KLF9-TXNRD2 axis in melanoma progression and metastasis

KLF9-TXNRD2 轴在黑色素瘤进展和转移中的作用

• 批准号: 9108882
• 负责人: Mikhail Nikiforov
• 金额: $ 40.68万
• 依托单位: ROSWELL PARK CANCER INSTITUTE CORP
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-07-10 至 2020-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9108882
• 关键词: Accounting; Affect; Anchorage-Independent Growth; Anoikis; Antineoplastic Agents; Antioxidants; Antirheumatic Agents; Apoptosis; Auranofin; BRAF gene; Benign; Cell Death; Cell Line; Cell Nucleus; Cell Survival; Cells; Cessation of life; Chemicals; Clinical Trials; Data; Databases; Development; Disease; Down-Regulation; Drug Metabolic Detoxication; Ectopic Expression; Extracellular Matrix; FDA approved; Genes; Genotype; Goals; Health; Human; In Vitro; Knockout Mice; Kruppel-like transcription factors; Lung; Maintenance; Malignant Neoplasms; Melanocytic nevus; Melanoma Cell; Messenger RNA; Metastatic Melanoma; Metastatic Neoplasm to Lymph Nodes; Mitochondria; Modeling; Molecular; Mus; Mutation; NRAS gene; Neoplasm Metastasis; Nevus; Nuclear; Oncogenes; Outcome; Oxidative Phosphorylation; Oxidative Stress; Patients; Phenotype; Process; Production; Proteins; Publishing; Reactive Oxygen Species; Regulation; Reporting; Research; Resistance; Role; Staging; Testing; The Cancer Genome Atlas; Transcription Coactivator; Transgenes; Transgenic Mice; Tumor Suppressor Proteins; Tumorigenicity; Up-Regulation; Xenograft procedure; cancer cell; cancer type; drug development; effective therapy; improved; inhibitor/antagonist; killings; melanocyte; melanoma; mouse model; mutant; novel; novel therapeutics; overexpression; response; senescence; thioredoxin reductase; treatment strategy; tumor; tumor progression; tumorigenesis

 DESCRIPTION (provided by applicant): Malignant melanoma is one of the most aggressive types of human cancer. Its ability to metastasize makes melanoma extremely difficult to cure, and consequently, the median survival of patients with metastatic melanoma is only 8.5 months. The molecular mechanisms underlying melanoma development are not well understood, and effective treatment is not currently available. Melanomas often originate from nevi, benign aggregates of senescent melanocytes harboring activating mutations in BRAF, NRAS genes. The causal role of BRAFV600E or NRASQ61R in induction of senescence (termed oncogene-induced senescence (OIS)) has been demonstrated in normal human melanocytes (NHM) and mouse models. Ectopic expression of these oncogenes in NHM leads to accumulation of reactive oxygen species (ROS), whereas chemical suppression of ROS abrogates senescence. Endogenous mechanisms suppressing OIS in NHM via down-regulation of excessive ROS have not been investigated. Metastasis is the most detrimental feature of melanoma. Detachment of cancer cells from the extracellular matrix often causes a specific form of apoptosis, termed anoikis. Invasion, anchorage-independent growth and abrogation of anoikis are critical steps of metastasis. Several independent studies have suggested that increase in ROS levels promotes anoikis and suppresses other transformed phenotypes. Mechanisms controlling these processes via regulation of intracellular ROS in melanoma cells are largely unknown. Tumors often develop adaptive mechanisms to suppress high levels of ROS. The most general one detected in multiple malignancies is an increase in the amounts of NRF2, a universal regulator of antioxidant response genes. Increase in NRF2 has also been shown in melanomas. Recently, we have discovered that paradoxically under conditions of high oxidative stress NRF2 amplifies ROS. It is well known that increase in ROS causes accumulation of NRF2 in the nucleus and activation of anti-oxidant genes. However, if ROS continue to elevate and exceed a critical threshold, NRF2 induces expression of a transcription factor KLF9 (Kruppel-like factor 9). KLF9 represses several anti-oxidant genes, which are not NRF2 targets, including mitochondrial thioredoxin reductase (TXNRD2), thus resulting in ROS amplification. TXNRD2 protein is critical for maintenance of intracellular red-ox status and ROS detoxification. We demonstrated that TXNRD2 depletion generates ROS, whereas its overexpression lowers KLF9-generated ROS. Accordingly, depletion of KLF9 inhibits oxidative stress in all tested cell lines. Thus, KLF9 downregulation or TXNRD2 upregulation could be beneficial for the malignancies where oxidative stress (that hinders tumor progression) overwhelms NRF2- dependent anti-oxidant defense. According to the TCGA database, high KLF9 or low TXNRD2 mRNA levels correlate with the overall survival of patients with lymph node metastases (major determinants of outcome for melanoma patients), suggesting the importance of both genes for melanoma progression. Furthermore, we confirmed that KLF9 directly regulates TXNRD2 in melanocytic cells. Combinational treatment is critical for overcoming resistance to BRAFV600E inhibitor vemurafenib (VEMU). Several independent reports characterized activation of oxidative phosphorylation (which is accompanied by oxidative stress) as an adaptive response to BRAFV600E inhibition in melanoma cells. Our preliminary data demonstrate that oxidative stress-inducing agent AUR (a FDA-approved antirheumatic drug) synergizes with VEMU in inducing cell death in BRAFV600E-melanoma cells. Additionally, AUR is equally potent in causing cell death as a single agent in wildtype and mutant BRAF melanoma cells. This finding is important since no effective treatment exists for wildtype BRAF melanoma patients. The proposed research will establish the mechanisms by which KLF9-TXNRD2 axis regulates OIS in normal melanocytic cells and multiple transformed phenotypes in melanoma cells. Our findings will have broad scientific appeal since the suppression of OIS is a prerequisite for tumorigenesis, and suppression of anoikis, increase in invasion and anchorage-independent growth are prerequisites for metastasis, the most detrimental feature of cancer. Additionally, repurposing of anti-rheumatic agent auranofin for the treatment of malignant melanomas may significantly improve management of this very aggressive disease.

