缺氧诱导因子（HIF-2α）通过TFDP3诱导肝癌细胞凋亡分子机制及临床研究
摘要
氧气是生化反应的底物,是生物体能量代谢不可缺少的底物,机体中缺氧会导致自由基的不正常积累,对DNA和蛋白产生损害,对细胞产生巨大的生存压力。缺氧会迫使细胞通过能量代谢、生物合成、和氧化还原等一系列改变来适应氧气浓度的改变；缺氧会导致细胞周期停滞,能量消耗减少；缺氧会刺激细胞生存因子和血管生成因子的分泌。尽管这些改变是由多种信号通路共同调控的,缺氧诱导因子(hypoxia inducible factosr, HIFs)在其中扮演着重要作用。研究发现HIFs的下游基因功能涉及到葡萄糖的代谢(GLUT1,LDH),凋亡相关分子(BNIP3),pH值调节(CA9),血管生成(VEGFA,PDGF)等过程。大量的临床数据表明HIFs在许多肿瘤中高表达,其表达通常与病人的预后较差,生存期短相关；试验室数据也表明HIFs丢失会导致肿瘤生长减慢,血管生成减少,转移能力降低,而过表达HIFs则可以挽救这些现象。
HIFs是由HIF-α和HIF—β亚基组成的异二聚体。哺乳动物有三种HIF-α亚基：HIF-1α、HIF-2α和HIF-3α,均可与HIF-β亚单位结合,其中HIF-1α和HIF-2α的功能研究较多。随着研究的深入,越来越多的数据表明：HIFs与肿瘤的相关性是非常复杂的。首先,HIF-1α亚基的功能在不同肿瘤类型会有所不同：在脑癌、乳腺癌和卵巢癌中HIF-1α过表达与病人的预后差相关,但在非小细胞肺癌、头颈癌中其表达与病人的预后较好相关。其次,HIF-1α和HIF-2α亚基虽然结构相似,但二者的功能并不重叠,甚至相反。研究发现,HIF-2α可与Myc复合物协同结合在目的基因启动子区的HIF(HRE)和Myc(Ebox)的结合位点,促进c-myc的转录活性。HIF-1α虽然也可以通过PAS B区域与Myc结合,但却抑制其与靶基因的启动子结合,从而下调c-myc激活的基因,上调c-myc抑制表达基因的活性。
我们组的前期研究发现HIF-1α在肝癌中广泛表达,HIF-1α高表达与病人的高BCLC分期相关,HIF-1α高表达意味着血管侵犯,是肝癌术后总体生存率和复发率的重要预测指标。目前,关于HIF-2α在肝癌的功能研究较少,本研究旨在通过临床标本的回顾性分析探索HIF-2α在肝癌的功能,通过ChIP on chip等试验手段筛选寻找HIF-2α下游效应分子,揭示分子机制,为HIF-2α在肝癌的临床应用提供证据。
第一部分HIF-2α在肝癌组织中的表达及其临床意义研究
本研究旨在分析HIF-2α在肝癌标本的表达情况,探索HIF-2α表达与肝癌发展的临床关系。通过对254例接受手术治疗和157例术前接受TACE治疗的肝癌临床标本进行免疫组化染色,根据染色的强度和密度将病人分成高表达和低表达组；分析病人的临床病理相关指标,研究HIF-2α表达水平与病人年龄、性别、肝炎病毒标志物、肝硬化、肿瘤大小、分化、包膜、TNM分期等临床病理特征及患者的生存时间,生存率等的关系,拟在发现HIF-2α与肝癌病人的预后、总体生存时间、生存率等之间的相互关系。
结果发现：与正常的肝脏相比,肿瘤中HIF-2α的RNA水平显著下降(p=0.04),254例接受手术治疗的肝癌病人的免疫组化染色结果表明HIF-2α在大部分肿瘤中均有表达。根据HIF-2α表达强度,254例病人分成低表达组(<50%)及高表达组(>50%)。高表达组的五年总体生存率是61.5%；低表达组的是46.6%,高表达组显著高于低表达组(p=0.023)。高表达组的平均生存时间是42.62个月,而低表达组的平均生存时间是36.33个月,二者差异显著(p=0.028)。单因素生存分析显示AFP (p=0.007), GGT (p=0.004),肿瘤直径(p<0.001),肿瘤数目(p=0.003),包膜完整性(p=0.011), TNM分期(p=0.018)及HIF-2α(p=0.023)均为影响病人5年总体生存的因素,其中AFP (p=0.045),肿瘤数目(p=0.009),TNM分期(p=0.050)也是影响病人无瘤生存的因素。高表达HIF-2α组的五年肿瘤复发率是60.7%；低表达组的五年肿瘤复发率是63.4%,两组的无瘤生存率无显著差异(p=0.929)；高表达组的平均无瘤生存时间是32.80个月,而低表达组的为30.00个月,二者差异不显著(p=0.355)。为进一步分析HIF-2α在病人亚群中的预测作用,我们将254例病人按照年龄、肿瘤大小、肿瘤数目、肿瘤包膜完整性、TNM等病理临床指标进行分层,发现HIF-2α的预测作用主要存在于单肿瘤,肿瘤包膜完整,EdmonsonⅠ-Ⅱ期,年纪大于50岁病人中。在单肿瘤病人中HIF-2α高表达的病人的生存率为67%,而低表达病人的生存率为48.7%(p=1.011)；在肿瘤包膜完整的病人中,HIF-2α高表达的病人的生存率为71.6%,而低表达病人的生存率为60.3%(p=0.005)；在EdmonsonⅠ-Ⅱ期的病人中,HIF-2α高表达的病人的生存率为66.7%,而低表达病人的生存率为50.9%(p=0.027)；在TNM I期病人中HIF-2α高表达的病人的生存率为65.5%,而低表达病人的生存率为50.0%(p=0.04)；在年纪大于50岁的病人中,HIF-2α高表达的病人的生存率为65.3%,而低表达病人的生存率为44.7%(p=1.01)。研究结果表明：HIF-2α在手术切除的肝癌标本中的表达情况与病人的总体生存相关,高表达意味着病人的预后较好,尤其是年龄大于50岁、肿瘤分期较早、包膜完整的单灶肝癌病人亚群中。
免疫组化染色结果表明HIF-2α在术前接受TACE治疗的肝癌病人中广泛表达,表达强度高于只接受手术治疗的病人。同样根据HIF-2α的染色强度对这部分病人分成高表达组和低表达组,结果发现高表达HIF-2α组病人的生存率为27.1%,平均生存时间为37.15个月；低表达组病人的生存率为20.4%,平均生存时间为29个月,高表达HIF-2α组病人的术后总体生存要显著高于低表达组病人(p=0.034)。术后复发分析结果发现高表达组的术后复发率为78%,低表达组的术后复发率为81.6%,二者之间无显著差异(p=0.379),高表达组的平均复发时间为术后25.93个月,低表达组的复发时间为19.20个月,二者无统计学差异。研究结果表明：HIF-2α在手术切除前接受TACE治疗的肝癌标本中的表达情况与病人的总体生存相关,高表达意味着病人的预后较好。
HIF-2α与临床病例特征之间的相关性数据表明在254例标本中HIF-2α与AFP (p= 0.025)、肿瘤直径(p=0.001)相关性显著。单因素分析表明HIF-2α、AFP、和肿瘤直径是影响病人术后总体生存时间的重要指标,多因素分析表明肿瘤直径是预测病人术后总体生存时间的独立预后指标。对术前接受TACE治疗的病人多因素分析结果表明HIF-2α可以作为总体生存的独立预测指标。这些数据提示HIF-2α可能通过影响肿瘤的生长而进一步影响病人的预后。
第二部分HIF-2α促进肝癌细胞凋亡,抑制肝癌的生长
本部分旨在通过研究HIF-2α对肝癌细胞生长的影响,分析HIF-2α影响肿瘤生长的原因。利用lipofection2000转染质粒,构建高表达及knockdown HIF-2α的稳转细胞,利用CCK8法检测HIF-2α对细胞生长的影响,利用免疫染色染色检测细胞增殖和凋亡比例的改变,应用Annexin V和PI染色法检测细胞凋亡等分析HIF-2α水平对肝癌细胞生长、增殖、及凋亡的影响。利用免疫缺陷的裸鼠模型研究HIF-2α水平对肝癌生长的影响。
结果发现：与正常的转入空载体的细胞相比,过表达HIF-2α可以显著抑制细胞的生长(p<0.001),而下调HIF-2α水平对细胞生长没有显著抑制。对细胞增殖和凋亡分析发现：在MHCC97H和MHCC97L细胞中,Ki67阳性细胞比例均没有随着HIF-2α的水平而有明显变化,但HIF-2α高表达显著提高了活化的Caspase3阳性细胞比例；Annexin V和PI染色,流式检测发现HIF-2α高表达组的细胞凋亡比例高达26%。Real time PCR结果发现凋亡相关基因,如Caspase7, Caspase8, BID的水平显著上调。小鼠体内试验显示：过表达HIF-2α组的肿瘤明显小于对照空载体组的肿瘤,约为其重量的1/10,差异显著(p<0.001), knockdown HIF-2α组的肿瘤重量稍小于空载体组肿瘤,差异不显著；对肿瘤的免疫组化染色结果显示：HIF-2α高表达组的Activated—Caspase3阳性细胞的比例明显增高。
