Shp2调控肝癌进展的信号网络及临床意义研究
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摘要
研究背景和目的
肝癌是当前世界上最常见的恶性肿瘤之一，在全球恶性肿瘤中其发病率排第五位，男性病死率更高居第二位。因其恶性程度高，预后差，素有癌中之王的称号[1]。随着肝癌外科治疗和生物治疗的不断发展，目前临床上肝癌的疗效较过去取得了一定提高，但由于大多数肝癌患者确诊时已是中晚期，失去了最佳手术时机，常规放化疗又不敏感，且肝癌术后常有复发转移，这些因素使得肝癌患者的预后仍然较差[2]。统计资料显示，肝癌术后5年复发率高达70%，复发转移已经成为提高肝癌患者远期预后、影响肝癌患者生存率和生活质量的重要制约，也成为治愈乃至最终攻克肝癌的重中之重[3]。因此，阐明肝癌生长、转移和化疗抵抗的分子机制，找到有效的预后标志物和干预靶点已成为当前肝癌研究领域的重中之重。
随着对癌基因和抑癌基因生物学功能研究的不断深入，肿瘤发生发展的分子机制不断得到阐释[4]。在肿瘤细胞中，有些关键的节点分子调控着多条肿瘤相关信号转导通路[5,6]。在生物化学水平上，一些节点分子的酪氨酸磷酸化和去磷酸化状态在肿瘤信号网络调控中非常关键，这一过程受到蛋白酪氨酸激酶和酪氨酸磷酸酶的精密调控。目前已在很多肿瘤中检测到了蛋白酪氨酸激酶和酪氨酸磷酸酶的异常表达[5,7]。Shp2（The Src homology phosphotyrosin phosphatase2）就是上世纪90年代本校特聘教授冯根生等人首先发现的一种非跨膜型蛋白酪氨酸磷酸酶，它由PTPN11基因编码，含有2个SH2结构域和1个酪氨酸蛋白磷酸酶（PTP酶）结构（图1）；
Shp2的SH2结构域与磷酸化的酪氨酸结合后，可使其自身的PTP酶活化并对其底物蛋白进行去磷酸，进而对细胞内其它信号分子发挥重要的调控作用[8-10]。二十年来的研究显示，Shp2分子可参与传递细胞外多种生长因子、细胞因子和激素产生的信号，从而正向调控与细胞存活和增殖相关的信号转导通路[11,12]。
已有研究表明Shp2可参与多种有丝分裂原对细胞中Erk信号分子的激活[11]。冯根生等利用Shp2肝细胞条件性敲除小鼠模型研究Shp2在肝再生中的作用时发现，肝部分切除术后Shp2敲除的肝细胞中Erk的活化和肝细胞的增殖被明显抑制[13]。在特定的条件下，Shp2还能活化PI3-K/Akt和Jak/STAT信号通路。利用小鼠进行的多项遗传学研究提示，Shp2在哺乳动物的发育和疾病中可能存在广泛的作用[17,18]。在发育障碍性疾病Noonan综合征中发现，大约50％的患者伴有Shp2突变。此外，Shp2突变被公认是青少年粒单细胞白血病（JMML）的高危因素[19]，在不同类型白血病中均检测到Shp2的突变激活，所以Shp2被认为是白血病的原癌基因[20,21]。有研究显示，在胃癌、间变性大细胞型淋巴瘤和神经胶质瘤中，Shp2可受到癌基因正调控而呈过度活化状态[22-24]，但是Shp2在肝癌等实体肿瘤中的突变鲜有报道[25-26]。
我们前期的研究发现，在肝癌发生过程中Shp2肝细胞条件性敲除的小鼠更容易发生肝癌，进一步研究证实Shp2可以通过下调IL-6/STAT3信号通路抑制炎症诱发的肝癌，这也提示Shp2在特定条件下可以发挥抑癌作用。该研究在Cancer Cell杂志发表后受到广泛关注，并被评为Cancer Cell杂志最受关注的20篇论著之一。促癌分子在特定条件下发挥抑癌作用之前也有报道，如肝癌研究权威Micheal Karin报道了肝细胞中敲除IKK后小鼠更易发生肝癌[27]。冯根生教授对这些发现进行总结后提出，在肝细胞内特异性敲除Shp2或IKK等促进细胞存活的分子后，促进了肝细胞损伤并引起慢性炎症。肝细胞具有强大的再生能力，长期的慢性炎症引起肝细胞过度的代偿性增殖最终诱发了肝癌（图2）。这一观点得到了国际同行的一致认同，相关综述发表在Cancer Cell杂志[28]。
有同行在我们工作的提示下，研究并提出Shp2在部分肝癌中低表达并与肝癌病人的预后呈负相关[29]。有趣的是在进行上述研究过程中我们发现，在大多数肝癌组织中Shp2呈高表达状态，与Shp2在白血病和乳腺癌中的表达一致；并且Shp2可以上调与肝癌生长和转移密切相关的Ras和PI3-K蛋白激酶活性，这些前期结果提示Shp2在肝癌的发展过程中可能仍然发挥促进作用[30]。综上所述，Shp2在肝癌中的生物学作用尚待进一步的研究来证实，但可以肯定的是作为重要的节点分子，Shp2在肝癌细胞中可能调控着多条重要的肿瘤相关信号通路，并且可能成为新的肝癌预后标志物和潜在的治疗靶点。
