【作者】 徐琰；

【导师】 姜军；

【作者基本信息】 第三军医大学 ， 外科学（专业学位）， 2013， 博士

【摘要】 乳腺癌是世界上女性患病率和死亡率最高的恶性肿瘤之一。尽管如ER，PR和HER2等一些分子标记物已经被广泛用于对乳腺癌病理类型的分析、治疗方案的选择以及预后判断的参考，但是，这些分子标记物对判断患者的质量反应及预后仍有相当的局限性。所以，我们仍需要发现和验证更多可靠的分子标记物，使之能够应用于临床诊治和预后判断。Twist1属于碱性螺旋-环-螺旋(basic helix-loop-helix, bHLH)转录因子超家族成员，它编码一种含有碱性螺旋-环-螺旋(bHLH)结构域的转录因子和氨基酸基序特异蛋白参与调节器官发生的过程。Twist1最初被发现在果蝇的胚胎中胚层分化中发挥关键作用。与其调节器官发生的主要作用一致的是，生理情况下，Twist1主要在中胚层来源的组织中的表达。作为一个胚胎形态发生的主要调节因子，Twist1也被认为在肿瘤细胞的上皮间质转化、侵袭、转移、癌细胞干性维持以及癌细胞化疗耐药等过程中起到非常重要的作用。Twist1在人乳腺癌中过表达往往与肿瘤淋巴结转移、远处转移以及不良预后密切相关。因此Twist1被看做是乳腺癌治疗的潜在靶标。第一部分：SRC-1与Twist1在人乳腺癌中的表达及与临床预后的关系类固醇受体辅助活化因子1(SRC-1或NCOA1)是p160家族成员之一，其作用是在性激素存在时辅助活化ERα和PR等核受体。之前的研究表明，SRC-1不仅在动物生殖器官的发育和生长过程中起到非常重要的作用，而且在肿瘤细胞的增殖、侵袭和转移的多种信号通路中扮演着异常重要的角色。尽管基础研究已经发现在乳腺癌细胞中，SRC-1通过共激活能结合于Twist1近端启动子的转录因子PEA3来激活Twist1的转录，但在人乳腺癌中，SRC-1与Twist1的关系以及它们与各临床因子的关系目前仍属未知。为了解在人乳腺癌中SRC-1与Twist1蛋白表达的关系，以及探索二者在乳腺癌患者临床预后判断中的价值，我们将对制作成组织芯片的137例乳腺癌患者的肿瘤组织进行SRC-1和Twist1免疫组化染色并评分，其中123例患者有中位时间长达5年的术后随访资料。我们采用Kaplan–Meier法制作了生存曲线，并采用COX比例风险回归模型进行单因素和多因素生存分析。在本研究中，我们发现：1.在人乳腺癌中SRC-1和Twist1的蛋白表达呈正相关(p=0.009)。2. SRC-1的表达与HER2的呈正相关(P=0.024)。Twist1的表达与淋巴转移呈正相关(p<0.001)，与PR阳性表达呈负相关(P=0.041)。3. SRC-1或Twist1阳性表达患者的总生存率(OS)和无病生存率(DFS)比均阴性表达的患者明显较差(各组p <0.05)。另外，SRC-1和Twist1同时阳性表达的患者在四组患者中预后最差，而SRC-1和Twist1同时阴性表达的患者预后最佳(各组p <0.01)。4. COX多因素生存分析发现，SRC-1的表达、肿瘤分期和PR状态是总生存率的独立预测因子(p=0.019,<0.001和0.020)；Twist1的表达、淋巴结转移情况和PR状态是无病生存率的独立预测因子(p=0.006,0.001和0.029)。第二部分：Twist1蛋白在成体小鼠全身组织器官中的分布及诱导性敲除Twist1基因对小鼠生理功能的影响由于Twist1缺失会导致小鼠在胚胎时期致死亡，Twist1在成年动物中的功能仍然未知。人类的Twist1等位基因突变是Saethre-Chotzen综合征(Saethre-Chotzensyndrome，SCS，尖头并指(趾)畸形综合征Ⅲ型，一种常染色体显性遗传综合征。临床表现为短头畸形、面部不对称、额部发际低、眉异常、眼睑下垂、睑裂斜向下、眶距增宽、鼻纵隔偏曲、面中部凹陷、小耳、错颌、并指，以及其它发育缺陷)发生的原因。针对癌症患者，如果靶向敲除Twist1后是否会导致严重的并发症？这是一个值得探讨的问题。首先，我们发现，Twist1蛋白在成体小鼠的乳腺成纤维细胞以及皮肤毛囊毛乳头细胞等一些组织器官中表达；随后，我们建立了他莫西芬诱导性Twist1基因敲除小鼠模型以研究Twist1敲除对成体小鼠的影响。我们在实验中发现：1．在鼠龄6周时敲除雌鼠的Twist1基因对其乳腺在青春期、怀孕期和哺乳期的发育及其在哺乳期的泌乳功能没有明显影响。2.尽管Twist1蛋白在皮肤毛乳头细胞中的表达并未随毛发生长周期而发生改变，我们在小鼠出生后第13天(此时毛囊已发育)时敲除Twist1基因，发现小鼠毛发的休止期明显缩短，生长期明显延长，因而毛发生长的速度明显加快。3．Twist1敲除小鼠的生长发育以及生育功能正常。分别在雄鼠和雌鼠中敲除Twist1基因，对其体重、心率、肌肉和脂肪含量等指标均没有明显影响，仅在雌鼠中发现Twist1基因敲除使其血压有轻度下降，但对雄鼠血压没有影响。综上所述，我们首次发现，在人乳腺癌中SRC-1的表达与Twist1的表达具有正相关，验证了二者间存在调控关系；我们也验证了SRC-1和Twist1蛋白表达可作为乳腺癌预后的预测因子；此外，SRC-1和Twist1的共同表达状态可以为患者的预后判断提供更好的参考。上述实验结果还表明，对于幼鼠和成体小鼠，Twist1并不是维持其健康生命所必需的基因；而在失去了Twist1的基因功能后，能够促使成鼠毛发生长。所以我们认为，敲除Twist1不会对机体产生严重的副作用，Twist1基因及蛋白可以作为乳腺癌治疗的靶标。更多还原

