细丝蛋白A调控表皮生长因子受体活性在乳腺癌发病中的作用
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摘要
目的:乳腺癌是严重威胁女性健康的第一位恶性肿瘤。随着经济发展和人民生活水平的提高,乳腺癌的发病率呈现上升的趋势,肿瘤复发和远处转移是乳腺癌患者主要的死亡原因。乳腺癌的发生和发展机制至今仍未完全阐明,临床尚缺乏有效的治疗措施。
表皮生长因子受体(epidermal growth factor receptor,EGFR)家族包括erbB-1/EGFR、erbB-2/HER2/neu、erbB-3和erbB-4四大成员,属于Ⅰ型酪氨酸激酶受体,酪氨酸磷酸化可使受体活化,控制着细胞的增殖、分化、移动和存活,受体的表达失调与肿瘤的发生相关。HER2是细胞来源的癌基因,在正常情况下处于非活化状态,参与细胞生长、分化的调节,在乳腺癌的研究中,HER2的扩增和过度表达研究最为深入,见于30%的乳腺癌,与乳腺癌的浸润和扩散有关,曲妥珠单抗(Trastuzumab、Herceptin、赫赛汀)是针对HER2强阳性表达乳腺癌首选的生物靶向治疗药物。但临床有19%乳腺癌的HER2、雌激素受体(estrogen receptor, ER)和孕激素受体(progesterone receptor, PR)均为阴性,且易发生复发和转移,预后较差,目前仍无有效的治疗靶点。研究发现EGFR蛋白在此类乳腺癌呈现过表达,且乳腺癌的表型与EGFR蛋白、淋巴结转移相关。
EGFR是细胞膜的跨膜受体,与细胞的增殖、分化、运动、存活有关。正常细胞表达适量的EGFR,用于维持细胞的生命活动,但其过表达或活性增强可持续启动细胞增生信号的传递系统,致细胞过度增殖和表型恶化。EGFR在超过35 %～50 %的乳腺癌组织中表达。EGFR的表达与乳腺癌患者的预后有关,其表达越多则乳腺癌的预后越差,有淋巴结转移的乳腺癌的EGFR表达明显高于无淋巴结转移者。EGFR酪氨酸的磷酸化是EGFR信号转导过程中的关键分子事件,EGFR的磷酸化程度决定着肿瘤的预后,EGFR磷酸化程度高,肿瘤的预后差。影响EGFR磷酸化的原因目前不清楚,因此本研究重点探讨影响EGFR磷酸化的分子,为寻找治疗肿瘤的新靶点奠定基础。
细丝蛋白A(Filamin A,FLNa)又称为Filamin-1或ABP-280,是细胞内一种肌动蛋白结合蛋白,同时又是脚手架蛋白,在细胞浆内广泛分布,也可以跨膜分布或转位至细胞核。FLNa蛋白是以“V”形的同型二聚体存在,每个FLNa分子内含一个肌动蛋白结合区(actin-binding domain,ABD)、24个重复单位及两个绞链区。特殊结构决定其具有强大的功能:FLNa与肌动蛋白丝结合,有助于细胞运动,与细胞增殖、迁徙、血管形成和器官发生有关;FLNa为脚手架蛋白,将细胞内多种蛋白分子与细胞骨架连接,并募集细胞膜的共刺激分子,参与细胞内的多种重要功能的信号转导和细胞对外界刺激的反应。
许多研究证实FLNa在恶性肿瘤细胞的表达增多。如肺癌细胞内FLNa的表达上调,与肺癌的迁徙、粘附和侵袭相关;前列腺癌细胞的胞浆内FLNa表达较正常前列腺组织显著增高,且有转移的前列腺癌FLNa的表达高于无转移者;乳腺癌细胞内也存在FLNa高表达。通过免疫组化、流式细胞技术已观察到FLNa的表达量与乳腺癌的恶性度和转移相关,FLNa的表达随乳腺癌恶性度的增高而增加,有淋巴结转移的乳腺癌的FLNa表达明显高于无淋巴结转移者。Fiori和Zhu等对不同转移能力的黑色素瘤细胞系研究发现FLNa可影响EGFR的磷酸化(见第二部分文献2)。但是,FLNa是否也影响乳腺癌细胞的EGFR的磷酸化目前尚未见报道。
为此,我们拟利用人乳腺癌细胞系,通过转染FLNa的siRNA降低FLNa表达、转染FLNa全长基因增加FLNa的表达后,观察EGFR的磷酸化水平及与EGFR磷酸化的相关的信号转导分子的变化;经免疫共沉淀(co-Immunoprecipitation,IP)证实FLNa与EGFR分子间的相互关系;利用MTT检测FLNa的表达对乳腺癌细胞增殖的影响。从组织、细胞和分子水平综合分析FLNa对EGFR磷酸化的调控,探讨FLNa调控EGFR磷酸化在乳腺癌发病中的作用,为有效治疗乳腺癌提供新靶点。
第一部分细丝蛋白A在浸润性乳腺癌组织中的表达及与临床病理特征的相关性
方法:采用链霉抗生物素蛋白-过氧化物酶(SP)免疫组织化学方法和流式细胞分析技术,检测46例分化程度不同的浸润性乳腺癌细胞中FLNa和增殖细胞核抗原(pro-liferating cell nuclear antigen, PCNA)的表达情况,以正常乳腺组织或乳腺良性增生为阴性对照。
结果:
1. FLNa在乳腺癌组织中的表达