越来越多的研究表明，肝癌中存在一群具有自我更新能力和多向分化潜能的干性细胞群即肝癌干细胞。肝癌干细胞在肝癌进程中发挥重要作用，它不但控制着肝癌的发生和进展，也参与了肝癌的复发和耐药，因此深入研究肝癌干细胞的调控机制并找到合适的干预靶点有望为肝癌的治疗提供新的思路。到目前为止，Shp2在干细胞中的功能研究还很少，冯根生课题组发现Shp2在胚胎干细胞中可以促进干细胞的分化，而在造血干细胞中可以促进干细胞的自我更新，但Shp2对肝癌干细胞的调控目前尚未见报道[31]。OV6是本研究中心发现并报道的肝癌干细胞标志物，目前已被国内外同行普遍接受[32]。我们以OV6为标志物从肝癌细胞中分选肝癌干细胞进行研究发现，β-catenin在肝癌干细胞中异常活化并对肝癌干细胞具有重要的调控作用。前期工作中，本课题组从病人新鲜肝癌样本中分离了原代肝癌细胞（其中存在一定比例的肝癌干细胞），分别感染表达shShp2和对照的慢病毒后进行体外成球实验（SpheroidFormation Assay），结果发现干扰Shp2后肝癌干细胞成球能力明显被抑制，这提示Shp2可能对肝癌干细胞自我更新具有调控作用。在Shp2肝细胞条件性敲除小鼠的肝脏中，我们观察到β-catenin的表达显著降低，这提示Shp2可能对β-catenin具有正调控效应，Shp2是否通过影响β-catenin的表达和活化对肝癌干细胞发挥调控作用值得进一步深入研究。
肝癌是对人类健康威胁最大的肿瘤之一，至今其调控机制仍不十分清楚，没有可用于临床诊断的预后标志物，治疗方面也缺乏有效的分子靶点。寻找肝癌治疗靶标和预后标志物是目前肝癌研究的重要内容。Shp2作为一个重要的肿瘤相关信号调节蛋白，其对肝癌及肝癌干细胞的调控尚无报道。在前期研究基础上，本课题将利用多种特征性肝癌细胞模型、肿瘤转移模型系统研究Shp2在肝癌生长和转移中的作用，揭示其对肝癌干细胞的调控及分子机制，并利用Shp2肝细胞条件性敲除小鼠进行验证。利用大规模肝癌样本及其临床病理资料和随访信息，系统分析Shp2与肝癌临床病理特征和患者预后的关系，探讨Shp2作为肝癌预后标志物的应用前景，提出Shp2在索拉菲尼用药指征和联合化疗中的价值，最终为肝癌的诊断和治疗提供新的靶标。
研究方法：
1.利用蛋白电泳（western-blot）、免疫组织化学染色和Realtime PCR法检测临床肝癌组织中Shp2分子的表达情况并运用统计学方法分析Shp2的表达情况与临床病理和预后资料的相关性；
2.构建过表达和干扰Shp2的质粒，并利用慢病毒系统建立稳定干扰Shp2的肝癌细胞系SMMC-7721shShp2和LM3shShp2及其对照细胞系；
3. SMMC-7721shShp2和LM3shShp2及其对照细胞系分别采用CCK-8法、细胞周期检测、克隆形成实验、衰老检测、粘附实验、划痕实验、Transwell、侵袭实验等方法观察干扰Shp2后对肝癌细胞系增殖、迁移、侵袭、转移能力等生物学特性的影响；
4.采用Spheroid形成实验、流式细胞分析等方法检测Shp2稳定干扰细胞系中肝癌干细胞表型的变化；
5.应用化疗药物ETO、5-FU和多靶点靶向性药物索拉菲尼刺激稳定细胞系，研究Shp2对肝癌细胞化疗敏感性和索拉菲尼用药敏感性的影响；
6.繁殖Shp2肝细胞条件性敲除小鼠，并建立DEN诱导小鼠肝癌模型，与对照组比较，进行Shp2调控信号转导机制的体内研究；
7.体外分离Shp2肝细胞条件性敲除小鼠和对照小鼠原代肝细胞，并离体培养，检测未加刺激情况下和加EGF刺激后Shp2下游信号通路的活性改变；
8.将SMMC-7721shShp2及其对照细胞分别进行裸鼠皮下荷瘤实验，观察干扰Shp2对肝癌细胞体内增殖能力和成瘤能力的影响；
9.将SMMC-7721shShp2及其对照细胞分别进行裸鼠尾静脉注射，建立肺转移模型，进行裸鼠脾注射，建立肝转移模型，观察Shp2对肝癌细胞体内转移能力的影响。
10.将SMMC-7721shShp2及其对照细胞分别进行裸鼠皮下荷瘤实验，并分别给予索拉菲尼和安慰剂进行灌胃，与安慰剂组相比，观察干预Shp2联合索拉菲尼治疗对肿瘤生长的影响；
11.将SMMC-7721shShp2及其对照细胞按照一定比例进行NOD-SCID鼠体内有限稀释成瘤实验，观察Shp2在体内对肝癌干细胞的影响；
12.将SMMC-7721shShp2及其对照细胞按照一定比例种于低粘附96孔板，进行体外有限稀释成球实验，观察Shp2在体外对肝癌干细胞的影响。
13.利用Realtime-PCR、Western-blot、免疫共沉淀、Ras活性检测和PI3-K活性检测等方法研究Shp2对Ras/Raf/MEK/Erk、PI-3K/Akt等信号通路活性的影响。
14.利用Realtime-PCR、Western-blot、报告基因检测、免疫共沉淀、激光共聚焦等方法研究Shp2影响肝癌细胞化疗敏感性及肝癌干细胞表型的分子机制，并探寻Shp2对Wnt/β-catenin信号转导通路的影响。
研究结果：
1.从转录水平、蛋白水平证实Shp2在肝细胞癌中表达显著高于癌旁，且其表达水平与肝癌组织的分化程度呈负相关。在门静脉癌栓等肝癌转移灶中Shp2的表达显著高于相应的肝癌及其癌旁组织，提示Shp2可能参与调控肝癌的恶性生物学行为，并可能与肝癌转移密切相关。
2.通过对带有详细病理资料和预后情况的肝癌组织芯片进行Shp2的免疫组化染色实验并评分，统计分析得出肝癌中Shp2表达越高，患者总体生存期和无瘤生存期越差，并与肿瘤病理类型（AFP水平、肿瘤包膜、分化程度、血管侵犯、子灶、肿瘤大小）密切相关。
3.通过增殖、周期、克隆形成、衰老和裸鼠荷瘤实验发现，与对照组相比，干扰Shp2表达后肝癌细胞系生长、增殖和成瘤能力明显减弱。
