Role of PALB2 in the DNA damage response and breast cancer suppression

描述(申请人提供)：BRCA2杂合胚系突变使女性携带者易患乳腺癌，也会增加患卵巢癌、男性乳腺癌、前列腺癌和胰腺癌的风险。双等位基因种系BRCA2突变导致D1型Fanconi贫血(FA-D1型)。BRCA2是一种非常大的蛋白质，其功能至少部分是作为基因组完整性的守护者。我们最近发现了一个由1,186个氨基酸组成的新蛋白质，我们将其命名为PALB2，它是BRCA2在DNA损伤反应中发挥作用所必需的。我们进一步发现，PALB2基因和BRCA2一样，在乳腺癌和Fanconi贫血(FA-N)中也发生了突变。因此，我们假设PALB2通过启动BRCA2在基因组完整性控制和肿瘤抑制中的功能而发挥肿瘤抑制作用。我们已经证明，PALB2在BRCA2与染色质结构的连接中起着至关重要的作用，从而使BRCA2在DNA损伤反应中发挥作用，如HR、S期DNA损伤检查点和链间交联修复。然而，PALB2如何履行其职能以及其职能如何受到监管的机制细节仍有待阐明。在这项计划中，我们计划进行全面的结构-功能分析，包括生化纯化、基于细胞的分析和体外分析，以进一步确定PALB2 DNA损伤反应功能的机制。此外，我们建议建立条件性PALB2基因敲除小鼠模型，以直接测试我们的假设，即PALB2通过调节BRCA2的功能在乳腺上皮细胞中发挥肿瘤抑制作用。人们将提出以下问题：1)乳腺上皮细胞中PALB2的失活是否导致乳腺肿瘤的形成？2)鉴于P53和BRCA2协同抑制乳腺癌的作用，P53是否在PALB2介导的肿瘤抑制中发挥作用？3)鉴于在PALB2相关的人类乳腺肿瘤中几乎没有发现杂合性丢失(LOH)的证据，PALB2是单倍体不足的乳腺癌抑制因子吗？4)PALB2仅仅通过支持BRCA2功能来抑制乳腺肿瘤的发生吗？公共卫生相关性：拟议的研究将对BRCA1-PALB2-BRCA2基因组完整性控制通路的运作产生重要的见解，该通路对于抑制乳腺癌和其他一些恶性肿瘤至关重要。从拟议的努力中获得的知识可能会转化为新的治疗策略，通过利用癌细胞无法修复某些类型的DNA损伤，例如双链断裂、链间交联等，选择性地靶向和消除癌细胞。最后，从长远来看，这里产生和表征的小鼠模型可能被证明对癌症药物的临床前测试有用。

DESCRIPTION (provided by applicant): Heterozygous germline mutations in BRCA2 predispose female carriers to breast cancer and also lead to increased risks of ovarian, male breast, prostate, and pancreatic cancers. Biallelic germline BRCA2 mutations cause the D1 subtype of Fanconi anemia (FA-D1). BRCA2 is a very large protein that functions, at least in part, as a guardian of genome integrity. We recently identified a novel protein of 1,186 amino acids, which we named PALB2, as a major BRCA2-binding protein, and established that PALB2 is required for BRCA2 functions in the DNA damage response. We further discovered that the PALB2 gene, like BRCA2, is also mutated in breast cancer and Fanconi anemia (FA-N). Thus, we hypothesize that PALB2 functions as a tumor suppressor by enabling BRCA2 functions in the genome integrity control and tumor suppression. We have shown that PALB2 plays an essential role in tethering BRCA2 to chromatin structures and thus enables BRCA2 functions in the DNA damage response, e.g. HR, S phase DNA damage check point, and interstrand crosslink (ICL) repair. However, the mechanistic details as to how PALB2 carries out its functions and how its functions are regulated remain to be elucidated. In this proposal, we plan to carry out comprehensive structure-function analysis, including biochemical purification, cell-based assays, and in vitro assays, to further define the mechanisms of PALB2 DNA damage response functions. Furthermore, we propose to generate conditional PALB2 knockout mouse models to directly test our hypothesis that PALB2 functions as a tumor suppressor in mammary epithelial cells by regulating the function of BRCA2. The following questions will be asked: 1) Does PALB2 inactivation in breast epithelial cells lead to mammary tumor formation? 2) Does p53 play a role in PALB2-mediated tumor suppression, given that p53 and BRCA2 function synergistically to suppress breast cancer? 3) Is PALB2 a haploinsufficient breast cancer suppressor, given that little evidence of loss of heterozygosity (LOH) has been found in PALB2-associated human breast tumors? 4) Does PALB2 suppress tumorigenesis in the mammary gland solely through its support of BRCA2 function? PUBLIC HEALTH RELEVANCE: The proposed studies will generate important insights into the operation of the BRCA1-PALB2- BRCA2 genome integrity control pathway that is crucial for the suppression of breast cancer and a number of other malignancies. The knowledge that will be gained from the proposed efforts may potentially translate into new therapeutic strategies to selectively target and eliminate cancer cells by exploiting their inability to repair certain types of DNA damage, e.g. double strand breaks, interstrand crosslinks, etc. Finally, in the longer term the mouse models generated and characterized here may prove useful for pre-clinical testing of cancer drugs.