免疫组化结果:FLNa在正常乳腺上皮细胞的胞浆内少量表达(Fig. 1-1);FLNa在浸润性乳腺癌细胞阳性表达的程度较强,且随组织分化程度的降低而增高(Fig.1-2～4、Table 1-1),差异有统计学意义(P<0.05);有淋巴结转移乳腺癌细胞的FLNa的表达明显强于无转移组(Table 1-1),差异有统计学意义(P<0.05)。FLNa在浸润性乳腺癌细胞中的表达与组织学分型无关。
流式细胞检测结果: FLNa在正常乳腺组织的表达量较少;在低分化乳腺癌细胞中FLNa的表达(1.22±0.13)明显高于中高分化的乳腺癌细胞(1.10±0.11),差异有统计学意义(P<0.05),见Fig.1-6、Table 1-2。无淋巴结转移者(1.21±0.11)明显高于有淋巴结转移者(1.10±0.10),差异有统计学意义(P<0.05),见Table 1-2。FLNa在浸润性乳腺癌细胞中的表达与组织学分型无关。
2. PCNA在乳腺癌组织中的表达
免疫组化结果:PCNA在浸润性乳腺癌细胞核呈阳性表达,PCNA的表达随乳腺癌分化程度的降低而增强,在低分化浸润性乳腺癌细胞的表达呈强阳性,见Fig.1-5。
流式细胞检测结果:低分化浸润性乳腺癌细胞中PCNA的表达(1.65±0.22)明显高于中高分化乳腺癌(1.35±0.15),差异有统计学意义(P<0.05),无淋巴结转移的乳腺癌细胞PCNA的表达(1.42±0.15)明显低于有淋巴结转移者(1.56±0.16),差异有统计学意义(P<0.05),见Fig.1-7、Table 1-3。PCNA的表达与FLNa呈正相关(P<0.05)。
第二部分FLNa对乳腺癌细胞表皮生长因子受体活化的影响
方法:
1.沉默FLNa的表达对乳腺癌细胞EGFR活化的影响
用含10%胎牛血清的低糖DMEM培养乳腺癌MDA-MB-231细胞。实验分组:FLNa-siRNA转染组;通用HK对照转染组。每组分别经EGF(20nM)刺激后0min、5min和10min后收集细胞,提取蛋白,Western-blot检测各时间点FLNa、EGFR、磷酸化EGFR (p-EGFR)、ERK (extracellular signal-regulated kinase)和磷酸化ERK(p-ERK)蛋白的表达。
2. FLNa过表达对乳腺癌细胞EGFR活化的影响
细胞培养同方法1。实验分组:人FLNa-full(hfilamin A/pREP4质粒,内含FLNa全长基因)转染组;空质粒对照pcDNA3.1转染组。每组分别经EGF(20nM)刺激0min、5min和10min后收集细胞,提取蛋白, Western-blot检测各时间点FLNa、EGFR、p-EGFR、ERK和p-ERK蛋白的表达。
3.免疫共沉淀证实FLNa对乳腺癌细胞EGFR的活化影响
细胞培养同方法1。用含10%胎牛血清的DMEM培养液调细胞浓度为3.5×105/ml,接种至60mm培养皿。实验分组:转染FLNa-siRNA组;转染通用HK对照组。每组分别经EGF(20nM)刺激0min和10min后,收集细胞,提取蛋白,对提取的蛋白分别用抗EGFR抗体和抗磷酸化酪氨酸的4G10抗体免疫共沉淀,Western-blot分别检测沉淀蛋白内p-EGFR和EGFR的水平。
结果:
1.沉默FLNa的表达对乳腺癌细胞EGFR活化的影响
1.1 FLNa蛋白的表达:FLNa-siRNA转染组FLNa的表达(1.40±0.10、1.27±0.06)较HK对照组(3.97±0.29、3.33±0.06)明显减少,差异有统计学意义(均P<0.01),见Fig.2-1、Table 2-1。
1.2 EGFR蛋白磷酸化水平:EGF刺激5min、10min后,FLNa-siRNA转染组的EGFR磷酸化水平(0.73±0.01、0.37±0.05)明显低于HK对照组(1.08±0.01、1.30±0.06),差异有统计学意义(均P<0.01),见Fig.2-2、Table 2-2。
1.3 ERK磷酸化水平:EGF刺激5min、10min后,FLNa的siRNA转染组ERK磷酸化水平(0.17±0.00、0.12±0.04)明显低于HK对照组(0.32±0.02、0.54±0.04),差异有统计学意义(均P<0.01),见Fig.2-3、Table 2-3。
2. FLNa过表达对乳腺癌细胞EGFR活化的影响
2.1 FLNa的表达:FLNa-full质粒转染组FLNa的表达(5.93±0.38、6.07±0.32)明显高于空质粒pcDNA3.1对照组组(4.50±0.20、4.60±0.17),差异有统计学意义(均P<0.01),见Fig.2-4、Table 2-4。
