Adrenocortical Cancer and Thyroid Carcinomas: Models with Unique Properties

肾上腺皮质癌和甲状腺癌：具有独特特性的模型

• 批准号: 7733117
• 负责人: Antonio Fojo
• 金额: $ 51.76万
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/7733117
• 关键词: Accounting; Adenovirus Infections; Adenovirus Vector; Adenoviruses; Adrenal Cortex; Adrenal Glands; Adrenocortical carcinoma; Adriamycin PFS; Anaplastic Carcinoma Cell; Anaplastic Carcinomas; Biochemical; Biological Markers; CYP11B1 gene; Carcinoma; Cell Line; Cells; Cessation of life; Cholesterol; Cisplatin/Mitotane; Clinic; Clinical; Condition; Corticosterone; Cortodoxone; Cyclic AMP; Depsipeptides; Diagnosis; Disease; Dose; Endocrine; Endocrine Gland Neoplasms; Endothelial Cells; Enhancers; Enzymes; Excision; FK228; Ganciclovir; Gene Expression; Gene Transfer; Genes; Genetic Enhancer Element; Genetic Transcription; HSV-Tk Gene; Hematopoietic; Hepatotoxicity; Histone Deacetylase Inhibitor; Human; Hydrocortisone; In Vitro; Incidence; Infection; Integrins; Investigation; Iodine; Liver; Malignant Epithelial Cell; Malignant Neoplasms; Malignant neoplasm of adrenal cortex; Malignant neoplasm of thyroid; Mammalian Cell; Mediating; Messenger RNA; Mixed Function Oxygenases; Modeling; Molecular Profiling; Mus; Normal tissue morphology; Oncogenes; Operative Surgical Procedures; Organ; Ovary; P-Glycoprotein; P-Glycoproteins; Patients; Personal Satisfaction; Pharmaceutical Preparations; Phase; Plasmids; Population; Predisposition; Prodrugs; Property; RNA; Rate; Recurrent disease; Refractory; Relative (related person); Resistance; Side; Sodium Butyrate; Specificity; Steroid 11-beta-Monooxygenase; Suicide; Suicide Gene Therapy; TK Gene; Testing; Testis; Therapeutic; Thinking; Thyroglobulin; Thyroid Gland; Thyroid carcinoma; Time; Tissues; Transfection; Umbilical vein; Xenograft procedure; cancer cell; cell type; chemotherapy; cytotoxic; cytotoxicity; day; experience; gene therapy; in vivo; interest; malignant endocrine gland neoplasm; melanoma; neoplastic cell; preference; promoter; research study; response; steroid metabolism; success; suicide gene; symporter; transgene expression; tumor; vector

Adrenocortical carcinoma (ACC) is a highly malignant tumor with an incidence of 1 to 1.6 cases per million per year. It presents with metastatic disease in up to 40% of cases. In advanced or recurrent disease treatment options are limited, and therapies using agents such as mitotane, cisplatin and adriamycin effect a tumor response rate of less than 30%. Thyroid carcinoma is the most common endocrine malignancy, accounting for the majority of deaths from endocrine cancers. Each year in the US, approximately 14,000 new cases of thyroid carcinoma are diagnosed and 1200 patients die from this disease. Conventional therapy consists of surgical resection and radioiodine (131I) therapy. However, for poorly differentiated thyroid carcinomas (PDTCs) and anaplastic carcinomas that do not concentrate iodine, 