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

描述（由申请人提供）：放疗通常单独或联合化疗或手术治疗头颈癌。几乎所有接受局部放射治疗的患者都会出现口咽并发症。辐射对正常组织的损害会导致一系列副作用，包括口腔黏膜炎、溃疡、唾液功能减退、吞咽困难、感染和蛀牙。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处理主要是姑息性的，预防对正常组织的辐射毒性提供了最好的治疗前景，以减少对患者健康的影响。尽管辐射传输技术有所改进，但辐射引起的毒性仍然是癌症治疗的一个限制因素。由于临床使用的辐射防护剂很少，人们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，在体外，Toulsed-like kinase 1B （TLK1B）的表达增加可以保护正常的唾液细胞免受辐射损伤，并且在体内唾液腺中直接转移细胞可渗透的TAT-TLK1B蛋白可以有效减轻辐射诱导的唾液功能减退。但是，与大多数旨在保护正常组织的放射性保护剂类似，TLK1B具有不可避免的保护肿瘤和限制癌症治疗效果的风险。因此，为了在不影响癌症治愈率的情况下降低发病率，该提案的中心目标是开发TLK1B作为正常细胞的放射保护剂，但作为肿瘤的放射增敏剂。包括基质金属蛋白酶（MMPs）在内的蛋白酶降解细胞外基质是肿瘤生长、侵袭和转移的标志。蛋白水解活性MMPs，特别是MT1（膜型1）-MMP、MMP-2和MMP-9的表达升高与包括头颈癌（HNSCC）在内的多种癌症有关。利用癌细胞周围富含蛋白水解酶的环境，我们将设计带有MT1-MMP切割基序的TAT-TLK1B，并在体外验证重组蛋白在头颈部鳞状细胞癌（HNSCC）细胞系中的转导和有效性（目的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MT1-MMP易感的dat - tlk1b的转导显着减少。我们有信心，我们提出的蛋白质工程方法有潜力有效地区分蛋白质进入

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.