2.2 EGFR蛋白磷酸化水平:EGF刺激5min、10min后,FLNa-full质粒转染组EGFR的磷酸化水平(1.53±0.11、0.86±0.14)明显高于空质粒pcDNA3.1对照组(0.57±0.01、0.44±0.02),差异有统计学意义(分别P<0.01,P<0.05),见Fig.2-5、Table 2-5。
2.3 ERK磷酸化水平:EGF刺激5min、10min后,FLNa-full质粒转染组ERK的磷酸化水平(1.83±0.07、1.06±0.07)明显高于空质粒pcDNA3.1对照组(0.74±0.01、0.59±0.03),差异有统计学意义(均P<0.01),见Fig.2-6、Table 2-6。
3.免疫共沉淀证实FLNa对乳腺癌细胞EGFR活化的影响
3.1 FLNa-siRNA转染后FLNa的表达:FLNa-siRNA转染组FLNa的表达(3.53±1.08、4.33±1.33)较HK对照组(7.23±2.20、7.53±1.86)组明显减少,差异有统计学意义(均P<0.01)。见Fig.2-7、Table 2-7。
3.2经抗EGFR抗体免疫沉淀后的EGFR磷酸化的水平: EGF刺激10min后,FLNa-siRNA转染组的EGFR磷酸化水平(1.09±0.09)明显低于HK对照组(2.32±0.22),差异有显著性(P<0.01)。见Fig.2-8、Table 2-8。
3.3经抗酪氨酸磷酸化4G10抗体免疫沉淀后EGFR的表达: EGF刺激10min时,FLNa-siRNA转染组的EGFR表达(0.42±0.01)明显低于HK对照组(1.07±0.01),差异有显著性(P<0.01),见Fig.2-9、Table 2-9。
第三部分细丝蛋白A对乳腺癌细胞增殖的影响
方法:细胞培养见第二部分的方法1。用含10%胎牛血清的低糖DMEM培养液调细胞浓度为9×103/ml,按100μL /孔体积接种于96孔板。实验分组:FLNa-siRNA转染组和HK对照组;FLNa-full质粒转染组和空质粒pcDNA3.1对照组。分别用不同浓度的EGF(4nM、20nM、100nM)刺激过夜,用MTT法检测细胞的增殖水平。
结果:
1.FLNa沉默后MDA-MB-231细胞增殖水平:在4nM、20nM、100nM浓度的EGF刺激下,FLNa-siRNA转染组细胞的增殖水平随刺激浓度的增加而增高, FLNa沉默后细胞的增殖水平(1.28±0.09、1.46±0.02、1.58±0.06)均低于对照组(1.55±0.12、1.62±0.06、1.68±0.12),差异有显著性(p<0.01),见Fig.3-1、Table 3-1。
2. FLNa过表达后MDA-MB-231的细胞增殖水平:在4nM、20nM、100nM浓度的EGF刺激下,FLNa-full质粒转染组细胞的增殖水平随刺激浓度的增加而增高,但FLNa-full质粒转染组细胞的增殖水平(1.14±0.08、1.28±0.11、1.51±0.03)高于对照组(1.09±0.17、1.21±0.17、1.25±0.05),差异有显著性(P<0.01),见Fig.3-2、Table 3-2。
结论:
1. FLNa在正常乳腺组织少量表达;FLNa的表达随浸润性乳腺癌分化程度的降低而增高,且与淋巴结转移有关;FLNa的表达与乳腺癌细胞的增殖能力呈正相关。
2. FLNa的表达可调控乳腺癌细胞EGFR的磷酸化,磷酸化的EGFR又通过MAPK增殖信号传导通路活化ERK,影响乳腺癌细胞的增殖。
3.沉默FLNa的表达导致乳腺癌细胞的增殖水平降低;FLNa的过表达则使乳腺癌细胞的增殖水平升高,表明FLNa可通过调控EGFR的活化影响乳腺癌的发生和发展。
Objective:Breast Carcinoma is the leading fetal cancer that affects the health of women now. With the development of economy and the improve- ment of peoples’life, the mortality of breast carcinoma is going up. Breast carcinoma recurrence and metastasis are the main death causes of these patients. However, the mechanism of occurrence and development remains obscure. Patients suffering from breast cancer have a poor prognosis because of deficiency in effective treatment measures in clinical practices.
