人乳腺癌组织中mTOR-Notch通路的验证及靶向治疗方案探讨

【作者】 张舒；

【导师】 张宏冰； 孙强；

【作者基本信息】 北京协和医学院 ， 临床医学， 2009， 博士

【摘要】 乳腺癌是女性最常见的恶性肿瘤之一,不但严重威胁着女性的生命,对患者的心理也造成巨大的创伤。我国属于乳腺癌低发地区,但发病率正在以高于世界平均水平1-2%的速度增长,在部分城市乳腺癌已占女性恶性肿瘤之首位。虽然已知乳腺癌与遗传、激素影响、生活方式等因素有关,但具体发病机制尚不清楚。膜受体酪氨酸激酶(RTK)-PI3K-AKT-mTOR通路可以调节细胞增殖、分化、死亡等功能。Notch通路则是调控细胞分化的重要通路。大量研究表明在乳腺癌中这两条通路常存在异常活化,我们的前期工作表明mTOR高度活化的MEF细胞中Notch信号通路通过mTOR上调,但在肿瘤组织中尚未验证此通路存在。为了在乳腺癌组织中验证mTOR与Notch表达的相关性,本研究运用WesternBlot及免疫组化染色的方法检测了乳腺癌肿瘤组织中mTOR与Notch的表达水平,结果表明,在所有的样本中(Western Blot30例；免疫组化57例),mTOR与Notch的表达水平正相关,二者相关性在低分化乳腺癌和早期乳腺癌中更为显著,在具有淋巴结转移、ER(-)、HER2(+)、Ki67指数≥50%及p53(-)的乳腺癌组织中更显著,支持了乳腺癌中存在mTOR-Notch通路的假设,同时表明在恶性程度越高的乳腺癌中,mTOR与Notch的相关性越高。ERK/MAPK通路也是典型的受体酪氨酸激酶调节通路,与PI3K-AKT-mTOR通路之间存在着密切的联系,对肿瘤的发生发展起重要的作用。由于mTOR的抑制可以反馈性激活ERK/MAPK通路及PI3K-AKT-mTOR通路,为了探讨更好的联合靶向治疗方案,本研究第二部分以PTEN-/-MEF细胞作为肿瘤的细胞模型,联合应用PI3K、mTOR双重抑制剂BEZ235及ERK激酶MEK1/2抑制剂U0126,观察其对细胞增殖的抑制效果。结果显示：BEZ235及U0126对PTEN-/-细胞均有抑制作用,二者IC50值分别为：6.257nM及22.85μM。但联合应用BEZ235与U0126,二者表现为拮抗的作用方式,因此在PTEN-/-细胞中或PTEN突变的肿瘤的联合靶向治疗中,不推荐应用BEZ235与U0126联合使用。本研究进一步验证了mTOR通过上调Notch进而调节分化导致肿瘤发生的机理,并通过联合用药实验检验了常用通路抑制剂的抗肿瘤效果,将有助于加强临床治疗的针对性和有效性。更多还原

【Abstract】 Breast cancer is one of the most common malignant tumors among women. Although the morbidity rate of breast cancer in China is relatively low comparing to the one in western countries, it is growing at a rate1-2%higher than the world average. Although genetic factors, hormonal influence and life style play important roles in the development of breast cancer, the underlying mechanism of the tumorigenesis is still largely unknown.The receptor tyrosine kinase (RTK)-phosphatidylinositol-3-kinase (PI3K)-AKT-mammalian target of rapamycin (mTOR) pathway regulates multiple cellular functions including proliferation, differentiation, and cell death, whereas Notch pathway plays an important role in controlling cell differentiation. Previous study in our laboratory indicates that Notch receptor is up-regulated in mTOR hyperactivated MEFs through mTOR. A large body of evidence suggests that both mTOR and Notch pathways are frequently aberrant regulated in breast cancer, however, the interaction between these two pathways exists in vivo has yet to be tested.Therefore Western Blot and immunohistochemistry (IHC) staining were used to examine the correlation between mTOR activation and Notch expression in breast cancer tissues. Our data confirm that mTOR and Notch have significant relevance in all breast cancer samples examined (30samples using Western Blot and57samples using IHC), especially in low grade differentiated group and early stage group. Furthermore, higher significance was observed in the sample groups with indicators of poor prognosis, such as higher lymph node metastasis, negative ER expression, positive HER2expression, higher Ki67index, and p53mutation. These. results are in agreement with the hypothesis that there is an mTOR-Notch cascade existing in breast cancer. We also conclude that concurrent activation of mTOR and Notch is more prevalent in tumors with higher malignancy. ERK/MAPK pathway is another classic receptor tyrosine kinase pathway which plays essential role in tumorigenisis, and has close relationship with PI3K-AKT-mTOR pathway. It was newly identified that the efficacy of mTOR inhibition could be limited by mTOR-mediated negative feed back regulation on ERK/MAPK and RTK-PI3K-AKT pathway. To develop better combined target therapy, PTEN knockout MEF cell lines were used as cellular model for malignant tumor. BEZ235, the dual inhibitor of PI3K and mTOR, and U0126, the inhibitor of MEK1/2were combined to threat the cells. The inhibitory effects to cell proliferation were monitored by MTT. Our results show that both BEZ235and U0126suppress PTEN knockout cell proliferation, and their IC50values are6.257nM and22.85μM, respectively. Surprisingly, these two drugs had antagonistic rather than synergistic effect on cell proliferation, suggesting BEZ235and U0126are not suitable for a combined target therapy regimen.This study provides further evidence of oncogenically activated mTOR up-regulating Notch signaling as the possible mechanism for the inhibition of differentiation and the subsequent tumorigenisis. The combinational usage of pathway inhibitors investigated the possible cancer treatment with improved specificity and effectiveness, and provided information for clinical therapy.更多还原