Metabolic Mechanisms for Programmed Cell Death

在以前发表的研究中，我们已经表明，POX的过度表达可以通过稳定的转基因(Liu Y，&lt；I&gt；et al.，Carcinationallt；/I&gt；et al.，26：1335,2005；Liu Y，&lt；I&gt；et al.，Oncogene&lt；/I&gt；，25：5640,2006)或通过增加药物的表达(Pandhare J，&lt；I&gt；et al.，J.Biol)来诱导细胞凋亡。化学/I&GT；，281：2044,2006)。此外，我们还发现POX可以激活内源性(线粒体)和外源性(死亡受体)途径(Liu Y，&lt；i&gt；et al.，Oncogene&lt；/I&gt；，25：5640,2006)。这些作用是基于依赖于Pro的超氧化物的产生；在线粒体中共表达MnSOD可消除依赖POX的细胞凋亡效应。POX的过度表达不仅诱导了细胞的凋亡级联反应，而且破坏了细胞周期的进程。控制G2/M检查点的调节蛋白发生了显著变化。RT-PCR检测发现GADD45亚型增加。在Western blotts上，GADD45pha在POX抑制后不能被检测到，显示出强烈的POX表达信号。这些发现清楚地表明，POX的过度表达不仅诱导细胞凋亡，而且可能通过诱导GADD(Liu，Y，&lt；I&gt；et al.，Cancer Res.&lt；/I&gt；，69：6414,2009)阻止G2/M转变。我们还发现POX信号下调COX2/PGE2(Liu，Y，&lt；I&gt；et.，Oncogene&lt；/I&gt；，27：6729,2008)，最有趣的是，通过增加α-酮戊二酸来降低HIF-1α的水平，从而增加HIF-1α的Pro羟化及其蛋白酶体降解(Liu，Y，&lt；I&gt；et al.，Cancer Res.&lt；/I&gt；69：6414,2009)。因此，POX的表达可通过多种机制抑制细胞生长和启动细胞凋亡。为了测试这种POX依赖的细胞凋亡和生长抑制机制是否可以转化为动物模型，我们使用DLD-TET-OFF-POX在裸鼠体内形成异种移植瘤，并在它们的饮用水中不同地给予多西环素(100微克/毫升)。我们发现，当多西环素从饮用水中移除并表达POX时，肿瘤的植入明显受到抑制(Liu，Y，&lt；I&gt；et al.，Cancer Res.&lt；/I&gt；，69：6414,2009)。POX在人类肿瘤中作为肿瘤抑制蛋白发挥作用的证据是通过免疫组织化学检查(IHC)在各种人类肿瘤和来自同一患者的配对正常组织中提供的。在36对来自消化道的肿瘤中，28对与正常组织相比，肿瘤中的水平显著降低或检测不到。从来自肾脏的6对肿瘤/正常组织中，所有6个肿瘤的POX免疫组织化学水平都降低(Liu，Y，&lt；I&gt；et al，Cancer Res.&lt；/I&gt；，69：6414,2009)。因此，痘似乎是一种肿瘤抑制蛋白。我们寻求这种肿瘤抑制蛋白丢失的机制，但没有发现任何更常见的遗传或表观遗传机制。相反，我们发现一种名为miRNA-23B*的microRNA通过与其3-UTR结合而抑制POX翻译。我们首先证实了肿瘤组织中的痘明显减少。选择透明细胞肾癌，我们从档案收集中获得样本，其中肿瘤和邻近的非恶性组织都在同一张幻灯片上。我们通过IHC检查了符合这些标准(6)的每个切片；与正常组织相比，每个肿瘤都有明显的减少或检测不到痘。此外，我们获得了16对冷冻的透明细胞肾肿瘤和配对的非恶性组织，IHC结果显示，与配对的非恶性组织相比，16个肿瘤中有13个肿瘤的POX表达显著降低。为了研究miRNAs的作用机制，我们首先利用计算机识别了一组miRNAs，然后通过比较3个肾癌细胞系和正常肾上皮细胞对它们进行了体外测试。MiRNA-23b*在肿瘤细胞中的表达水平较高，且在肿瘤细胞与正常细胞之间的差异最大。模拟miRNA-23B*可显著降低POX的表达，而反义miRNA-23B*可增加POX的表达。重要的是，通过使用模拟miRNA-23B*Pr anttimiRNA-23B*可以产生对POX表达的功能性反应(ROS产生、细胞凋亡增加或抑制增殖)。这些体外研究在人类肿瘤中得到了验证，方法是检测POX的表达与16对POX表达的肿瘤组织中miRNA-23B*的表达进行比较。我们发现POX水平与miRNA-23B*呈负相关。此外，通过原位杂交，miRNA-23b*在肿瘤中的表达增加。这些研究首次表明，Pro作为一种肿瘤抑制蛋白的微环境底物，其功能可以受到一种特定的miRNA-miRNA-23b*的调节。因此，miRNA-23b*在肿瘤中的高表达及其对POX的抑制使其成为一个功能性癌基因。我们感到兴奋的是，这些机制可以恢复P53依赖、ROS依赖的程序性细胞死亡。阻断miRNA-23B*激活肿瘤的POX功能可作为化疗的辅助手段。测试这种可能性的研究正在进行中。

In previously published studies, we have shown that apoptosis can be induced by overexpression of POX by stable transfection ( Liu Y, <I>et al., Carcinogenesis</I>, 26:1335, 2005; Liu Y, <I>et al., Oncogene</I>, 25:5640, 2006) or by increased expression with pharmacologic agents (Pandhare J, <I>et al., J. Biol. Chem.</I>, 281:2044, 2006 ). Furthermore, we showed that POX activates both the intrinsic (mitochondrial) and extrinsic (death receptor) pathways (Liu Y, <I>et al., Oncogene</I>, 25:5640, 2006) for apoptosis. These effects are based on the generation of proline-dependent superoxide; co-expression of MnSOD in mitochondria abolished the POX-dependent apoptotic effects. Not only is the apoptotic cascade induced by overexpression of POX, but also progression through the cell cycle is disrupted. There were marked changes in regulatory proteins governing the G2/M checkpoint. Using RT-PCR we found that isoforms of GADD45 were increased. On Western blots, GADD45alpha which was undetectable with POX suppressed, showed a robust signal with POX expression. These findings clearly showed that POX overexpression not only induces apoptosis, but also blocks the G2/M transition, perhaps by inducing GADD (Liu, Y, <I>et al., Cancer Res. </I>, 69:6414, 2009). We also found that POX signaling downregulates COX2/PGE2 (Liu, Y, <I>et al., Oncogene </I>, 27:6729, 2008), and most interestingly, decreases the level of HIF-1alpha by increasing alpha ketoglutarate which increases prolyl hydroxylation of HIF-1alpha and its proteasomal