THE ROLE OF AP-1 AND OTHER TRANSCRIPTION FACTORS IN CANCER CAUSE AND PREVENTION

本研究的总体目的是确定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，与特定基因的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+）而非转化抗性（P-）小鼠JB6细胞通过激活AP-1依赖性转录对肿瘤启动子有反应，这表明AP-1激活可能是肿瘤前表型向肿瘤表型发展所必需的。对这一假设的检验表明，药理学抑制剂、糖皮质激素和类维生素a以及基因治疗抑制剂dominant negative jun （TAM67）阻断了AP- 1的激活和转化反应（Dong et al PNAS, 1994）。这项研究已经扩展到表明，在不反激活视黄酸反应元件（RARE）依赖基因转录的情况下，转抑AP- 1活性的特定类维生素a也能阻止肿瘤转化（Li等人，Cancer Res, 1996）。令人惊讶的是，TPA诱导，而不是肿瘤坏死因子α诱导，AP-1对类维生素a的转抑制很敏感（Li et, Cancer Res 1997）。目前的研究旨在确定类维生素a调节AP-1转抑制的分子相互作用。由角蛋白14（K14）启动子驱动的显性负jun突变体（TAM67）转基因在小鼠角化细胞系中表达时，可抑制AP-1和NFkB转录因子的活性，并诱导其侵入基质（Dong et al., Molec）。Carcinog。，1997)，提示第二种转录因子NFkB在导致和预防进展方面可能具有重要意义。AP-1和NFkB活性和DNA结合在人角化细胞进展模型中显示进行性升高。当转基因K14-TAM67或四环素调控的TAM结构在更进展的致瘤性或不依赖于锚定的人类细胞系中表达时，肿瘤细胞表型被抑制（Li et al., Oncogene, 1998 and Li et al.，提交）。当小鼠JB6 P+细胞在裸鼠移植床上生长时，TAM67在小鼠JB6 P+细胞中的表达使细胞表型恢复到P-表型（Strickland et al., Carcinog. 1997）。已经产生了表达K14-TAM67基因的转基因小鼠。在dba - tpa启动-促进皮肤癌变实验中，这些K14-TAM 67小鼠显示出90%的AP-1激活抑制和95%的防止皮肤癌变的保护作用，即预防癌前乳头状瘤的形成（Young等，PNAS, 1999）。没有证据表明肿瘤启动子对增生反应有毒性或抑制作用。最近，我们发现JB6 P-变体的转化抗性是由于MAPK激酶Erks 1和2的缺乏，这也限制了AP-1的转激活（Huang等人，PNAS, 1998）。AP-1应答细胞中显性负Erk 2的表达证明了Erk对AP-1激活和转化应答的要求（Watts et al . 1998）。目前的研究旨在确定激活AP-1所需的Erk依赖分子事件。最近的观察发现NFkB无响应性是JB6模型中转化无响应性的一种解释（Hsu等人，已提交）。因此，针对AP-1和NFkB的升高可以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角质细胞进展模型，以及移植和转基因小鼠模型。对限制分子相互作用的新认识正在出现。人角质形成细胞和表达AP-1抑制剂TAM 67的转基因小鼠都为鉴定AP-1靶基因提供了宝贵的机会，这些基因的表达对肿瘤转化至关重要。-癌症成因，癌症预防，基因调控，转录因子，AP-1， MAPK, NFkappaB，

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. Testing of this hypothesis revealed that the pharmacologic inhibitors, glucocorticoids and retinoids and the gene therapy inhibitor dominant negative jun (TAM67) blocked both AP- 1 activation and transformation response (Dong et al PNAS, 1994). This has been extended to show that specific retinoids that transrepress AP- 1 activity without transactivating retinoic acid response element (RARE) dependent gene transcription, also prevent neoplastic transformation (Li et al., Cancer Res, 1996). Surprisingly TPA induced, but not tumor necrosis factor alpha induced, AP-1 is sensitive to transrepression by retinoid(Li et, Cancer Res 1997). Current research is directed to defining the molecular interactions that regulate AP-1 transrepression by retinoids. The dominant negative jun mutant(TAM67) transgene driven by a keratin 14(K14)promoter, when expressed in a mouse keratinocyte line suppressed both AP-1 and NFkB transcription factor activities as well as induced invasion into matrigel (Dong et al., Molec. Carcinog.,1997)suggesting the possible importance of a second transcription factor NFkB in both the cause and prevention of progression. Both AP-1 and NFkB activities and DNA binding show progressive elevation in a human keratinocyte progression model. When the transgene K14-TAM67 or a tetracycline regulated TAM construct was expressed in the more progressed human cell lines that are tumorigenic or anchorage independent, tumor cell phenotype was suppressed (Li et al., Oncogene, 1998 and Li et al., submitted). Expression of TAM67 in mouse JB6 P+ cells produced phenotypic reversion to P- phenotype when cells were grown on a nude mouse graft bed (Strickland et al., Carcinog. 1997). Transgenic mice expressing the K14-TAM67 transgene have been generated. In DMBA-TPA initiation-promotion skin carcinogenesis experiments these K14-TAM 67 mice show 90% inhibition of AP-1 activation and 95% protection against promotion of skin carcinogenesis, i.e., prevention of premalignant papilloma formation(Young et al., PNAS, 1999). There is no evidence of toxicity or inhibition of the hyperplasia respons to tumor promoter. Recently we have found that the transformation resistance of a JB6 P- variant is due to a shortage of the MAPK 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 for AP-1 activation and transformation responses (Watts et al 1998). Current research is directed to identifying Erk dependent molecular events that are required for activating AP-1. Recent observations have identified NFkB non-responsiveness as an explanation for transformation non- responsiveness in the JB6 model(Hsu et al, submitted). 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 grafting and transgenic mouse models. New understanding of limiting molecular interactions is emerging. Both the human keratinocytes and the transgenic mice expressing AP-1 inhibitor TAM 67 present valuable opportunities to identify AP-1 target genes whose expression is critical to neoplastic transformation. - Cancer Cause, Cancer Prevention, Gene regulation, Transcription Factors, AP-1, MAPK, NFkappaB,