世界生命科学前沿动态周报（六十六）

（11.14-11.20/2011）
美宝国际集团:陶国新 

　　主要内容：发现SHARPIN蛋白是内源性β1整合素激活的抑制剂；癌症联合治疗的“万能药”；终止皮肤癌形成的信号；人体多能干细胞代谢的调节机制；会分泌红细胞生成素EPO的血管；肿瘤生长离不开分子伴侣介导的自噬。

　　焦点动态：人体多能干细胞代谢的调节机制。

1. 发现SHARPIN蛋白是内源性β1整合素激活的抑制剂
【动态】
　　有序激活整合素对细胞附着、运动和组织的稳定都是至关重要的。蛋白Talin 和 kindlin激活β1整合素，但是抵消激活的抑制机制还不清楚。芬兰科学家通过RNAi筛选发现SHARPIN蛋白是β1整合素的重要抑制剂。SHARPIN蛋白在人体癌细胞和主要的白血球中抑制β1整合素的功能。在SHARPIN缺陷的老鼠中纤维细胞、白血球和角质细胞表现出增强了的β1整合素作用，而通过重新表达SHARPIN蛋白能够完全解除这种增强。SHARPIN蛋白直接结合到整合素α亚基的一个保守的胞质区，抑制整合素招募激活蛋白Talin 和 kindlin。因此，SHARPIN蛋白抑制了β1整合素从无活性到有活性的关键的构象转变。

【点评】
　　该研究在已知β1整合素的激活蛋白基础上发现了抑制其激活的蛋白，进一步完善了β1整合素的调控机制，对于研究人体组织如何保持自身的平衡和稳定有重要意义。

【参考论文】
Nature Cell Biology, 2011; 13 (11): 1315 DOI: 10.1038/ncb2340
SHARPIN is an endogenous inhibitor of β1-integrin activation
Juha K. Rantala, Jeroen Pouwels, Teijo Pellinen, et al.
Regulated activation of integrins is critical for cell adhesion, motility and tissue homeostasis. Talin and kindlins activate β1-integrins, but the counteracting inhibiting mechanisms are poorly defined. We identified SHARPIN as an important inactivator of β1-integrins in an RNAi screen. SHARPIN inhibited β1-integrin functions in human cancer cells and primary leukocytes. Fibroblasts, leukocytes and keratinocytes from SHARPIN-deficient mice exhibited increased β1-integrin activity, which was fully rescued by re-expression of SHARPIN. We found that SHARPIN directly binds to a conserved cytoplasmic region of integrin α-subunits and inhibits recruitment of talin and kindlin to the integrin. Therefore, SHARPIN inhibits the critical switching of β1-integrins from inactive to active conformations.

2. 癌症联合治疗的“万能药”
【动态】
　　由于每种癌症都是一些处于不同发展阶段的癌细胞的非均匀混合物，其治疗就面对一次同时治疗多种不同的病变细胞。一种可信的办法是建立个体化的基因和蛋白图谱，找出目标癌基因并开出专门针对单个病人的靶向治疗药物的组合。但是，个体化医学还存在很多实际问题：1）肿瘤经常演变出抗药的类型；2）治疗会极其昂贵；3）很多靶向治疗药物还没有开发出来。美国科学家就此提出了已在动物实验中见效的另一种策略：2-脱氧葡萄糖（2DG）与Bcl-2拮抗剂（如ABT）的联合治疗。促凋亡蛋白Bak通常被Mcl-1 和 Bcl-xL抑制。只有从Mcl-1 和 Bcl-xL释放出来后，Bak才能诱导凋亡。2DG通过解离Bak-Mcl-1 复合物能够使大量分解糖的细胞，一般是一些脑细胞和大部分癌细胞，做好准备。之后，ABT能够结合Bcl-xL，解离Bak-Bcl-xL复合物，解放Bak去诱导细胞凋亡。ABT不能穿过血脑屏障，只有位于脑部以外大量分解糖的癌细胞能够接触2DG和ABT两种物质。由于ABT在非常接近凋亡末期的阶段直接触发细胞凋亡，2DG-ABT联合治疗适用于所有发展阶段的很多种癌症，副作用很小。