131I therapy is ineffective. In these patients, therapeutic options are few and largely ineffective. Since treatment options for endocrine cancers are limited, we have begun to examine alternate therapeutic approaches. Transfer of a suicide gene into tumor cells is one strategy for cancer gene therapy. One example of a suicide gene is the herpes simplex virus thymidine kinase gene (HSV-TK). HSV-TK encodes an enzyme absent in mammalian cells that catalyzes the conversion of a nontoxic prodrug, such as ganciclovir (GCV), to its toxic phosphorylated metabolite. The key to successful suicide gene therapy is to restrict gene expression to a defined cell population using specific promoter(s). An approach that restricts expression to a given cell type is applicable to endocrine cancers, because they possess numerous genes with very restricted or specific expression. We began by seeking a molecular marker of adrenocortical tissue. CYP11B1 (11<=-hydroxylase) catalyzes conversion of 11-deoxy-corticosterone and 11-deoxycortisol to corticosterone and cortisol, respectively. Although steroid metabolism is not unique to the adrenal gland, biochemical studies have suggested CYP11B1 activity is confined to the adrenal cortex. The specificity of CYP11B1 expression was confirmed using 30 normal tissues. Expression was found only in normal adrenal tissue at an arbitrary level of 10, with a level of 0.1 in testes and ovary and undetectable expression in 27 other normal tissues. More importantly, expression was documented in 32/32 ACCs, with very high levels of expression in about half. Expression was not detected in other tumors. This expression profile makes this gene an attractive choice for a target specific therapy. Since expression of CYP11B1 was highly specific for adrenal tissue and ACCs, the putative CYP11B1 promoter was cloned proximal to an HSV-TK gene. In ACC cells a plasmid containing the HSV-TK gene under the control of the CYP11B1 promoter resulted in HSV-TK expression and enhanced sensitivity to GCV. These findings suggested the CYP11B1 promoter could be used to target ACC cells in suicide gene therapy. Although CYP11B1 promoter activity was preferentially observed in ACC cells, we set out to enhance it while maintaining its specificity for adrenal cells. Placing a putative enhancer element from the cholesterol side-chain cleavage (P450scc) gene upstream of the CYP11B1 promoter (SCC/11<= construct) enhanced its activity and specificity for ACC cells. A second construct using the P450scc promoter and enhancer (SCC/SCC) was also shown to have a high adrenal preference. Using the SCC/11<= and SCC/SCC vectors we constructed suicide HSV-TK adenoviruses and demonstrated their utility in vitro. GCV treatment demonstrated at least a 100-fold greater sensitivity of H295 cells at all concentrations of GCV, and exclusive cytotoxicity at clinically achievable concentrations. Encouraged by our success using