EGFR family encompasses members of erbB-1/EGFR, erbB-2/ HER2/ neu, erbB-3 and erbB-4. They belong to tyrosine kinase receptor which can be activated by tyrosine phosphorylation. HER2 is unactived oncogene in normal correlating with regulating to growth and differentiation. Amplification and overexpression of HER2 have been examined in a deep-going way and associated with invasion and diffusion. Trastuzumab (Herceptin), a humanized monoclonal antibody, targets activated HER2 and is clinically effective in HER2-over-expressing breast cancers. However, Nineteen percent of breast carcinoma which has no EGFR, ER and PR, is prone to recurrence and metastasis. Total survival rate is lower than that of other breast carcino ma.These patients have worse prognisis. But effective targeted therapy has not been emerged now. Studies have shown that overexpression of EGFR exists in this kind of breast carcinoma whose phenotype is significantly correlated with EGFR and lymphnode metastasis.
EGFR distributes in the way of transmembrane controlling proliferation, differentiation, motility and survival. Appropriate amount expression in normal tissue is necessary for normal vital movement.Overexpression or strong activity of EGFR can promote proliferation system. The excessive proliferation and malignant phenotype will be emerged. Overexpression of EGFR has been in 35%～50 % breast carcinoma and correlates with prognosis. The expression of EGFR in metastasis is significantly higher than that of non-metastasis one. No recurrence and high survival rate result from negative expression of EGFR. Tyrosine phosphorylation is the key molecular event in the EGFR signaling pathway. The level of EGFR phosphorylation decides the prognosis of breast carcinoma. Higher phosphorylation of EGFR will lead to poor prognosis. However, the mechanisms accounting for the upregulation of EGFR activity are largely unknown. So searching the factors that affect EGFR activity is the key purpose and provides foundation for new target in tumor.
Filamins A (Filamin-1, FLNa) are large actin-binding and scaffolding proteins expressed widely in cytoplasm. FLNa can distribute in the way of transmembrane or localize to the nucleus. Vertebrate filamins are elongated dimeric V-shaped proteins with two large polypeptide chains. Each monomeric chain of filamins consists of an N-terminal actin-binding domain (ABD), a long rod-like domain of twenty-four repeated and two hinges. FLNa is endued with powerful functions because of special structure. FLNa stabilizes cortical three-dimensional F-actin networks, anchors cytoskeleton to cellular membranes by binding to transmembrane receptors and integrate cell architecture and functions signaling, which is essential for cell motility correlating with proliferation, migration, organ development and potential role in oncogenesis;Acting as cross-linking proteins, Filamins not only link various signaling proteins to the cytoskeleton,but also collect costimulus molecule of cytomembrane. So they can take part in many important signaling transduction and make cell response to external stimulus in time;
Overexpression of FLNa has been confirmed in many malignancy tumors. It correlates with migration, adhesion and invasion in lung and prostate carcinoma. High expression of FLNa exists in breast carcinoma, too. The expression of FLNa correlating with the malignancy degree and metastasis has been proved with immunohistochemistry and Flow cytometry. Fiori and Zhu et al have shown that FLNa can regulate the activity of EGFR in melanoma cells and the expressions of FLNa and EGFR in melanoma with highly metastatic potentiality are higher than that of lower one. Up to now, FLNa influencing EGFR phosphorylation or not has not been reported.
Thus, the phosphorylation level of EGFR and correlated signaling molecules in breast carcinoma cells were observed after FLNa siRNA and full-lengh FLNa transfection, then the relationship between FLNa and EGFR phosphorylation as well as the effection of FLNa on proliferation were detected by Immunoprecipitation and MTT, respectively. Analysis was performed aggregately about the role of FLNa regulating to EGFR plosphorylation in breast carcinoma genesis from the levels of tissue, cell and molecule. In this study, the putative connection between FLNa and EGFR plosphorylation was explored and would provide a new target for effective treatment of breast carcinoma.
Part one The expression of filaminA in invasive breast carcinoma and its relationship with clinicopathological features
Methods:
Streptavidin-biotin-peroxidase (SP) Immunohistochemistry and Flow Cytometry were applied to detect the expression of FLNa and PCNA in 46 cases invasive breast cancer with different differentiation, using normal breast tissue or benign breast hyperplasia as negative control.
