Increasing Effects of Interferons for Melanoma

必须为手术后的高风险患者和转移性疾病患者确定消除黑色素瘤的新疗法。干扰素（ifn）在两种临床环境中都具有治疗活性，但对黑色素瘤或其他恶性肿瘤的临床疗效机制的了解却很少。潜在的临床抗肿瘤作用必须是在转录水平上发生的强效和多效基因调节作用。利用寡核苷酸阵列分析，已经确定了黑色素瘤细胞系中由ifn诱导的100多个基因。在等摩尔的基础上，这些基因被ifn - β诱导的程度比被ifn - α 2诱导的程度大得多。基于IFNs对黑色素瘤的抗肿瘤作用可能是直接作用于肿瘤细胞的假设，我们的研究重点是IFNs对肿瘤细胞的凋亡作用。细胞凋亡的诱导，加上阵列研究，导致蛋白质TRAIL （tnf相关凋亡诱导配体）成为部分解释黑色素瘤细胞死亡的机制。在敏感的黑色素瘤细胞系中，ifn - β，而非IFN-alpha2，诱导与TRAIL相关的caspase依赖性凋亡。耐药黑色素瘤细胞系的特点是缺乏IFN- α 2或IFN- β的增殖抑制或凋亡诱导，以及缺乏TRAIL诱导。这些数据进一步表明，TRAIL激活NFkappaB（核因子κ B）对细胞凋亡的诱导有负面影响。为了了解ifn作为抗肿瘤细胞因子的作用，并以黑色素瘤为模型，我们的工作假设是，尚未确定的基因和功能机制在很大程度上促进了ifn的抗肿瘤作用。我们基于基因组大小的估计表明，仍有超过500个干扰素刺激基因（isg）有待鉴定。该提案的目标是：1)通过对敏感和耐药黑色素瘤的评估，发现可能有助于介导ifn诱导的凋亡反应和耐药的新基因，2)确定和确认新发现的ISG TRAIL对ifn诱导的凋亡的功能作用，3)确定NF- kappaB或其他诱导的抗凋亡途径如何影响ifn和TRAIL的作用，4)作为小鼠和TRAIL的有效诱导因子，开展ifn - β的II期试验，以确定转移性黑色素瘤的疾病反应。这些数据将有助于了解ifn在黑色素瘤和其他肿瘤中的作用，并有助于初步了解TRAIL的治疗潜力。

New therapies to eliminate melanoma must be identified both for the high-risk patient after surgery and for metastatic disease. Interferons (IFNs) have therapeutic activity in both clinical settings but little expansion has occurred in understanding of mechanisms underlying clinical effectiveness in melanoma -- or indeed other malignancies. Underlying clinical antitumor effects must be the potent and pleiotropic gene modulatory effects that occur at a transcriptional level. Utilizing oligonucleotide array analysis, over 100 genes that are induced by IFNs in a melanoma cell line have been identified. On an equimolar basis these genes are induced to a substantially greater extent by IFN-beta than by IFN-alpha2. Based upon the hypothesis that the antitumor effects of IFNs in melanoma may result from direct effects on tumor cells, apoptosis, an understudied effect of IFNs, is a focus of our studies. Induction of apoptosis, coupled with array studies, led to the protein TRAIL (TNF-Related Apoptosis Inducing Ligand) as a mechanism that in part accounts for melanoma cell death. In sensitive melanoma cell lines, IFN-beta, but not IFN-alpha2, induces caspase dependent apoptosis that was associated with TRAIL. Resistant melanoma cell lines are characterized by lack of proliferative inhibition or apoptosis induction by either IFN- alpha2 or IFN-beta and lack of TRAIL induction. The data further suggest that apoptosis induction is negatively influenced by NFkappaB (Nuclear Factor kappa B) activation by TRAIL. With a goal of understanding of IFNs as antitumor cytokines and using melanoma as a model, our working hypothesis is that genes and functional mechanisms, yet to be identified, contribute substantially to the antitumor effects of IFNs. Our estimates based upon genome size suggest that greater than 500 interferon- stimulated genes (ISGs) remain to be identified. The goals of the proposal are: 1) By assessment of sensitive and resistant melanomas to identify new genes that may contribute to mediating apoptosis response and resistance in response to IFNs, 2) To define and confirm functional effects of the newly identified ISG, TRAIL, on IFN-induced apoptosis, 3) To determine how NF- kappaB or other induced anti-apoptotic pathways influence actions of IFNs and TRAIL, and 4) As an effective inducer of of apoptosis in mice and of TRAIL, to conduct a Phase II trial of IFN-beta to identify disease response in metastatic melanoma. The data should contribute to understanding of IFNs effects in melanoma but also other neoplasms and to a beginning understanding of the therapeutic potential of TRAIL.