Therapeutic targets and novel anticancer agents for endocrine cancers

内分泌癌的治疗靶点和新型抗癌药物

• 批准号: 10926174
• 负责人: Naris Nilubol
• 金额: $ 89.15万
• 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/10926174
• 关键词: 3-Dimensional; Acetylcysteine; Adherent Culture; Adrenal Cortex Hormones; Adrenocortical carcinoma; Apoptosis; Auranofin; Autophagocytosis; BRCA1 gene; Biological Assay; Blood Vessels; CASP3 gene; CCNB1 gene; CHEK1 gene; CTNNB1 gene; Cancer Patient; Caspase; Cell Cycle Arrest; Cell Death; Cell Line; Cell Proliferation; Cells; Client; Clinical; Clinical Trials; Coculture Techniques; Combination Drug Therapy; Combined Modality Therapy; DNA Double Strand Break; Data; Databases; Development; Disease-Free Survival; Dose; Down-Regulation; Drug Combinations; Drug Delivery Systems; Enzymes; Exclusion; Extracellular Matrix; Extracellular Matrix Proteins; Extravasation; FDA approved; Family; Fibroblasts; Gene Expression; Gene Expression Profiling; Genes; Glutathione; Goals; Gold; Heat-Shock Proteins 90; High Pressure Liquid Chromatography; Human; Hyaluronan; Hydrocortisone; ITGB3 gene; In Vitro; Induction of Apoptosis; Intercellular Fluid; Invaded; Iodine; LOX gene; Label; Lipid Peroxidation; Malignant Neoplasms; Malignant neoplasm of thyroid; Manuscripts; Matrix Metalloproteinases; Messenger RNA; Migration Assay; Mitotic; Molecular; Mutation; Nuclear Translocation; Operative Surgical Procedures; Oxidative Stress; PIK3CG gene; Paclitaxel; Pathway interactions; Patients; Penetration; Pharmaceutical Preparations; Pilot Projects; Preparation; Prognostic Marker; Protein Isoforms; Proteins; Publishing; Quality of life; Radiation Tolerance; Radiation therapy; Radiation-Sensitizing Agents; Recombinants; Recurrence; Resistance; Rheumatoid Arthritis; Role; SMARCA4 gene; Sampling; Scheme; Strategic Planning; Survivors; TIMP1 gene; TNF gene; The Cancer Genome Atlas; Toxic effect; Treatment Efficacy; Vascular Endothelial Cell; Vascular Endothelial Growth Factors; Vascular Permeabilities; Xenograft procedure; anaplastic thyroid cancer; biological adaptation to stress; cancer care; cancer cell; clinically relevant; computerized; cytokine; cytotoxic; differential expression; efficacy testing; experimental study; high-throughput drug screening; hyaluronan synthase 1; improved; in vivo; inhibitor; interstitial; mRNA Expression; malignant endocrine gland neoplasm; molecular targeted therapies; nanoGold; novel; novel anticancer drug; overexpression; particle; pressure; public database; repaired; resistance mechanism; screening; synergism; therapeutic target; thioredoxin reductase; transcriptome sequencing; treatment group; treatment response; tumor; tumor microenvironment