degradation ((Liu, Y, <I>et al., Cancer Res. </I>, 69:6414, 2009). Thus, by a variety of mechanisms, the expression of POX can inhibit growth and initiate apoptosis by a variety of mechanisms. To test whether this POX-dependent apoptotic and growth-inhibition mechanism can be translated to animal models, we performed studies using DLD-tet-off-POX to form xenograft tumors in athymic mice differentially administered doxycycline (100 micrograms/ml) in their drinking water. We found that when doxycycline was removed from the drinking water and POX expressed, the engraftment of tumors was markedly inhibited (Liu, Y, <I>et al., Cancer Res. </I>, 69:6414, 2009). Evidence that POX functions as a tumor suppressor protein in human tumors was provided by immunohisto-chemical examination (IHC) of POX expression in a variety of human tumors with paired normal tissue from the same patient. Out of 36 pairs of tumors from the digestive tract, 28 showed marked decrease or undetectable levels in tumors as compared to normal tissue. From 6 pairs of tumors/ normal tissue from the kidney, all 6 tumors had decreased immunohistochemical levels of POX (Liu, Y, <I>et al., Cancer Res. </I>, 69:6414, 2009). Thus, POX appears to function as a tumor suppressor protein. We sought the mechanism for the loss of this tumor suppressor protein, but did not find any of the more common genetic or epigenetic mechanisms. Instead, we found that a microRNA, miRNA-23B*, suppressed POX translation by binding to its 3-UTR. We first confirmed that POX is markedly decreased in tumor tissue. Choosing clear cell renal carcinoma, we obtained samples from an archival collection, in which both tumor and adjacent nonmalignant tissue are on the same slide. We examined every slice fitting these criteria (6) by IHC; every tumor had marked decrease or undetectable POX as compared to normal tissue. In addition, we obtained 16 pairs of frozen clear cell renal tumors with paired nonmalignant tissue and showed by IHC that 13 of the 16 tumors had markedly decreased POX expression as compared to the paired nonmalignant tissues. To investigate the mechanistic role of miRNAs, we first identified a group of miRNAs by computer and then tested them in vitro by comparing 3 renal cancer cell lines with normal kidney epithelial cells. Levels of miRNA-23b* were high in tumor cells and showed the highest differences between tumor and normal cells. Mimic miRNA-23B* markedly decreased the expression of POX and anti-miRNA-23B* increased the expression of POX. Importantly, the functional responses to POX expression (ROS generation, increased apoptosis, or inhibition of prolfieration) could be produced by using mimic miRNA-23B* pr antimiRNA-23B*. These in vitro studies were validated in human tumors by examining the expression of POX compared to the expression of miRNA-23B* in the 16 paired tumor tissues examined for POX expression. We found there was negative correlation between POX levels and miRNA-23B*. Furthermore, using in situ hybridization, the expression of miRNA-23b* was increased in tumors. These studies are the first to show that the function of proline as a microenvironmental substrate for proline oxidase, a tumor suppressor protein, could be regulated by a specific miRNA, miRNA-23b*. Thus, the increased expression of miRNA-23b* in tumors and its inhibition of POX makes it a functional oncogene. We are excited that these mechanisms can restore a p53-independent, ROS-dependent programmed cell death. The blockade of miRNA-23B* to activate POX function in tumors may be used as an adjunct in chemotherapy. Studies are underway to test this possibility.