【点评】
　　该研究通过两种物质的联合使用分两步特异性的使脑外的癌细胞首先失去葡萄糖能量代谢供应继而触发细胞凋亡，可以适用于不同阶段的多种癌症，副作用是有时候会引起淋巴细胞和血小板减少。

【参考论文】
Cancer Research, 2011; DOI: 10.1158/0008-5472.CAN-11-3091
Finding a Panacea Among Combination Cancer Therapies
R. Yamaguchi, G. Perkins.
Since each cancer is a heterogeneous mix of cancer cells at different stages of development, we are faced with trying to treat many different diseased cells all at once. An authentic approach is to build a genomic and proteomic profile of a patient, identify the target oncogenes and prescribe the combination of targeted drugs tailored for that patient. However, there are many practical problems with this personalized medicine approach: (1) cancers often generate treatment-resistant phenotypes, (2) the treatment could be enormously expensive, and (3) most of the targeted drugs have not been developed yet. We propose a different approach: therapies that combine 2-deoxyglucose (2DG) with Bcl-2 antagonists such as ABT-263/737 (ABT). Pro-apoptotic protein Bak is normally sequestered by Mcl-1 and Bcl-xL. Only when Bak is released from both Mcl-1 and Bcl-xL, can it induce apoptosis. 2DG can prime highly glycolytic cells by dissociating Bak-Mcl-1 complex. Cells primed by 2DG are some brain cells and most cancer cells. ABT can bind to Bcl-xL, dissociating Bak-Bcl-xL complex, freeing Bak and inducing apoptosis. Since ABT cannot cross blood-brain barrier, only cells exposed to both agents are highly glycolytic cancer cells located outside the brain. Because ABT directly triggers apoptosis at the step very near the terminal point of apoptosis, 2DG-ABT combination therapies are applicable to many types of cancer at all stages of development, with little side effect.

3. 终止皮肤癌形成的信号
【动态】
　　鳞状细胞癌（SCC）虽然很常见，但是其分子机理仍不明了。澳大利亚、波兰和美国科学家的合作研究在老鼠中发现了高效的SCC抑制物--发育转录因子Grhl3，并证明以Grhl3为目标的miR-21依赖的原癌基因网络加强了人体的SCC。成体表皮中删除Grhl3会引起一种GRHL3的直接靶标PTEN蛋白表达的丧失，导致PI3K/AKT/mTOR信号激活诱导的侵袭性SCC。恢复Pten表达能够完全终止SCC的形成。人体皮肤和头颈SCC中明显减少的GRHL3和PTEN水平与以此二者为靶标的miR-21水平增加相关联。他们的数据将GRHL3-PTEN轴定义为SCC的关键肿瘤抑制途径。
【点评】
　　该研究发现了抑制鳞状皮肤癌形成的分子机制，对于鳞状皮肤癌的预防和治疗有重要价值。

【参考论文】
Cancer Cell, Volume 20, Issue 5, 635-648, 15 November 2011
Targeting of the Tumor Suppressor GRHL3 by a miR-21-Dependent Proto-Oncogenic Network Results in PTEN Loss and Tumorigenesis
Charbel Darido, Smitha R. Georgy, Tomasz Wilanowski,  et al.
Despite its prevalence, the molecular basis of squamous cell carcinoma (SCC) remains poorly understood. Here, we identify the developmental transcription factor Grhl3 as a potent tumor suppressor of SCC in mice, and demonstrate that targeting of Grhl3 by a miR-21-dependent proto-oncogenic network underpins SCC in humans. Deletion of Grhl3 in adult epidermis evokes loss of expression of PTEN, a direct GRHL3 target, resulting in aggressive SCC induced by activation of PI3K/AKT/mTOR signaling. Restoration of Pten expression completely abrogates SCC formation. Reduced levels of GRHL3 and PTEN are evident in human skin, and head and neck SCC, associated with increased expression of miR-21, which targets both tumor suppressors. Our data define the GRHL3-PTEN axis as a critical tumor suppressor pathway in SCC.