4.通过粘附实验、划痕实验、Transwell、侵袭实验、建立裸鼠肺转移模型和肝转移模型等方法发现，干扰Shp2表达后肝癌细胞系粘附、迁移和体内外转移侵袭能力明显减弱。
5.通过Spheroid形成实验、化疗抵抗实验、体外和体内有限稀释实验发现干扰Shp2表达后肝癌干细胞自我更新能力明显减弱。
6.通过对干扰Shp2肝癌细胞系及其对照细胞系进行裸鼠荷瘤实验，并给予小分子靶向药索拉菲尼进行治疗，我们发现干扰Shp2可以提高肝癌细胞对索拉菲尼的敏感性，并在临床随访病例中得到验证。
7.通过对干扰Shp2肝癌细胞系及其对照细胞系进行Ras活性检测、免疫共沉淀、Western blot蛋白电泳检测以及DEN诱导的Shp2肝细胞条件性敲除小鼠及其对照小鼠肝癌切片免疫组织化学染色分析等实验发现， Shp2可能通过调控Ras/Raf/MEK/Erk信号转导通路促进肝癌细胞的生长增殖。
8.通过对干扰Shp2肝癌细胞系及其对照细胞系进行PI3-K激酶活性检测，Western blot蛋白电泳检测及在裸鼠肺转移灶和肝转移灶上的免疫组织化学染色分析等实验发现，Shp2可能通过调控PI3-K/Akt/mTOR信号转导通路并促进肝癌细胞的转移能力。
9.通过报告基因检测、DEN诱导的Shp2肝细胞条件性敲除小鼠及其对照小鼠肝癌切片免疫组织化学染色分析、对干扰Shp2肝癌细胞系及其对照细胞系行免疫共沉淀、激光共聚焦等实验以及放线菌酮（CHX）和蛋白酶体抑制剂（MG132）刺激实验发现，Shp2可能通过与β-catenin相互结合抑制β-catenin的降解进而调控Wnt/β-catenin信号转导通路影响肝癌干细胞。
结论：
我们前期的工作报道了Shp2在肝癌发生中的作用，但是Shp2在肝癌复发转移过程中的生物学作用尚不清楚。本课题研究发现Shp2促进了肝癌的转移且其表达水平与肝癌患者临床预后密切相关。实验结果显示肝癌组织中Shp2表达显著升高，在其转移灶中的表达水平更高。临床病理分析提示，Shp2高表达预示着肝癌的预后不良。体内和体外实验显示Shp2可能通过调控Ras/Raf/MEK/Erk通路促进肝癌细胞的生长。通过调控PI3-K/Akt/mTOR通路促进肝癌细胞的转移。Shp2还可以直接结合β-catenin抑制β-catenin的降解进而促进β-catenin的核转位和活化，从而增强了肝癌细胞干性基因的表达以及化疗抵抗和自我更新能力；这提示Shp2可能通过活化Wnt/β-catenin信号转导通路参与调控肝癌干细胞的生物学功能。进一步的实验结果还提示我们干扰Shp2可增强索拉菲尼的治疗效果且Shp2分子在肝癌细胞中的表达水平可以预测肝癌患者对索拉菲尼治疗的敏感性。综上所述，Shp2在肝癌的发展过程中起到了重要的作用，可能是肝癌复发转移的诊断标志物和潜在的临床治疗靶点。
Hepatocellular carcinoma (HCC) is the fifth most common cancer in the worldand the second leading cause of cancer death in men[1]. Despite the current advance inthe hepatic resection and transplantation, long-term survival of HCC patients remainsunsatisfactory due to the high incidence of recurrence and metastasis after surgicalresection[2]. It has been reported that HCC recurrence rate exceeds70%at5years[3].Therefore, it is urgent to identify novel therapeutic targets so that new strategies forHCC treatment can be developed. Accumulating genetic and functional studies ofoncogenes and tumor suppressor genes have greatly contributed to the advance ofknowledge on the molecular mechanism of tumorigenesis[4]. Inside the cancer cells,various signaling pathways are regulated by some predominant hub molecules at thebiochemical levels[5,6]. Tyrosine phosphorylation and dephosphorylation of these keymolecules plays critical roles in the regulatory network and aberrant tyrosinephosphorylation has been frequently detected in various cancers[5,7].