Results:
1. FLNa expression in breast cancer cells
1.1 FLNa expression with immunohistochemical detection in breast cancer cells:
FLNa expression was mainly distributed in the cytoplasm of mammary epithelial cells, staining shallow was regarded as negative in normal breast tissue (Fig.1-1); however, FLNa positive expression was gradually increased with the reducing of differentiation in invasive breast cancer. There were significant differences between the results of different differentiations (P <0.05); FLNa expression in the metastasis group was significantly stronger than that of non-metastasis one, the differences between them was statistically significant (P <0.05). FLNa has nothing to do with the histologic types in invasive breast cancer cells. (Fig.1-2～4 and Table1-1)
1.2 FLNa expression with Flow cytometry in breast cancer cells:
There was little expression of FLNa in normal breast tissue. It was gradually increasing with the reducing of differentiation. The highest expres- sion was in poorly differentiated breast cancer. The expression of FLNa in poorly differentiated breast carcinomas (1.22±0.13) was higher than that of well differentiated ones (1.10±0.11); Metastasis group (1.21±0.11) was higher than that of non-metastasis group (1.11±0.10), too. It has nothing to do with the histological types. There was a statistical difference between two groups (P <0.05). (Fig.1-6 and Table1-2)
2. PCNA expression in breast cancer cells
2.1 The results of PCNA with immunohistochemical detection in breast cancer cells:
The positive expression of PCNA in nucleus of breast cancer cell was very similar to the expression tendency of FLNa. PCNA expression had also changed with the differentiation. PCNA expression showed strong positive expression in poorly differentiated invasive breast cancer. (Fig.1-5)
2.2 The results of PCNA with Flow Cytometry in breast cancer cells
The expression of PCNA increased with differentiation decreased. The expression of PCNA in poorly differentiated breast carcinoma (1.65±0.22) was significantly higher than that of well-differentiated ones (1.35±0.15); Metastasis group (1.56±0.16) was higher than that of non-metastasis group (1.42±0.15), too. There was a statistical difference between two groups (P <0.05). (Fig.1-7 and Table 1-3). The expression of PCNA was correlated positively with that of FLNa (P <0.05).
Part two Effect of FLNa on the EGFR phosphorylation in breast carcinoma Methods:
1. Effect of FLNa silence on EGFR phosphorylation in Human breast carcinoma.
Human breast carcinoma MDA-MB-231 cells were cultured at 37℃, 5%CO2 in DMEM media containing 10% fetal calf serum and divided into two groups: FLNa-siRNA group and HK group. Each group was stimulated for 0min, 5min and 10min by 20nM EGF. The expression levels of FLNa, EGFR, p-EGFR, ERK and p-ERK were assessed by western-blot at the end.
2. Effect of FLNa over expression on EGFR phosphorylation in Human breast carcinoma.
MDA-MB-231 cells were cultured and divided into two groups: FLNa-full group and pcDNA3.1 group. Each group was stimulated for 0min, 5min and 10min by 20nM EGF. The expression levels of FLNa, EGFR, p-EGFR, ERK and p-ERK were examined by western-blot at last.
3. FLNa regulating to EGFR phosphorylation was demonstrated by co- immunoprecipitation in human breast carcinoma.
MDA-MB-231 cells were cultured and divided into two groups: FLNa-siRNA group and HK group.Each group was stimulated for 0min and 10min by 20nM EGF. Then 4G10 or EGFR protein immuneprecipitation was performed. The expressions levels of EGFR and p-EGFR were assessed by western-blot at the end.
Results:
1. Effect of FLNa silence on EGFR phosphorylation in Human breast carcinoma.
(1)The expression of FLNa
After FLNa-siRNA transfection,the expressions of FLNa in FLNa- siRNA group (1.40±0.10, 1.27±0.06) were lower than that of control group of HK. (3.97±0.29, 3.33±0.06). There was a statistical difference between two groups(P<0.01).(Fig. 2-1, Table 2-1)
(2)The expression of phosphorylated EGFR
After stimulation of EGF at 5min and 10min, the expressions of phosphorylated EGFR in FLNa-siRNA group (0.73±0.01, 0.37±0.05) were lower than that of control group of HK (1.08±0.01, 1.30±0.06). There was statistical difference between two groups(P<0.01). (Fig. 2-2, Table 2-2)
(3) The expression of phosphorylated ERK
After stimulation of EGF at 5min and 10min, the expressions of phosphorylated ERK in FLNa-siRNA group (0.17±0.00, 0.12±0.04) were lower than that of control group of HK (0.32±0.02, 0.54±0.04). There was a statistical difference between two groups(P<0.01). (Fig. 2-3, Table 2-3).
2. Effect of FLNa over expression on EGFR phosphorylation in Human breast carcinoma.
(1)The expression of FLNa
After FLNa-full transfection, the expressions of FLNa in FLNa-full group (5.93±0.38, 6.07±0.32) were higher than that of control group of pcDNA3.1 (4.50±0.20, 4.60±0.17). There was a statistical difference between two groups (P<0.01). (Fig. 2-4, Table 2-4).
(2)The expression of phosphorylated EGFR
After stimulation of EGF at 5min and 10min, the expressions of phosphorylated EGFR in FLNa-full group ( 1.53±0.11,0.86±0.14) were higher than that of control group of pcDNA3.1(0.57±0.01, 0.44±0.02).There was statistical difference between two groups(P<0.01, P<0.05). (Fig. 2-5, Table 2-5).
(3) The expression of phosphorylated ERK after FLNa-full transfection
After stimulation of EGF at 5min and 10min, the expressions of phosphorylated ERK in FLNa-full group (1.83±0.07, 1.06±0.07) were higher than that of control group of pcDNA3.1 (0.74±0.01, 0.59±0.03). There was statistical difference between two groups(P<0.01). (Fig. 2-6, Table 2-6). 3. FLNa regulating to EGFR phosphorylation was proved by Immunoprecipi- tation in human breast carcinoma.