研究表明：HIF-2α与肝癌细胞的生长关系密切,对肝癌细胞增殖没有影响,但可以促进肝癌细胞的凋亡,抑制肝癌细胞的生长,进而抑制肿瘤的生长。
第三部分HIF-2α抑制TFDP3基因的表达,促进E2F1信号通路,诱导肝癌细胞凋亡
本部分旨在应用ChIP on chip技术检测筛选HIF-2α在肝癌细胞系MHCC97H中调控的下游靶基因,探索HIF-2α促进肝癌细胞凋亡,抑制肿瘤生长的作用机理。利用KEGG数据库将所得到的基因进行功能和信号归类,挑选其中影响细胞的增殖、凋亡及转移等的基因,并在MHCC97L,与MHCC97H同源的另一个肝癌细胞系中,利用ChIP PCR验证ChIP on chip结果；利用实时定量PCR在过表达HIF-2α的MHCC97H细胞系和对应的空载体细胞中检测下游靶基因的转录水平变化；挑选变化较大的基因,通过上调或下调该靶基因的水平,分析其在HIF-2α诱导肝癌细胞凋亡过程中的作用。
结果发现：通过ChIP on chip,我们筛选到330个HIF-2α的靶基因；通过功能分析及信号通路分析,发现这些基因主要涉及到ErbB、p53、MAPK、TGF-β、Wnt、和Hedgehog信号通路；根据对基因富集的峰值得分和峰值误差排序,结合基因的功能及信号通路归类,我们挑选了了部分基因(GZF1、RUNX1、RADIL、TFDP3、MMP16、FNDC1、GSPT1)在MHCC97L细胞中对其进行验证。Chip-PCR结果表明：与对照IgG相比,大部分基因在HIF-2α组中高富集,与ChIP on ChIP的一致。Real time PCR结果显示在高表达HIF-2α的MHCC97H细胞中FNDC1、TFDP3基因的表达水平显著下降,其中TFDP3基因的下降幅度高达11倍。我们发现细胞中过表达HIF-2α导致TP73和ARF基因的RNA水平上调2倍以上,14-3-3σ、NOXA、GADD45α、和BID的上调倍数也在2倍以上,TP53、TP53INP2、P21、Cyclin D1和BAK1的RNA水平有少量变化。当细胞中TFDP3和HIF-2α两基因的表达水平同时上调时,ARF、14-3-3σ、NOXA、GADD45α、和BID基因的表达水平与转入空载体的相当；与单独高表达HIF-2α组的相比,ARF、14-3-3σ、NOXA、GADD45α、和BID基因的表达水平显著下降；TP53、TP53INP2、P21、Cyclin D1和BAK1基因的转录水平均有不同程度的下调。在过表达HIF-2α的MHCC97H和MHCC97L细胞中同时过表达TFDP3基因,通过免疫染色我们发现Activated-caspase 3细胞阳性比例显著下降,流式检测结果显示早期凋亡细胞的比例从20%降到12%。
研究结果表明：TFDP3是HIF-2α的下游靶基因之一,其表达水平受到HIF-2α的抑制；细胞中过表达HIF-2α可以提高E2F1通路的下游靶基因的转录水平,细胞中同时过表达HIF-2α和TFDP3可以部分抑制E2F1信号通路,减少HIF-2α过表达引发的细胞凋亡。以上结果显示HIF-2α通过抑制TFDP3基因的转录而促进E2F1信号通路活性,从而导致肝癌细胞的凋亡
结论
1.在本研究中,我们回顾性研究分析了254例接受手术治疗的肝癌标本,结果发现血清中AFP水平较高、肿瘤直径较大的病人的HIF-2α表达水平较低。肿瘤标本的HIF-2α表达水平低的病人与表达高的病人相比,其总体生存时间较短,生存率低。在肝癌中高表达HIF-2α的病人,预后较好。
2. HIF-2α在术前接受TACE治疗的肝癌病人中的预测作用与只接受手术治疗的病人相同。肝癌中HIF-2α(?)氐表达的病人的生存时间和生存率显著低于高表达的病人。单因素分析结果表明肿瘤中低表达HIF-2α与病人的总体生存率低相关,多因素分析结果表明肿瘤中HIF-2α的表达水平是病人总体生存率的独立预后指标。
3.体内试验结果表明过表达HIF-2α的MHCC97H细胞在裸鼠中产生的肿瘤显著小于转入空载体的细胞产生的肿瘤,体外实验结果也表明过表达HIF-2α显著降低肝癌细胞的生长速度。这些数据与我们的临床数据是一致的,均表明HIF-2α与肿瘤的生长关系密切,高表达HIF-2α的肿瘤直径较小
4.增殖和凋亡的相关指标检测结果表明HIF-2α可以促进肝癌细胞的凋亡,对细胞的增殖影响不明显。
5.通过ChIP on chip技术,我们发现在MHCC97H细胞中存在330个HIF-2α的潜在下游靶基因。TFDP3是HIF-2α的下游靶基因之一,其转录水平受到HIF-2α的抑制,通过外源性转入恢复TFDP3基因的表达水平可以显著降低HIF-2α引发的细胞凋亡,E2F1信号通路中的相关凋亡基因水平显著下调。这些数据表明TFDP3在HIF-2α引发的凋亡中起着重要的作用。
创新点
1.我们首次发现肝癌组织中HIF-2α表达水平较正常组织的降低,肝癌组织中HIF-2α低表达的病人的总体生存时间和生存率明显低于高表达病人,肝癌组织中的HIF-2α表达水平与病人的总体生存相关。
2.我们首次发现TFDP3是HIF-2α的下游靶基因。
3.我们首次发现HIF-2α通过抑制TFDP3基因的表达促进E2F1信号通路引发的凋亡
潜在应用价值
1.我们结果表明高表达HIF-2α与肝癌病人的生存时间、生存率相关,在术前接受FACE治疗的肝癌病人中可以作为总体生存的独立预后指标,因此HIF-2α可以用做肝癌病人总体生存的一个预测指标。
2. TFDP3,作为HIF-2α的下游靶基因,可能可以成为肝癌治疗的一个靶点。
3.本研究数据进一步证实了HIF a在肿瘤中功能的复杂性,因此选择HIFα的抑制剂作为肿瘤治疗的靶点需要更多的研究。
Hypoxia inducible factors (HIFs) is a critical transcriptional regulator of cellular responses to a variety of stressful conditions[1]. Oxygen sensing is a core feature of HIF regulation[2].Under normoxic conditions, the HIF-αsubunits are hydroxylated on key proline residues by the von Hippel-Lindau protein complex that targets HIF-αfor degradation by the proteasome[3]. The hydroxylated modification is mediated by PHDs, whose activities are regulated by O2 availability [4]. HIF-αsubunits are also substrates for an asparaginyl hydroxylase:factor-inhibiting HIF-1α(FIH), this enzyme is also O2 dependent, and represents another component of the oxygen sensing machinery[5]. There are some other stress factors inducing HIFs response, including microRNAs, oncogenic signal such as micro-107[6], P3K/Akt [7], Ras[8]. HIFs are expressed at high levels in a variety of solid tumors and tumor cell lines [9-10], regulate hundreds of gene's expression, including glycolysis, angiogenesis, erythropoiesis, cell death, and differentiation[11].
