Functional and Clinical Analysis of Rak in Breast Cancer Suppression

描述（由申请人提供）：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癌症发展所需的两个基本变化。Rak是一种非受体酪氨酸激酶，通过我们增强的逆转录病毒突变筛选发现，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DNA损伤信号的传递中具有额外的功能，并且是完整DNA损伤检查点所必需的。此外，Rak位于染色体6q21-23上，该区域在30%的乳腺癌病例中发生杂合性缺失。通过DNA阵列、实时PCR和蛋白质分析，乳腺癌组织和癌细胞系中Rak的表达也显著降低。因此，我们假设Rak可能通过双重功能在乳腺癌中发挥肿瘤抑制基因的作用：抑制细胞永生化和维持基因组稳定性。这一假设将在以下步骤中进行检验。(1)我们将利用正常人乳腺上皮细胞条件表达小发夹RNA对抗Rak，确定Rak在阻止细胞永生化中的作用，并探讨Rak对hTERT表达的作用机制。(2)我们将利用Rak缺失的细胞来研究Rak在基因毒性应激反应、基因组完整性维持和细胞转化中的作用；在这些研究中，我们将通过使用比较基因组杂交（CGH）和光谱核型（SKY）来可视化染色体畸变来评估基因组不稳定性；我们将研究Rak如何直接参与DNA损伤信号的传递；我们将使用敲除小鼠模型来评估Rak在体内维持基因组完整性和抑制肿瘤形成方面的功能。(3)我们将在乳腺癌患者样本中识别和表征Rak的畸变，以确定Rak是否可以作为癌症的预后标志物或治疗靶点。因此，分析Rak的功能及其在癌症患者中的畸变将有助于提高对癌症发生和发展的关键病理改变的理解，并有助于开发新的、有效的癌症治疗方法。相关性：Rak在阻止细胞永生化和维持基因组稳定性方面起双重作用。我们的研究已经确定Rak在乳腺癌中是一个潜在的重要肿瘤抑制基因。对Rak的功能及其相互作用蛋白网络的分析将阐明癌症发生和发展的关键病理改变，并最终有助于开发新的、有效的乳腺癌治疗方法。

DESCRIPTION (provided by applicant): Loss of cell cycle checkpoint control and acquisition of the ability to proliferate indefinitely constitute 2 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 repressor of hTERT function, has been implicated in cellular immortalization. Our preliminary data indicates 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.