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

描述(申请人提供)：在头颈部癌症的治疗中，放射治疗经常单独使用或与化疗或手术联合使用。几乎所有接受区域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好的治疗前景，以减少对患者福祉的影响。尽管辐射传递技术有所改进，但辐射引起的毒性仍然是癌症治疗中的一个限制因素。由于临床上使用的放射防护剂很少，人们正在探索预防或治疗辐射损伤的新干预措施。我们已经证明，Toulsed-like kinase1B(TLK1B)的高表达在体外可以保护正常唾液细胞免受辐射损伤，在体内直接将TAT-TLK1B蛋白转移到唾液腺中可以有效地缓解辐射所致的唾液功能低下。但是，与大多数旨在保护正常组织的放射保护剂一样，TLK1B存在着保护肿瘤和限制癌症治疗效果的不可避免的风险。因此，为了在不影响癌症治愈的情况下降低发病率，该提案的中心目标是开发TLK1B作为正常细胞的辐射保护剂，但作为肿瘤的放射增敏剂。细胞外基质的降解是肿瘤生长、侵袭和转移的标志。蛋白水解性MMPs的高表达，尤其是MT1(膜型1)-MMP1、MMP2和MMP9的表达与包括头颈癌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，我们将构建带有MT1-MMP裂解基序的TAT-TLK1B，并在体外验证重组蛋白在头颈部鳞状细胞癌(HNSCC)细胞系中的转导和疗效(目标1)。我们的方法的功能将在裸鼠HNSCC异种移植模型(AIM 2)中进行活体测试。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敏感的TAT-TLK1B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。在体内的肿瘤环境中，MT1-MMPs敏感的TAT-TLK1B蛋白被活性蛋白酶切割，将在肿瘤细胞中转导一种显性负性蛋白，使其对电离辐射敏感。TLK1B作为一种放射防护剂的发展，不仅通过对正常组织的选择性保护，而且通过对肿瘤组织的增敏来提高治疗指数，将极大地增强其临床应用的潜力。

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.