Mammals possess three isoforms of asubunits, of which HIF-1a and HIF-2αare most structurally similar and best characterized[2]. Despite the structurally similarities between HIF-1αand HIF-2α, genetic studies have provided firm evidence for their non-redundant functions[12].Targeted inactivation of HIF-1αand HIF-2αin embryonic stem cells is associated with different response to hypoxia, low glucose stress and different development defects[12]. HIF-1αantagonizes c-MYC function, whereas HIF-2αpromotes c-MYC function [2; 13]. The role of HIF-1αhas been studied in many cancers, and associations between HIF-1aover expression and poor patient survival has been shown in brain[14], breast[15], and ovarian cancers[16]. In contrast, association between HIF-1αover expression and improved patient survival was reported for patients with non-small cell lung carcinoma [17], and head and neck squamous cell carcinoma[18]. It appears the clinical significance of HIF-1αexpression depends on the cancer type and associated genetic alterations [19-20]. There is no clear role for HIF-2αin influencing patient outcome[21]. Knock-in replacement of HIF-1aby HIF-2αstimulated teratoma growth [22], stable expression of HIF-2αoverrode the tumor suppressing activity of the von Hippel-Lindau(VHL) protein [23], and it directly induces the expression of the cell cycle regulatory protein cyclin D1 in RCC cells[24]. However, there are also studies suggesting a tumor suppressor role of HIF-2α. Over-expression of HIF-2a has been reported to suppress tumor growth in breast cancer cell [25] and rat glioma, knockdown of HIF-2a expression reduced apoptosis in hypoxic human malignant glioblastoma cells[21]. Loss of the VHL gene, resulting in increased activation of both HIF-1αand HIF-2α, also impaired the growth of teratomas[26]. Mounting evidence suggests like HIF-1α, HIF-2α's effects may be tissue and cell context dependent.
In this report, we followed up hundreds of hepatocelluar carcinoma patients for more than 5 years to study the correlation between HIF-2αand clinical outcome. We also studied the effect of HIF-2αin hepatocellular carcinoma through gain and loss of experiments. Finally, we uncovered a novel mechanism by which HIF-2αcan promote the transcriptional activities of E2F1/DP complex, influence cellular apoptosis. Our findings open a new avenue for targeted therapies in patients with hepatocellular carcinoma.
Part One High HIF-2αexpression correlates significantly with good prognosis in patients with hepatocellular carcinoma
Initially, we compared the expression levels of HIF2-αin normal non-cirrhotic liver to those of HCC patients who had undergone tumor resection. Compared to normal liver, HIF-2αmRNA expression was significantly lower in HCC's tumors, while there was no significant difference in HIF-1αexpression. Next we aimed to determine the role of HIF-2αin HCC patients by consecutive TMAs slides including 254 HCC samples that were immunostained by HIF-2αantibody. The clinical/pathological characteristics of patients were listed in table 1. Immunohistochemical results revealed that HIF-2αlocated on the cytoplasm and nuclear of tumor cells and low expressed in 131 cases(51.97%), We further analyzed the molecules expression in this 254 HCC samples annotated with extensive clinical follow up data. HCC patients characterized by high AFP level and large tumor size were prone to have low HIF-2αexpression. Patients with low levels of HIF-2αhad a significantly shorter overall survival time than those with high level of HIF-2α.Kaplan-Meier analysis revealed that the patients with low levels of HIF-2αhad a significantly lower overall survival rate in the five years although the difference became smaller after 5 years. Univariate analysis revealed that low expression of HIF-2a showed a prognostic significance with poor prognisis for overall survival in five year. Multivariate analysis indicated that HIF-2αwas not an independent predictor for overall survival. There was no significant difference in the recurrence free time and recurrence free rate between different levels of HIF-2a. We further studied the role of HIF-2αin another separate subset of consecutive TMAs slides including 157 HCC patients who received a preoperative transareterial chemoembolization (TACE) before surgery. The TACE group showed younger age, larger tumor size, and multiple tumor than the other group, the correlation between HIF-2αand clinicopathological characteristics were shown. Patients with low levels of HIF-2αhad a significantly shorter overall survival time than those with high level of HIF-2a.Kaplan-Meier analysis revealed that the patients with low levels of HIF-2αhad a significantly lower overall survival rate. Univariate analysis revealed that low expression of HIF-2αshowed a prognostic significance with poor prognisis for overall survival. Multivariate analysis indicated that HIF-2αwas an independent predictor for overall survival in TACE patients. There was no significant difference in the recurrence free time and recurrence free rate in TACE patients.
Part Two
HIF-2a reduces hepatocellular carcinoma cell growth and augments apoptosis in HCC cells in vitro and in vivo
We began to investigate how elevated HIF-2αexpression in HCC might lead to better clinical outcome by selecting a hepatocellular carcinoma cell line (MHCC97H and MHCC97L. Hypoxia strongly induced HIF-2a mRNA and nuclear HIF-2a accumulation compared to cells cultured under normoxic conditions There was no significant change in HIF-1a regardless of the oxygen conditions.
