The Role of AP-1 and Other Transcription Factors in Cancer Cause and Prevention

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。尽管基础AP-1活性是正常功能所必需的，但在一些人类癌症部位，AP-1活性升高可驱动肿瘤进展。在与人类癌变相关的多种小鼠模型中，显性负Jun在转基因小鼠中角化细胞特异性表达可抑制诱导的AP-1和肿瘤发生，而不抑制细胞增殖或细胞存活（Young et al.， PNAS, 1999）。在这些小鼠中，人类乳头瘤病毒E7的表达提高了小鼠的皮肤肿瘤促进反应（Young等Molec Carc 2002），以及通过反复暴露于UVB诱导形成鳞状癌的小鼠（Cooper等Molec Cancer Res 2003）。与powell Brown （Shen等人Dev Biol 2006）合作，四环素调控的TAM 67的表达已直接作用于乳腺上皮细胞，并在最近进行了预防HER2/Neu诱导的乳腺癌发生的有效性测试。结果显示AP-1阻滞剂对肿瘤前期以及早期和晚期肿瘤发展有实质性的抑制作用（Shen et al . Ca Prev Res 2008）。重要的是，TAM67的表达在乳腺肿瘤中被抑制。与Jay Tichelaar实验室（Cancer Prev Research 2010）合作发现，可诱导的TAM67也可抑制化学诱导的肺癌发生。转录因子NFkappa B与AP-1协同调节，表明这两个因子在转化过程中可能很重要（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种解释（Hsu等人，Cancer Res 2001， Hu等人Carcinogenesis 2004）。转化抗性细胞的无反应性是由于无法激活NFkappa B p65蛋白。p65 S536位点的磷酸化对于DNA结合和抑制剂IkappaB α的泛素化和降解很重要（Hu et al Molec Carcinog 2005）。针对AP-1和NFkB的升高可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，以及转基因小鼠模型。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TAM67靶基因在驱动癌变中的功能意义。这些靶基因可能是预防癌症的有希望的新分子靶点（Young等人，2003年分子医学趋势）。最近的研究已经确定了染色质结构蛋白HMGA1 （Dhar et al Oncogene 2004）、COX-2、骨桥蛋白、尿激酶纤溶酶原激活剂、Cxcl1和MMP-10 （Matthews et al Cancer Res 2007）作为具有重要功能的TAM67靶点的重要性。最近发现的TAM67/AP-1靶基因之一是硫氧还蛋白（sulfiredoxin），这是一种氧化还原酶，在肿瘤促进和侵袭中都具有重要的功能，并且在人类癌症中升高（Wei Q等，PNAS 2008和PNAS 2011）。最近发现的AP-1阻滞剂的靶点是Wnt5a，它通过涉及蛋白激酶C和STAT3的非规范途径发出信号，在几种模型中驱动肿瘤发生（Kang et al Genes and Cancer, 2012 in press）。与分子靶标实验室合作的一个药物发现项目已经确定了模拟TAM67特异性的化合物，即抑制AP-1和/或NFkB，在不抑制细胞增殖或细胞存活的情况下防止致癌。初级AP-1高通量筛选30万种合成和天然产物，结合细胞增殖（XTT）试验产生了一小组AP-1命中（Ruocco等J . Biomolec筛选，2007年）。二级分析评估了NFkB的抑制作用，并确定了一种AP-1抑制剂，对NFkappa B的抑制作用更强（Kang等人Mol Ca Ther， 2009）。新的化学生物学实验室，CCR正在生成这种NFkappaB抑制剂的类似物，并与董事创新奖得主Cristina Annunziata （MOB， CCR）合作，评估其对人类卵巢癌的可能有效性。综上所述，本项目最近的成就包括：1)发现新的AP-1调控基因，其衰减负责抑制AP-1阻滞剂TAM67的癌变活性，特别是Sulfiredoxin和Wnt5a； 2)发现新的靶向AP-1或NFkappaB的小分子，而不抑制细胞活力。

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. Although basal AP-1 activity is needed for normal function, elevated AP-1 activity drives tumor progression in several human cancer sites. Keratinocyte-specific expression of Dominant Negative Jun in transgenic mice inhibits induced AP-1 and tumorigenesis without inhibiting cell proliferation or cell survival in multiple mouse models relevant to human carcinogenesis (Young et al., PNAS, 1999). Among these are mice whose skin tumor promotion response is elevated by expression of Human Papilloma Virus E7 (Young et al Molec Carc 2002) and mice induced to form squamous carcinomas by repeated exposure to UVB (Cooper et al Molec Cancer Res 2003). Tetracycline regulated expression of TAM 67 has been directed to mammary epithelia in collaboration with Powel Brown (Shen et al Dev Biol 2006) and has recently been tested for efficacy in preventing HER2/Neu induced mammary carcinogenesis. The results show substantial inhibition by the AP-1 blocker of preneoplastic as well as early and later stage tumor development (Shen et al Ca Prev Res 2008). Importantly, TAM67 expression was silenced in mammary tumors that developed. Inducible TAM67 has been found also to inhibit chemically induced lung carcinogenesis in collaboration with the laboratory of Jay Tichelaar (Cancer Prev Research 2010). 