Shp2, encoded by PTPN11, was first identified by us and other groups in the early1990s as a non-receptor protein tyrosine phosphatases containing two Src-homology2(SH2) domains[8-10]. In the following two decades, accumulating studies documentedthat Shp2acts as a transducer of extracellular pro-survival and proliferation signalsfrom various cytokines, growth factors and hormones[11,12]. Shp2is required for theamplification of ERK signaling upon distinct mitogenic stimuli[11]. In previous work,we generated a hepatocyte-specific Shp2knockout (Shp2hep△) mouse model, andreported that Shp2deletion attenuated Erk activation and hepatocyte proliferationfollowing partial hepatectomy[13]. Nevertheless, effect of Shp2on the modulation ofPI3-K/Akt pathways and Jak/STAT cascade is cell specific or stimuli specific[14-16]. Inaddition, mouse genetics and sequencing studies have also indicated a broad role forShp2in development and disease[17,18].
Genetically, Ptpn11mutation has been detected in up to50%of patients withNoonan syndrome, a kind of developmental disorder, and the patients possess strkingly higher risk of juvenile myelomonocytic leukemia (JMML)[19]. Constitutiveactivation of Shp2by somatic mutations are present in different types of leukemia, andShp2has been considered as a proto-oncogene in leukemia[20,21]. Shp2has beendocumented to hyperactivated by oncogenes in gastric carcinoma, anaplastic large-celllymphoma and glioblastoma[22-24]. However, Ptpn11mutation has been scarcelydetected in solid tumors including hepatoma[25,26]. Our most current data indicated thetumor-inhibiting effect of Ptpn11/Shp2in carcinogenesis via downregulation ofinflammatory signaling. We found that Shp2deficience in hepatocytes causes chronichepatic damage and injury, which triggers inflammation and compensatory hepatocyteproliferation and thus promotes tumorigenesis in the liver, after a long latent period oftime[27]. Nevertheless, it is interesting that overexpression of Shp2was detected in thevast majority of HCC samples. Herein, we found Shp2acts as hub molecule regulatingseveral key cancer-related signaling in hepatoma cells and could serve as a promisingprognosis biomarker and therapeutic target for HCC therapy.
So our purpose of this project is to design a series of related experiments toobserve the role of Shp2on HCC recurrence and metastasis,clarify the molecularbiological mechanism and the clinical significance of Shp2on HCC development, andprovided a theoretical and experimental basis for targeted therapy of HCC.
肝癌是当前世界上最常见的恶性肿瘤之一，在全球恶性肿瘤中其发病率排第五位，男性病死率更高居第二位。因其恶性程度高，预后差，素有癌中之王的称号[1]。随着肝癌外科治疗和生物治疗的不断发展，目前临床上肝癌的疗效较过去取得了一定提高，但由于大多数肝癌患者确诊时已是中晚期，失去了最佳手术时机，常规放化疗又不敏感，且肝癌术后常有复发转移，这些因素使得肝癌患者的预后仍然较差[2]。统计资料显示，肝癌术后5年复发率高达70%，复发转移已经成为提高肝癌患者远期预后、影响肝癌患者生存率和生活质量的重要制约，也成为治愈乃至最终攻克肝癌的重中之重[3]。因此，阐明肝癌生长、转移和化疗抵抗的分子机制，找到有效的预后标志物和干预靶点已成为当前肝癌研究领域的重中之重。
