Selective transduction of radioprotector protein, Tousled, for treatment of cance

描述（由申请人提供）：放射疗法经常单独使用或与化疗或手术联合使用治疗头颈部癌症。几乎所有接受区域放射治疗的患者都会出现口咽并发症。对落在辐射入口内的正常组织的损伤导致许多副作用，包括口腔粘膜炎、溃疡、唾液功能减退、吞咽困难、感染和龋齿。放射治疗的衰弱后遗症影响患者的生活质量，并对癌症治疗的继续产生负面影响。目前对这些并发症的治疗主要是姑息性的，预防对正常组织的辐射毒性提供了最佳的治疗前景，以减少对患者健康的影响。尽管放射递送技术有所改进，但放射诱导的毒性仍然是癌症治疗的限制因素。由于临床使用的辐射防护剂很少，正在探索新的干预措施来预防或治疗辐射损伤。我们已经证明，增加表达的Toulsed样激酶1B（TLK 1B）保护正常的唾液腺细胞免受辐射损伤在体外，和细胞可渗透的TAT-TLK 1B蛋白在唾液腺中的直接转移在体内有效地减轻辐射诱导的唾液腺功能减退。但是，与大多数旨在保护正常组织的辐射防护剂类似，TLK 1B具有保护肿瘤和限制癌症治疗效果的不可避免的风险。因此，为了降低发病率而不损害癌症的可治愈性，该提案的中心目标是开发TLK 1B作为正常细胞的辐射保护剂，但作为肿瘤的辐射增敏剂。细胞外基质被包括基质金属蛋白酶（MMP）的蛋白酶降解是肿瘤生长、侵袭和转移的标志。具有蛋白水解活性的MMP，特别是MT 1（膜1型）-MMP、MMP-2和MMP-9的表达升高，与包括头颈癌（HNSCC）在内的多种癌症相关。利用癌细胞周围富含蛋白水解酶的环境，我们将用MT 1-MMP切割基序改造TAT-TLK 1B，并在体外验证重组蛋白在头颈部鳞状细胞癌（HNSCC）细胞系中的转导和功效（Aim 1）。我们的方法的功能将在裸鼠HNSCC异种移植模型中进行体内测试（目的2）。我们最近获得了令人鼓舞的初步数据，显示在培养的HNSCC细胞中MT 1-MMP-敏感性TAT-TLK 1B的转导显著减少。我们有信心，我们提出的蛋白质工程方法有可能有效地区分蛋白质进入， 癌细胞在体内肿瘤环境中，活性蛋白酶对MT 1-MMP-敏感的TAT-TLK 1B蛋白的切割将在肿瘤细胞中激活一种激酶缺陷的显性阴性蛋白，该蛋白可使肿瘤细胞对电离辐射敏感。TLK 1B作为一种放射防护剂的开发，不仅通过选择性保护正常组织，而且通过肿瘤组织的敏化来提高治疗指数，这将大大增加其临床应用的潜力。

DESCRIPTION (provided by applicant): Radiotherapy is frequently used alone or in combination with chemotherapy or surgery in the treatment of head and neck cancer. Nearly all patients receiving regional radiotherapy with curative intent develop oropharyngeal complications. The damage to normal tissues that fall within the portals of radiation result in a number of side- effects that include oral mucositis, ulcerations, salivary hypofunction, dysphagia, infections, and dental decay. The debilitating sequelae of radiotherapy impact the patient's quality of life and negatively affect the continuation of cancer therapy. Current management of these complications is largely palliative, and prevention of radiation toxicity to normal tissues provides the best treatment prospect to reduce the impact on patient's well-being. Despite improvements in radiation delivery techniques, radiation-induced toxicity continues to be a limiting factor in cancer treatment. With few radioprotectors in clinical use, novel interventions are being explored to prevent or treat radiation injury. We have demonstrated that increased expression of Toulsed-like kinase 1B (TLK1B) protects normal salivary cells against radiation damage in vitro, and the direct transfer of cell-permeable TAT-TLK1B protein in salivary glands in vivo effectively mitigates irradiation-induced salivary hypofunction. But, similar to most radioprotectors aimed at protecting normal tissues, TLK1B carries the unavoidable risk of protecting tumors and limiting the efficacy of cancer treatment. Therefore, to reduce morbidity without compromising cancer curability, the central goal of the proposal is to develop TLK1B as a radioprotector for normal cells, but a radiosensitizer for tumors. Degradation of the extracellular matrix by proteases including matrix metalloproteinases (MMPs) is a hallmark of tumor growth, invasion, and metastasis. Elevated expression of proteolytically active MMPs, especially MT1 (membrane-type 1)-MMP, MMP-2, and MMP-9, is associated with a variety of cancers including head and neck cancer (HNSCC). Capitalizing on the proteolytic enzyme-rich milieu around cancer cells, we will engineer TAT-TLK1B with MT1-MMP cleavage motif and validate recombinant protein transduction and efficacy in head and neck squamous cell carcinoma (HNSCC) cell lines in vitro (Aim 1). The functionality of our approach will be tested in vivo in a nude mouse HNSCC xenograft model (Aim 2). We recently obtained encouraging preliminary data showing significantly reduced transduction of MT1-MMP- susceptible TAT-TLK1B in cultured HNSCC cells. We have confidence that our proposed protein engineering approach has the potential to effectively discriminate against protein entry in cancer cells. The cleavage of MT1-MMP-sensitive TAT-TLK1B protein by active proteases in a tumor milieu in vivo will transduce a kinase-deficient, dominant-negative protein in tumor cells that could sensitize them to ionizing radiation. The development of TLK1B as a radioprotector that enhances the therapeutic index not only by selective protection of normal tissue, but also by sensitization of tumor tissue will greatly augment its potential for clinical use.