A Novel ROS Controlling Kinase

描述（由申请人提供）：丝氨酸/苏氨酸激酶Mirk/dyrk1B在约90%的胰腺导管腺癌和大多数胰腺癌细胞系中表达，介导其克隆生长。Mirk可能为胰腺癌细胞提供了一些生存优势，因为在胰腺癌中，Mirk基因位于19q13扩增子持续扩增的660 kb亚区内的16个基因中，而附近的基因Akt2则不在其中。突变的K-ras是大多数胰腺癌的起始病变，并通过突变的K-ras/Rac1/MKK3信号通路在胰腺癌细胞系中导致Mirk激酶的组成性激活。因此，Mirk在胰腺癌中广泛表达，并在克隆实验中介导胰腺癌细胞存活，而Mirk基因在胰腺癌的一个亚群中被扩增。Mirk最近被证明在癌细胞中有两个主要功能。由于代谢增加和致癌刺激，癌细胞通常表现出比正常细胞更高的ROS水平，Mirk降低了ROS水平，至少部分是通过增加抗氧化基因的表达。Mirk在癌细胞中的第二个功能是与p27kip1一起阻止细胞从G0中退出。我们采用双参数流式细胞术单独测量DNA和RNA（主要是核糖体）含量，发现所有胰腺癌细胞培养至少有5%的细胞处于G0期，DNA含量为2N， RNA含量较低。当细胞在次优条件下培养时，G0分数增加了10倍以上，这也使Mirk水平升高了10倍。Mirk是G0中最丰富、最活跃的激酶。Mirk的ROS抑制功能及其限制细胞循环的能力都是在一个激酶中结合的不寻常的能力，特别是在胚胎小鼠敲除研究中被证明不是必需基因的激酶。我们推测，循环癌细胞可能由于代谢升高而短暂进入G0以修复氧化应激造成的损伤，然后在有利的环境条件下重新进入循环。具体目的是：(1)测试G0癌细胞中Mirk的缺失是否会导致细胞DNA损伤未修复，使其对同样损伤DNA的药物敏感，确定Mirk缺失细胞中积累的ROS类型，并将qRT-PCR检测结果扩展到其他几种胰腺癌细胞系中，该检测结果确定了SU86.86细胞中Mirk上调的一小组抗氧化基因。(2)两种具有多西环素诱导Mirk shRNA的人胰腺癌细胞系将被用作SCID/NOD小鼠体内异种移植模型，以评估Mirk单独消耗或与吉西他滨或天然化合物PEITC （β -苯乙基异硫氰酸酯）联合治疗的效果，后者已知可提高ROS水平。

DESCRIPTION (provided by applicant): The serine/threonine kinase Mirk/dyrk1B is expressed in about 90% of pancreatic ductal adenocarcinomas and in most pancreatic cancer cell lines where it mediates their clonogenic growth. Mirk may provide some survival advantage for pancreatic cancer cells as the Mirk gene was among 16 genes within the consistently amplified 660 kb subregion of the 19q13 amplicon in pancreatic cancers, while the nearby gene Akt2 was not. Mutant K-ras is the initiating lesion in most pancreatic cancers and leads to constitutive activation of Mirk kinase through the mutant K-ras/Rac1/MKK3 signaling pathway in pancreatic cancer cell lines. Thus Mirk is widely expressed in pancreatic cancers and mediated pancreatic cancer cell survival in clonogenic assays, while the Mirk gene was amplified in a subset of pancreatic cancers. Mirk was recently shown to have two primary functions in cancer cells. While cancer cells often exhibit higher levels of ROS than normal cells because of increased metabolism and oncogenic stimulation, Mirk lowered ROS levels, at least in part by increasing expression of antioxidant genes. The second function of Mirk in cancer cells was to work together with p27kip1 to block the exit of cells from G0. We adapted a two-parameter flow cytometry assay to measure DNA and RNA (predominately ribosomal) content independently, and found that all cultures of pancreatic cancer cells examined had at least 5% of cells in a G0 phase with a 2N DNA content and a low RNA content. The G0 fraction increased over 10-fold when cells were cultured under suboptimal conditions, which also elevated Mirk levels up to 10 fold. Mirk was a kinase most abundant and active in G0. Both the ROS suppressing function of Mirk and its capacity to limit cell cycling are unusual capacities to be combined in one kinase, particularly one which was shown not to be an essential gene in embryonic mouse knock-out studies. We hypothesize that cycling cancer cells may transiently enter G0 to repair damage incurred by oxidative stress from their elevated metabolism, and then re-enter cycle under favorable environmental conditions. The specific aims are (1) to test whether depletion of Mirk in cancer cells in G0 leaves cells with unrepaired DNA damage, sensitizing them to drugs which also damage DNA, to determine the types of ROS which accumulate in Mirk-depleted cells, and to extend the results of qRT-PCR assays which identified a small group of antioxidant genes as targets of upregulation by Mirk in SU86.86 cells to several additional pancreatic cancer cell lines. (2) Two human pancreatic cancer cell lines with a doxycycline-inducible shRNA to Mirk will be used as in vivo xenograft models in SCID/NOD mice to assess the effect of Mirk depletion alone, or together with treatment with either gemcitabine or with the natural compound PEITC (beta-phenylethyl isothiocyanate), which is known to raise ROS levels. PUBLIC HEALTH RELEVANCE: Pancreatic ductal adenocarcinoma is the fourth leading cause of cancer deaths in the U.S. A large fraction of these cancer cells in vivo are noncycling. Some of these cells may be reversibly blocked in a quiescent G0 state and can re-enter the cell cycle under favorable clues from the microenvironment, enabling tumor spread. Noncycling quiescent cancer cells are more resistant than cycling cells to most chemotherapeutic drugs and radiation. Factors which allow the prolonged survival of quiescent tumor cells in vivo are of clinical relevance, and include antioxidant proteins and other factors which repress the production of reactive oxygen species (ROS). The serine/threonine kinase Mirk is upregulated in quiescent pancreatic cancer cells and mediates their survival by increasing transcription of a cohort of antioxidant genes which detoxify superoxides and which prevent the generation of hydroxyl radicals. The proposed studies will document the effect of combining Mirk depletion with drugs that raise ROS levels, define a larger set of antioxidant genes upregulated by Mirk, measure DNA damage caused by Mirk depletion, and create murine models to define Mirk's actions in vivo.