随着对癌基因和抑癌基因生物学功能研究的不断深入，肿瘤发生发展的分子机制不断得到阐释[4]。在肿瘤细胞中，有些关键的节点分子调控着多条肿瘤相关信号转导通路[5,6]。在生物化学水平上，一些节点分子的酪氨酸磷酸化和去磷酸化状态在肿瘤信号网络调控中非常关键，这一过程受到蛋白酪氨酸激酶和酪氨酸磷酸酶的精密调控。目前已在很多肿瘤中检测到了蛋白酪氨酸激酶和酪氨酸磷酸酶的异常表达[5,7]。Shp2（The Src homology phosphotyrosin phosphatase2）就是上世纪90年代本校特聘教授冯根生等人首先发现的一种非跨膜型蛋白酪氨酸磷酸酶，它由PTPN11基因编码，含有2个SH2结构域和1个酪氨酸蛋白磷酸酶（PTP酶）结构（图1）；
Shp2的SH2结构域与磷酸化的酪氨酸结合后，可使其自身的PTP酶活化并对其底物蛋白进行去磷酸，进而对细胞内其它信号分子发挥重要的调控作用[8-10]。二十年来的研究显示，Shp2分子可参与传递细胞外多种生长因子、细胞因子和激素产生的信号，从而正向调控与细胞存活和增殖相关的信号转导通路[11,12]。
已有研究表明Shp2可参与多种有丝分裂原对细胞中Erk信号分子的激活[11]。冯根生等利用Shp2肝细胞条件性敲除小鼠模型研究Shp2在肝再生中的作用时发现，肝部分切除术后Shp2敲除的肝细胞中Erk的活化和肝细胞的增殖被明显抑制[13]。在特定的条件下，Shp2还能活化PI3-K/Akt和Jak/STAT信号通路。利用小鼠进行的多项遗传学研究提示，Shp2在哺乳动物的发育和疾病中可能存在广泛的作用[17,18]。在发育障碍性疾病Noonan综合征中发现，大约50％的患者伴有Shp2突变。此外，Shp2突变被公认是青少年粒单细胞白血病（JMML）的高危因素[19]，在不同类型白血病中均检测到Shp2的突变激活，所以Shp2被认为是白血病的原癌基因[20,21]。有研究显示，在胃癌、间变性大细胞型淋巴瘤和神经胶质瘤中，Shp2可受到癌基因正调控而呈过度活化状态[22-24]，但是Shp2在肝癌等实体肿瘤中的突变鲜有报道[25-26]。
我们前期的研究发现，在肝癌发生过程中Shp2肝细胞条件性敲除的小鼠更容易发生肝癌，进一步研究证实Shp2可以通过下调IL-6/STAT3信号通路抑制炎症诱发的肝癌，这也提示Shp2在特定条件下可以发挥抑癌作用。该研究在Cancer Cell杂志发表后受到广泛关注，并被评为Cancer Cell杂志最受关注的20篇论著之一。促癌分子在特定条件下发挥抑癌作用之前也有报道，如肝癌研究权威Micheal Karin报道了肝细胞中敲除IKK后小鼠更易发生肝癌[27]。冯根生教授对这些发现进行总结后提出，在肝细胞内特异性敲除Shp2或IKK等促进细胞存活的分子后，促进了肝细胞损伤并引起慢性炎症。肝细胞具有强大的再生能力，长期的慢性炎症引起肝细胞过度的代偿性增殖最终诱发了肝癌（图2）。这一观点得到了国际同行的一致认同，相关综述发表在Cancer Cell杂志[28]。
有同行在我们工作的提示下，研究并提出Shp2在部分肝癌中低表达并与肝癌病人的预后呈负相关[29]。有趣的是在进行上述研究过程中我们发现，在大多数肝癌组织中Shp2呈高表达状态，与Shp2在白血病和乳腺癌中的表达一致；并且Shp2可以上调与肝癌生长和转移密切相关的Ras和PI3-K蛋白激酶活性，这些前期结果提示Shp2在肝癌的发展过程中可能仍然发挥促进作用[30]。综上所述，Shp2在肝癌中的生物学作用尚待进一步的研究来证实，但可以肯定的是作为重要的节点分子，Shp2在肝癌细胞中可能调控着多条重要的肿瘤相关信号通路，并且可能成为新的肝癌预后标志物和潜在的治疗靶点。
越来越多的研究表明，肝癌中存在一群具有自我更新能力和多向分化潜能的干性细胞群即肝癌干细胞。肝癌干细胞在肝癌进程中发挥重要作用，它不但控制着肝癌的发生和进展，也参与了肝癌的复发和耐药，因此深入研究肝癌干细胞的调控机制并找到合适的干预靶点有望为肝癌的治疗提供新的思路。到目前为止，Shp2在干细胞中的功能研究还很少，冯根生课题组发现Shp2在胚胎干细胞中可以促进干细胞的分化，而在造血干细胞中可以促进干细胞的自我更新，但Shp2对肝癌干细胞的调控目前尚未见报道[31]。OV6是本研究中心发现并报道的肝癌干细胞标志物，目前已被国内外同行普遍接受[32]。我们以OV6为标志物从肝癌细胞中分选肝癌干细胞进行研究发现，β-catenin在肝癌干细胞中异常活化并对肝癌干细胞具有重要的调控作用。前期工作中，本课题组从病人新鲜肝癌样本中分离了原代肝癌细胞（其中存在一定比例的肝癌干细胞），分别感染表达shShp2和对照的慢病毒后进行体外成球实验（SpheroidFormation Assay），结果发现干扰Shp2后肝癌干细胞成球能力明显被抑制，这提示Shp2可能对肝癌干细胞自我更新具有调控作用。在Shp2肝细胞条件性敲除小鼠的肝脏中，我们观察到β-catenin的表达显著降低，这提示Shp2可能对β-catenin具有正调控效应，Shp2是否通过影响β-catenin的表达和活化对肝癌干细胞发挥调控作用值得进一步深入研究。
肝癌是对人类健康威胁最大的肿瘤之一，至今其调控机制仍不十分清楚，没有可用于临床诊断的预后标志物，治疗方面也缺乏有效的分子靶点。寻找肝癌治疗靶标和预后标志物是目前肝癌研究的重要内容。Shp2作为一个重要的肿瘤相关信号调节蛋白，其对肝癌及肝癌干细胞的调控尚无报道。在前期研究基础上，本课题将利用多种特征性肝癌细胞模型、肿瘤转移模型系统研究Shp2在肝癌生长和转移中的作用，揭示其对肝癌干细胞的调控及分子机制，并利用Shp2肝细胞条件性敲除小鼠进行验证。利用大规模肝癌样本及其临床病理资料和随访信息，系统分析Shp2与肝癌临床病理特征和患者预后的关系，探讨Shp2作为肝癌预后标志物的应用前景，提出Shp2在索拉菲尼用药指征和联合化疗中的价值，最终为肝癌的诊断和治疗提供新的靶标。
研究方法：
1.利用蛋白电泳（western-blot）、免疫组织化学染色和Realtime PCR法检测临床肝癌组织中Shp2分子的表达情况并运用统计学方法分析Shp2的表达情况与临床病理和预后资料的相关性；
2.构建过表达和干扰Shp2的质粒，并利用慢病毒系统建立稳定干扰Shp2的肝癌细胞系SMMC-7721shShp2和LM3shShp2及其对照细胞系；
3. SMMC-7721shShp2和LM3shShp2及其对照细胞系分别采用CCK-8法、细胞周期检测、克隆形成实验、衰老检测、粘附实验、划痕实验、Transwell、侵袭实验等方法观察干扰Shp2后对肝癌细胞系增殖、迁移、侵袭、转移能力等生物学特性的影响；