3.1The expressions of FLNa with Western blot detection after FLNa-siRNA transfection
After FLNa-siRNA transfection,the expressions of FLNa in FLNa- siRNA group (3.53±1.08,4.33±1.33) were lower than that of control group of HK (7.23±2.20,7.53±1.86). There was statistical difference between two groups(P<0.01). (Fig. 2-7, Table 2-7)
3.2 The expression of phosphorylated EGFR after EGFR IP:
After stimulation of EGF at 10min,the expression of phosphorylated EGFR in FLNa-siRNA group (1.09±0.09) was lower than that of control group of HK (2.32±0.22).There was statistical difference between two groups(P<0.01). (Fig. 2-8, Table 2-8)
3.3 The expression of EGFR after 4G10 IP:
After stimulation of EGF at 10min,the expression of EGFR in FLNa- siRNA group(0.42±0.01) was lower than that of control group of HK (1.07±0.01). There was statistical difference between two groups(P<0.01). (Fig. 2-9, Table 2-9)
Part Three Effect of FLNa on proliferation of breast carcinoma Methods:
MDA-MB-231 cells were cultured and transferred cells into 96 holes plate. MDA-MB-231 cells were divided into two groups: FLNa-siRNA and HK-siRNA transfection group;FLNa-full and pcDNA3.1 plasmid transfection group.EGF stimulating was done with concentration of 4nM, 20nM and 100nM on the two groups overnight. The MDA-MB-231 cells proliferation was examined by MTT after FLNa-siRNA and FLNa-full plasmid trans- fection.
Results:
1. MTT detection after FLNa-siRNA transfection
Under the EGF stimulation with concentration of 4nM, 20nM and 100nM, the proliferation level of FLNa-siRNA group (1.28±0.09, 1.46±0.02, 1.58±0.06 was lower than that of control group of HK(1.55±0.12, 1.62±0.06, 1.68±0.12).There was statistical difference between two groups (P<0.01).The proliferation level of two groups was increasing following concentration of EGF.
2. MTT detection after FLNa-full transfection
Under the EGF stimulation with concentration of 4nM, 20nM and 100nM, the proliferation level of FLNa-full group (1.14±0.08, 1.28±0.11,1.51±0.03) were higher than that of control group of pcDNA3.1 (1.09±0.17, 1.21±0.17, 1.25±0.05). There was statistical difference between two groups(P<0.01). The proliferation level of two groups was rising following concentration of EGF.
Conclusion:
1. The expression of FLNa is little in normal breast tissue. It is gradually increasing with the reducing of differentiation and correlated with lymphnode metastasis in breast carcinoma. The expression of FLNa is correlated positive- ly with proliferation of breast carcinoma.
