Metabolic Mechanisms for Programmed Cell Death

在先前发表的研究中，我们已经表明，通过稳定转染，POX可以通过过表达诱导细胞凋亡（Liu Y, et al., Carcinogenesis, 26:1335, 2005; Liu Y, et al., Oncogene, 25:5640, 2006）或通过药物增加表达诱导细胞凋亡（Pandhare J, et al.， J. Biol.）。化学。， 281:2044, 2006)。此外，我们发现痘激活了细胞凋亡的内在（线粒体）和外在（死亡受体）途径（Liu Y, et al., Oncogene, 25:5640, 2006）。这些影响是基于脯氨酸依赖性超氧化物的产生；线粒体中MnSOD的共表达消除了pox依赖性的凋亡效应。过度表达痘蛋白不仅会引起细胞凋亡级联反应，还会破坏细胞周期的进程。G2/M检查点的调节蛋白有明显变化。通过RT-PCR，我们发现GADD45亚型增加。在Western blots上，在POX抑制下无法检测到的GADD45alpha在POX表达时显示出强大的信号。这些发现清楚地表明，POX过表达不仅诱导细胞凋亡，而且可能通过诱导GADD来阻断G2/M转变（Liu， Y, et ., Cancer Res., 69: 6414,2009）。我们还发现痘信号下调COX2/PGE2 (Liu， Y, et al., Oncogene, 27:7 729, 2008)，最有趣的是，通过增加α酮戊二酸增加hif -1 α的脯氨酰羟基化及其蛋白酶体降解来降低hif -1 α水平（Liu， Y, et al., Cancer Res., 69:6414, 2009）。因此，通过多种机制，痘的表达可以通过多种机制抑制生长和启动细胞凋亡。为了测试这种依赖于pox的凋亡和生长抑制机制是否可以转化到动物模型中，我们在胸腺小鼠的饮用水中使用不同剂量的多西环素（100微克/毫升），使用DLD-tet-off-POX形成异种移植肿瘤。我们发现，当饮用水中去除强力霉素和POX表达时，肿瘤的植入受到明显抑制（Liu， Y, et al., Cancer Res., 69:6414, 2009）。通过免疫组化检查（IHC），通过对来自同一患者的配对正常组织的多种人类肿瘤中的POX表达，提供了POX在人类肿瘤中作为肿瘤抑制蛋白功能的证据。在来自消化道的36对肿瘤中，28对肿瘤中显示出与正常组织相比明显减少或无法检测到的水平。从6对肾脏肿瘤/正常组织中，6个肿瘤的免疫组化水平均降低（Liu， Y, et al., Cancer Res., 69:6414, 2009）。因此，POX似乎是一种肿瘤抑制蛋白。我们寻找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microRNA miR-23b*通过结合其3'-UTR抑制痘翻译。我们首先证实肿瘤组织中痘蛋白明显降低。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在同一张载玻片上。我们通过免疫组化检查了符合这些标准的每个切片；与正常组织相比，每个肿瘤的痘都明显减少或无法检测到。此外，我们获得了16对冷冻的透明细胞肾癌，与配对的非恶性组织相比，Western免疫印迹显示，16个肿瘤中有13个肿瘤的POX表达明显降低。为了研究mirna的作用机制，我们首先通过计算机鉴定了一组mirna，然后在体外将3株肾癌细胞系与正常肾上皮细胞进行比较。miR-23b*在肿瘤细胞中表达水平较高，且在肿瘤细胞与正常细胞间差异最大。Mimic miR-23b*显著降低POX的表达，anti-miR-23b*显著升高POX的表达。重要的是，通过模拟miR-23b*或anti-miR-23b*处理，对POX表达的功能反应（ROS生成、细胞凋亡增加或增殖抑制）受到影响。这些体外研究在人类肿瘤中得到验证，通过检测POX的表达与miR-23b*在检测POX表达的16对肿瘤组织中的表达进行比较。我们发现POX水平与miR-23b*呈负相关。此外，通过原位杂交，miR-23b*在肿瘤中的表达增加。这些研究首次表明脯氨酸作为肿瘤抑制蛋白脯氨酸氧化酶的微环境底物的功能可以受到特定miRNA miR-23b*的调节。因此，miR-23b*在肿瘤中的表达增加及其对POX的抑制作用使其成为功能性癌基因。我们很兴奋地发现这些机制可以恢复不依赖p53、ros的程序性细胞死亡。阻断miR-23b*激活肿瘤中的POX功能可作为化疗的辅助手段。研究人员正在测试这种可能性。我们还发现癌基因c-Myc通过miR-23b*调控POX。在稳定转染了抑制型c-Myc构建体的细胞中，随着c-Myc的表达，POX降低。重要的是，c-Myc增加miR-23b*的水平，从而抑制POX mRNA的翻译。这些关于痘的发现补充了c-Myc对谷氨酰胺酶的作用，谷氨酰胺酶在细胞增殖中起重要作用。因此，c-Myc对谷氨酰胺酶和痘的作用是相反的。此外，谷氨酰胺酶由miR-23a/b控制，miR-23b*的互补链。谷氨酰胺酶的升高是由miR-23a/b水平的降低介导的，而其在痘中的降低是由于miR-23b*水平的升高。

In previously published studies, we have shown that apoptosis can be induced by overexpression of POX by stable transfection ( Liu Y, et al., Carcinogenesis, 26:1335, 2005; Liu Y, et al., Oncogene, 25:5640, 2006) or by increased expression with pharmacologic agents (Pandhare J, et al., J. Biol. Chem., 281:2044, 2006 ). Furthermore, we showed that POX activates both the intrinsic (mitochondrial) and extrinsic (death receptor) pathways (Liu Y, et al., Oncogene, 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, et al., Cancer Res. , 69:6414, 2009). We also found that POX signaling downregulates COX2/PGE2 (Liu, Y, et al., Oncogene , 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, et al., Cancer Res. , 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, et al., Cancer Res. , 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, et al., Cancer Res. , 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, miR-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 carcinomas with paired nonmalignant tissue and showed by Western immunoblots that 13 of the 16 tumors had markedly decreased POX expression as compared to 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 miR-23b* were high in tumor cells and showed the highest differences between tumor and normal cells. Mimic miR-23b* markedly decreased the expression of POX and anti-miR-23b* increased the expression of POX. Importantly, the functional responses to POX expression (ROS generation, increased apoptosis, or inhibition of prolfieration) were affected by treating with mimic miR-23b* or anti-miR-23b*. These in vitro studies were validated in human tumors by examining the expression of POX compared to the expression of miR-23b* in the 16 paired tumor tissues examined for POX expression. We found there was negative correlation between POX levels and miR-23b*. Furthermore, using in situ hybridization, the expression of miR-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, miR-23b*. Thus, the increased expression of miR-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 miR-23b* to activate POX function in tumors may be used as an adjunct in chemotherapy. Studies are underway to test this possibility. We have also shown that the oncogene c-Myc regulates POX through miR-23b*. In cells stably transfected with a tet-off c-Myc construct, POX decreases as c-Myc is expressed. Importantly, c-Myc increases the level of miR-23b* which inhibits the translation of POX mRNA. These findings on POX complement the effects of c-Myc on glutaminase, an enzyme which plays an important role in cell proliferation. Thus, the effect of c-Myc on glutaminase and POX are oppositely directed. Furthermore, glutaminase is controlled by miR-23a/b, the complementary strand to miR-23b*. The increase in glutaminase is mediated by a decrease in the level of miR-23a/b while its decrease in POX is due to an increase in the level of miR-23b*.