To begin to uncover the mechanism by which HIF-2a leads to improved outcome in HCC patients, we made clones that over express and suppress HIF-2a in the HCC cell line (MHCC97H). Transfection of MHCC97H cells with pcDNA3-HIF-2a induced a fourfold increase in HIF-2a mRNA expression while HIF-1awas essentially unchanged compared to control vector transfection. There was a 5 fold suppression of HIF-2a mRNA and 2.6 fold increase in HIF-1a in cells transfected with pT2sh-HIF-2a plasmid compared to control cells. Western blot analysis showed a similar trend as was seen in the quantitative RT-PCR. We found over expression of HIF-2αsignificantly reduced HCC tumor weight. Cells that suppressed HIF-2αshowed no significant change in tumor weight. These results are consistent with the clinical observation that elevated HIF-2αis a negative regulator of hepatocellular carcinogenesis.
We found no difference in the proliferative index between control and HIF-2a over expressing cells. However, we did find significantly more cleaved-caspase-3, a key executer of apoptosis, in cells over expressing HIF-2αcompared to mock tranfected 97H control. We used propidium iodide and annexin-v to determine the proportion of apoptotic cells in the HIF-2αover expressing cells. We found more than 20% apoptotic cells in the high HIF-2a group, while there was only 1% in the control group. To further confirm this effect, we derived another set of clones in a related but different cell line (MHCC97L) and determined the proliferative and apoptotic index. Over expression of HIF-2αin this cell line also showed significantly greater apoptosis, as determined by the activated caspase 3 staining, while suppression of HIF-2αwith shRNA plasmid had little effect on apoptosis. There was no significant effect on the proliferative index in cells expressing either high or low HIF-2α.This data further confirms that high levels of HIF-2αinduce apoptosis in HCC cell lines and has little effect on cell proliferation. In vivo data also showed that overexpression of HIF-2αcan induce more cleaved caspased 3 expression in tumor cells compared to mock transfected 97H cells. Together the in vitro and in vivo data support HIF-2αrole inducing HCC apoptosis while having little effect on cell proliferation.
Part Three
HIF-2αaugments apoptosis by directly inhibiting the expression of TFDP3
Having established HIF-2αas a mediator of HCC apoptosis, we sought to uncover the mechanism by which it induces apoptosis. We performed a chromatin immunoprecipitation (ChIP) combined with a DNA microarray (ChIP on chIP) screen with MHCC97H cells to uncover HIF-2αtarget genes. We identified 330 genes in the screen and grouped them according to the KEGG pathway database. HIF-2αtargets genes were found in several pathways. We listed the genes with the highest peakscore and low peak false discovery rate and focused upon genes that were implicated in cellular apoptosis. We sought to confirm the ChIP on chIP screen with an independent cell line, MHCC97L. We found the genes GSPT1, FNDC1, MMP16, TFDP3, RUNX1, GZF1 and RADIL were highly enriched in the anti-HIF-2αprecipitated fraction compared to control precipitation. Since these appear to be bono-fide HIF-2αtargets, we used Q-RT-PCR to determine if HIF-2αcan regulate the transcription of a subset of these genes. Interestingly, FNDC1, MMP16 and TFDP3 transcription were repressed by HIF-2αwhile RUNX1 was upregulated. We noted the particularly strong 10 fold suppression of transcription factor DP3 (TFDP3) transcription by HIF-2a. The TFDP3 gene belongs to the DP (dimerization parter) family [27], which exerts a regulatory function by dimerzing with E2F proteins. The heterodimerization of E2F-DP complex is essential for both high affinities DNA binding and efficient transcriptional regulation by E2Fs. It is known that TFDP3 can suppress apoptosis mediated through E2F and p53 [28]. We hypothesized that HIF-2αrepressed TFDP3 expression could account for the high rate of apoptosis seen in HCC cell lines which had high level of HIF-2a. To test this idea, we over-expressed TFDP3 and HIF-2αin the MHCC97H cell line. First we confirmed the expression levels of TFDP3 and HIF-2αmRNA in each transfectant. As expected, HIF-2αexpression significantly increased apoptosis while re-expression of TFDP3 significantly reversed this effect. We also studied actived-caspase-3 expression and found significant suppression of apoptosis when TFDP3 expression was re-established in HIF-2αover expressing cells. We found identical results in an additional cell line. We next investigated the cellular outcome of TFDP3-mediated E2F1 inhibition. Transcript levels for several E2F targets were measured in HIF-2a expression cells and re-expression of TFDP3 gene in these cells. Compared to control cells, HIF-2αexpression cells showed a marked increase in TP73 (2.7 fold), ARF(2.5 fold),14-3-3σ(2 fold), Noxa (5 fold), GADD45α(3.8 fold), and BID (2 fold) mRNAs and a subtle increase in TP53, TP53INP2, P21, and BAK1 transcripts, while the expression of a number of other known target/downstream genes did not change significantly. When we reexpressed TFDP3 gene in these cells, the expression of genes such as ARF,14-3-3σ, Noxa, GADD45α, and BID were down and the levels were near to that in mock cells.
Take as a whole, the results support that TFDP3 is a mediator of HIF-2αinduced apoptosis.
Conlusion
1. In this study, we followed 254 hepatocellular carcinoma patients treated with resection, and found that HCC patients characterized by high AFP level and large tumor size were prone to have low HIF-2a expression. Patients with low levels of HIF-2αhad a significantly shorter overall survival time than those with high level of HIF-2α, HIF-2αexpression in HCC correlated with improved clinical outcome.
2. The role of HIF-2αin 157 HCC patients who received a preoperative transareterial chemoembolization (TACE) before surgery showed the same results as that in 254 patients treated with resection only Patients with low levels of HIF-2αhad a significantly shorter overall survival time than those with high level of HIF-2α. Univariate analysis revealed that low expression of HIF-2αshowed a prognostic significance with poor prognisis for overall survival. Multivariate analysis indicated that HIF-2αwas an independent predictor for overall survival in TACE patients
3. We found over expression of HIF-2αin MHCC97H cell line which injected into nude mice, significantly reduced the tumor weight. Cells that suppressed HIF-2αshowed no significant change in tumor weight. In vitro data also showed that over expression of HIF-2αdecreased the cell growth significantly. These results are consistent with the clinical observation that elevated HIF-2αwas a negative regulator of hepatocellular carcinogenesis.
4. By activated-caspase -3, KI67 immunoflurecent staining and FACS sort confirmation, we found that high levels of HIF-2αinduced hepatocellualr carcinoma cell apoptosis and had little effect on cell proliferation in vivo and in vitro.
5. Screening by performed a chromatin immunoprecipitation (ChIP) combined with a DNA microarray (ChIP on chIP), we got 330 potential target genes of HIF-2αin MHCC97H cell. Confirmed by chip PCR and real time PCR, we found that TFDP3 was one of HIF-2αtarget genes and was downregulated by HIF-2α. By re-expression of TFDP3 in HIF-2αoverexpression liver cancer cell, we found that less cells were apoptosis and the expression of genes in the E2F1 was dowe-regulated than that of HIF-2αoverexpression, these data showed that TFDP3 was a mediator of HIF-2αinducing apoptosis.
Novelty
1. First we showed that HCC patients with low levels of HIF-2αhad a significantly shorter overall survival time and a lower overall survival rate than those patients with high level of HIF-2α, and HIF-2αexpression in HCC correlated with improved clinical outcome in hepatocellular carcinoma patients.
