Re-activation of maspin tumor suppressor gene by designed transcription factors

描述（申请人提供）：在肿瘤进展过程中，癌细胞会发生动态转化，包括肿瘤生长、血管生成和转移性传播。在癌症治疗中，迫切需要开发靶向肿瘤进展期间的关键（理想地是多个）步骤的新方法。在这个提议中，我们设计了由锌指（ZF）结构域组成的人工转录因子（ATFs），作为抑制肿瘤进展过程中多个过程的新治疗策略。我们已经靶向了乳腺丝氨酸蛋白酶抑制剂（maspin）基因（SERPINB5），Maspin是一个重要的治疗靶点，因为它的过表达与乳腺和前列腺肿瘤模型中的肿瘤抑制、血管生成减少、运动性和转移相关。转移性细胞已经发展了几种下调maspin的机制。Maspin在侵袭性肿瘤中很少突变。相反，maspin的沉默涉及转录调控和异常启动子甲基化。我们的目标是构建能够特异性地重新激活转移性乳腺细胞系中maspin的ATF，克服表观遗传沉默。我们的假设是，ATFs上调maspin，通过自身或与增加染色质可及性的药物（甲基转移酶和组蛋白脱乙酰酶抑制剂）组合，将能够重新激活肿瘤细胞中的maspin功能，减少裸鼠中的肿瘤生长和转移扩散。首先，我们设计了高度特异性的ATF，由6ZF结构域靶向maspin启动子中的18个碱基对（bp）位点。ZF与有效的转录激活域连接，并使用逆转录病毒载体在肿瘤细胞中表达。我们将研究ATFs是否能够特异性地在几种乳腺癌细胞中重新激活maspin，这些细胞包含甲基化和沉默的maspin启动子。我们将研究ATFs是否与甲基转移酶和组蛋白去乙酰化酶抑制剂协同作用，以上调maspin。我们将评估这些ATF是否能够下调细胞侵袭并诱导转移性细胞系的凋亡。最后，将使用腺相关病毒（AAV）表达ATF，并且我们将研究它们在裸鼠中的乳腺癌原位异种移植模型中下调肿瘤生长和转移的能力。将使用生物发光成像（BLI）对转移性病变进行真实的监测。这项工作将导致新的抗癌调节剂的特征，能够重新编程肿瘤抑制因子的异常表观遗传沉默

DESCRIPTION (provided by applicant): During tumor progression, cancer cells undergo dynamic transformations, including tumor growth, angiogenesis, and metastatic dissemination. There is a crucial need in cancer therapeutics to develop novel approaches that target critical, ideally multiple steps, during tumor progression. In this proposal, we have designed artificial transcription factors (ATFs) made of zinc finger (ZF) domains as novel therapeutic strategy to inhibit multiple processes during tumor progression. We have targeted the mammary serine protease inhibitor (maspin) gene (SERPINB5), Maspin is an important therapeutic target since its overexpression is associated with tumor suppression, decreased angiogenesis, motility and metastasis in breast and prostate tumor models. Metastatic cells have developed several mechanisms to down-regulate maspin. Maspin is rarely mutated in aggressive tumors. Instead, silencing of maspin involves both, transcriptional regulation and aberrant promoter methylation. Our objective is to construct ATFs able to specifically re-activate maspin in metastatic breast cell lines, overcoming epigenetic silencing. Our hypothesis is that ATFs up-regulating maspin, by themselves or in combination with drugs that increase chromatin accessibility (methyltransferase and histone deacetylase inhibitors), will be able to re-activate maspin functions in tumor cells, reduce tumor growth and metastatic spread in nude mice. First, we have engineered highly specific ATFs made of 6ZF domains targeting 18-base pairs (bp) sites in the maspin promoter. The ZFs are linked to a potent transcriptional activator domain and expressed in tumor cells using retroviral vectors. We will investigate if the ATFs are able to specifically reactivate maspin in several breast cancer cells comprising a methylated and silenced maspin promoter. We will study if the ATFs synergize with methyltransferase and histone deacetylase inhibitors, to up-regulate maspin. We will assess if these ATFs are able to down-regulate cell invasion and to induce apoptosis in metastatic cell lines. Finally, the ATFs will be expressed using Adeno- Associated Viruses (AAVs) and we will study their capability to down-regulate tumor growth and metastasis in an orthotopic xenograft model of breast cancer in nude mice. Metastatic lesions will be monitored in real time using Bioluminescence Imaging (BLI). This work should lead to the characterization of novel anti-cancer regulators, able to reprogram the aberrant epigenetic silencing of tumor suppressors