【作者】 于洋；

【导师】 黄倩；

【作者基本信息】 上海交通大学 ， 肿瘤学， 2016， 博士

【摘要】 胰腺癌是目前致死率较高的恶性肿瘤之一。尽管医务人员在胰腺癌研究上投入巨大,但是其5年生存率仍然停留在7%左右。由于目前仍缺少针对胰腺癌早期诊断的有效手段且胰腺癌缺少典型的体征和症状,大多数胰腺癌病人得到明确诊断时已处于晚期。对于不能进行手术切除的晚期患者,放射治疗是一种行之有效的治疗手段。然而,尽管在放射治疗的过程中大量的肿瘤细胞能够被有效的清除,但仍会有少量的肿瘤细胞能够存活下来,这些残存的肿瘤细胞的加速增殖最终导致放射治疗失败或肿瘤的复发,这一过程被称之为放疗后的肿瘤细胞再增殖。肿瘤细胞再增殖在放疗后肿瘤复发过程中起着重要作用,但是人们对其分子机制仍然缺乏了解。本课题组前期研究阐述了乳腺癌中caspase 3介导的肿瘤细胞再增殖的分子机制:caspase3作为一个重要的凋亡效应因子,其在X射线诱导的凋亡乳腺癌细胞中被激活,从而激活不依赖钙离子的磷脂酶A₂（iPLA2）,活化的不依赖钙离子的磷脂酶A₂能够进一步促进凋亡细胞合成并释放前列腺素E₂（PGE₂）。本课题组近期一项针对胰腺癌的研究表明,蛋白激酶δ（PKCδ）能够被凋亡细胞中激活的caspase 3切割并激活,活化的蛋白激酶δ能够通过进一步激活p38促进肿瘤细胞再增殖。p38是一种能够调控细胞内生长因子合成的蛋白激酶。上述研究表明caspase 3/PKCδ/p38通路是放疗后肿瘤细胞再增殖过程当中的起始信号通路,其下游的效应因子值得我们深入的探索和研究。针对恶性转化相关的基因mRNA翻译水平的调控被认为是细胞恶性转化过程中关键步骤。多数情况下,针对翻译水平的调控都是在翻译起始过程中完成的。真核细胞翻译起始因子4E（eIF4E）被认为是翻译起始过程中的限速因子。越来越多的研究表明,eIF4E的过度活化导致了肿瘤治疗过程中放化疗抵抗的出现。eIF4E活性受到MNK1（Mitogen-activated Protein Kinases Interacting Kinases 1）的调控,MNK1能够磷酸化eIF4E第209位丝氨酸使其激活。而MNK1则是有丝分裂原激活的蛋白激酶p38和ERK1/2（Extracellular Signal-regulated Protein Kinase）的直接磷酸化底物。我们的实验结果证明X射线照射后caspase 3/PKCδ/p38能够激活MNK1,激活后的MNK1可导致eIF4E磷酸化水平的升高。通过在肿瘤再增殖体外模型中应用eIF4E抑制剂,我们证明X-射线照射后濒死的肿瘤细胞对少量的存活肿瘤细胞的促增殖作用依赖于eIF4E磷酸化水平的升高。Sox2被认为是调控胚胎发育过程中的细胞分化、形态发生和谱系特异性的关键转录因子。近期的研究结果表明,Sox2与某些上皮细胞肿瘤密切相关,尤其能够通过促进肿瘤细胞中干性相关基因的表达来调控皮肤磷状上皮细胞癌中肿瘤干细胞的功能。由于X射线照射后的肿瘤细胞中被证明存在着干细胞样的基因表达特征,我们推测X射线照射能够诱导肿瘤细胞中干性相关转录因子表达增高。我们发现在X射线处理后的胰腺癌细胞当中Sox2的表达显著升高。同时,我们意外的发现Sox2的表达受到eIF4E的调控。为了探索Sox2在肿瘤细胞再增殖中的作用,我们构建了稳定表达Sox2的胰腺癌细胞系,并应用与体外再增殖模型当中。结果表明,稳定过表达Sox2的胰腺癌细胞在经过X射线照射后相比于野生型胰腺癌细胞能够更加有效的刺激报告细胞的增殖,而这种刺激作用是通过旁分泌途径实现的。本研究揭示在X射线处理过的胰腺癌细胞中caspase 3/PKCδ/p38/MNK1信号通路的激活诱导eIF4E磷酸化,eIF4E磷酸化进一步导致Sox2表达升高;X射线照射后胰腺癌细胞中Sox2表达水平增高能够通过旁分泌的形式有效地促进残存肿瘤细胞的增殖。本研究结果提示Sox2有可能是潜在的针对放疗后肿瘤细胞再增殖的治疗靶点。更多还原