2. First we showed that TFDP3, a member of DP family, was one of HIF-2αtarget genes.
3. First we showed that TFDP3 was a mediator of HIF-2αinducing apoptosis by upregulating the E2F1 pathway.
Potential Application
1. Our data showed HIF-2αoverexpression was correlated with longer survival time and high survival rate in HCC patients treated with resection, HIF-2αwas an independent predictor for overall survival in HCC patients treated with TACE before resection, and HIF-2αcan be used as a predictor for HCC overall survival.
2. TFDP3, HIF-2αtarget gene, was a potential therapeutic target in liver cancer.
3. Our research confirmed the complex role of HIFαsubunit in cancer further and the screening of inhibitor about HIFαsubunit used for cancer therapy need more studies.
HIFs是由HIF-α和HIF—β亚基组成的异二聚体。哺乳动物有三种HIF-α亚基：HIF-1α、HIF-2α和HIF-3α,均可与HIF-β亚单位结合,其中HIF-1α和HIF-2α的功能研究较多。随着研究的深入,越来越多的数据表明：HIFs与肿瘤的相关性是非常复杂的。首先,HIF-1α亚基的功能在不同肿瘤类型会有所不同：在脑癌、乳腺癌和卵巢癌中HIF-1α过表达与病人的预后差相关,但在非小细胞肺癌、头颈癌中其表达与病人的预后较好相关。其次,HIF-1α和HIF-2α亚基虽然结构相似,但二者的功能并不重叠,甚至相反。研究发现,HIF-2α可与Myc复合物协同结合在目的基因启动子区的HIF(HRE)和Myc(Ebox)的结合位点,促进c-myc的转录活性。HIF-1α虽然也可以通过PAS B区域与Myc结合,但却抑制其与靶基因的启动子结合,从而下调c-myc激活的基因,上调c-myc抑制表达基因的活性。
我们组的前期研究发现HIF-1α在肝癌中广泛表达,HIF-1α高表达与病人的高BCLC分期相关,HIF-1α高表达意味着血管侵犯,是肝癌术后总体生存率和复发率的重要预测指标。目前,关于HIF-2α在肝癌的功能研究较少,本研究旨在通过临床标本的回顾性分析探索HIF-2α在肝癌的功能,通过ChIP on chip等试验手段筛选寻找HIF-2α下游效应分子,揭示分子机制,为HIF-2α在肝癌的临床应用提供证据。
第一部分HIF-2α在肝癌组织中的表达及其临床意义研究
本研究旨在分析HIF-2α在肝癌标本的表达情况,探索HIF-2α表达与肝癌发展的临床关系。通过对254例接受手术治疗和157例术前接受TACE治疗的肝癌临床标本进行免疫组化染色,根据染色的强度和密度将病人分成高表达和低表达组；分析病人的临床病理相关指标,研究HIF-2α表达水平与病人年龄、性别、肝炎病毒标志物、肝硬化、肿瘤大小、分化、包膜、TNM分期等临床病理特征及患者的生存时间,生存率等的关系,拟在发现HIF-2α与肝癌病人的预后、总体生存时间、生存率等之间的相互关系。
结果发现：与正常的肝脏相比,肿瘤中HIF-2α的RNA水平显著下降(p=0.04),254例接受手术治疗的肝癌病人的免疫组化染色结果表明HIF-2α在大部分肿瘤中均有表达。根据HIF-2α表达强度,254例病人分成低表达组(<50%)及高表达组(>50%)。高表达组的五年总体生存率是61.5%；低表达组的是46.6%,高表达组显著高于低表达组(p=0.023)。高表达组的平均生存时间是42.62个月,而低表达组的平均生存时间是36.33个月,二者差异显著(p=0.028)。单因素生存分析显示AFP (p=0.007), GGT (p=0.004),肿瘤直径(p<0.001),肿瘤数目(p=0.003),包膜完整性(p=0.011), TNM分期(p=0.018)及HIF-2α(p=0.023)均为影响病人5年总体生存的因素,其中AFP (p=0.045),肿瘤数目(p=0.009),TNM分期(p=0.050)也是影响病人无瘤生存的因素。高表达HIF-2α组的五年肿瘤复发率是60.7%；低表达组的五年肿瘤复发率是63.4%,两组的无瘤生存率无显著差异(p=0.929)；高表达组的平均无瘤生存时间是32.80个月,而低表达组的为30.00个月,二者差异不显著(p=0.355)。为进一步分析HIF-2α在病人亚群中的预测作用,我们将254例病人按照年龄、肿瘤大小、肿瘤数目、肿瘤包膜完整性、TNM等病理临床指标进行分层,发现HIF-2α的预测作用主要存在于单肿瘤,肿瘤包膜完整,EdmonsonⅠ-Ⅱ期,年纪大于50岁病人中。在单肿瘤病人中HIF-2α高表达的病人的生存率为67%,而低表达病人的生存率为48.7%(p=1.011)；在肿瘤包膜完整的病人中,HIF-2α高表达的病人的生存率为71.6%,而低表达病人的生存率为60.3%(p=0.005)；在EdmonsonⅠ-Ⅱ期的病人中,HIF-2α高表达的病人的生存率为66.7%,而低表达病人的生存率为50.9%(p=0.027)；在TNM I期病人中HIF-2α高表达的病人的生存率为65.5%,而低表达病人的生存率为50.0%(p=0.04)；在年纪大于50岁的病人中,HIF-2α高表达的病人的生存率为65.3%,而低表达病人的生存率为44.7%(p=1.01)。研究结果表明：HIF-2α在手术切除的肝癌标本中的表达情况与病人的总体生存相关,高表达意味着病人的预后较好,尤其是年龄大于50岁、肿瘤分期较早、包膜完整的单灶肝癌病人亚群中。
免疫组化染色结果表明HIF-2α在术前接受TACE治疗的肝癌病人中广泛表达,表达强度高于只接受手术治疗的病人。同样根据HIF-2α的染色强度对这部分病人分成高表达组和低表达组,结果发现高表达HIF-2α组病人的生存率为27.1%,平均生存时间为37.15个月；低表达组病人的生存率为20.4%,平均生存时间为29个月,高表达HIF-2α组病人的术后总体生存要显著高于低表达组病人(p=0.034)。术后复发分析结果发现高表达组的术后复发率为78%,低表达组的术后复发率为81.6%,二者之间无显著差异(p=0.379),高表达组的平均复发时间为术后25.93个月,低表达组的复发时间为19.20个月,二者无统计学差异。研究结果表明：HIF-2α在手术切除前接受TACE治疗的肝癌标本中的表达情况与病人的总体生存相关,高表达意味着病人的预后较好。
HIF-2α与临床病例特征之间的相关性数据表明在254例标本中HIF-2α与AFP (p= 0.025)、肿瘤直径(p=0.001)相关性显著。单因素分析表明HIF-2α、AFP、和肿瘤直径是影响病人术后总体生存时间的重要指标,多因素分析表明肿瘤直径是预测病人术后总体生存时间的独立预后指标。对术前接受TACE治疗的病人多因素分析结果表明HIF-2α可以作为总体生存的独立预测指标。这些数据提示HIF-2α可能通过影响肿瘤的生长而进一步影响病人的预后。
第二部分HIF-2α促进肝癌细胞凋亡,抑制肝癌的生长
本部分旨在通过研究HIF-2α对肝癌细胞生长的影响,分析HIF-2α影响肿瘤生长的原因。利用lipofection2000转染质粒,构建高表达及knockdown HIF-2α的稳转细胞,利用CCK8法检测HIF-2α对细胞生长的影响,利用免疫染色染色检测细胞增殖和凋亡比例的改变,应用Annexin V和PI染色法检测细胞凋亡等分析HIF-2α水平对肝癌细胞生长、增殖、及凋亡的影响。利用免疫缺陷的裸鼠模型研究HIF-2α水平对肝癌生长的影响。