4.采用Spheroid形成实验、流式细胞分析等方法检测Shp2稳定干扰细胞系中肝癌干细胞表型的变化；
5.应用化疗药物ETO、5-FU和多靶点靶向性药物索拉菲尼刺激稳定细胞系，研究Shp2对肝癌细胞化疗敏感性和索拉菲尼用药敏感性的影响；
6.繁殖Shp2肝细胞条件性敲除小鼠，并建立DEN诱导小鼠肝癌模型，与对照组比较，进行Shp2调控信号转导机制的体内研究；
7.体外分离Shp2肝细胞条件性敲除小鼠和对照小鼠原代肝细胞，并离体培养，检测未加刺激情况下和加EGF刺激后Shp2下游信号通路的活性改变；
8.将SMMC-7721shShp2及其对照细胞分别进行裸鼠皮下荷瘤实验，观察干扰Shp2对肝癌细胞体内增殖能力和成瘤能力的影响；
9.将SMMC-7721shShp2及其对照细胞分别进行裸鼠尾静脉注射，建立肺转移模型，进行裸鼠脾注射，建立肝转移模型，观察Shp2对肝癌细胞体内转移能力的影响。
10.将SMMC-7721shShp2及其对照细胞分别进行裸鼠皮下荷瘤实验，并分别给予索拉菲尼和安慰剂进行灌胃，与安慰剂组相比，观察干预Shp2联合索拉菲尼治疗对肿瘤生长的影响；
11.将SMMC-7721shShp2及其对照细胞按照一定比例进行NOD-SCID鼠体内有限稀释成瘤实验，观察Shp2在体内对肝癌干细胞的影响；
12.将SMMC-7721shShp2及其对照细胞按照一定比例种于低粘附96孔板，进行体外有限稀释成球实验，观察Shp2在体外对肝癌干细胞的影响。
13.利用Realtime-PCR、Western-blot、免疫共沉淀、Ras活性检测和PI3-K活性检测等方法研究Shp2对Ras/Raf/MEK/Erk、PI-3K/Akt等信号通路活性的影响。
14.利用Realtime-PCR、Western-blot、报告基因检测、免疫共沉淀、激光共聚焦等方法研究Shp2影响肝癌细胞化疗敏感性及肝癌干细胞表型的分子机制，并探寻Shp2对Wnt/β-catenin信号转导通路的影响。
研究结果：
1.从转录水平、蛋白水平证实Shp2在肝细胞癌中表达显著高于癌旁，且其表达水平与肝癌组织的分化程度呈负相关。在门静脉癌栓等肝癌转移灶中Shp2的表达显著高于相应的肝癌及其癌旁组织，提示Shp2可能参与调控肝癌的恶性生物学行为，并可能与肝癌转移密切相关。
2.通过对带有详细病理资料和预后情况的肝癌组织芯片进行Shp2的免疫组化染色实验并评分，统计分析得出肝癌中Shp2表达越高，患者总体生存期和无瘤生存期越差，并与肿瘤病理类型（AFP水平、肿瘤包膜、分化程度、血管侵犯、子灶、肿瘤大小）密切相关。
3.通过增殖、周期、克隆形成、衰老和裸鼠荷瘤实验发现，与对照组相比，干扰Shp2表达后肝癌细胞系生长、增殖和成瘤能力明显减弱。
4.通过粘附实验、划痕实验、Transwell、侵袭实验、建立裸鼠肺转移模型和肝转移模型等方法发现，干扰Shp2表达后肝癌细胞系粘附、迁移和体内外转移侵袭能力明显减弱。
5.通过Spheroid形成实验、化疗抵抗实验、体外和体内有限稀释实验发现干扰Shp2表达后肝癌干细胞自我更新能力明显减弱。
6.通过对干扰Shp2肝癌细胞系及其对照细胞系进行裸鼠荷瘤实验，并给予小分子靶向药索拉菲尼进行治疗，我们发现干扰Shp2可以提高肝癌细胞对索拉菲尼的敏感性，并在临床随访病例中得到验证。
7.通过对干扰Shp2肝癌细胞系及其对照细胞系进行Ras活性检测、免疫共沉淀、Western blot蛋白电泳检测以及DEN诱导的Shp2肝细胞条件性敲除小鼠及其对照小鼠肝癌切片免疫组织化学染色分析等实验发现， Shp2可能通过调控Ras/Raf/MEK/Erk信号转导通路促进肝癌细胞的生长增殖。
8.通过对干扰Shp2肝癌细胞系及其对照细胞系进行PI3-K激酶活性检测，Western blot蛋白电泳检测及在裸鼠肺转移灶和肝转移灶上的免疫组织化学染色分析等实验发现，Shp2可能通过调控PI3-K/Akt/mTOR信号转导通路并促进肝癌细胞的转移能力。
9.通过报告基因检测、DEN诱导的Shp2肝细胞条件性敲除小鼠及其对照小鼠肝癌切片免疫组织化学染色分析、对干扰Shp2肝癌细胞系及其对照细胞系行免疫共沉淀、激光共聚焦等实验以及放线菌酮（CHX）和蛋白酶体抑制剂（MG132）刺激实验发现，Shp2可能通过与β-catenin相互结合抑制β-catenin的降解进而调控Wnt/β-catenin信号转导通路影响肝癌干细胞。
结论：
我们前期的工作报道了Shp2在肝癌发生中的作用，但是Shp2在肝癌复发转移过程中的生物学作用尚不清楚。本课题研究发现Shp2促进了肝癌的转移且其表达水平与肝癌患者临床预后密切相关。实验结果显示肝癌组织中Shp2表达显著升高，在其转移灶中的表达水平更高。临床病理分析提示，Shp2高表达预示着肝癌的预后不良。体内和体外实验显示Shp2可能通过调控Ras/Raf/MEK/Erk通路促进肝癌细胞的生长。通过调控PI3-K/Akt/mTOR通路促进肝癌细胞的转移。Shp2还可以直接结合β-catenin抑制β-catenin的降解进而促进β-catenin的核转位和活化，从而增强了肝癌细胞干性基因的表达以及化疗抵抗和自我更新能力；这提示Shp2可能通过活化Wnt/β-catenin信号转导通路参与调控肝癌干细胞的生物学功能。进一步的实验结果还提示我们干扰Shp2可增强索拉菲尼的治疗效果且Shp2分子在肝癌细胞中的表达水平可以预测肝癌患者对索拉菲尼治疗的敏感性。综上所述，Shp2在肝癌的发展过程中起到了重要的作用，可能是肝癌复发转移的诊断标志物和潜在的临床治疗靶点。
Hepatocellular carcinoma (HCC) is the fifth most common cancer in the worldand the second leading cause of cancer death in men[1]. Despite the current advance inthe hepatic resection and transplantation, long-term survival of HCC patients remainsunsatisfactory due to the high incidence of recurrence and metastasis after surgicalresection[2]. It has been reported that HCC recurrence rate exceeds70%at5years[3].Therefore, it is urgent to identify