世界生命科學(xué)前沿動態(tài)周報（六十六）

（11.14-11.20/2011）
美寶國際集團(tuán):陶國新 

　　主要內(nèi)容：發(fā)現(xiàn)SHARPIN蛋白是內(nèi)源性β1整合素激活的抑制劑；癌癥聯(lián)合治療的“萬能藥”；終止皮膚癌形成的信號；人體多能干細(xì)胞代謝的調(diào)節(jié)機(jī)制；會分泌紅細(xì)胞生成素EPO的血管；腫瘤生長離不開分子伴侶介導(dǎo)的自噬。

　　焦點(diǎn)動態(tài)：人體多能干細(xì)胞代謝的調(diào)節(jié)機(jī)制。

1. 發(fā)現(xiàn)SHARPIN蛋白是內(nèi)源性β1整合素激活的抑制劑
【動態(tài)】
　　有序激活整合素對細(xì)胞附著、運(yùn)動和組織的穩(wěn)定都是至關(guān)重要的。蛋白Talin 和 kindlin激活β1整合素，但是抵消激活的抑制機(jī)制還不清楚。芬蘭科學(xué)家通過RNAi篩選發(fā)現(xiàn)SHARPIN蛋白是β1整合素的重要抑制劑。SHARPIN蛋白在人體癌細(xì)胞和主要的白血球中抑制β1整合素的功能。在SHARPIN缺陷的老鼠中纖維細(xì)胞、白血球和角質(zhì)細(xì)胞表現(xiàn)出增強(qiáng)了的β1整合素作用，而通過重新表達(dá)SHARPIN蛋白能夠完全解除這種增強(qiáng)。SHARPIN蛋白直接結(jié)合到整合素α亞基的一個保守的胞質(zhì)區(qū)，抑制整合素招募激活蛋白Talin 和 kindlin。因此，SHARPIN蛋白抑制了β1整合素從無活性到有活性的關(guān)鍵的構(gòu)象轉(zhuǎn)變。

【點(diǎn)評】
　　該研究在已知β1整合素的激活蛋白基礎(chǔ)上發(fā)現(xiàn)了抑制其激活的蛋白，進(jìn)一步完善了β1整合素的調(diào)控機(jī)制，對于研究人體組織如何保持自身的平衡和穩(wěn)定有重要意義。

【參考論文】
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. 癌癥聯(lián)合治療的“萬能藥”
【動態(tài)】
　　由于每種癌癥都是一些處于不同發(fā)展階段的癌細(xì)胞的非均勻混合物，其治療就面對一次同時治療多種不同的病變細(xì)胞。一種可信的辦法是建立個體化的基因和蛋白圖譜，找出目標(biāo)癌基因并開出專門針對單個病人的靶向治療藥物的組合。但是，個體化醫(yī)學(xué)還存在很多實際問題：1）腫瘤經(jīng)常演變出抗藥的類型；2）治療會極其昂貴；3）很多靶向治療藥物還沒有開發(fā)出來。美國科學(xué)家就此提出了已在動物實驗中見效的另一種策略：2-脫氧葡萄糖（2DG）與Bcl-2拮抗劑（如ABT）的聯(lián)合治療。促凋亡蛋白Bak通常被Mcl-1 和 Bcl-xL抑制。只有從Mcl-1 和 Bcl-xL釋放出來后，Bak才能誘導(dǎo)凋亡。2DG通過解離Bak-Mcl-1 復(fù)合物能夠使大量分解糖的細(xì)胞，一般是一些腦細(xì)胞和大部分癌細(xì)胞，做好準(zhǔn)備。之后，ABT能夠結(jié)合Bcl-xL，解離Bak-Bcl-xL復(fù)合物，解放Bak去誘導(dǎo)細(xì)胞凋亡。ABT不能穿過血腦屏障，只有位于腦部以外大量分解糖的癌細(xì)胞能夠接觸2DG和ABT兩種物質(zhì)。由于ABT在非常接近凋亡末期的階段直接觸發(fā)細(xì)胞凋亡，2DG-ABT聯(lián)合治療適用于所有發(fā)展階段的很多種癌癥，副作用很小。

【點(diǎn)評】
　　該研究通過兩種物質(zhì)的聯(lián)合使用分兩步特異性的使腦外的癌細(xì)胞首先失去葡萄糖能量代謝供應(yīng)繼而觸發(fā)細(xì)胞凋亡，可以適用于不同階段的多種癌癥，副作用是有時候會引起淋巴細(xì)胞和血小板減少。

【參考論文】
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. 終止皮膚癌形成的信號
【動態(tài)】
　　鱗狀細(xì)胞癌（SCC）雖然很常見，但是其分子機(jī)理仍不明了。澳大利亞、波蘭和美國科學(xué)家的合作研究在老鼠中發(fā)現(xiàn)了高效的SCC抑制物--發(fā)育轉(zhuǎn)錄因子Grhl3，并證明以Grhl3為目標(biāo)的miR-21依賴的原癌基因網(wǎng)絡(luò)加強(qiáng)了人體的SCC。成體表皮中刪除Grhl3會引起一種GRHL3的直接靶標(biāo)PTEN蛋白表達(dá)的喪失，導(dǎo)致PI3K/AKT/mTOR信號激活誘導(dǎo)的侵襲性SCC?；謴?fù)Pten表達(dá)能夠完全終止SCC的形成。人體皮膚和頭頸SCC中明顯減少的GRHL3和PTEN水平與以此二者為靶標(biāo)的miR-21水平增加相關(guān)聯(lián)。他們的數(shù)據(jù)將GRHL3-PTEN軸定義為SCC的關(guān)鍵腫瘤抑制途徑。
【點(diǎn)評】
　　該研究發(fā)現(xiàn)了抑制鱗狀皮膚癌形成的分子機(jī)制，對于鱗狀皮膚癌的預(yù)防和治療有重要價值。