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, Hu et al Carcinogenesis 2004). Transformation resistant cells owe their nonresponsiveness to an inability to activate NFkappa B p65 protein. p65 phosphorylation at S536 is important for DNA binding and for ubiquitination and degradation of inhibitor IkappaB alpha (Hu et al Molec Carcinog 2005). 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. Transgenic mice expressing AP-1/ NFkB inhibitor TAM 67 present a valuable opportunity to identify AP-1 or NFkB target genes whose expression is critical to neoplastic transformation. Expression microarray analysis has revealed TAM67 target genes that are being queried for functional significance in driving carcinogenesis. Such target genes may be promising new molecular targets for cancer prevention (Young et al Trends in Molec Medicine 2003). Recent studies have established the importance of chromatin architectural protein HMGA1 (Dhar et al Oncogene 2004), COX-2, osteopontin, urokinase plasminogen activator, Cxcl1 and MMP-10 (Matthews et al Cancer Res 2007) as functionally significant TAM67 targets. One of the recently discovered TAM67/AP-1 target genes is sulfiredoxin, a redox enzyme that is functionally significant both in tumor promotion and invasion as well as elevated in human cancer (Wei Q, et al, PNAS 2008 and PNAS 2011). The most recently discovered target of the AP-1 blocker is Wnt5a which signals through a non-canonical pathway involving protein kinase C and STAT3 to drive oncogenesis in several models (Kang et al Genes and Cancer, 2012 in press. A drug discovery project in collaboration with the Molecular Targets Laboratory has identified compounds that mimic the specificity of TAM67, i.e. that inhibit AP-1 and/or NFkB and prevent carcinogenesis without inhibiting cell proliferation or cell survival. The primary AP-1 high throughput screen of 300,000 synthetic and natural products coupled to a cell proliferation (XTT) assay yielded a small set of AP-1 hits(Ruocco et al J Biomolec Screening 2007). The secondary assay has assessed inhibition of NFkB and identified an AP-1 inhibitor that is even more active against NFkappa B (Kang et al Mol Ca Ther, 2009). The new Chemical Biology Laboratory, CCR is generating analogs of this NFkappaB inhibitor and collaborating with Cristina Annunziata (MOB, CCR), winner of a Directors Innovation Award to assess its possible effectivenss against human ovarian cancer. In summary, recent accomplishments for this Project include 1) the discovery of new AP-1 regulated genes whose attenuation is responsible for the carcingenesis suppressing activity of AP-1 blocker TAM67, notably Sulfiredoxin and Wnt5a, and 2) the discovery of new small molecules that target AP-1 or NFkappaB without inhibiting cell viability.