Therapeutic/Mechanistic Role of Ape1 in Germ Cell Tumors

描述（申请人提供）：播散性生殖细胞肿瘤（GCT）的治疗取得了成功，70-80%的患者通过一线化疗治愈。然而，对于那些20-30%的性腺外原发或难治性疾病的患者，治疗反应很差，只有3-30%的患者在二线药物治疗后无病存活。治疗耐药疾病的一种方法是开发策略来增加化疗药物，这种药物在大多数GCT患者中已经非常成功。我们对DNA修复系统在GCT中的作用知之甚少，只知道有效的修复似乎使肿瘤细胞对治疗产生耐药性。我们观察到，与正常组织相比，GCT表达高水平的Ape1/ref-1。Ape1/ref-1是一种多功能蛋白，具有DNA碱基切除修复（BER）活性和氧化还原活性，需要激活特定的转录因子，包括Fos、Jun、NFkappaB、hif -1 α（缺氧诱导因子）、p53和PAX5。这种新的功能组合将Ape1/ref-1与许多用于治疗GCT的治疗剂（博来霉素、顺铂、放疗和VP-16）的耐药性联系起来，这些治疗剂作为BER的直接底物或通过Ape1/ref-1调节的转录因子间接改变信号传导。基于这些信息，我们假设：Ape1/ref-1在GCT中的高水平表达是一种疾病的功能标志物，1)可以预测高风险疾病，2)可以被操纵以获得治疗优势。该提案的主要目的是表征GCT中Ape1/ref-1的分子生物学特征，因为它与患者的临床过程和GCT细胞对治疗剂的反应有关。为了实现这一目标，我们制定了四个特异性目标：特异性目标1：确定Ape1/ref-1在预后良好和高危GCT中的相对表达。这充分利用了印第安纳大学丰富的临床GCT材料。特异性目标2:Ape1/ref-1在GCT的进展和发展，包括细胞生长、凋亡、细胞周期和分化中的作用是什么？这就是Ape1/ref-1的高水平表达，包括修复、氧化还原和核定位域突变体的过表达是如何独立影响GCT细胞作为癌细胞生长的能力的。特定目标3:Ape1/ref-1氧化还原状态的变化如何影响修复功能？使用位点特异性突变，确定哪些半胱氨酸残基特异性控制Ape1/ref- 1的修复功能？特异性目标4:Ape1/ref-1修复和氧化还原功能的改变如何影响GCT细胞对治疗剂的反应？利用我们在目标2和3中学到的知识，我们如何改变GCT细胞对治疗剂的耐药性。通过这些分析，我们希望确定Ape1/ref-1功能与睾丸癌进展相关的潜在机制。如预期的那样，如果任何突变体显示出使GCT细胞系对化疗/IR药物敏感，这将为未来基因治疗方法使GCT对治疗敏感奠定基础。

DESCRIPTION (provided by applicant): Therapy for disseminated germ cell tumors (GCT) has been successful with 70-80% of patients being cured with front line chemotherapy. However, for those 20-30% of patients with extra-gonadal primaries or refractory disease, the response to therapy is poor with only 3-30% surviving disease-free after second line agents. One approach to treating resistant disease is the development of strategies to augment the chemotherapeutic agents that have been so successful in the majority of GCT patients. Little is known about the role of DNA repair systems in GCT's except that efficient repair appears to make tumor cells resistant to therapy. We have observed that GCT's express high levels of Ape1/ref-1 compared to normal tissues. Ape1/ref-1 is a multifunctional protein with DNA base excision repair (BER) activity and redox activity required for activation of specific transcription factors including Fos, Jun, NFkappaB, HIF-1alpha (hypoxia inducible factor), p53, and PAX5. This novel combination of functions links Ape1/ref-1 with resistance to many of the therapeutic agents (bleomycin, cisplatin, radiation, and VP-16) used to treat GCT's by acting as direct substrates for BER or indirectly by altering signaling through transcription factors regulated by Ape1/ref-l. Based on this information, we hypothesize: High level expression of Ape1/ref-1 in GCT's is a functional marker of disease which 1) is predictive of high risk disease and 2) can be manipulated to gain a therapeutic advantage. The major thrust of this proposal is to characterize the molecular biology of Ape1/ref-1 in GCT's as it relates to the clinical course of patients and the response of GCT cells to therapeutic agents. To approach this goal, we have developed four Specific Aims: Specific Aim1: Determine the relative expression of, Ape1/ref-1in good prognosis and high-risk GCT's. This takes advantage of the wealth of clinical GCT material available at Indiana University. Specific Aim 2: What is the role of Ape1/ref-1 in GCT progression and development, including cell growth, apoptosis, cell cycle, and differentiation? That is how does the high level expression of Ape1/ref-1, including over-expression of repair, redox, and nuclear localization domain mutants independently affect the ability of GCT cells to grow as cancer cells. Specific Aim 3: How do changes in the redox status of Ape1/ref-1 affect repair function? Using site-specific mutants, determine which cysteine residues specifically control repair function of Ape1/ref-l? Specific Aim 4: How do alterations in the repair and redox functioning of Ape1/ref-1 affect the response of GCT cells to therapeutic agents? Using what we learn in Aims 2 & 3, how can we alter the resistance of GCT cells to therapeutic agents. Through these analyses, we hope to determine the underlying mechanisms by which Ape1/ref-1 function is linked to the progression of testicular cancer. If any mutants are shown to sensitize the GCT cell lines to chemo-/IR agents, as expected, this will set the stage for future gene therapy approaches to sensitize GCT's to therapy.