1. Identification of novel synergistic drug combination therapy for ACC 1.1 Status: Published [J Exp Clin Cancer Res 2022]. The synergy of MELK and CDK inhibitors targeting multiple clinically-relevant molecules in ACC. Clinical trial development is ongoing. 1.2 The novel synergistic combination of PI3K and HSP90 inhibitors in ACC Aim: To identify and validate the effective novel synergistic drug combinations using PI3K inhibitor and Heat-shock protein 90 inhibitors and to study the molecular mechanisms of synergy identified by the computerized drug combination matrix screening in ACC cells. We analyzed publicly available databases and found that ACC overexpressed several isoforms of HSP90 and its clients involved in ACC initiation and progression. We validated the synergy in two ACC cell lines in a cell proliferation assay. ACC 3D tumor spheroid assays showed increased efficacy in the combination treatment group as compared to the single-drug groups. Because PIK75 is not yet available for a clinical trial, we identified and validated the synergy between BGT226 and STA9090 or with HSP990 in monolayer culture. The combination of BGT226 and one of the HSP90 inhibitors was more effective in ACC 3D tumor spheroids, clonogenic assays, and invasion/migration assays than single-drug groups at clinically achievable concentrations. We discovered that the synergistic mechanisms of cell death were completely different between PIK75 and BGT226. PIK75 combination induced G2M cell cycle arrest, followed by caspase-3/7 dependent apoptosis. However, the combination of BGT226 and STA9090 did not cause cell cycle arrest nor it induced apoptosis. We performed RNA-seq and compare gene expression profiles of ACC cells treated with these combinations and found that BGT226 combination induced autophagy-related genes and pathways, which were validated at a protein level. We are currently determining whether the induction of autophagy is the mechanism of cell death or it is the resistance mechanism responding to treatments. The in vivo efficacy in ACC xenografts is in progress. 2. The identification of radiosensitizer and oxidative stress response in ACC. Aim: To evaluate the effects and mechanism of action of auranofin in combination with radiotherapy (RT) in ACC, Summary: Most patients with ACC develop locoregional recurrence after surgery. We identified auranofin from a quantitative high-throughput drug screening in two ACC cell lines as a novel radiosensitizer with a potent cytotoxic effect. Auranofin is an FDA-approved gold particle-based drug with low toxicity for rheumatoid arthritis. The analyses of the independent databases of ACC samples showed the mRNAs of several genes involved in oxidative stress response including the TXNRD pathway, targetable by auranofin, are differentially expressed in ACC.TXNRD is the pro-survival reducing enzyme of TXN. In addition, patients with low TXNRD1 mRNA expression in ACC had shorter overall survival and disease-free survival (OS and DFS). We showed downregulation of several anti-oxidative stress-related genes was inversely correlated with overexpression of MKi67 and CCNB1 (poor prognostic markers). We confirmed the synergistic activity of auranofin and RTin monolayer culture. The combination treatment of auranofin and RT was more effective than single treatments in 3D ACC spheroids and clonogenic assays in two cell lines at clinically achievable concentrations. After pilot studies to optimize the dosing scheme, the in vivo experiment to test the efficacy will start soon. We confirmed that the effective concentrations inhibited thioredoxin reductase activity in ACC. We confirmed that the combination treatments induced a higher level of gH2AX, activated p-CHK1, and induction of p-BRCA1 in both cell lines consistent with double-strand DNA break and repair, respectively. We observed that NCI-H295R (cortisol-producing cells with activating