 描述（由申请人提供）：恶性黑色素瘤是最具侵袭性的人类癌症类型之一。其转移能力使得黑色素瘤极难治愈，因此，转移性黑色素瘤患者的中位生存期仅为8.5个月。黑色素瘤发展的分子机制尚不清楚，目前尚无有效的治疗方法。黑色素瘤通常起源于痣，即带有 BRAF、NRAS 基因激活突变的衰老黑色素细胞的良性聚集体。 BRAFV600E 或 NRASQ61R 在诱导衰老（称为癌基因诱导衰老 (OIS)）中的因果作用已在正常人黑素细胞 (NHM) 和小鼠模型中得到证实。 NHM 中这些癌基因的异位表达会导致活性氧 (ROS) 的积累，而化学抑制 ROS 则会消除衰老。尚未研究通过下调过量 ROS 抑制 NHM 中 OIS 的内源机制。转移是黑色素瘤最有害的特征。癌细胞从细胞外基质脱离通常会导致特定形式的细胞凋亡，称为失巢凋亡。侵袭、不依赖锚定的生长和失巢凋亡的消除是转移的关键步骤。几项独立研究表明，ROS 水平的增加会促进失巢凋亡并抑制其他转化表型。通过调节黑色素瘤细胞中的细胞内 ROS 来控制这些过程的机制在很大程度上尚不清楚。肿瘤通常会形成适应性机制来抑制高水平的 ROS。在多种恶性肿瘤中检测到的最普遍的现象是 NRF2 数量的增加，NRF2 是一种抗氧化反应基因的通用调节因子。 NRF2 的增加也在黑色素瘤中得到证实。最近，我们发现矛盾的是，在高氧化应激条件下，NRF2 会放大 ROS。众所周知，ROS的增加会导致NRF2在细胞核中积累并激活抗氧化基因。然而，如果 ROS 继续升高并超过临界阈值，NRF2 会诱导转录因子 KLF9（Kruppel 样因子 9）的表达。 KLF9 抑制多种抗氧化基因，包括线粒体硫氧还蛋白还原酶 (TXNRD2)，这些基因不是 NRF2 的靶标，从而导致 ROS 扩增。 TXNRD2 蛋白对于维持细胞内氧化还原状态和 ROS 解毒至关重要。我们证明，TXNRD2 缺失会产生 ROS，而其过表达会降低 KLF9 产生的 ROS。因此，KLF9 的消耗会抑制所有测试细胞系的氧化应激。因此，KLF9 下调或 TXNRD2 上调可能对氧化应激（阻碍肿瘤进展）压倒 NRF2 依赖性抗氧化防御的恶性肿瘤有益。根据 TCGA 数据库，高 KLF9 或低 TXNRD2 mRNA 水平与淋巴结转移患者的总体生存率（黑色素瘤患者预后的主要决定因素）相关，这表明这两种基因对黑色素瘤进展的重要性。此外，我们证实KLF9直接调节黑素细胞中的TXNRD2。联合治疗对于克服 BRAFV600E 抑制剂维莫非尼 (VEMU) 耐药性至关重要。几份独立报告将氧化磷酸化的激活（伴随氧化应激）描述为黑色素瘤细胞中对 BRAFV600E 抑制的适应性反应。我们的初步数据表明，氧化应激诱导剂 AUR（FDA 批准的抗风湿药）与 VEMU 协同作用，诱导 BRAFV600E 黑色素瘤细胞死亡。此外，AUR 作为单一药物在野生型和突变型 BRAF 黑色素瘤细胞中同样有效地导致细胞死亡。这一发现很重要，因为野生型 BRAF 黑色素瘤患者尚无有效的治疗方法。 拟议的研究将建立 KLF9-TXNRD2 轴调节正常黑色素细胞中的 OIS 和黑色素瘤细胞中多种转化表型的机制。我们的研究结果将具有广泛的科学吸引力，因为抑制 OIS 是肿瘤发生的先决条件，而抑制失巢凋亡、增加侵袭和不依赖贴壁的生长是转移（癌症最有害的特征）的先决条件。此外，重新利用抗风湿药金诺芬治疗恶性黑色素瘤可能会显着改善这种侵袭性疾病的治疗。