结果发现：与正常的转入空载体的细胞相比,过表达HIF-2α可以显著抑制细胞的生长(p<0.001),而下调HIF-2α水平对细胞生长没有显著抑制。对细胞增殖和凋亡分析发现：在MHCC97H和MHCC97L细胞中,Ki67阳性细胞比例均没有随着HIF-2α的水平而有明显变化,但HIF-2α高表达显著提高了活化的Caspase3阳性细胞比例；Annexin V和PI染色,流式检测发现HIF-2α高表达组的细胞凋亡比例高达26%。Real time PCR结果发现凋亡相关基因,如Caspase7, Caspase8, BID的水平显著上调。小鼠体内试验显示：过表达HIF-2α组的肿瘤明显小于对照空载体组的肿瘤,约为其重量的1/10,差异显著(p<0.001), knockdown HIF-2α组的肿瘤重量稍小于空载体组肿瘤,差异不显著；对肿瘤的免疫组化染色结果显示：HIF-2α高表达组的Activated—Caspase3阳性细胞的比例明显增高。
研究表明：HIF-2α与肝癌细胞的生长关系密切,对肝癌细胞增殖没有影响,但可以促进肝癌细胞的凋亡,抑制肝癌细胞的生长,进而抑制肿瘤的生长。
第三部分HIF-2α抑制TFDP3基因的表达,促进E2F1信号通路,诱导肝癌细胞凋亡
本部分旨在应用ChIP on chip技术检测筛选HIF-2α在肝癌细胞系MHCC97H中调控的下游靶基因,探索HIF-2α促进肝癌细胞凋亡,抑制肿瘤生长的作用机理。利用KEGG数据库将所得到的基因进行功能和信号归类,挑选其中影响细胞的增殖、凋亡及转移等的基因,并在MHCC97L,与MHCC97H同源的另一个肝癌细胞系中,利用ChIP PCR验证ChIP on chip结果；利用实时定量PCR在过表达HIF-2α的MHCC97H细胞系和对应的空载体细胞中检测下游靶基因的转录水平变化；挑选变化较大的基因,通过上调或下调该靶基因的水平,分析其在HIF-2α诱导肝癌细胞凋亡过程中的作用。
结果发现：通过ChIP on chip,我们筛选到330个HIF-2α的靶基因；通过功能分析及信号通路分析,发现这些基因主要涉及到ErbB、p53、MAPK、TGF-β、Wnt、和Hedgehog信号通路；根据对基因富集的峰值得分和峰值误差排序,结合基因的功能及信号通路归类,我们挑选了了部分基因(GZF1、RUNX1、RADIL、TFDP3、MMP16、FNDC1、GSPT1)在MHCC97L细胞中对其进行验证。Chip-PCR结果表明：与对照IgG相比,大部分基因在HIF-2α组中高富集,与ChIP on ChIP的一致。Real time PCR结果显示在高表达HIF-2α的MHCC97H细胞中FNDC1、TFDP3基因的表达水平显著下降,其中TFDP3基因的下降幅度高达11倍。我们发现细胞中过表达HIF-2α导致TP73和ARF基因的RNA水平上调2倍以上,14-3-3σ、NOXA、GADD45α、和BID的上调倍数也在2倍以上,TP53、TP53INP2、P21、Cyclin D1和BAK1的RNA水平有少量变化。当细胞中TFDP3和HIF-2α两基因的表达水平同时上调时,ARF、14-3-3σ、NOXA、GADD45α、和BID基因的表达水平与转入空载体的相当；与单独高表达HIF-2α组的相比,ARF、14-3-3σ、NOXA、GADD45α、和BID基因的表达水平显著下降；TP53、TP53INP2、P21、Cyclin D1和BAK1基因的转录水平均有不同程度的下调。在过表达HIF-2α的MHCC97H和MHCC97L细胞中同时过表达TFDP3基因,通过免疫染色我们发现Activated-caspase 3细胞阳性比例显著下降,流式检测结果显示早期凋亡细胞的比例从20%降到12%。
研究结果表明：TFDP3是HIF-2α的下游靶基因之一,其表达水平受到HIF-2α的抑制；细胞中过表达HIF-2α可以提高E2F1通路的下游靶基因的转录水平,细胞中同时过表达HIF-2α和TFDP3可以部分抑制E2F1信号通路,减少HIF-2α过表达引发的细胞凋亡。以上结果显示HIF-2α通过抑制TFDP3基因的转录而促进E2F1信号通路活性,从而导致肝癌细胞的凋亡
结论
1.在本研究中,我们回顾性研究分析了254例接受手术治疗的肝癌标本,结果发现血清中AFP水平较高、肿瘤直径较大的病人的HIF-2α表达水平较低。肿瘤标本的HIF-2α表达水平低的病人与表达高的病人相比,其总体生存时间较短,生存率低。在肝癌中高表达HIF-2α的病人,预后较好。
2. HIF-2α在术前接受TACE治疗的肝癌病人中的预测作用与只接受手术治疗的病人相同。肝癌中HIF-2α(?)氐表达的病人的生存时间和生存率显著低于高表达的病人。单因素分析结果表明肿瘤中低表达HIF-2α与病人的总体生存率低相关,多因素分析结果表明肿瘤中HIF-2α的表达水平是病人总体生存率的独立预后指标。
3.体内试验结果表明过表达HIF-2α的MHCC97H细胞在裸鼠中产生的肿瘤显著小于转入空载体的细胞产生的肿瘤,体外实验结果也表明过表达HIF-2α显著降低肝癌细胞的生长速度。这些数据与我们的临床数据是一致的,均表明HIF-2α与肿瘤的生长关系密切,高表达HIF-2α的肿瘤直径较小
4.增殖和凋亡的相关指标检测结果表明HIF-2α可以促进肝癌细胞的凋亡,对细胞的增殖影响不明显。
5.通过ChIP on chip技术,我们发现在MHCC97H细胞中存在330个HIF-2α的潜在下游靶基因。TFDP3是HIF-2α的下游靶基因之一,其转录水平受到HIF-2α的抑制,通过外源性转入恢复TFDP3基因的表达水平可以显著降低HIF-2α引发的细胞凋亡,E2F1信号通路中的相关凋亡基因水平显著下调。这些数据表明TFDP3在HIF-2α引发的凋亡中起着重要的作用。
创新点
1.我们首次发现肝癌组织中HIF-2α表达水平较正常组织的降低,肝癌组织中HIF-2α低表达的病人的总体生存时间和生存率明显低于高表达病人,肝癌组织中的HIF-2α表达水平与病人的总体生存相关。
2.我们首次发现TFDP3是HIF-2α的下游靶基因。
3.我们首次发现HIF-2α通过抑制TFDP3基因的表达促进E2F1信号通路引发的凋亡
潜在应用价值
1.我们结果表明高表达HIF-2α与肝癌病人的生存时间、生存率相关,在术前接受FACE治疗的肝癌病人中可以作为总体生存的独立预后指标,因此HIF-2α可以用做肝癌病人总体生存的一个预测指标。
2. TFDP3,作为HIF-2α的下游靶基因,可能可以成为肝癌治疗的一个靶点。
3.本研究数据进一步证实了HIF a在肿瘤中功能的复杂性,因此选择HIFα的抑制剂作为肿瘤治疗的靶点需要更多的研究。
Hypoxia inducible factors (HIFs) is a critical transcriptional regulator of cellular responses to a variety of stressful conditions[1]. Oxygen sensing is a core feature of HIF regulation[2].Under normoxic conditions, the HIF-αsubunits are hydroxylated on key proline residues by the von Hippel-Lindau protein complex that targets HIF-αfor degradation by the proteasome[3]. The hydroxylated modification is mediated by PHDs, whose activities are regulated by O2 availability [4]. HIF-αsubunits are also substrates for an asparaginyl hydroxylase:factor-inhibiting HIF-1α(FIH), this enzyme is also O2 dependent, and represents another component of the oxygen sensing machinery[5]. There are some other stress factors inducing HIFs response, including microRNAs, oncogenic signal such as micro-107[6], P3K/Akt [7], Ras[8]. HIFs are expressed at high levels in a variety of solid tumors and tumor cell lines [9-10], regulate hundreds of gene's expression, including glycolysis, angiogenesis, erythropoiesis, cell death, and differentiation[11].