CTNNB1 mutation) was more sensitive to RT and auranofin than SW-13 (SMARCA4 mutations, non-steroid producing cell line). While we observed cleaved-PARP in NCI-H295R treated with the combination, we did not observe cleaved-caspase or cleaved PARP in SW13 treated with auranofin and RT. Auranofin and RT induced p21 and p27 in SW-13 cells, but not NCI-H295R. To further explore the mechanism of action in both cell lines, we found that SW-13 effectively overexpressed NRF2 and KEAP1 nuclear translocation as well as TXNRD1 and HMOX1 as compared to that of NCI-H295R, suggesting SW-13 had a more robust anti-oxidative stress response. Preliminary data suggest that the combination treatment caused mitotic catastrophe in NCI-H295R but not in SW-13. Because strong induction of HMOX1 and a reduction in GPX4, we assessed ferroptosis. We confirmed in both cell lines that auranofin and RT induced glutathione-oxidative stress, which was rescued with N-acetylcysteine. However, we did not observe an increased lipid peroxidation, a hallmark of ferroptosis, when NCI-H295R cells were treated with auranofin and RT but we confirmed that the combination treatment induced ferroptosis in SW-13. We excluded the role of corticosteroids in radiosensitivity in ACC using various treatments. The bulk RNA-sequencing to understand the genes and pathways involved in treatment response is in progress. 3. Mechanism of TNF-a-related changes in tumor microenvironment (TME) to enhance drug delivery Status: manuscript preparation is in-progress. Aim: to elucidate the mechanism of action of TNF-a in reducing tumor interstitial pressure leading to improved drug delivery efficiency. I. Summary: Because the effects of TNF-a on the expression and functions of several intratumoral extracellular matrix proteins and cytokines and the roles of these proteins and cytokines on intratumoral vascular permeability are not fully understood. Using ECM, human matrix metalloproteinase (MMP) arrays, and gene expression profiling of iodine-resistant thyroid cancer cells treated with TNF-a, we identified and validated that TNF-a down-regulated TGFb and LOX, and upregulated MMPs, TIMP1, and ITGB3 which have roles in vascular permeability in TME. siLOX-treated thyroid cancer xenograft recapitulated the intratumoral vascular leakage seen in xenografts similar to that caused by TNF-a. Next, we validated the effects of TNF-a on vascular endothelial cells and found that TNF-a treatment downregulated TGFb1, Lox, and VEGF-R (a key regulator of vascular permeability). To confirm the efficacy of the treatments using recombinant TNF-a and TGFb inhibition, we combined the TME treatments with paclitaxel in two iodine-resistant poorly differentiated (PDTC) (TPC1) and anaplastic thyroid cancer (ATC) (8505C) 3D tumor spheroids and found that the anti-tumor efficacy was higher in the combination treatment groups than single-treatment groups. Furthermore, we demonstrated the higher intratumoral penetration of fluorescent-labeled paclitaxel when thyroid cancer spheroids were treated with TGFb inhibitors and LOX inhibition (siLOX and BAPN which inhibits LOX enzymatic activity) and the intratumoral paclitaxel concentration was severalfold higher than the paclitaxel-only group. We validated the enhanced intratumoral paclitaxel delivery in vivo using HPLC by treating 8505C xenografts with nanogold particles carrying TNF-a and TGFb inhibitor. To investigate the role of TGFb and LOX in the increased tumor interstitial fluid pressure, we identified the correlation between these and the synthesis of hyaluronan (hyaluronan synthase 1 (HAS1) in TCGA thyroid cancer database and validated this finding in vitro by treating the thyroid cancer cells with TGFb inhibitor and siLOX. Similar improvements in drug delivery efficiency and treatment efficacy can be seen in much denser co-cultured thyroid cancer spheroids with cancer-associated fibroblasts.