【Abstract】 Pancreatic cancer is one of the most lethal cancers.In spite of decades of effort,the 5-year survival rate remains at only 7%.Due to the lack of effective early diagnostic approaches and typical symptoms or signs,most patients with pancreatic cancer are usually diagnosed at advanced stages.For patients with advanced pancreatic cancer,radiation is an effective therapy.However,even though radiation may kill the great majority of tumor cells,the accelerated repopulation of surviving tumor cells could reestablish the tumor.Tumor repopulation,of which the molecular mechanisms are poorly understood,plays a significant role in tumor recurrence after radiotherapy.Our previous study has demonstrated that caspase 3,which is a master executioner during cell apoptosis,promotes tumor repopulation in breast cancer through activating calcium-independent phospholipase A2(iPLA2)and increasing subsequent release of prostaglandin E2(PGE2)from apoptotic cells.Another recent study in our laboratory has manifested that PKCδ in apoptotic pancreatic cancer cells,which can be cleaved and activated by activated caspase 3,stimulates tumor repopulation by activating p38,an important kinase mediating growth factor production.These studies indicate that caspase 3/PKCδ/p38 axis is an initiating signaling pathway for tumor repopulation,but the downstream factors of this pathway is still unclear.Translational control of malignancy-related mRNAs has been considered as a major part in malignant progression.In most cases,translational control is conducted at the initiation step.eIF4E(eukaryotic initiation factor 4E)is thought to be the rate-limiting factor in translation initiation progression.Accumulating evidence suggested that eIF4 E plays a significant role in radiotherapy resistance and chemotherapy resistance.MNK1 regulates eIF4 E activity by phosphorylating Ser209 of eIF4 E.MNK1 is directly phosphorylated by mitogen-activated protein kinases p38 and ERK.We hypothesized that eIF4 E may be the downstream factor of caspase 3/PKCδ/p38 pathway.Our results demonstrated that caspase 3/PKCδ/p38/MNK1 signaling pathway leads to elevated phosphorylation eIF4 E.We subsequently investigated whether inhibition of eIF4 E activity mitigates tumor cell repopulation after irradiation.We used two drugs to inhibit eIF4 E activity through different mechanisms,results manifested that elevated eIF4 E activity is required for the stimulatory effect of irradiated dying cells on living tumor cells.Sox2 has been identified as a key transcription factor required for lineage specification,morphogenesis and differentiation in development of mouse embryos.Recently,accumulating evidence has suggested that dysregulation of Sox2 plays an important role in epithelial cancers.Sox2 regulates cancer stem cell(CSCs)function in skin squamous-cell carcinoma by directly promoting transcription of genes associated with stemness,proliferation and survival.As stem cell-like gene signature identified in ionizing radiation-treated cancer cells,we wondered whether IR induces the upregulation of some “stemness” related transcription factors in irradiated cancer cells.Results manifested that Sox2 expression significantly increased at 24 hours after irradiation in pancreatic cancer cells.Unexpectedly,our data from western blot and qPCR manifested that eIF4 E phosphorylation controls Sox2 expression in a translational manner.In order to find our the specific role of Sox2 in tumor cell repopulation after irradiation,we constructed pancreatic cancer cell lines stably overexpressing Sox2.We found that over-expression of Sox2 strongly enhanced the growthstimulating effect of irradiated dying tumor cells on living tumor cells through a paracrine modality.In conclusion,we have shown that activation of caspase 3/PKCδ/p38/MNK1 signaling induces eIF4 E phosphorylation,which mediates upregulation of Sox2 in irradiated tumor cells.Importantly,Sox2 upregulation in irradiated dying tumor cells promotes proliferation of living tumor cells through a paracrine modality,indicating that Sox2 may be a target to reduce tumor cell repopulation after radiotherapy.更多还原