The Role of AP-1 and Other Transcription Factors in Canc

这项研究的总体目标是识别和描述在肿瘤促进和肿瘤进展过程中推动限速步骤的基因调控事件。AP-1转录因子是Jun和Fos家族蛋白的异源二聚体，与某些基因转录启动子上的特定序列结合并驱动其转录。我们1989年的观察(Bernstein和Colburn，Science，1989)发现，转化敏感(P+)而不转化抵抗(P-)的小鼠JB6细胞通过激活AP-1依赖的转录来响应肿瘤促进剂，这表明AP-1的激活可能是从癌前表型发展到肿瘤(肿瘤)表型所必需的。通过观察到显性负性Jun(TAM67)的表达抑制了小鼠JB6细胞中AP-1的激活和转化反应，确立了AP-1依赖基因转录的要求(董氏等人，1994)。此外，表达K14-TAM67转基因基因的转基因小鼠显示出90%的AP-1活性抑制和90%的保护作用，防止皮肤癌变的促进，即防止癌前乳头状瘤的形成(Young等人，PNAS，1999)。这种对肿瘤形成的抑制是在没有毒性或抑制对肿瘤促进剂的增殖反应的情况下发生的。转基因小鼠角质形成细胞特异性表达显性阴性Jun抑制AP-1诱导的肿瘤形成，而不抑制与人类致癌相关的多种小鼠模型的生长、分化或增殖。其中，皮肤肿瘤促进反应因人类乳头瘤病毒E7的表达而升高的小鼠(Young等人Molec Carc 2002)，由磷酸酶抑制剂冈田酸促进的小鼠(Thompson等人的癌症RES 2002)，以及通过反复暴露于UVB而诱导形成鳞癌的小鼠(Cooper等人提交)。为了进一步研究，四环素调控的67在我们实验室的皮肤和宫颈，以及合作者鲍威尔·布朗和杰伊·蒂施拉尔的实验室中的乳腺和肺上皮细胞中的表达。当转基因K14-TAM67或四环素调控的TAM67结构在更先进的致瘤或锚定非依赖且显示AP-1和NFkB活性升高的人类细胞系中表达时，肿瘤细胞的表型被抑制(Li等人，Oncogene，1998和Li等人，Molec Cancog2000)。转录因子NFkappa B与AP-1协调调节，表明这两种因子在转化中可能具有重要作用(Li et，Cancer res 1997)。最近的观察发现，NFkB无反应性是JB6模型中转化无反应性的一个解释(Hsu等人，癌症研究报告2001)。P细胞的转化无反应归因于不能激活NFkappa B p65蛋白。缺乏p65的激活又归因于无法激活AP-1依赖的转录。因此，以AP-1和NFkB的上调为靶点阻止肿瘤的促进和进展的观察已经从小鼠JB6模型扩展到小鼠和人类角质形成细胞进展模型，以及转基因小鼠模型。对关键分子相互作用的新理解正在出现。小鼠JB6细胞、人角质形成细胞和表达AP-1/NFkB抑制因子67的转基因小鼠为寻找在肿瘤转化中起关键作用的AP-1或NFkB靶基因提供了宝贵的机会。表达芯片分析揭示了目前研究对象的有限数量的潜在靶基因。这些靶基因可能是癌症预防的新分子靶点。 JB6 P变异体之一的转化抗性是由于缺乏MAP-1和ERKS 1和2，这也限制了AP-1的反式激活(Huang，et al，PNAS，1998)。AP-1反应细胞中显性负性Erk2的表达表明ERK活性对AP-1的激活和转化反应是必需的(Watts等人，1998)。目前针对确定激活AP-1所需的Erk依赖的分子事件的研究已经确定Fos家族蛋白Fra-1的激活是一个关键事件(Young等人，Molec Cell Biol 2002)。缺乏Erk或Fra-1蛋白的JB6变异体为AP-1和转化无反应。ERK和Fra-1的表达分别恢复了细胞的反应性。AP-1依赖的基因启动子的一个子集预计需要Fra-1进行转录激活。

The overall aim of this research is to identify and characterize gene regulation events that propel rate-limiting steps during tumor promotion and tumor progression. The AP-1 transcription factor is a heterodimer of Jun and Fos family proteins that binds to a specific sequence on the transcriptional promoter of certain genes and drives their transcription. Our 1989 observation (Bernstein and Colburn, Science, 1989) that transformation sensitive (P+) but not transformation resistant (P-) mouse JB6 cells responded to tumor promoters by activating AP-1 dependent transcription, suggested that AP-1 activation might be required for progression from preneoplastic to neoplastic (tumor) phenotype. A requirement for AP-1 dependent gene transcription was established by the observation that expression of dominant negative jun (TAM67) blocked both AP-1 activation and transformation response in mouse JB6 cells (Dong et al PNAS, 1994). Furthermore transgenic mice expressing the K14-TAM67 transgene show 90% inhibition of AP-1 activation and 90% protection against promotion of skin carcinogenesis, i.e., prevention of premalignant papilloma formation (Young et al., PNAS, 1999). This inhibition of tumorigenesis occurred without evidence of toxicity or inhibition of the hyperplasia response to tumor promoter. Keratinocyte-specific expression of Dominant Negative Jun in transgenic mice inhibits induced AP-1 and tumorigenesis without inhibiting growth, differentiation or hyperplasia in multiple