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

描述（由申请人提供）：活性氧（ROS）如H2O2促进肿瘤发生已被广泛接受。因此，有必要对h2o2介导的癌症发展机制有更深入的了解。我们已经证明缺乏一个或两个过氧化物酶Prdx1拷贝的小鼠过早死于溶血性贫血和多种癌症。此外，缺乏Prdx1的细胞和小鼠转化为H-RasV12、ErbB-2和H-RasV12诱导的乳腺癌的发生率更高。缺乏Prdx1的成纤维细胞和乳腺上皮细胞显示Akt在Ser473上的磷酸化水平较高，肿瘤抑制因子PTEN的活性更低（氧化）。PTEN很容易通过其催化位点的低pKa半胱氨酸氧化而失活。我们发现Prdx1与PTEN的结合对于保护PTEN免受氧化诱导的失活至关重要。Prdx1:PTEN复合物被H2O2破坏，Akt及其下游信号被激活，导致细胞凋亡减少。因此，我们的工作假设是H2O2由于Prdx1:PTEN的复杂破坏而使PTEN脂质磷酸酶活性失活，从而促进Akt激酶驱动的肿瘤发生。我们进一步假设Prdx1:PTEN结合是正常PTEN功能所必需的，包括其脂质磷酸酶活性、核稳定性和诱导凋亡。特异性Aim1将确定H2O2调节Prdx1:PTEN相互作用改变PTEN脂质磷酸酶活性和Prdx1过氧化物酶活性的机制。我们将确定h2o2诱导的Prdx1或PTEN的氧化是否会破坏或阻止Prdx1:PTEN的相互作用，并调节PTEN脂质磷酸酶活性和Prdx1过氧化物酶活性。Specific Aim2将检测Prdx1:PTEN相互作用是否通过调节PTEN或Akt依赖机制促进肿瘤抑制。为了完成这项工作，我们将在体外和体内使用肽干扰来检测PTEN诱导的肿瘤抑制功能，包括破坏Prdx1:PTEN相互作用后的细胞凋亡。特异性Aim3将评估核Prdx1是否促进肿瘤抑制erbb -2诱导的小鼠乳腺癌。我们发现Prdx1的缺失降低了核PTEN蛋白的水平。因此，我们将Prdx1融合核定位信号序列导入Prdx1-/- mmtv - erbb -2小鼠分离的乳腺上皮细胞，并将细胞移植到NCR裸鼠的透明衬底中，观察核Prdx1是否降低乳腺癌发病率或肿瘤负荷。缺乏Prdx1的小鼠提供了第一个过氧化物酶缺失导致内源性H2O2升高从而导致癌症的小鼠模型。这个小鼠模型模拟了H2O2水平升高的情况，如衰老、吸烟、电离辐射和环境致癌物中发现的情况。通过研究缺乏Prdx1的乳腺癌易感小鼠，我们应该有助于找到更具体的预防h2o2诱导乳腺癌的治疗方法。

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.