细胞信号转导

1、MED12 Controls the Response to Multiple Cancer Drugs through Regulation of TGF- Receptor Signaling 肺癌是世界上最常见的恶性肿瘤之一，其中非小细胞肺癌（non-small cell lung cancer, NSCLC）约占80%。2007年Soda等在NSCLC患者肿瘤标本中首次发现由2号染色体短臂内转位inv（2）（p21p23）形成的棘皮动物微管相关类蛋白4（echinoderm microtubule-associated proteinlike4, EML4）与间变性淋巴瘤激酶（anap

2、lastic lymphoma kinase, ALK）的融合基因。已有研究表明EML4-ALK可诱导肿瘤生成，给予ALK抑制剂后肿瘤可迅速消退。在目前已报道的研究中EML4-ALK融合基因在NSCLC中的阳性率约为5%，EML4-ALK融合基因已成为肺癌临床治疗新靶点。 通过构建RNAi表达载体制备shRNA(发卡结构小干扰RNA)，已成为进行RNA干扰实验的常用手段之一。 而目前为了感染原代细胞和难感染的细胞系或者在动物水平进行RNAi，研究科学家更多的选择了慢病毒载体。在RNAi的研究中，为了感染原代细胞和难感染的细胞系，科学家开始借助于病毒载体实现RNAi；由于慢病毒可以同时感染分裂和

3、非分裂细胞、整合性以及免疫原性低等优点，目前在RNAi的研究中，基于慢病毒构建的shRNA被最广泛的使用。而在RNAi研究需要长期观察或者需要进行动物实验时，基于慢病毒构建的shRNA的优势更为明显。 large-scale RNAi screen BRAF是一种癌基因，它编码一种丝/苏氨酸特异性激酶，是RAS/RAF/MEK/ERK/MAPK通路重要的转导因子，参与调控细胞内多种生物学事件，如细胞生长、分化和凋亡等。研究表明，在多种人类恶性肿瘤中，如恶性黑色素瘤、结直肠癌、肺癌、甲状腺癌、肝癌及胰腺癌等均存在不同比例的BRAF基因突变，约66%恶性黑色素瘤和15的结肠癌中BRAF基因存在体细

4、胞错义突变。大约80-90%的BRAF基因突变发生在exon15的1799核苷酸上，T突变为A，导致其编码的谷氨酸由缬氨酸取代(V600E)。目前认为，V600E突变可模拟T599和S602两个位点的磷酸化过程，从而使BRAF蛋白异常激活. MEK（MAPK/ERK激酶）激酶） MEK分为分子量44kD和45kD的MEK1和MEK2两种。Raf被激活后，它的C端催化区能与MEK结合，并使其催化区第亚区中两个Ser磷酸化，从而使MEK激活。MEK属于少有的双重特异性激酶，使Tyr和Thr两个调节位点磷酸化而激活ERK。但MEK如何兼具Tyr和Thr双特异性磷酸化活性目前尚不清楚，不过MEK对ER

5、K的Tyr/Thr双特异性磷酸化具有重要的生理意义，因为ERK信号通路在细胞信号转导网络中处于枢纽地位，任何错误的活化都会对细胞生命活动产生深远的影响，而这种双特异性的识别和激活机制，大大提高了信号转导的准确性，防止了ERK的错误激活。EMT 上皮细胞在某些因素的作用下，细胞极性、细胞间密连接和黏附连接逐渐消失，而获得了游走和侵袭性的迁移能力，并产生了大量的细胞外基质成分，抑制了细胞凋亡，变成了具有间质细胞形态和特性的细胞，此改变称为EMT。上皮细胞具有非常典型的游离-基底面极性，而且细胞间紧密连接与黏附连接也限制了其任意迁移的能力。在EMT过程中，细胞功能发生了一些变化，与相邻的细胞分离开，

