KLF9-TXNRD2 axis in melanoma progression and metastasis

 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。它的转移能力使黑色素瘤极难治愈，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未完全了解，目前还没有有效的治疗方法。黑色素瘤通常起源于痣，痣是在BRAF、NRAS基因中携带激活突变的衰老黑色素细胞的良性聚集体。BRAFV 600 E或NRASQ 61 R在诱导衰老（称为癌基因诱导的衰老（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。这些癌基因在NHM中的异位表达导致活性氧（ROS）的积累，而ROS的化学抑制消除衰老。尚未研究通过下调过量ROS抑制NHM中OIS的内源性机制。转移是黑色素瘤最有害的特征。癌细胞与细胞外基质的分离通常会导致一种特定形式的细胞凋亡，称为失巢凋亡。侵袭、非锚定依赖性生长和失巢凋亡的消除是转移的关键步骤。一些独立的研究表明，ROS水平的增加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是未知的。肿瘤通常会产生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常见的一种是NRF 2的量增加，NRF 2是抗氧化反应基因的通用调节剂。在黑色素瘤中也显示出NRF 2的增加。最近，我们发现，矛盾的是，在高氧化应激的条件下，NRF 2放大ROS。众所周知，ROS的增加导致NRF 2在细胞核中的积累和抗氧化基因的激活。然而，如果ROS继续升高并超过临界阈值，则NRF 2诱导转录因子KLF 9（Kruppel样因子9）的表达。 KLF 9抑制几种抗氧化基因，这些基因不是NRF 2靶基因，包括线粒体硫氧还蛋白还原酶（TXNRD 2），从而导致ROS扩增。TXNRD 2蛋白是维持细胞内红氧状态和ROS解毒的关键。我们证明了TXNRD 2缺失会产生ROS，而其过表达会降低KLF 9产生的ROS。因此，KLF 9的耗竭抑制所有测试细胞系中的氧化应激。因此，KLF 9下调或TXNRD 2上调可能对氧化应激（阻碍肿瘤进展）破坏NRF 2依赖性抗氧化防御的恶性肿瘤有益。根据TCGA数据库，高KLF 9或低TXNRD 2 mRNA水平与淋巴结转移患者的总生存期相关（黑色素瘤患者结局的主要决定因素），表明两种基因对黑色素瘤进展的重要性。此外，我们证实KLF 9直接调节黑素细胞中的TXNRD 2。联合治疗对于克服对BRAFV 600 E抑制剂维罗非尼（VEMU）的耐药性至关重要。几份独立的报告将氧化磷酸化（伴随氧化应激）的活化表征为黑色素瘤细胞中对BRAFV 600 E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批准的抗风湿药物）协同VEMU诱导BRAFV 600 E-黑色素瘤细胞的细胞死亡。此外，AUR在野生型和突变型BRAF黑色素瘤细胞中作为单一药剂引起细胞死亡方面同样有效。这一发现很重要，因为野生型BRAF黑色素瘤患者没有有效的治疗方法。 该研究将建立KLF 9-TXNRD 2轴调节正常黑素细胞中OIS和黑色素瘤细胞中多种转化表型的机制。我们的发现将具有广泛的科学吸引力，因为OIS的抑制是肿瘤发生的先决条件，而失巢凋亡的抑制、侵袭的增加和锚定非依赖性生长是转移的先决条件，转移是癌症最有害的特征。此外，将抗风湿药金诺芬用于治疗恶性黑色素瘤可能会显着改善这种非常侵袭性疾病的管理。

 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.