2. The expression of FLNa can regulate EGFR phosphorylation which active the molecule of ERK through MAPK signaling pathway and effect the proliferation of breast carcinoma.
3. FLNa silence can lead to the proliferation level of breast carcinoma decreasing, while the overexpression of FLNa can lift the the proliferation level of breast carcinoma.The results show that FLNa can regulate phos- phorylation of EGFR and effect genesis and development of breast carcinoma.
表皮生长因子受体(epidermal growth factor receptor,EGFR)家族包括erbB-1/EGFR、erbB-2/HER2/neu、erbB-3和erbB-4四大成员,属于Ⅰ型酪氨酸激酶受体,酪氨酸磷酸化可使受体活化,控制着细胞的增殖、分化、移动和存活,受体的表达失调与肿瘤的发生相关。HER2是细胞来源的癌基因,在正常情况下处于非活化状态,参与细胞生长、分化的调节,在乳腺癌的研究中,HER2的扩增和过度表达研究最为深入,见于30%的乳腺癌,与乳腺癌的浸润和扩散有关,曲妥珠单抗(Trastuzumab、Herceptin、赫赛汀)是针对HER2强阳性表达乳腺癌首选的生物靶向治疗药物。但临床有19%乳腺癌的HER2、雌激素受体(estrogen receptor, ER)和孕激素受体(progesterone receptor, PR)均为阴性,且易发生复发和转移,预后较差,目前仍无有效的治疗靶点。研究发现EGFR蛋白在此类乳腺癌呈现过表达,且乳腺癌的表型与EGFR蛋白、淋巴结转移相关。
EGFR是细胞膜的跨膜受体,与细胞的增殖、分化、运动、存活有关。正常细胞表达适量的EGFR,用于维持细胞的生命活动,但其过表达或活性增强可持续启动细胞增生信号的传递系统,致细胞过度增殖和表型恶化。EGFR在超过35 %～50 %的乳腺癌组织中表达。EGFR的表达与乳腺癌患者的预后有关,其表达越多则乳腺癌的预后越差,有淋巴结转移的乳腺癌的EGFR表达明显高于无淋巴结转移者。EGFR酪氨酸的磷酸化是EGFR信号转导过程中的关键分子事件,EGFR的磷酸化程度决定着肿瘤的预后,EGFR磷酸化程度高,肿瘤的预后差。影响EGFR磷酸化的原因目前不清楚,因此本研究重点探讨影响EGFR磷酸化的分子,为寻找治疗肿瘤的新靶点奠定基础。
细丝蛋白A(Filamin A,FLNa)又称为Filamin-1或ABP-280,是细胞内一种肌动蛋白结合蛋白,同时又是脚手架蛋白,在细胞浆内广泛分布,也可以跨膜分布或转位至细胞核。FLNa蛋白是以“V”形的同型二聚体存在,每个FLNa分子内含一个肌动蛋白结合区(actin-binding domain,ABD)、24个重复单位及两个绞链区。特殊结构决定其具有强大的功能:FLNa与肌动蛋白丝结合,有助于细胞运动,与细胞增殖、迁徙、血管形成和器官发生有关;FLNa为脚手架蛋白,将细胞内多种蛋白分子与细胞骨架连接,并募集细胞膜的共刺激分子,参与细胞内的多种重要功能的信号转导和细胞对外界刺激的反应。
许多研究证实FLNa在恶性肿瘤细胞的表达增多。如肺癌细胞内FLNa的表达上调,与肺癌的迁徙、粘附和侵袭相关;前列腺癌细胞的胞浆内FLNa表达较正常前列腺组织显著增高,且有转移的前列腺癌FLNa的表达高于无转移者;乳腺癌细胞内也存在FLNa高表达。通过免疫组化、流式细胞技术已观察到FLNa的表达量与乳腺癌的恶性度和转移相关,FLNa的表达随乳腺癌恶性度的增高而增加,有淋巴结转移的乳腺癌的FLNa表达明显高于无淋巴结转移者。Fiori和Zhu等对不同转移能力的黑色素瘤细胞系研究发现FLNa可影响EGFR的磷酸化(见第二部分文献2)。但是,FLNa是否也影响乳腺癌细胞的EGFR的磷酸化目前尚未见报道。
为此,我们拟利用人乳腺癌细胞系,通过转染FLNa的siRNA降低FLNa表达、转染FLNa全长基因增加FLNa的表达后,观察EGFR的磷酸化水平及与EGFR磷酸化的相关的信号转导分子的变化;经免疫共沉淀(co-Immunoprecipitation,IP)证实FLNa与EGFR分子间的相互关系;利用MTT检测FLNa的表达对乳腺癌细胞增殖的影响。从组织、细胞和分子水平综合分析FLNa对EGFR磷酸化的调控,探讨FLNa调控EGFR磷酸化在乳腺癌发病中的作用,为有效治疗乳腺癌提供新靶点。
第一部分细丝蛋白A在浸润性乳腺癌组织中的表达及与临床病理特征的相关性
方法:采用链霉抗生物素蛋白-过氧化物酶(SP)免疫组织化学方法和流式细胞分析技术,检测46例分化程度不同的浸润性乳腺癌细胞中FLNa和增殖细胞核抗原(pro-liferating cell nuclear antigen, PCNA)的表达情况,以正常乳腺组织或乳腺良性增生为阴性对照。
结果:
1. FLNa在乳腺癌组织中的表达
免疫组化结果:FLNa在正常乳腺上皮细胞的胞浆内少量表达(Fig. 1-1);FLNa在浸润性乳腺癌细胞阳性表达的程度较强,且随组织分化程度的降低而增高(Fig.1-2～4、Table 1-1),差异有统计学意义(P<0.05);有淋巴结转移乳腺癌细胞的FLNa的表达明显强于无转移组(Table 1-1),差异有统计学意义(P<0.05)。FLNa在浸润性乳腺癌细胞中的表达与组织学分型无关。