novel therapeutic targets so that new strategies forHCC treatment can be developed. Accumulating genetic and functional studies ofoncogenes and tumor suppressor genes have greatly contributed to the advance ofknowledge on the molecular mechanism of tumorigenesis[4]. Inside the cancer cells,various signaling pathways are regulated by some predominant hub molecules at thebiochemical levels[5,6]. Tyrosine phosphorylation and dephosphorylation of these keymolecules plays critical roles in the regulatory network and aberrant tyrosinephosphorylation has been frequently detected in various cancers[5,7].
Shp2, encoded by PTPN11, was first identified by us and other groups in the early1990s as a non-receptor protein tyrosine phosphatases containing two Src-homology2(SH2) domains[8-10]. In the following two decades, accumulating studies documentedthat Shp2acts as a transducer of extracellular pro-survival and proliferation signalsfrom various cytokines, growth factors and hormones[11,12]. Shp2is required for theamplification of ERK signaling upon distinct mitogenic stimuli[11]. In previous work,we generated a hepatocyte-specific Shp2knockout (Shp2hep△) mouse model, andreported that Shp2deletion attenuated Erk activation and hepatocyte proliferationfollowing partial hepatectomy[13]. Nevertheless, effect of Shp2on the modulation ofPI3-K/Akt pathways and Jak/STAT cascade is cell specific or stimuli specific[14-16]. Inaddition, mouse genetics and sequencing studies have also indicated a broad role forShp2in development and disease[17,18].
Genetically, Ptpn11mutation has been detected in up to50%of patients withNoonan syndrome, a kind of developmental disorder, and the patients possess strkingly higher risk of juvenile myelomonocytic leukemia (JMML)[19]. Constitutiveactivation of Shp2by somatic mutations are present in different types of leukemia, andShp2has been considered as a proto-oncogene in leukemia[20,21]. Shp2has beendocumented to hyperactivated by oncogenes in gastric carcinoma, anaplastic large-celllymphoma and glioblastoma[22-24]. However, Ptpn11mutation has been scarcelydetected in solid tumors including hepatoma[25,26]. Our most current data indicated thetumor-inhibiting effect of Ptpn11/Shp2in carcinogenesis via downregulation ofinflammatory signaling. We found that Shp2deficience in hepatocytes causes chronichepatic damage and injury, which triggers inflammation and compensatory hepatocyteproliferation and thus promotes tumorigenesis in the liver, after a long latent period oftime[27]. Nevertheless, it is interesting that overexpression of Shp2was detected in thevast majority of HCC samples. Herein, we found Shp2acts as hub molecule regulatingseveral key cancer-related signaling in hepatoma cells and could serve as a promisingprognosis biomarker and therapeutic target for HCC therapy.