4. 人体多能干细胞代谢的调节机制
【动态】
　　主要根据形态学证据，假设人体多能干细胞（hPSCs）含有未成熟的、不能进行生物能量代谢的线粒体。相反，已分化的成体细胞拥有枝状的线粒体网络进行氧化磷酸化作为主要的能量来源。线粒体在hPSCs的生物能学和细胞分化中的作用还不确定。美国科学家的最新研究表明hPSCs有起作用的呼吸复合物能够最大量的消耗氧气。尽管如此，hPSCs的ATP生产主要还是靠糖酵解，F1F0 ATP合成酶消耗ATP来部分维持hPSC线粒体膜电位和细胞活力。解耦联蛋白2（UCP2）通过预防线粒体葡萄糖氧化和促进经由底物分流机制的糖酵解调节hPSC的能量代谢。早期的分化使得hPSC增殖变慢，能量代谢降低，UCP2被抑制，导致糖酵解减少，维持或提高了线粒体葡萄糖氧化。异常的UCP2表达扰乱了这一代谢转换损害了hPSC分化。总之，hSPCs具有有功能的线粒体也需要UCP2抑制以进行完全的分化。

【点评】
　　该研究表明人体多能干细胞拥有能够进行氧化磷酸化的线粒体，但是其主要能量来源是糖酵解，其正常分化需要抑制UCP2的作用，使其能量代谢途径从糖酵解向氧化磷酸化转变。

【参考论文】
The EMBO Journal, 2011; DOI: 10.1038/emboj.2011.401
UCP2 regulates energy metabolism and differentiation potential of human pluripotent stem cells
Jin Zhang, Ivan Khvorostov, Jason S Hong, et al. 
It has been assumed, based largely on morphologic evidence, that human pluripotent stem cells (hPSCs) contain underdeveloped, bioenergetically inactive mitochondria. In contrast, differentiated cells harbour a branched mitochondrial network with oxidative phosphorylation as the main energy source. A role for mitochondria in hPSC bioenergetics and in cell differentiation therefore remains uncertain. Here, we show that hPSCs have functional respiratory complexes that are able to consume O2 at maximal capacity. Despite this, ATP generation in hPSCs is mainly by glycolysis and ATP is consumed by the F1F0 ATP synthase to partially maintain hPSC mitochondrial membrane potential and cell viability. Uncoupling protein 2 (UCP2) plays a regulating role in hPSC energy metabolism by preventing mitochondrial glucose oxidation and facilitating glycolysis via a substrate shunting mechanism. With early differentiation, hPSC proliferation slows, energy metabolism decreases, and UCP2 is repressed, resulting in decreased glycolysis and maintained or increased mitochondrial glucose oxidation. Ectopic UCP2 expression perturbs this metabolic transition and impairs hPSC differentiation. Overall, hPSCs contain active mitochondria and require UCP2 repression for full differentiation potential.

5. 会分泌红细胞生成素EPO的血管
【动态】
　　几十年来，自身间接体内基因治疗被假定为注射重组蛋白的替代方法。然而，有效的将先前取自患者的细胞再移植回去挑战很大。美国科学家通过基因工程将特定指令引入了血管组织细胞中，构建了人体血源内皮细胞系形成细胞（ECFCs）在四环素调节系统控制下表达EPO，并在免疫缺陷老鼠中创建了皮下血管网络能够系统的释放EPO。这些以ECFCs为内表面的血管网络形成了与老鼠脉管系统的有效吻合，使得重组的EPO可以直接释放到血流中。EPO表达激活后，在正常和贫血老鼠中都诱导了红细胞生成。这一过程能够完全逆转。该方法使得病人从频繁的EPO注射中解放出来，减少治疗贫血的医疗费用。