Mammals possess three isoforms of asubunits, of which HIF-1a and HIF-2αare most structurally similar and best characterized[2]. Despite the structurally similarities between HIF-1αand HIF-2α, genetic studies have provided firm evidence for their non-redundant functions[12].Targeted inactivation of HIF-1αand HIF-2αin embryonic stem cells is associated with different response to hypoxia, low glucose stress and different development defects[12]. HIF-1αantagonizes c-MYC function, whereas HIF-2αpromotes c-MYC function [2; 13]. The role of HIF-1αhas been studied in many cancers, and associations between HIF-1aover expression and poor patient survival has been shown in brain[14], breast[15], and ovarian cancers[16]. In contrast, association between HIF-1αover expression and improved patient survival was reported for patients with non-small cell lung carcinoma [17], and head and neck squamous cell carcinoma[18]. It appears the clinical significance of HIF-1αexpression depends on the cancer type and associated genetic alterations [19-20]. There is no clear role for HIF-2αin influencing patient outcome[21]. Knock-in replacement of HIF-1aby HIF-2αstimulated teratoma growth [22], stable expression of HIF-2αoverrode the tumor suppressing activity of the von Hippel-Lindau(VHL) protein [23], and it directly induces the expression of the cell cycle regulatory protein cyclin D1 in RCC cells[24]. However, there are also studies suggesting a tumor suppressor role of HIF-2α. Over-expression of HIF-2a has been reported to suppress tumor growth in breast cancer cell [25] and rat glioma, knockdown of HIF-2a expression reduced apoptosis in hypoxic human malignant glioblastoma cells[21]. Loss of the VHL gene, resulting in increased activation of both HIF-1αand HIF-2α, also impaired the growth of teratomas[26]. Mounting evidence suggests like HIF-1α, HIF-2α's effects may be tissue and cell context dependent.
In this report, we followed up hundreds of hepatocelluar carcinoma patients for more than 5 years to study the correlation between HIF-2αand clinical outcome. We also studied the effect of HIF-2αin hepatocellular carcinoma through gain and loss of experiments. Finally, we uncovered a novel mechanism by which HIF-2αcan promote the transcriptional activities of E2F1/DP complex, influence cellular apoptosis. Our findings open a new avenue for targeted therapies in patients with hepatocellular carcinoma.
Part One High HIF-2αexpression correlates significantly with good prognosis in patients with hepatocellular carcinoma
Initially, we compared the expression levels of HIF2-αin normal non-cirrhotic liver to those of HCC patients who had undergone tumor resection. Compared to normal liver, HIF-2αmRNA expression was significantly lower in HCC's tumors, while there was no significant difference in HIF-1αexpression. Next we aimed to determine the role of HIF-2αin HCC patients by consecutive TMAs slides including 254 HCC samples that were immunostained by HIF-2αantibody. The clinical/pathological characteristics of patients were listed in table 1. Immunohistochemical results revealed that HIF-2αlocated on the cytoplasm and nuclear of tumor cells and low expressed in 131 cases(51.97%), We further analyzed the molecules expression in this 254 HCC samples annotated with extensive clinical follow up data. HCC patients characterized by high AFP level and large tumor size were prone to have low HIF-2αexpression. Patients with low levels of HIF-2αhad a significantly shorter overall survival time than those with high level of HIF-2α.Kaplan-Meier analysis revealed that the patients with low levels of HIF-2αhad a significantly lower overall survival rate in the five years although the difference became smaller after 5 years. Univariate analysis revealed that low expression of HIF-2a showed a prognostic significance with poor prognisis for overall survival in five year. Multivariate analysis indicated that HIF-2αwas not an independent predictor for overall survival. There was no significant difference in the recurrence free time and recurrence free rate between different levels of HIF-2a. We further studied the role of HIF-2αin another separate subset of consecutive TMAs slides including 157 HCC patients who received a preoperative transareterial chemoembolization (TACE) before surgery. The TACE group showed younger age, larger tumor size, and multiple tumor than the other group, the correlation between HIF-2αand clinicopathological characteristics were shown. Patients with low levels of HIF-2αhad a significantly shorter overall survival time than those with high level of HIF-2a.Kaplan-Meier analysis revealed that the patients with low levels of HIF-2αhad a significantly lower overall survival rate. Univariate analysis revealed that low expression of HIF-2αshowed a prognostic significance with poor prognisis for overall survival. Multivariate analysis indicated that HIF-2αwas an independent predictor for overall survival in TACE patients. There was no significant difference in the recurrence free time and recurrence free rate in TACE patients.
Part Two
HIF-2a reduces hepatocellular carcinoma cell growth and augments apoptosis in HCC cells in vitro and in vivo
We began to investigate how elevated HIF-2αexpression in HCC might lead to better clinical outcome by selecting a hepatocellular carcinoma cell line (MHCC97H and MHCC97L. Hypoxia strongly induced HIF-2a mRNA and nuclear HIF-2a accumulation compared to cells cultured under normoxic conditions There was no significant change in HIF-1a regardless of the oxygen conditions.
To begin to uncover the mechanism by which HIF-2a leads to improved outcome in HCC patients, we made clones that over express and suppress HIF-2a in the HCC cell line (MHCC97H). Transfection of MHCC97H cells with pcDNA3-HIF-2a induced a fourfold increase in HIF-2a mRNA expression while HIF-1awas essentially unchanged compared to control vector transfection. There was a 5 fold suppression of HIF-2a mRNA and 2.6 fold increase in HIF-1a in cells transfected with pT2sh-HIF-2a plasmid compared to control cells. Western blot analysis showed a similar trend as was seen in the quantitative RT-PCR. We found over expression of HIF-2αsignificantly reduced HCC tumor weight. Cells that suppressed HIF-2αshowed no significant change in tumor weight. These results are consistent with the clinical observation that elevated HIF-2αis a negative regulator of hepatocellular carcinogenesis.