6、迁移到邻近组织，其主要分子特征如角蛋白、E-钙黏素等上皮标记物表达了下调，和波形蛋白、N-钙黏素等间质标记物表达了上调。此外，如果肿瘤细胞发生了转移，在微环境等某些因素的刺激下，转化的上皮细胞又可再分化为上皮细胞，即可以再次发生间质-上皮转化，又获得了上皮细胞的特性，即E-钙黏素表达的上调。EMT不仅存在于胚胎发育的过程中，同时还和多种慢性病，如肾纤维化的发生与肿瘤侵袭及转移有着密切的联系。在EMT过程中有时发生功能的改变，有时只以上皮表型为主，但是同时都会存在间质表型。CONTENTS ABSTRATE INTRODUCTION RESULTS SUMMERY DISCUSSIONABSTR

7、ATE 在包含EML4-ALK易位或激活EGFR突变体的肺癌中，ALK和EGF受体酪氨酸激酶的抑制子刺激产生强烈而短暂的反应。我们利用大范围的RNAi筛选方法鉴定到MED12，它是癌症中被突变的转录调节复合物，可以作为ALK和EGFR抑制子应答的一个决定性因子。MED12部分存在于细胞质中，在这里它可以通过生理相互作用负调控TGF-R2。MED12被抑制时，TGF-R信号通路被激活，而这对于耐药性是必须且足够的。TGF-信号引起MEK/ERK激活作用，所以MED12抑制作用在其他癌症中也可以对MEK和BRAF的抑制子产生抗性。MED12的缺失诱导类EMT表型，这种表型在结肠癌患者产生的化学疗法

8、抗性和肺癌患者中产生的对吉非替尼抗性相关联。在MED12KD 细胞中，TGF-R信号的抑制作用修复了药物应答性，这是一种缺失MED12条件下治疗耐药性肿瘤的策略。INTRODUCTION Cancer therapy is often hampered by the rapid emergence of drug resistance. This is true not only for the conventional chemotherapies but also for the new generation of drugs targeting those components that

9、 are mutated or deregulated in tumor cells. NSCLCs（非小细胞肺癌非小细胞肺癌）、EGFR、 ALK inhibitor 、responses are often short-lived、 CML(慢性髓性白血病)、 BRAF EGFR mutant 、NSCLCs 、secondary mutations in the EGFR gene . Similarly, acquired resistance to BRAF inhibition in melanoma can result from an activating mutation i

10、n the MEK1 kinase that was not detectable in the primary tumor and overexpression of COT, leading to activation of MEK, can be a causal agent in BRAF resistance in melanoma. . At present, some 30% of the resistance to EGFR-targeted therapies in NSCLCs cannot be explained by any of the mechanisms des

11、cribed above. Through functional genetic screens， we find that suppression of MED12 also confers resistance to a range of cancer drugs, including chemotherapy, in colon cancer, melanoma, and liver cancer. We identify an unexpected activity of MED12 in regulating transforming growth factor (TGF-) rec

12、eptor signaling, as the major mechanism of drug-resistance induction。 实验流程图MED12的发现探究MED12的功能TGF-R2的发现及MED12的loss对TGF-通路的影响TGF-通路在抗癌药物中的地位MED12 TGF- 通路 TGF-R2的关系MED12特异性基因都有EMT特点TGF-R Inhibitor and TKIs协同抑制MED12KD NSCLC CELLRESULTS 1.MED12 Suppression Confers Resistance to Multiple Tyrosine Kinase I

13、nhibitors in NSCLCs. 2.MED12 Loss Leads to MEK/ERK Activation and Drug Resistance in Different Cancer Types. Our finding that MED12 suppression confers resistance to both ALK and EGFR inhibitors suggests that MED12 might act on a core pathway downstream of both ALK and EGFR, such as RAS signaling. 慢

14、病毒载体（复制缺陷型慢病毒载体，它可以直接通过转染操作被导入细胞，作为常规RNAi载体使用，也可被包装成慢病毒颗粒，然后感染细胞）MED12的抑制导致了下游通路的激活ALK抑制剂EGFR抑制剂 3.TGF- Signaling Is Required for Drug Resistance Caused by MED12 Loss. To address through which pathway MED12 acts to mediate these drug-resistance effects, we screened a lentiviral shRNA library represe