【点评】
　　通过基于内皮细胞系形成细胞的基因输送策略，创建了可以调控的EPO体内表达和直接释放入血流的体系，成为注射重组蛋白的替代方法。

【参考论文】
Blood, 2011; DOI: 10.1182/blood-2011-08-372946
Induction of erythropoiesis using human vascular networks genetically-engineered for controlled erythropoietin release
R.-Z. Lin, A. Dreyzin, K. Aamodt, et al.
For decades, autologous ex vivo gene therapy has been postulated as a potential alternative to parenteral administration of recombinant proteins. However, achieving effective cellular engraftment of previously retrieved patient cells is challenging. Recently, our ability to engineer vasculature in vivo has allowed for the introduction of instructions into tissues by genetically modifying the vascular cells that build these blood vessels. In the present study, we genetically engineered human blood–derived endothelial colony-forming cells (ECFCs) to express erythropoietin (EPO) under the control of a tetracycline-regulated system, and generated subcutaneous vascular networks capable of systemic EPO release in immunodeficient mice. These ECFC-lined vascular networks formed functional anastomoses with the mouse vasculature, allowing direct delivery of recombinant human EPO into the bloodstream. After activation of EPO expression, erythropoiesis was induced in both normal and anemic mice, a process that was completely reversible. This approach could relieve patients from frequent EPO injections, reducing the medical costs associated with the management of anemia. We propose this ECFC-based gene-delivery strategy as a viable alternative technology when routine administration of recombinant proteins is needed.

6. 肿瘤生长离不开分子伴侣介导的自噬
【动态】
　　细胞自噬对于维持哺乳动物细胞的稳定和生物能学非常重要。两种研究的最清楚的自噬机制是大自噬和分子伴侣介导的自噬（CMA）。大自噬的行为变化在癌细胞和实体瘤中已有描述，抑制大自噬会促进肿瘤形成。正常细胞通过上调CMA途径来对抑制大自噬作出反应。美国科学家的最新研究表明在不同类型的癌细胞中都有CMA上调且与大自噬状态无关，另外在不同类型和来源的人体肿瘤中CMA组分都有增加。体外癌细胞增殖需要CMA，因为它维持恶变细胞的代谢变化特征。在老鼠中用人肺癌细胞异种移植，这些科学家证明了体内癌细胞对CMA的依赖性。抑制CMA延缓了抑制肿瘤的生长，减少了癌转移的数量，并诱导老鼠中已移植的人肺癌组织的萎缩。类似的处理也减少了两种不同类型的黑色素瘤细胞系的生长的事实意味着针对该自噬途径可能有广谱的抗肿瘤潜力。

【点评】
　　该研就结果表明肿瘤生长离不开分子伴侣介导的细胞自噬，从而为癌症治疗提供了新的研究方向。

【参考论文】
Science Translational Medicine, 2011; 3 (109): 109ra117
Chaperone-Mediated Autophagy Is Required for Tumor Growth
Maria Kon, Roberta Kiffin, Hiroshi Koga, et al.
The cellular process of autophagy (literally “self-eating”) is important for maintaining the homeostasis and bioenergetics of mammalian cells. Two of the best-studied mechanisms of autophagy are macroautophagy and chaperone-mediated autophagy (CMA). Changes in macroautophagy activity have been described in cancer cells and in solid tumors, and inhibition of macroautophagy promotes tumorigenesis. Because normal cells respond to inhibition of macroautophagy by up-regulation of the CMA pathway, we aimed to characterize the CMA status in different cancer cells and to determine the contribution of changes in CMA to tumorigenesis. Here, we show consistent up-regulation of CMA in different types of cancer cells regardless of the status of macroautophagy. We also demonstrate an increase in CMA components in human cancers of different types and origins. CMA is required for cancer cell proliferation in vitro because it contributes to the maintenance of the metabolic alterations characteristic of malignant cells. Using human lung cancer xenografts in mice, we confirmed the CMA dependence of cancer cells in vivo. Inhibition of CMA delays xenograft tumor growth, reduces the number of cancer metastases, and induces regression of existing human lung cancer xenografts in mice. The fact that similar manipulations of CMA also reduce tumor growth of two different melanoma cell lines suggests that targeting this autophagic pathway may have broad antitumorigenic potential.