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

本研究的总体目标是识别和表征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，其与某些基因的转录启动子上的特定序列结合并驱动其转录。我们在1989年观察到（伯恩斯坦和科尔本，Science，1989）转化敏感（P+）而非转化抗性（P-）小鼠JB 6细胞通过激活AP-1依赖性转录对肿瘤促进剂作出反应，这表明AP-1激活可能是从癌前发展到肿瘤（肿瘤）表型所必需的。通过观察到显性负性jun（TAM 67）的表达阻断了小鼠JB 6细胞中AP-1的活化和转化反应，确定了AP-1依赖性基因转录的需要（Dong等PNAS，1994）。此外，表达K14-TAM 67转基因的转基因小鼠显示出90%的AP-1活化抑制和90%的防止促进皮肤癌发生的保护，即，预防癌前乳头状瘤形成（Young等，PNAS，1999）。这种肿瘤发生的抑制没有毒性或抑制对肿瘤促进剂的增生反应的证据。转基因小鼠中显性负性Jun的角质形成细胞特异性表达抑制诱导的AP-1和肿瘤发生，而不抑制与人类致癌相关的多种小鼠模型中的生长、分化或增生。其中包括通过表达人乳头瘤病毒E7而提高皮肤肿瘤促进反应的小鼠（Young et al Molec Carc 2002）、通过磷酸酶抑制剂冈田酸促进的小鼠（Thompson et al Cancer Res 2002）和通过反复暴露于UVB而诱导形成鳞状细胞癌的小鼠（库珀et al提交）。对于进一步的研究，四环素调节的TAM 67表达在我们的实验室中被定向到皮肤和子宫颈，在合作者Powel Brown和Jay Tischlaar的实验室中被定向到乳腺和肺上皮。当转基因K14-TAM 67或四环素调节的TAM 67构建体在更进展的人细胞系中表达时，肿瘤细胞表型被抑制，所述人细胞系是致瘤性或锚定非依赖性的，并且显示出升高的AP-1和NF κ B活性（Li et al.，Oncogene，1998和Li等人，Molec Carcinog 2000）。转录因子NF κ B与AP-1协同调节，表明两种因子在转化中的可能重要性（Li et，Cancer Res 1997）。最近的观察发现NFkB无反应性可以解释JB 6模型中转化无反应性（Hsu et al，Cancer Res 2001）。P-细胞的转化无反应性归因于不能激活NF κ B p65蛋白. p65活化的缺乏又可归因于不能活化AP-1依赖性转录。因此，靶向AP-1和NFkB升高防止肿瘤促进和进展的观察结果已经从小鼠JB 6模型扩展到小鼠和人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认识正在出现。小鼠JB 6细胞、人角质形成细胞和表达AP-1/ NFkB抑制剂TAM 67的转基因小鼠为鉴定AP-1或NFkB靶基因提供了宝贵的机会，这些靶基因的表达对肿瘤转化至关重要。表达微阵列分析揭示了有限数量的潜在TAM靶基因，是目前研究的主题。这些靶基因可能成为癌症预防的新分子靶点。 JB 6P-变体之一的转化抗性可归因于MAP激酶Erks 1和2的缺乏，这也限制了AP-1的反式激活（Huang等人，PNAS，1998）。显性失活Erk 2在AP-1应答细胞中的表达已经证明AP-1活化和转化应答需要Erk活性（Watts等1998）。目前针对鉴定活化AP-1所需的Erk依赖性分子事件的研究已经鉴定Fos家族蛋白Fra-1的活化为关键事件（Young等，Molec Cell Biol 2002）。Erk或Fra-1蛋白缺陷的JB 6变体是AP-1和转化无响应的。分别通过Erk或Fra-1的表达恢复反应性。预期AP-1依赖性基因启动子的子集需要Fra-1用于转录激活。

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.