Molecular mechanism of ID1 function in advanced breast cancer

描述（由申请人提供）：2008年将有20多万美国妇女被诊断患有乳腺癌，其中许多人将接受抗雌激素治疗。不幸的是，许多最初对抗雌激素有反应的乳腺肿瘤随着时间的推移会变得无反应，导致转移和死亡。乳腺癌进展的分子机制在很大程度上是未知的，这阻碍了晚期癌症靶向治疗的发展。我们最近的研究表明，分化抑制剂ID1在促进乳腺癌细胞采用侵袭性的、激素不依赖的表型中起作用，导致抗雌激素抵抗。最近的报道强调了我们发现的重要性，即ID1在低分化转移性肿瘤中高度表达。ID1的主要功能是抑制包含基本螺旋-环-螺旋（bHLH）结构域的转录因子家族的DNA结合。为了确定乳腺癌细胞中可能受ID1调控的bHLH转录因子，我们对已发表的人类乳腺肿瘤基因表达数据进行了全面的生物信息学分析。meta分析显示bHLH转录因子BHLHB2的表达与激素依赖性乳腺肿瘤密切相关，BHLHB2共表达基因与乳腺癌激素依赖性表型的关键转录因子ESR1、FOXA1和GATA3共表达基因显著重叠。此外，我们发现E-box motif， BHLHB2识别的顺式调控序列，在ESR1的直接靶基因中被过度代表。我们假设BHLHB2是决定乳腺癌激素依赖性表型的转录调控网络的一个组成部分，而ID1通过抑制BHLHB2的功能促进肿瘤细胞获得低分化表型。我们将通过对BHLHB2在乳腺癌细胞中的功能和调控进行系统详细的研究，在以下三个具体目标中验证这一假设。具体来说，我们将：1)通过检测BHLHB2和ID1在人乳腺癌细胞中功能的获得或丧失的后果，研究BHLHB2在维持激素依赖性表型中的作用及其通过ID1的调节；2)利用基因表达和BHLHB2 DNA结合的高通量全基因组分析，鉴定和表征BHLHB2和ID1调控的基因；3)研究氟维司汀耐药相关miRNA的调控和功能，重点研究氟维司汀耐药相关miRNA与激素依赖性乳腺癌细胞核心转录因子之间的交叉调控。公共卫生相关性：乳腺癌治疗的一个主要障碍是耐药性的发展。转录调节因子通过改变基因表达模式在耐药过程中发挥重要作用。因此，鉴定和表征晚期乳腺癌细胞中发生改变的转录调控因子对于更好地了解肿瘤进展非常重要，并可能最终为有效的靶向治疗提供候选药物。

DESCRIPTION (provided by applicant): In 2008 more than 200,000 American women will be diagnosed with breast cancer and many will be treated with antiestrogens. Unfortunately, many breast tumors initially respond to antiestrogens will become unresponsive over time, leading to metastasis and mortality. The molecular mechanisms of breast cancer progression are largely unknown, which hinders the development of targeted therapies for advanced cancer. Our recent studies showed that ID1, an inhibitor of differentiation, played a role in promoting breast cancer cells to adopt an aggressive, hormone-independent phenotype, resulting in antiestrogen resistance. The significance of our finding is underscored by recent reports that ID1 is highly expressed in poorly differentiated, metastatic tumors. The primary function of ID1 is to inhibit DNA binding of a family of transcription factors that contain a basic helix-loop-helix (bHLH) domain. To identify the potential bHLH transcription factor that is regulated by ID1 in breast cancer cells, we performed a comprehensive bioinformatic analysis of published gene expression data of human breast tumors. Meta-analysis revealed that the expression of BHLHB2, a bHLH transcription factor and putative target of ID1, is strongly associated with hormone-dependent breast tumors, and BHLHB2-coexpressed genes significantly overlap with genes coexpressed with ESR1, FOXA1 and GATA3, the key transcription factors that specify the hormone-dependent phenotype of breast cancer. In addition, we found that E-box motif, the cis-regulatory sequence recognized by BHLHB2, was overrepresented in ESR1 direct target genes. We hypothesize that BHLHB2 is an integral component of a transcription regulatory network that dictates the hormone-dependent phenotype of breast cancer, and ID1 facilitates tumor cells to acquire a poorly differentiated phenotype by inhibiting BHLHB2 function. We will test this hypothesis in the following three specific aims by conducting a systematic and detailed study on the function and regulation of BHLHB2 in breast cancer cells. Specifically, we will: 1) investigate the role of BHLHB2 in maintaining hormone-dependent phenotype and its regulation by ID1 by examining the consequences of gain- or loss-of- function of BHLHB2 and ID1 in human breast cancer cells; 2) identify and characterize genes regulated by BHLHB2 and ID1 using high-throughput genome-wide analysis of gene expression and BHLHB2 DNA binding; and 3) investigate the regulation and function of fulvestrant resistance-related miRNAs, with emphasis on the cross-regulation between the fulvestrant resistant-relate miRNA and core transcription factors of hormone- dependent breast cancer cells. PUBLIC HEALTH RELEVANCE: One major obstacle of breast cancer treatment is the development of drug resistance. Transcription regulators play an important role in drug resistance by altering gene expression patterns. Thus, identification and characterization of transcription regulators that altered in advanced breast cancer cells are important for a better understanding of tumor progression and may ultimately provide candidates for effective targeted therapies.