adrenal specific promoters, and to determine whether expression could be modulated in other endocrine tumors, we carried out similar studies in thyroid cancer cells. These studies have shown that the thyroglobulin (Tg) promoter can direct specific expression of HSV-TK in thyroid cancer cells. Furthermore, a putative enhancer element for the Tg gene, augmented the activity and specificity of the Tg. In transient or stably-transfected thyroid carcinoma cells, treatment with the histone deacetylase (HDAC) inhibitors, depsipeptide (FR-901228, FK228) or sodium butyrate, alone or in combination with 8-Br-cAMP, resulted in further enhancement. Similar results were seen in two follicular and two anaplastic carcinoma cell lines, suggesting efficacy in the full spectrum of thyroid malignancies. The drug-mediated enhancement was not found in the non-thyroid cells. Enhancement of GCV sensitivity of as much as 100,000- fold was achieved despite low transfection efficiencies. Our studies have demonstrated that depsipeptide increases the efficiency of adenoviral transgene expression in vitro in cancer cells, in hematopoietic cells and in human umbilical vein endothelial cells (HUVEC) and may be useful in cancer gene therapy. Treatment with minimally cytotoxic doses of depsipeptide increases CAR and av integrin RNA preferentially in cancer cells. Ongoing experiments have demonstrated that depsipeptide can increase the efficiency of adenovirus infection in vivo and this occurs in a variety of models. Finally, surprised by the change following the addition of HDAC inhibitors, we examined the ability of depsipeptide to modulate expression of thyroid specific genes. Two follicular (FTC 132 and FTC 136) and two anaplastic thyroid carcinoma cell lines (SW 1736 and KAT-4) were treated with a sub-cytotoxic concentration of depsipeptide (1 ng/ml). After three days, Tg and Na+/I- symporter (NIS).mRNA levels approached those of a normal thyroid. 125I accumulations indicated a functional NIS was induced. These in vitro results suggest depsipeptide or another HDAC inhibitor might be used clinically in thyroid carcinomas that do not to trap iodine, as an adjunct to radioiodine therapy a strategy currently under investigation in the clinic It is thought that CAR is important in adenovirus selectivity. In contrast to other vectors adenovirus infect a variety of organs with high efficiency, with transducibility depending to a certain extent on CAR expression. The lack of CAR expression limits the efficacy of adenovirus-mediated gene transfer for cancer cells and increased CAR levels increase cancer cell susceptibility to adenovirus mediated gene transfer. CAR expression has been demonstrated in human and mouse liver, and is likely responsible for the liver toxicity of Ad-TK gene therapy, especially when non-specific promoters are used. We will optimize conditions for drug dose and infection time relative to drug treatment