【Abstract】 Breast cancer is one of most common causes of cancer deaths worldwide. Althoughsome pathological factors such as ER, PR and HER2have been wildly used for referencesin clinical diagnosis and treatment, their predictive ability of disease prognosis in breastcancer still have limitations. Therefore, it is necessary to identify reliable molecularprognostic markers in clinical practice for the treatment of breast cancer.The transcription factor Twist1belongs to the basic helix–loop–helix (bHLH)superfamily. It encodes a transcription factor that contains a basic helix-loop-helix(bHLH)domain and an amino-acid motif specific to proteins that are involved in the regulation oforganogenesis. Twist1was originally identified as one of the zygotic genes in Drosophilaresponsible for mesoderm development during embryonic morphogenesis. Consistent withits major role in organogenesis, under physiological circumstances, Twist1is primarilyexpressed in mesoderm-derived tissues. As a master regulator of embryonic morphogenesis,Twist1is also known to contribute to epithelialemesenchymal transition, invasion,metastasis, stemness, and chemotherapy resistance in cancer cells. Twist1is over-expressedin human breast cancers and usually associated with lymph-node and distant metastases andpoor prognosis, and thus is a potential target for breast cancer therapy.Part1SRC-1and Twist1are positively correlated and associated with poorprognosis in human breast cancerThe steroid receptor coactivator1(SRC-1, also known as NCOA1) is a member ofp160family, it has the ability to coactivate nuclear receptors such as ERα and PR in thepresence of hormones. Previous studies revealed that SRC-1plays a important role indevelopment and growth of reproductive organs and SRC-1also plays a critical role in cancer cell proliferation, invasion and metastasis through multiple pathways. Twist1hasbeen shown to induce EMT and plays a critical role in cancer cell metastasis. Althoug basicstudies have revealed that SRC-1serves as a coactivator for the transcription factor PEA3to enhance Twist1expression in breast cell lines, the correlation between SRC-1andTwist1in human breast cancer and their relationship with clinical parameters still remainunclear.In order to analyze the expression profiles of SRC-1and Twist1in human breastcancer and evaluate their possible prognostic values in breast cancer patients,immunohistochemistry was performed for detecting SRC-1and Twist1proteins on breastcancer with tissue microarray containing137specimens, in which123breast cancerpatients were followed up for a median of5years after surgery. Survival curves weregenerated using the Kaplan–Meier method. Multivariate analysis was performed by usingthe Cox proportional hazard regression model to assess the prognostic values of SRC-1andTwist1.Below we present the results of our study:1. There was an positive correlation between SRC-1and Twist1expression at proteinlevels (p=0.009).2. SRC-1-positive expression was correlated with HER2expression (P=0.024). Theprotein expression of Twist1was positively correlated with lymph node metastasis (p<0.001) and inversely associated with PR status(P=0.041).3. Patients with SRC-1or Twist1-positive expression had a poorer overall survival(OS) and disease-free survival (DFS) than those with SRC-1or Twist1-negative expression(p <0.05for all). In addition, SRC-1-negativeive/Twist1-negative patients had the best OSand DFS (p <0.01for both).4. In multivariate survival analysis, SRC-1expression, tumor stage and PR wereindependent prognostic factors related to OS (p=0.019,<0.001and0.020, respectively),Twist1expression, lymph node status and PR were found as independent predictors of DFS(p=0.006,0.001and0.029, respectively).Part2Studies on the distribution of Twist1protein in mouse organs and tissuesand the effects of inducible Twist1gene knockout on the physiological functions of miceBecause Twist1-null mice are embryonic lethal, the function of Twist1in adultanimals remains unknown. People with one Twist1germline mutant allele developSaethre-Chotzen syndrome. It is questionable whether Twist1can be targeted in cancerpatients without severe adverse effects.In this study, We found that Twist1is expressed in several tissues of mouse, includingfibroblasts of the mammary glands and dermal papilla cells of the hair follicles. We thendeveloped a tamoxifen-inducible Twist1knockout mouse model to knock out Twist1allelesin adult mice to define the effects of Twist1inactivation on the health of the adult animal.Our further studies were outlined as follows:1. Twist1knockout in6-week-old female mice did not affect mammary glandmorphogenesis and function during pregnancy and lactation.2. Although Twist1is not cyclically expressed in dermal papilla cells, knockout ofTwist1at postnatal day13(when hair follicles have developed) drastically shortened thetelogen phase and extended the anagen phase and thus accelerated hair growth.3. The Twist1null mice grew normally and were fertile. In both males and females,the knockout did not influence body weight gain, heart rate, or total lean and fatcomponents. The knockout also did not alter blood pressure in males, although it slightlyreduced blood pressure in females.In conclusion, we demonstrated for the first time that SRC-1and Twist1expressionsare positively correlated in human breast cancer. We also confirmed that SRC-1and Twist1expression are prognostic factors in human breast cancer. Moreover, combined expressionof SRC-1/Twist1could improve prognostic judgment for breast cancer patients.These results also indicate that Twist1is not essential for maintaining an overallhealthy condition in young and adult mice and that loss of function facilitates hair growth inadulthood, supporting Twist1gene or protein could be used as specific targets for breastcancer therapy without severe side effects.更多还原