We found no difference in the proliferative index between control and HIF-2a over expressing cells. However, we did find significantly more cleaved-caspase-3, a key executer of apoptosis, in cells over expressing HIF-2αcompared to mock tranfected 97H control. We used propidium iodide and annexin-v to determine the proportion of apoptotic cells in the HIF-2αover expressing cells. We found more than 20% apoptotic cells in the high HIF-2a group, while there was only 1% in the control group. To further confirm this effect, we derived another set of clones in a related but different cell line (MHCC97L) and determined the proliferative and apoptotic index. Over expression of HIF-2αin this cell line also showed significantly greater apoptosis, as determined by the activated caspase 3 staining, while suppression of HIF-2αwith shRNA plasmid had little effect on apoptosis. There was no significant effect on the proliferative index in cells expressing either high or low HIF-2α.This data further confirms that high levels of HIF-2αinduce apoptosis in HCC cell lines and has little effect on cell proliferation. In vivo data also showed that overexpression of HIF-2αcan induce more cleaved caspased 3 expression in tumor cells compared to mock transfected 97H cells. Together the in vitro and in vivo data support HIF-2αrole inducing HCC apoptosis while having little effect on cell proliferation.
Part Three
HIF-2αaugments apoptosis by directly inhibiting the expression of TFDP3
Having established HIF-2αas a mediator of HCC apoptosis, we sought to uncover the mechanism by which it induces apoptosis. We performed a chromatin immunoprecipitation (ChIP) combined with a DNA microarray (ChIP on chIP) screen with MHCC97H cells to uncover HIF-2αtarget genes. We identified 330 genes in the screen and grouped them according to the KEGG pathway database. HIF-2αtargets genes were found in several pathways. We listed the genes with the highest peakscore and low peak false discovery rate and focused upon genes that were implicated in cellular apoptosis. We sought to confirm the ChIP on chIP screen with an independent cell line, MHCC97L. We found the genes GSPT1, FNDC1, MMP16, TFDP3, RUNX1, GZF1 and RADIL were highly enriched in the anti-HIF-2αprecipitated fraction compared to control precipitation. Since these appear to be bono-fide HIF-2αtargets, we used Q-RT-PCR to determine if HIF-2αcan regulate the transcription of a subset of these genes. Interestingly, FNDC1, MMP16 and TFDP3 transcription were repressed by HIF-2αwhile RUNX1 was upregulated. We noted the particularly strong 10 fold suppression of transcription factor DP3 (TFDP3) transcription by HIF-2a. The TFDP3 gene belongs to the DP (dimerization parter) family [27], which exerts a regulatory function by dimerzing with E2F proteins. The heterodimerization of E2F-DP complex is essential for both high affinities DNA binding and efficient transcriptional regulation by E2Fs. It is known that TFDP3 can suppress apoptosis mediated through E2F and p53 [28]. We hypothesized that HIF-2αrepressed TFDP3 expression could account for the high rate of apoptosis seen in HCC cell lines which had high level of HIF-2a. To test this idea, we over-expressed TFDP3 and HIF-2αin the MHCC97H cell line. First we confirmed the expression levels of TFDP3 and HIF-2αmRNA in each transfectant. As expected, HIF-2αexpression significantly increased apoptosis while re-expression of TFDP3 significantly reversed this effect. We also studied actived-caspase-3 expression and found significant suppression of apoptosis when TFDP3 expression was re-established in HIF-2αover expressing cells. We found identical results in an additional cell line. We next investigated the cellular outcome of TFDP3-mediated E2F1 inhibition. Transcript levels for several E2F targets were measured in HIF-2a expression cells and re-expression of TFDP3 gene in these cells. Compared to control cells, HIF-2αexpression cells showed a marked increase in TP73 (2.7 fold), ARF(2.5 fold),14-3-3σ(2 fold), Noxa (5 fold), GADD45α(3.8 fold), and BID (2 fold) mRNAs and a subtle increase in TP53, TP53INP2, P21, and BAK1 transcripts, while the expression of a number of other known target/downstream genes did not change significantly. When we reexpressed TFDP3 gene in these cells, the expression of genes such as ARF,14-3-3σ, Noxa, GADD45α, and BID were down and the levels were near to that in mock cells.
Take as a whole, the results support that TFDP3 is a mediator of HIF-2αinduced apoptosis.
Conlusion
1. In this study, we followed 254 hepatocellular carcinoma patients treated with resection, and found that HCC patients characterized by high AFP level and large tumor size were prone to have low HIF-2a expression. Patients with low levels of HIF-2αhad a significantly shorter overall survival time than those with high level of HIF-2α, HIF-2αexpression in HCC correlated with improved clinical outcome.
2. The role of HIF-2αin 157 HCC patients who received a preoperative transareterial chemoembolization (TACE) before surgery showed the same results as that in 254 patients treated with resection only Patients with low levels of HIF-2αhad a significantly shorter overall survival time than those with high level of HIF-2α. Univariate analysis revealed that low expression of HIF-2αshowed a prognostic significance with poor prognisis for overall survival. Multivariate analysis indicated that HIF-2αwas an independent predictor for overall survival in TACE patients
3. We found over expression of HIF-2αin MHCC97H cell line which injected into nude mice, significantly reduced the tumor weight. Cells that suppressed HIF-2αshowed no significant change in tumor weight. In vitro data also showed that over expression of HIF-2αdecreased the cell growth significantly. These results are consistent with the clinical observation that elevated HIF-2αwas a negative regulator of hepatocellular carcinogenesis.
4. By activated-caspase -3, KI67 immunoflurecent staining and FACS sort confirmation, we found that high levels of HIF-2αinduced hepatocellualr carcinoma cell apoptosis and had little effect on cell proliferation in vivo and in vitro.
5. Screening by performed a chromatin immunoprecipitation (ChIP) combined with a DNA microarray (ChIP on chIP), we got 330 potential target genes of HIF-2αin MHCC97H cell. Confirmed by chip PCR and real time PCR, we found that TFDP3 was one of HIF-2αtarget genes and was downregulated by HIF-2α. By re-expression of TFDP3 in HIF-2αoverexpression liver cancer cell, we found that less cells were apoptosis and the expression of genes in the E2F1 was dowe-regulated than that of HIF-2αoverexpression, these data showed that TFDP3 was a mediator of HIF-2αinducing apoptosis.
Novelty
1. First we showed that HCC patients with low levels of HIF-2αhad a significantly shorter overall survival time and a lower overall survival rate than those patients with high level of HIF-2α, and HIF-2αexpression in HCC correlated with improved clinical outcome in hepatocellular carcinoma patients.
2. First we showed that TFDP3, a member of DP family, was one of HIF-2αtarget genes.
3. First we showed that TFDP3 was a mediator of HIF-2αinducing apoptosis by upregulating the E2F1 pathway.
Potential Application
1. Our data showed HIF-2αoverexpression was correlated with longer survival time and high survival rate in HCC patients treated with resection, HIF-2αwas an independent predictor for overall survival in HCC patients treated with TACE before resection, and HIF-2αcan be used as a predictor for HCC overall survival.
2. TFDP3, HIF-2αtarget gene, was a potential therapeutic target in liver cancer.
3. Our research confirmed the complex role of HIFαsubunit in cancer further and the screening of inhibitor about HIFαsubunit used for cancer therapy need more studies.
引文
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