1. 新型协同药物联合治疗ACC的研究进展[J]。MELK和CDK抑制剂在ACC中靶向多种临床相关分子的协同作用。临床试验正在进行中。1.2 ACC中PI3K与HSP90抑制剂的新型协同组合目的：鉴定并验证PI3K抑制剂与热休克蛋白90抑制剂有效的新型协同药物组合，研究计算机化药物组合基质筛选在ACC细胞中鉴定的协同作用的分子机制。我们分析了公开可用的数据库，发现ACC过表达几种HSP90亚型及其客户参与ACC的起始和进展。我们在细胞增殖试验中验证了两种ACC细胞系的协同作用。ACC 3D肿瘤球体测定显示，与单药组相比，联合治疗组的疗效更高。由于PIK75尚未用于临床试验，我们在单层培养中确定并验证了BGT226与STA9090或与HSP990之间的协同作用。在临床可达到的浓度下，BGT226与一种HSP90抑制剂联合使用在ACC 3D肿瘤球体、克隆性试验和侵袭/迁移试验中比单药组更有效。我们发现PIK75和BGT226细胞死亡的协同机制完全不同。PIK75联合诱导G2M细胞周期阻滞，随后出现caspase-3/7依赖性凋亡。然而，BGT226和STA9090联合使用不会引起细胞周期阻滞，也不会引起细胞凋亡。我们进行了RNA-seq并比较了这些组合处理的ACC细胞的基因表达谱，发现BGT226组合诱导了自噬相关的基因和途径，并在蛋白水平上得到了验证。我们目前正在确定诱导自噬是细胞死亡的机制还是对治疗的抗性机制。ACC异种移植物的体内疗效尚在研究中。2. ACC中放射增敏剂及氧化应激反应的鉴定。目的：探讨金糠蛋白联合放疗（RT）治疗ACC的疗效及作用机制。我们从两种ACC细胞系的定量高通量药物筛选中鉴定出金糠蛋白是一种具有强细胞毒性作用的新型放射增敏剂。Auranofin是一种经fda批准的基于金颗粒的低毒性类风湿性关节炎药物。通过对ACC样本独立数据库的分析，我们发现ACC中有几个参与氧化应激反应的基因mrna存在差异表达，包括被金嘌呤靶向的TXNRD通路。TXNRD是TXN的促生存还原酶。此外，ACC中TXNRD1 mRNA低表达的患者总生存期和无病生存期（OS和DFS）较短。我们发现，一些抗氧化应激相关基因的下调与MKi67和CCNB1（不良预后标志物）的过表达呈负相关。我们证实了金糠蛋白和RTin单层培养物的协同作用。在临床上可达到的浓度下，在三维ACC球体和两种细胞系的克隆测定中，金糠蛋白和RT联合治疗比单独治疗更有效。在初步研究优化给药方案后，体内试验将很快开始。我们证实了有效浓度抑制ACC中硫氧还蛋白还原酶的活性。我们证实，在两种细胞系中，联合处理分别诱导更高水平的gH2AX，激活p-CHK1，并诱导p-BRCA1，这与双链DNA断裂和修复一致。我们观察到NCI-H295R（激活CTNNB1突变的皮质醇产生细胞）比SW-13 （SMARCA4突变，非类固醇产生细胞系）对RT和金糠蛋白更敏感。我们在NCI-H295R细胞中观察到裂解的PARP，而在SW13细胞中，我们没有观察到裂解的caspase或裂解的PARP。在SW-13细胞中，auranofin和RT诱导了p21和p27，但NCI-H295R细胞中没有。为了进一步探讨其在两种细胞系中的作用机制，我们发现，与NCI-H295R相比，SW-13有效过表达NRF2和KEAP1核易位以及TXNRD1和HMOX1，表明SW-13具有更强的抗氧化应激反应。初步资料显示，联合用药可引起NCI-H295R细胞的有丝分裂突变，但对SW-13细胞无影响。由于HMOX1的强烈诱导和GPX4的减少，我们评估了铁下垂。我们在两种细胞系中证实，金糠蛋白和RT诱导谷胱甘肽氧化应激，并通过n -乙酰半胱氨酸修复。然而，我们没有观察到当NCI-H295R细胞用金烷fin和RT处理时，脂质过氧化增加，这是铁死亡的标志，但我们证实了联合处理诱导了w -13的铁死亡。我们排除了使用各种治疗方法的皮质类固醇在ACC放射敏感性中的作用。大量rna测序以了解参与治疗反应的基因和途径正在进行中。3. 肿瘤微环境（tumor microenvironment， TME）中tnf -a相关变化促进药物传递的机制研究现状：稿件准备正在进行中。目的：阐明TNF-a降低肿瘤间质压力，提高给药效率的作用机制。1 .小结：由于TNF-a对肿瘤内几种细胞外基质蛋白和细胞因子的表达和功能的影响以及这些蛋白和细胞因子对肿瘤内血管通透性的作用尚不完全清楚。利用ECM、人基质金属蛋白酶（MMP）阵列和TNF-a治疗的碘耐药甲状腺癌细胞的基因表达谱，我们发现并验证了TNF-a下调TGFb和LOX，上调MMPs、TIMP1和ITGB3，它们在TME的血管通透性中起作用。silox治疗的甲状腺癌异种移植物再现了肿瘤内血管渗漏，类似于TNF-a引起的异种移植物。接下来，我们验证了TNF-a对血管内皮细胞的作用，发现TNF-a治疗下调了TGFb1、Lox和VEGF-R（血管通透性的关键调节因子）。为了证实重组TNF-a和TGFb抑制治疗的疗效，我们将TME与紫杉醇联合治疗2个耐碘低分化（PDTC） （TPC1）和间变性甲状腺癌（ATC） （8505C） 3D肿瘤球体，发现联合治疗组的抗肿瘤疗效高于单一治疗组。此外，我们证明，当甲状腺癌球体用TGFb抑制剂和LOX抑制剂（siLOX和BAPN抑制LOX酶活性）治疗时，荧光标记的紫杉醇在肿瘤内的渗透性更高，瘤内紫杉醇浓度比仅使用紫杉醇组高几倍。我们使用HPLC验证了携带TNF-a和TGFb抑制剂的纳米金颗粒处理8505C异种移植物，增强了肿瘤内紫杉醇的体内递送。为了研究TGFb和LOX在肿瘤间质液压力升高中的作用，我们在TCGA甲状腺癌数据库中确定了它们与透明质酸（透明质酸合成酶1 (HAS1)）合成之间的相关性，并通过TGFb抑制剂和siLOX在体外治疗甲状腺癌细胞验证了这一发现。在密度更大的甲状腺癌球体与癌相关成纤维细胞共培养中，药物递送效率和治疗效果也有类似的改善。

