Functional and Clinical Analysis of Rak in Breast Cancer Suppression

细胞周期检查点控制的丧失和无限增殖能力的获得构成了两个因素 癌症发展所需的根本性变化。RAK，一种非受体酪氨酸激酶 通过我们的增强型逆转录病毒突变筛查发现，它是hTERT功能的抑制因子 与细胞永生有关。我们的初步数据表明，RAK在 传递DNA损伤信号，是完整的DNA损伤检查点所必需的。此外，Rak是 位于染色体6q21-23，该区域在30%的乳腺癌中经历杂合性丢失 案子。RAK在乳腺癌组织和癌细胞系中的表达也显著降低， 通过DNA阵列、实时荧光定量聚合酶链式反应和蛋白质分析进行分析。因此，我们假设RAK可能起作用 作为乳腺癌中肿瘤抑制基因的双重功能：抑制细胞永生化和 保持基因组的稳定性。这一假设将在以下步骤中得到检验。(1)我们将确定 RAK在利用正常人乳腺上皮细胞防止细胞永生化中的作用 有条件地表达抗RAK的小发夹RNA，我们将研究其介导的机制 RAK对hTERT表达的影响。(2)我们将使用RAK耗竭的细胞来研究RAK在 对基因毒性应激的反应，保持基因组完整性，以及细胞转化； 研究中，我们将使用比较基因组杂交(CGH)和光谱来评估基因组的不稳定性 核型分析(SKY)以可视化染色体异常；我们将研究RAK如何直接参与 传递DNA损伤信号；我们将使用基因敲除小鼠模型来评估Rak在体内的功能 在保持基因组完整性和抑制肿瘤形成方面的作用。(3)我们将识别和描述 乳腺癌患者样本中RAK基因的异常以确定RAK是否可作为预后指标 癌症的标记物或治疗靶点。作为推论，分析了RAK的功能和它的像差 癌症患者将有助于更好地了解癌症的关键病理变化。 对癌症的启动和进展以及新的、有效的治疗方法的开发具有重要意义。 相关性：RAK在防止细胞永生化和维持基因组稳定性方面具有双重作用。 我们的研究已经确定RAK基因是乳腺癌中一个潜在的重要抑癌基因。一个 对RAK及其相互作用蛋白网络的功能分析将阐明关键的病理变化 在癌症的发生和发展中起作用，最终将有助于开发新的、有效的治疗方法 乳腺癌的治疗方法。

Loss of cell cycle checkpoint control and acquisition of the ability to proliferate indefinitely constitute two of the fundamental changes required for the development of cancer. Rak, a non-receptor tyrosine kinase identified through our enhanced retroviral mutation screen as a represser of hTERT function, has been implicated in cellular immortalization. Our preliminary data indicate that Rak has additional functions in the transmission of DNA damage signals and is required for intact DNA damage checkpoints. Moreover, Rak is located on chromosome 6q21-23, a region that undergoes loss of heterozygosity in 30% of breast cancer cases. Rak expression is also significantly reduced in breast cancer tissues and cancer cell lines as analyzed by DNA arrays, real-time PCR, and protein analysis. Thus, we hypothesize that Rak may function as a tumor suppressor gene in breast cancer through dual functions: repressing cellular immortalization and maintaining genomic stability. This hypothesis will be tested in the following steps. (1) We will determine the role of Rak in preventing cellular immortalization by using normal human mammary epithelial cells that conditionally express small hairpin RNA against Rak, and we will investigate the mechanisms mediating Rak's function on hTERT expression. (2) We will use Rak-depleted cells to study the role of Rak in the response to genotoxic stress, maintenance of genomic integrity, and cellular transformation; for these studies, we will assess genomic instability by using comparative genomic hybridization (CGH) and spectral karyotyping (SKY) to visualize chromosomal aberrations; we will study how Rak may participate directly in transmitting DNA damage signals; and we will use a knockout mouse model to assess Rak's function in vivo in maintaining genomic integrity and suppressing tumor formation. (3) We will identify and characterize aberrations in Rak in samples from patients with breast cancer to determine if Rak serves as a prognostic marker or a therapeutic target in cancer. As a corollary, analyzing the function of Rak and its aberrations in patients with cancer will contribute to an improved understanding of the key pathologic alterations in cancer initiation and progression and to the development of novel, effective therapeutic approaches for cancer. Relevance: Rak plays a dual role in preventing cell immortalization and maintaining genomic stability. Our studies have identified Rak as a potentially important tumor suppressor gene in breast cancer. An analysis of the function of Rak and its network of interacting proteins will clarify the key pathologic alterations in cancer initiation and progression and eventually will aid in the development of novel, effective therapeutic approaches for breast cancer.