in healthy adult tissues. SALL4 binds to the retinoblastoma binding protein 4 (RBBp4) of the nucleosome remodeling and deacetylase (NuRD) complex and thereby represses various genes, including tumor suppressors, eventually leading to cancer. Recently, it was demonstrated that disruption of the SALL4-RBBp4/NuRD interaction by a synthesized peptide (FFW) results in decreased HCC growth in murine xenografts, indicating that it represents a very promising target for HCC patients. However, since RBBp4 is additionally present in other nuclear complexes, blocking RBBp4 is a nonselective therapeutic approach. To overcome this obstacle, the proposed research project aims to specifically target the SALL4-RBBp4/NuRD complex at promoters of specific repressed tumor suppressors. In the first part of the project, the most promising tumor suppressor genes that are affected by the SALL4-RBBp4/NuRD complex will be validated. Therefore, their effect on the tumorigenic properties of HCC cells, such as proliferation as well as migration, will be tested in culture. In the second part, the SALL4-RBBp4/NuRD interaction will be disrupted with the novel CRISPR/dCas9 technology at the specific promoter of a validated tumor suppressor demonstrating strong antitumor effects in part one. By designing a specific guide RNA and fusing the dCas9 protein with the FFW peptide, FFW will specifically and exclusively disrupt the SALL4-RBBp4/NuRD complex at the intended promoter and not nonspecifically at other genomic sites. Consequently, this particular tumor suppressor will become reactivated. In the third part, the antitumor effect of the activated tumor suppressor will be analyzed in HCC cells. Finally, the therapeutic potential of the dCas9-FFW construct to treat liver tumors will be investigated in vivo in a xenograft mouse model. In summary, the proposed research project will target relevant tumor suppressors downregulated in SALL4-positive HCC, providing a very novel and innovative technique to specifically reactivate repressed tumor suppressors in HCC patients. This represents a very promising strategy for personalized cancer therapy. Due to the high abundance of SALL4-positive cancers, this strategy can additionally be transferred to a variety of other cancer types.