mouse models relevant to human carcinogenesis. Among these are mice whose skin tumor promotion response is elevated by expression of Human Papilloma Virus E7 (Young et al Molec Carc 2002), mice promoted by phosphatase inhibitor okadaic acid (Thompson et al Cancer Res 2002), and mice induced to form squamous carinomas by repeated exposure to UVB (Cooper et al submitted). For further studies, tetracycline regulated expression of TAM 67 is being directed to skin and cervix in our laboratory, and to mammary and lung epithelia in the laboratories of collaborators Powel Brown and Jay Tischlaar. When the transgene K14-TAM67 or a tetracycline regulated TAM67 construct was expressed in the more progressed human cell lines that are tumorigenic or anchorage independent and show elevated AP-1 and NFkB activities, tumor cell phenotype was suppressed (Li et al., Oncogene, 1998 and Li et al., Molec Carcinog 2000). The transcription factor NFkappa B is coordinately regulated with AP-1, suggesting the possible importance of both factors in transformation (Li et, Cancer Res 1997). Recent observations have identified NFkB non-responsiveness as an explanation for transformation non-responsiveness in the JB6 model (Hsu et al, Cancer Res 2001). P- cells owe their transformation nonresponsiveness to an inability to activate NFkappa B p65 protein. Lack of p65 activation is in turn attributable to an inability to activate AP-1 dependent transcription. Thus the observation that targeting AP-1 and NFkB elevation prevents tumor promotion and progression has been extended from the mouse JB6 model to mouse and human keratinocyte progression models, and to transgenic mouse models. New understanding of critical molecular interactions is emerging. The mouse JB6 cells, the human keratinocytes and the transgenic mice expressing AP-1/ NFkB inhibitor TAM 67 present valuable opportunities to identify AP-1 or NFkB target genes whose expression is critical to neoplastic transformation. Expression microarray analysis has revealed a limited number of potential TAM target genes that are subjects of current research. Such target genes may be promising new molecular targets for cancer prevention. The transformation resistance of one of the JB6 P- variants is attributable to a shortage of the MAP kinases Erks 1 and 2 which are also limiting for AP-1 transactivation (Huang, et al, PNAS, 1998). Expression of dominant negative Erk 2 in AP-1 responsive cells has demonstrated the requirement of Erk activity for AP-1 activation and transformation responses (Watts et al 1998). Current research directed to identifying Erk dependent molecular events required for activating AP-1 has identified the activation of Fos family protein Fra-1 as a pivotal event (Young et al, Molec Cell Biol 2002). JB6 variants deficient in either Erk or Fra-1 proteins are AP-1 and transformation nonresponsive. Responsiveness is restored by expression of Erk or Fra-1 respectively. A subset of AP-1 dependent gene promoters is expected to require Fra-1 for transcriptional activation.