项目成果

Do patients with familial nonmedullary thyroid cancer present with more aggressive disease? Implications for initial surgical treatment.

• DOI: 10.1016/j.surg.2018.05.075
• 发表时间: 2019-01
• 期刊: Surgery
• 影响因子: 3.8
• 作者: El Lakis M;Giannakou A;Nockel PJ;Wiseman D;Gara SK;Patel D;Sater ZA;Kushchayeva YY;Klubo-Gwiezdzinska J;Nilubol N;Merino MJ;Kebebew E
• 通讯作者: Kebebew E

ONCOGENE PANEL SEQUENCING ANALYSIS IDENTIFIES CANDIDATE ACTIONABLE GENES IN ADVANCED WELL-DIFFERENTIATED GASTROENTEROPANCREATIC NEUROENDOCRINE TUMORS.

癌基因组测序分析确定了晚期分化良好的胃肠胰腺神经内分泌肿瘤中的候选作用基因。

• DOI: 10.4158/ep-2018-0603
• 发表时间: 2019
• 期刊: Endocrine practice : official journal of the American College of Endocrinology and the American Association of Clinical Endocrinologists
• 作者: Tirosh,Amit;Killian,JKeith;Zhu,YuelinJack;Petersen,David;Walling,Jennifer;Mor-Cohen,Ronit;Neychev,Vladimir;Stevenson,Holly;Keutgen,XavierM;Patel,Dhaval;Nilubol,Naris;Meltzer,Paul;Kebebew,Electron
• 通讯作者: Kebebew,Electron

Markers of Systemic Inflammatory Response are Prognostic Factors in Patients with Pancreatic Neuroendocrine Tumors (PNETs): A Prospective Analysis.

• DOI: 10.1245/s10434-017-6241-4
• 发表时间: 2018-01
• 期刊: Annals of surgical oncology
• 影响因子: 3.7
• 作者: Gaitanidis A;Patel D;Nilubol N;Tirosh A;Sadowski S;Kebebew E
• 通讯作者: Kebebew E

Incidence and management of postoperative hyperglycemia in patients undergoing insulinoma resection.

• DOI: 10.1007/s12020-018-1633-1
• 发表时间: 2018-09
• 期刊: Endocrine
• 影响因子: 3.7
• 作者: Nockel P;Tirosh A;El Lakis M;Gaitanidis A;Merkel R;Patel D;Nilubol N;Sadowski SM;Cochran C;Gorden P;Kebebew E
• 通讯作者: Kebebew E

Prognosis after surgery for multiple endocrine neoplasia type 1-related pancreatic neuroendocrine tumors: Functionality matters.

• DOI: 10.1016/j.surg.2020.09.037
• 发表时间: 2021-04
• 期刊: SURGERY
• 影响因子: 3.8
• 作者: van Beek, Dirk-Jan;Nell, Sjoerd;Verkooijen, Helena M.;Rinkes, Inne H. M. Borel;Valk, Gerlof D.;Vriens, Menno R.
• 通讯作者: Vriens, Menno R.