in order to obtain maximum transgene expression in the adenovirus infected melanoma xenograft-bearing mice. Finally with both poorly differentiated thyroid carcinomas and anaplastic thyroid carcinomas, therapeutic options are limited and largely unsuccessful. Their inability to trap iodine is thought to be a consequence of a loss of expression of the Na+/I- symporter (NIS). Our results suggest HDAC inhibitors up-regulate NIS transcription. Our clinical experience with depsipeptide has found it to be well tolerated, and the levels achieved greatly exceed those that modulated expression of the thyroid genes. A phase one trial to test this observation is ongoing.

肾上腺皮质癌（ACC）是一种高度恶性肿瘤，发病率为每年百万分之1至1.6例。它在高达40%的病例中表现为转移性疾病。在晚期或复发性疾病中，治疗选择有限，使用米托坦、顺铂和阿霉素等药物的治疗效果的肿瘤缓解率低于30%。甲状腺癌是最常见的内分泌恶性肿瘤，占内分泌癌死亡的大多数。在美国，每年大约有14000例新的甲状腺癌病例被诊断出来，1200名患者死于这种疾病。常规治疗包括手术切除和放射性碘（131I）治疗。然而，对于不浓缩碘的低分化甲状腺癌（PDTCs）和间变性癌，131I治疗无效。在这些患者中，治疗选择很少，而且大多无效。由于内分泌癌的治疗选择有限，我们已经开始研究替代治疗方法。将自杀基因转移到肿瘤细胞中是癌症基因治疗的一种策略。自杀基因的一个例子是单纯疱疹病毒胸苷激酶基因（HSV-TK）。HSV-TK编码哺乳动物细胞中不存在的一种酶，该酶催化无毒前药（如更昔洛韦（GCV））转化为其有毒的磷酸化代谢物。自杀基因治疗成功的关键是使用特定的启动子将基因表达限制在特定的细胞群中。一种限制特定细胞类型表达的方法适用于内分泌癌，因为它们拥有许多表达非常有限或特定的基因。我们从寻找肾上腺皮质组织的分子标记开始。CYP11B1（11<=-羟化酶）分别催化11-去氧皮质酮和11-去氧皮质醇转化为皮质酮和皮质醇。虽然类固醇代谢并非肾上腺独有，但生化研究表明CYP11B1活性仅限于肾上腺皮质。用30个正常组织证实CYP11B1表达的特异性。仅在正常肾上腺组织中任意水平表达10，在睾丸和卵巢中表达0.1，在其他27个正常组织中未检测到表达。更重要的是，在32/32个acc中记录了表达，大约有一半的表达水平很高。在其他肿瘤中未见表达。这种表达谱使该基因成为靶向特异性治疗的有吸引力的选择。由于CYP11B1的表达对肾上腺组织和acc具有高度特异性，因此推测的CYP11B1启动子克隆在HSV-TK基因的近端。在ACC细胞中，含有HSV-TK基因的质粒在CYP11B1启动子的控制下，导致HSV-TK的表达和对GCV的敏感性增强。这些发现表明，CYP11B1启动子可以在自杀基因治疗中靶向ACC细胞。虽然CYP11B1启动子活性优先在ACC细胞中观察到，但我们开始增强它，同时保持其对肾上腺细胞的特异性。在CYP11B1启动子（SCC/11<= construct）上游放置一个假定的胆固醇侧链切割（P450scc）基因增强元件，增强了其对ACC细胞的活性和特异性。使用P450scc启动子和增强子的第二种构建体（SCC/SCC）也显示出高度的肾上腺偏好。利用SCC/11<=和SCC/SCC载体构建自杀性单纯疱疹病毒- tk腺病毒，并对其体外应用效果进行了验证。GCV治疗显示，在所有浓度的GCV下，H295细胞的敏感性至少提高100倍，并且在临床可达到的浓度下具有排他的细胞毒性。受我们成功使用肾上腺特异性启动子的鼓舞，并确定是否可以在其他内分泌肿瘤中调节表达，我们在甲状腺癌细胞中进行了类似的研究。这些研究表明，甲状腺球蛋白（Tg）启动子可以指导HSV-TK在甲状腺癌细胞中的特异性表达。此外，一种推测的Tg基因增强元件增强了Tg的活性和特异性。在瞬时或稳定转染的甲状腺癌细胞中，组蛋白去乙酰化酶（HDAC）抑制剂、抑郁肽（FR-901228、FK228）或丁酸钠单独或与8-Br-cAMP联合治疗可进一步增强。在两种滤泡癌和两种间变性癌细胞系中也观察到类似的结果，表明该药物对甲状腺恶性肿瘤全谱有效。在非甲状腺细胞中没有发现药物介导的增强。尽管转染效率较低，但GCV敏感性的提高高达10万倍。我们的研究表明，在体外，沉积肽可提高腺病毒在癌细胞、造血细胞和人脐静脉内皮细胞（HUVEC）中的转基因表达效率，并可能在癌症基因治疗中发挥作用。用最低细胞毒性剂量的沉积肽治疗会优先增加癌细胞中的CAR和av整合素RNA。正在进行的实验表明，沉积肽可以提高体内腺病毒感染的效率，这种情况发生在多种模型中。最后，对添加HDAC抑制剂后的变化感到惊讶，我们检查了抑郁肽调节甲状腺特异性基因表达的能力。用亚细胞毒性浓度（1 ng/ml）的沉积肽处理2个滤泡细胞（FTC 132和FTC 136）和2个间变性甲状腺癌细胞（SW 1736和KAT-4）。3 d后，Tg和Na+/I-同调体（NIS）。mRNA水平接近正常甲状腺。125I积累表明诱导了功能性NIS。这些体外实验结果表明，抑郁肽或另一种HDAC抑制剂可能在临床上用于不捕获碘的甲状腺癌，作为放射性碘治疗的辅助手段，这是目前正在临床研究的一种策略。与其他载体不同，腺病毒能高效感染多种器官，其可转导性在一定程度上取决于CAR的表达。CAR表达的缺乏限制了腺病毒介导的癌细胞基因转移的效果，而CAR水平的增加增加了癌细胞对腺病毒介导的基因转移的易感性。CAR表达已在人类和小鼠肝脏中得到证实，并且可能是Ad-TK基因治疗的肝毒性的原因，特别是当使用非特异性启动子时。我们将优化药物剂量和感染时间相对于药物治疗的条件，以便在腺病毒感染的黑色素瘤异种移植小鼠中获得最大的转基因表达。最后，对于低分化甲状腺癌和间变性甲状腺癌，治疗选择有限且大部分不成功。它们无法捕获碘被认为是Na+/I-同调体（NIS）表达缺失的结果。我们的研究结果表明HDAC抑制剂上调NIS转录。我们对抑郁肽的临床经验表明，它具有良好的耐受性，并且达到的水平大大超过了那些调节甲状腺基因表达的水平。检验这一观察结果的第一阶段试验正在进行中。