The Role of Peroxiredoxin1 & Reactive Oxygen Species in Breast Tumor Initiation

描述（由申请人提供）：广泛认为活性氧（ROS），如H2 O2促进肿瘤发生。因此，迫切需要更好地了解H2 O2介导的癌症发展机制。我们已经证明，缺乏一个或两个拷贝的过氧化物酶Prdx 1的小鼠过早死于溶血性贫血和多种癌症。此外，缺乏Prdx 1的细胞和小鼠表现出更高的转化为H-RasV 12和ErbB-2以及H-RasV 12诱导的乳腺癌的发生率。缺乏Prdx 1的成纤维细胞和乳腺上皮细胞显示Ser 473上Akt的更高磷酸化和肿瘤抑制因子PTEN的更无活性（氧化）形式。PTEN容易通过其催化位点中的低pKa半胱氨酸的氧化而失活。我们发现Prdx 1与PTEN的结合对于保护其免受氧化诱导的失活至关重要。Prdx 1：PTEN复合物被H2 O2破坏，随后Akt包括其下游信号传导被激活，导致细胞凋亡减少。因此，我们的工作假设是，过氧化氢失活PTEN脂质磷酸酶活性，由于复杂的破坏Prdx 1：PTEN，从而促进Akt激酶驱动的肿瘤发生。我们进一步假设Prdx 1：PTEN结合是正常PTEN功能所必需的，包括其脂质磷酸酶活性、核稳定性和凋亡诱导。特异性Aim 1将决定H2 O2调节Prdx 1：PTEN相互作用以改变PTEN脂质磷酸酶活性和Prdx 1过氧化物酶活性的机制。我们将确定是否H2 O2诱导的Prdx 1或PTEN的氧化破坏或阻止Prdx 1：PTEN相互作用，并调节PTEN脂质磷酸酶活性和Prdx 1过氧化物酶活性。特异性Aim 2将检查Prdx 1：PTEN相互作用是否通过调节PTEN或Akt依赖性机制促进肿瘤抑制。为了完成这一点，我们将检查PTEN诱导的肿瘤抑制功能，包括细胞凋亡后，通过使用肽干扰在体外和体内破坏Prdx 1：PTEN的相互作用。特异性Aim 3将评估核Prdx 1是否促进小鼠中ErbB-2诱导的乳腺癌的肿瘤抑制。我们已经发现Prdx 1的缺失降低了核PTEN蛋白水平。因此，我们将融合到核定位信号序列的Prdx 1导入从Prdx 1-/-MMTV-ErbB-2小鼠分离的乳腺上皮细胞中，并将细胞移植到NCR裸鼠的透明fad垫中，以观察核Prdx 1是否降低乳腺癌的发病率或肿瘤负荷。缺乏Prdx 1的小鼠提供了第一个小鼠模型，其中过氧化物酶的损失导致内源性H2 O2升高，从而导致癌症。该小鼠模型模拟了在衰老、吸烟、电离辐射和环境致癌物中发现的H2 O2水平升高的条件。通过研究缺乏Prdx 1的乳腺癌易感小鼠，我们应该有助于在H2 O2诱导的乳腺癌中找到更具体的预防性治疗方法。 公共卫生相关性：研究过氧化物酶Prdx 1在抑制乳腺癌促进信号通路中的作用，将是未来开发最终肿瘤预防性治疗的重要工具。

DESCRIPTION (provided by applicant): It is widely accepted that reactive oxygen species (ROS), such as H2O2 promote tumorigenesis. Consequently, there is a crucial need for a greater understanding of the mechanism of H2O2-mediated cancer development. We have shown that mice lacking one or two copies of the peroxidase Prdx1 die prematurely of hemolytic anemia and multiple cancers. Furthermore, cells and mice lacking Prdx1 show a higher incidence of transformation to H-RasV12 and ErbB-2 and H-RasV12-induced breast cancer. Fibroblasts and mammary epithelial cells lacking Prdx1 show higher phosphorylation of Akt on Ser473 and more inactive (oxidized) forms of the tumor suppressor PTEN. PTEN is readily inactivated via oxidation of the low pKa cysteine in its catalytic site. We found that Prdx1 binding to PTEN is essential to protect it from oxidation-induced inactivation. The Prdx1:PTEN complex is disrupted by H2O2, upon which Akt including its downstream signaling is activated resulting in a decrease in apoptosis. Therefore, our working hypothesis is that H2O2 inactivates PTEN lipid phosphatase activity due to a complex disruption of Prdx1:PTEN, thereby promoting Akt kinase-driven oncogenesis. We further hypothesize that Prdx1:PTEN binding is required for normal PTEN function, including its lipid phosphatase activity, nuclear stability and apoptosis induction. Specific Aim1 will determine the mechanisms by which H2O2 regulates the Prdx1:PTEN interaction to alter PTEN lipid phosphatase activity and Prdx1 peroxidase activity. We will define if H2O2-induced oxidation of either Prdx1 or PTEN disrupts or prevents the Prdx1:PTEN interaction and modulates PTEN lipid phosphatase activity and Prdx1 peroxidase activity. Specific Aim2 will examine if the Prdx1:PTEN interaction promotes tumor suppression by regulating PTEN or Akt dependent mechanisms. To complete this we will examine PTEN-induced tumor suppressive functions including apoptosis upon disrupting the Prdx1:PTEN interactions by using peptide interference in vitro and in vivo. Specific Aim3 will evaluate if nuclear Prdx1 promotes tumor suppression ErbB-2-induced breast cancer in mice. We have found that loss of Prdx1 decreases nuclear PTEN protein levels. We will therefore introduce Prdx1 fused to nuclear localization signal sequence into mammary epithelial cells isolated from Prdx1-/-MMTV-ErbB-2 mice and transplant cells into clear fad pads of NCR nude mice, to observe if nuclear Prdx1 decreases incidence of breast cancer or tumor burden. Mice lacking Prdx1 offer the first mouse model where loss of a peroxidase results in elevation of endogenous H2O2 thereby causing cancer. This mouse model mimics conditions where H2O2 levels are elevated as found in aging, tobacco smoking, ionizing radiation and environmental carcinogens. By studying breast cancer prone mice lacking Prdx1 we should help to find more specific preventive treatments in H2O2-induced breast cancer. PUBLIC HEALTH RELEVANCE: Studying the role of the peroxidase Prdx1 is inhibiting breast cancer promoting signaling pathways, will be an important tool in the future to develop eventually tumor preventive treatments.