15、nting all 518 human kinases for genes whose inhibition restores sensitivity to ALK inhibitors in MED12KDcells.This suggests that suppression of TGF-R2 synergizes (协同)with ALK inhibition in MED12KDcells.These findings indicate that suppression ofTGF-R2 resensitizes（使再敏感） the MED12KD cells to ALK inhi

16、bition and suggest that TGF- signaling is required for the drug resistance caused by MED12 loss。MED12的存在提高了TGF- 4.TGF- Signaling Is Sufficient to Confer Resistance to a Variety of Cancer DrugsThese results demonstrate that activation of TGF- signaling is sufficient to confer resistance to multiple c

17、ancer drugs in the cancer types in which MED12KD also confers drug resistance. 5.Downregulation of MED12 Activates TGF- Signaling by Elevating TGF-R2 Protein LevelsIt is well-established that TGF- induces an epithelial mesenchymal transition (EMT), leading to the induction of several mesenchymal mar

18、kers such as Vimentin (VIM)(波形蛋白) and N-cadherin (CDH2)（钙粘蛋白）,MED12KDalso induced expression of VIM and CDH2, indicating that an EMT-like process is initiated in MED12KD cells. Accordingly, the protein products of these mesenchymal-specific genes were also detected in MED12KD cells at levels similar

19、 to those induced by TGF- treatment in the same cells Expression of the epithelial marker E-cadherin (CDH1) was not lost in MED12KDcells ，suggesting that MED12KD induces a partial EMT. Together these unbiased gene-expression studies support the notion that MED12 is a suppressor of TGF- signaling in

20、a wide range of cancer types and that its loss activates TGF- signaling。These results suggest that MED12 predominantly suppresses TGF-R2 in a posttranscriptional manner. H3122通过临位连接技术和免疫共沉淀技术发现：位于细胞质中的MED12与TGF-之间的相互作用。 The observation that MED12KD caused a strong increase of cell-surface TGF-R2 sug

21、gests that MED12 could inhibit TGF-R signaling by preventing the maturation of TGF-R（MED12 interferes with the proper glycosylation of TGF-R2 and hence blocks cell-surface expression of the receptor.） 6.A MED12KDGene Signature Has Features of EMT and Is Both Prognostic and Predictive.MED12 suppressi

22、on leads to activation of TGF- signaling and expression of mesenchymal（胞质） markers, suggestive of a partial EMT-like process. Recently, EMT has been identified as a program in human CRC that correlates with poor prognosis We therefore asked whether MED12KD indeed induces an EMT-like process and whet

23、her the processes induced by MED12KD are likewise associated with poor prognosis in CRC.（237upegulation,229,31部分结直肠癌，231patients MED12KD These results indicate that the processes induced by MED12KD are associated with a poor survival in CRC patients. ） Next, we examined a second cohort of 270 stage

24、III CRC patients, only some of whom were treated with 5-FU-based chemotherapy and whose responses to chemotherapy are known, to determine whether the MED12KD signature could also predict responses to chemotherapy in patients.使用微阵列技术，将MED12分为野生型和MED12KD Identified by RNA-Seq, Thus the group of genes

25、that is upregulated upon MED12KD predicts response to MEK inhibitors in a very heterogeneous(多样化) panel of cancer cell lines, consistent with our finding that MED12 acts independently of cellular context to influence cancer drug response（独立行使功能）. Finally, we asked whether expression of MED12KD Signa

26、ture genes is associated with drug resistance to targeted agents in the clinic. We obtained pairs of tumor samples derived from three patients that have NSCLC tumors With EGFR-activating mutations both before and after development of resistance to gefitinib . Two of the resistant tumors did have the

27、 EGFR T790M gatekeeper mutation. For the tumor pair without the EGFR T790M mutation ,we did observe a significant overlap of genes upregulated after acquisition of gefitinib resistance with the MED12KDsignature genes but not for the two tumor pairs with EGFR T790M mutation . This result indicates that in the patient of case 10, a gene