KLF9-TXNRD2 axis in melanoma progression and metastasis

 描述(申请人提供)：恶性黑色素瘤是人类最具侵袭性的癌症类型之一。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。黑色素瘤发生的分子机制尚不清楚，目前也没有有效的治疗方法。黑色素瘤通常起源于色素痣，这是一种良性的衰老黑素细胞聚集体，含有BRAF和NRAS基因的激活突变。BRAFV600E或NRASQ61R在正常人类黑素细胞(NHM)和小鼠模型中诱导衰老(称为癌基因诱导衰老(OIS))中的因果作用已被证明。这些癌基因在NHM中的异位表达导致了活性氧物种(ROS)的积累，而ROS的化学抑制则阻止了衰老。通过下调过量的ROS来抑制NHM中的OIS的内源性机制尚未被研究。转移是黑色素瘤最有害的特征。癌细胞从细胞外基质中分离出来通常会导致一种特殊形式的细胞凋亡，称为失巢凋亡。侵袭、非锚定生长和消除失巢细胞是肿瘤转移的关键步骤。一些独立的研究表明，ROS水平的增加促进了失巢失眠，并抑制了其他转化的表型。通过调节黑色素瘤细胞内ROS来控制这些过程的机制在很大程度上还不清楚。肿瘤通常会发展出适应性机制来抑制高水平的ROS。在多种恶性肿瘤中发现的最常见的一种现象是NRF2的数量增加，NRF2是抗氧化反应基因的普遍调节因子。黑色素瘤中也显示出NRF2的增加。最近，我们发现在高氧化应激条件下，NRF2反常地放大ROS。众所周知，ROS的增加导致NRF2在细胞核内积聚，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抑制几个非NRF2靶点的抗氧化基因，包括线粒体硫氧还蛋白还原酶(TXNRD2)，从而导致ROS扩增。TXNRD2蛋白是维持细胞内红牛状态和ROS解毒的关键蛋白。我们证明了TXNRD2缺失会产生ROS，而其过表达则会降低KLF9产生的ROS。因此，KLF9的缺失抑制了所有受试细胞系的氧化应激。因此，KLF9下调或TXNRD2上调对于氧化应激(阻碍肿瘤进展)压倒NRF2依赖的抗氧化防御的恶性肿瘤可能是有益的。根据TCGA数据库，高KLF9或低TXNRD2基因水平与淋巴结转移患者的总体生存相关(黑色素瘤患者预后的主要决定因素)，表明这两个基因在黑色素瘤进展中的重要性。此外，我们还证实了KLF9直接调节黑素细胞中的TXNRD2。联合治疗是克服对BRAFV600E抑制剂维莫拉非尼(Vemu)耐药性的关键。一些独立的报告将氧化磷酸化的激活(伴随着氧化应激)描述为对黑色素瘤细胞中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批准的抗风湿药物)与Vemu在诱导BRAFV600E黑色素瘤细胞死亡方面具有协同作用。此外，在野生型和突变型BRAF黑色素瘤细胞中，AUR作为单一药物在导致细胞死亡方面同样有效。这一发现很重要，因为目前还没有针对野生型BRAF黑色素瘤患者的有效治疗方法。这项拟议的研究将建立KLF9-TXNRD2轴调节正常黑素细胞OIS和黑色素瘤细胞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，因为抑制OIS是肿瘤发生的先决条件，而抑制失巢细胞、增加侵袭和非锚定生长是转移的先决条件，而转移是癌症最有害的特征。此外，将抗风湿剂Auranofin用于治疗恶性黑色素瘤可能会显著改善这种非常侵袭性疾病的治疗。

 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.