【參考論文】
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. 人體多能干細(xì)胞代謝的調(diào)節(jié)機(jī)制
【動態(tài)】
　　主要根據(jù)形態(tài)學(xué)證據(jù)，假設(shè)人體多能干細(xì)胞（hPSCs）含有未成熟的、不能進(jìn)行生物能量代謝的線粒體。相反，已分化的成體細(xì)胞擁有枝狀的線粒體網(wǎng)絡(luò)進(jìn)行氧化磷酸化作為主要的能量來源。線粒體在hPSCs的生物能學(xué)和細(xì)胞分化中的作用還不確定。美國科學(xué)家的最新研究表明hPSCs有起作用的呼吸復(fù)合物能夠最大量的消耗氧氣。盡管如此，hPSCs的ATP生產(chǎn)主要還是靠糖酵解，F(xiàn)1F0 ATP合成酶消耗ATP來部分維持hPSC線粒體膜電位和細(xì)胞活力。解耦聯(lián)蛋白2（UCP2）通過預(yù)防線粒體葡萄糖氧化和促進(jìn)經(jīng)由底物分流機(jī)制的糖酵解調(diào)節(jié)hPSC的能量代謝。早期的分化使得hPSC增殖變慢，能量代謝降低，UCP2被抑制，導(dǎo)致糖酵解減少，維持或提高了線粒體葡萄糖氧化。異常的UCP2表達(dá)擾亂了這一代謝轉(zhuǎn)換損害了hPSC分化。總之，hSPCs具有有功能的線粒體也需要UCP2抑制以進(jìn)行完全的分化。

【點(diǎn)評】
　　該研究表明人體多能干細(xì)胞擁有能夠進(jìn)行氧化磷酸化的線粒體，但是其主要能量來源是糖酵解，其正常分化需要抑制UCP2的作用，使其能量代謝途徑從糖酵解向氧化磷酸化轉(zhuǎn)變。

【參考論文】
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. 會分泌紅細(xì)胞生成素EPO的血管
【動態(tài)】
　　幾十年來，自身間接體內(nèi)基因治療被假定為注射重組蛋白的替代方法。然而，有效的將先前取自患者的細(xì)胞再移植回去挑戰(zhàn)很大。美國科學(xué)家通過基因工程將特定指令引入了血管組織細(xì)胞中，構(gòu)建了人體血源內(nèi)皮細(xì)胞系形成細(xì)胞（ECFCs）在四環(huán)素調(diào)節(jié)系統(tǒng)控制下表達(dá)EPO，并在免疫缺陷老鼠中創(chuàng)建了皮下血管網(wǎng)絡(luò)能夠系統(tǒng)的釋放EPO。這些以ECFCs為內(nèi)表面的血管網(wǎng)絡(luò)形成了與老鼠脈管系統(tǒng)的有效吻合，使得重組的EPO可以直接釋放到血流中。EPO表達(dá)激活后，在正常和貧血老鼠中都誘導(dǎo)了紅細(xì)胞生成。這一過程能夠完全逆轉(zhuǎn)。該方法使得病人從頻繁的EPO注射中解放出來，減少治療貧血的醫(yī)療費(fèi)用。

【點(diǎn)評】
　　通過基于內(nèi)皮細(xì)胞系形成細(xì)胞的基因輸送策略，創(chuàng)建了可以調(diào)控的EPO體內(nèi)表達(dá)和直接釋放入血流的體系，成為注射重組蛋白的替代方法。

【參考論文】
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. 腫瘤生長離不開分子伴侶介導(dǎo)的自噬
【動態(tài)】
　　細(xì)胞自噬對于維持哺乳動物細(xì)胞的穩(wěn)定和生物能學(xué)非常重要。兩種研究的最清楚的自噬機(jī)制是大自噬和分子伴侶介導(dǎo)的自噬（CMA）。大自噬的行為變化在癌細(xì)胞和實體瘤中已有描述，抑制大自噬會促進(jìn)腫瘤形成。正常細(xì)胞通過上調(diào)CMA途徑來對抑制大自噬作出反應(yīng)。美國科學(xué)家的最新研究表明在不同類型的癌細(xì)胞中都有CMA上調(diào)且與大自噬狀態(tài)無關(guān)，另外在不同類型和來源的人體腫瘤中CMA組分都有增加。體外癌細(xì)胞增殖需要CMA，因為它維持惡變細(xì)胞的代謝變化特征。在老鼠中用人肺癌細(xì)胞異種移植，這些科學(xué)家證明了體內(nèi)癌細(xì)胞對CMA的依賴性。抑制CMA延緩了抑制腫瘤的生長，減少了癌轉(zhuǎn)移的數(shù)量，并誘導(dǎo)老鼠中已移植的人肺癌組織的萎縮。類似的處理也減少了兩種不同類型的黑色素瘤細(xì)胞系的生長的事實意味著針對該自噬途徑可能有廣譜的抗腫瘤潛力。

【點(diǎn)評】
　　該研就結(jié)果表明腫瘤生長離不開分子伴侶介導(dǎo)的細(xì)胞自噬，從而為癌癥治療提供了新的研究方向。

【參考論文】
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.