So our purpose of this project is to design a series of related experiments toobserve the role of Shp2on HCC recurrence and metastasis,clarify the molecularbiological mechanism and the clinical significance of Shp2on HCC development, andprovided a theoretical and experimental basis for targeted therapy of HCC.
引文
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[5]. Pawson, T.&Kofler, M. Kinome signaling through regulated protein-protein interactions innormal and cancer cells. Curr. Opin. Cell Biol.21,147–153(2009).
[6]. Vogelstein, B.&Kinzler, K.W. Cancer genes and the pathways they control. Nat. Med.10,789–799(2004).
[7]. Hunter, T. Tyrosine phosphorylation: thirty years and counting. Curr. Opin. Cell Biol.21,140–146(2009).
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[11]. Lai, L.A., Zhao, C., Zhang, E.E., and Feng, G.S.(2004). The Shp-2tyrosine phosphatase. InProtein Phosphatases, J. Arino and D. Alexander, eds.(Berlin: Springer-Verlag), pp.275–299.
[12]. Neel, B.G., Gu, H., and Pao, L.(2003). The ‘Shp’ing news: SH2domain-containing tyrosinephosphatases in cell signaling. Trends Biochem. Sci.28,284–293.
[13]. Bard-Chapeau, E.A., Yuan, J., Droin, N., Long, S., Zhang, E.E., Nguyen, T.V., and Feng, G.S.(2006). Concerted functions of Gab1and Shp2in liver regeneration and hepatoprotection. Mol.Cell. Biol.26,4664–4674.
[14]. Shi, Z.Q., Yu, D.H., Park, M., Marshall, M.&Feng, G.S. Molecular mechanism for the Shp-2tyrosine phosphatase function in promoting growth factor stimulation of Erk activity. Mol. Cell.Biol.20,1526–1536(2000).
[15]. Chan, G., Kalaitzidis, D.&Neel, B.G. The tyrosine phosphatase Shp2(PTPN11) in cancer.Cancer Metastasis Rev.2008;27,179–192.
[16]. Ostman, A., Hellberg, C.&Bohmer, F.D. Protein-tyrosine phosphatases and cancer. Nat. Rev.Cancer2006;6,307–320.
[17]. Feng, G.S. Shp2-mediated molecular signaling in control of embryonic stem cell self-renewaland differentiation. Cell Res.2007;17,37–41.
[18]. Grossmann, K.S., Rosario, M., Birchmeier, C.&Birchmeier, W. The tyrosine phosphataseShp2in development and cancer. Adv. Cancer Res.2010;106,53–89.
[19]. Tartaglia, M. et al. Mutations in PTPN11, encoding the protein tyrosine phosphatase SHP-2,cause Noonan syndrome. Nat. Genet.2001;29,465–468.
[20]. Tartaglia, M., and Gelb, B.D. Germ-line and somatic PTPN11mutations in human disease.Eur. J. Med. Genet.2005;48,81–96.
[21]. Chan, R.J., and Feng, G.S. PTPN11is the first identified proto-oncogene that encodes atyrosine phosphatase. Blood.2007;109,862–867.
[22]. Zhan, Y., Counelis, G.J.&O’Rourke, D.M. The protein tyrosine phosphatase SHP-2isrequired for EGFRvIII oncogenic transformation in human glioblastoma cells. Exp. Cell Res.315,2343–2357(2009).
[23]. Voena, C. et al. The tyrosine phosphatase Shp2interacts with NPM-ALK and regulatesanaplastic lymphoma cell growth and migration. Cancer Res.2007;67,4278–4286.
[24]. Nicola Aceto1, Nina Sausgruber1, Heike Brinkhaus1, Dimos Gaidatzis1, GeorgMartiny-Baron2, Giovanni Mazzarol3, et al. Tyrosine phosphatase Shp2promotes breast cancerprogression and maintains tumor-initiating cells via activation of key transcription factors and apositive feedback signaling loop. Nature Med.2012;18(4):529-37.
[25]. Xu, R., Yu, Y., Zheng, S., Zhao, X., Dong, Q., He, Z., Liang, Y., Lu, Q., Fang, Y., Gan, X., etal. Overexpression of Shp2tyrosine phosphatase is implicated in leukemogenesis in adult humanleukemia. Blood2005;106,3142–3149.
[26]. Zhou, X., Coad, J., Ducatman, B., and Agazie, Y.M.(2008). Shp2is up-regulated in breastcancer cells and in infiltrating ductal carcinoma of the breast, implying its involvement in breastoncogenesis. Histopathology53,389–402.
[27]. Shin Maeda, Hideaki Kamata, Jun-Li Luo, eCytokine-Driven Compensatory Proliferation that Promotes Chemical Hepatocarcinogenesis. Cell2005;121:977–990
[28]. Emilie A, Shuang L, Ding J, et al. PTPN11/Shp2Acts as a Tumor Suppressor inHepatocellular Carcinogenesis. Cancer Cell2011;19,629-639.
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