流式细胞检测结果: FLNa在正常乳腺组织的表达量较少;在低分化乳腺癌细胞中FLNa的表达(1.22±0.13)明显高于中高分化的乳腺癌细胞(1.10±0.11),差异有统计学意义(P<0.05),见Fig.1-6、Table 1-2。无淋巴结转移者(1.21±0.11)明显高于有淋巴结转移者(1.10±0.10),差异有统计学意义(P<0.05),见Table 1-2。FLNa在浸润性乳腺癌细胞中的表达与组织学分型无关。
2. PCNA在乳腺癌组织中的表达
免疫组化结果:PCNA在浸润性乳腺癌细胞核呈阳性表达,PCNA的表达随乳腺癌分化程度的降低而增强,在低分化浸润性乳腺癌细胞的表达呈强阳性,见Fig.1-5。
流式细胞检测结果:低分化浸润性乳腺癌细胞中PCNA的表达(1.65±0.22)明显高于中高分化乳腺癌(1.35±0.15),差异有统计学意义(P<0.05),无淋巴结转移的乳腺癌细胞PCNA的表达(1.42±0.15)明显低于有淋巴结转移者(1.56±0.16),差异有统计学意义(P<0.05),见Fig.1-7、Table 1-3。PCNA的表达与FLNa呈正相关(P<0.05)。
第二部分FLNa对乳腺癌细胞表皮生长因子受体活化的影响
方法:
1.沉默FLNa的表达对乳腺癌细胞EGFR活化的影响
用含10%胎牛血清的低糖DMEM培养乳腺癌MDA-MB-231细胞。实验分组:FLNa-siRNA转染组;通用HK对照转染组。每组分别经EGF(20nM)刺激后0min、5min和10min后收集细胞,提取蛋白,Western-blot检测各时间点FLNa、EGFR、磷酸化EGFR (p-EGFR)、ERK (extracellular signal-regulated kinase)和磷酸化ERK(p-ERK)蛋白的表达。
2. FLNa过表达对乳腺癌细胞EGFR活化的影响
细胞培养同方法1。实验分组:人FLNa-full(hfilamin A/pREP4质粒,内含FLNa全长基因)转染组;空质粒对照pcDNA3.1转染组。每组分别经EGF(20nM)刺激0min、5min和10min后收集细胞,提取蛋白, Western-blot检测各时间点FLNa、EGFR、p-EGFR、ERK和p-ERK蛋白的表达。
3.免疫共沉淀证实FLNa对乳腺癌细胞EGFR的活化影响
细胞培养同方法1。用含10%胎牛血清的DMEM培养液调细胞浓度为3.5×105/ml,接种至60mm培养皿。实验分组:转染FLNa-siRNA组;转染通用HK对照组。每组分别经EGF(20nM)刺激0min和10min后,收集细胞,提取蛋白,对提取的蛋白分别用抗EGFR抗体和抗磷酸化酪氨酸的4G10抗体免疫共沉淀,Western-blot分别检测沉淀蛋白内p-EGFR和EGFR的水平。
结果:
1.沉默FLNa的表达对乳腺癌细胞EGFR活化的影响
1.1 FLNa蛋白的表达:FLNa-siRNA转染组FLNa的表达(1.40±0.10、1.27±0.06)较HK对照组(3.97±0.29、3.33±0.06)明显减少,差异有统计学意义(均P<0.01),见Fig.2-1、Table 2-1。
1.2 EGFR蛋白磷酸化水平:EGF刺激5min、10min后,FLNa-siRNA转染组的EGFR磷酸化水平(0.73±0.01、0.37±0.05)明显低于HK对照组(1.08±0.01、1.30±0.06),差异有统计学意义(均P<0.01),见Fig.2-2、Table 2-2。
1.3 ERK磷酸化水平:EGF刺激5min、10min后,FLNa的siRNA转染组ERK磷酸化水平(0.17±0.00、0.12±0.04)明显低于HK对照组(0.32±0.02、0.54±0.04),差异有统计学意义(均P<0.01),见Fig.2-3、Table 2-3。
2. FLNa过表达对乳腺癌细胞EGFR活化的影响
2.1 FLNa的表达:FLNa-full质粒转染组FLNa的表达(5.93±0.38、6.07±0.32)明显高于空质粒pcDNA3.1对照组组(4.50±0.20、4.60±0.17),差异有统计学意义(均P<0.01),见Fig.2-4、Table 2-4。
2.2 EGFR蛋白磷酸化水平:EGF刺激5min、10min后,FLNa-full质粒转染组EGFR的磷酸化水平(1.53±0.11、0.86±0.14)明显高于空质粒pcDNA3.1对照组(0.57±0.01、0.44±0.02),差异有统计学意义(分别P<0.01,P<0.05),见Fig.2-5、Table 2-5。
2.3 ERK磷酸化水平:EGF刺激5min、10min后,FLNa-full质粒转染组ERK的磷酸化水平(1.83±0.07、1.06±0.07)明显高于空质粒pcDNA3.1对照组(0.74±0.01、0.59±0.03),差异有统计学意义(均P<0.01),见Fig.2-6、Table 2-6。
3.免疫共沉淀证实FLNa对乳腺癌细胞EGFR活化的影响
3.1 FLNa-siRNA转染后FLNa的表达:FLNa-siRNA转染组FLNa的表达(3.53±1.08、4.33±1.33)较HK对照组(7.23±2.20、7.53±1.86)组明显减少,差异有统计学意义(均P<0.01)。见Fig.2-7、Table 2-7。
3.2经抗EGFR抗体免疫沉淀后的EGFR磷酸化的水平: EGF刺激10min后,FLNa-siRNA转染组的EGFR磷酸化水平(1.09±0.09)明显低于HK对照组(2.32±0.22),差异有显著性(P<0.01)。见Fig.2-8、Table 2-8。
3.3经抗酪氨酸磷酸化4G10抗体免疫沉淀后EGFR的表达: EGF刺激10min时,FLNa-siRNA转染组的EGFR表达(0.42±0.01)明显低于HK对照组(1.07±0.01),差异有显著性(P<0.01),见Fig.2-9、Table 2-9。
第三部分细丝蛋白A对乳腺癌细胞增殖的影响
方法:细胞培养见第二部分的方法1。用含10%胎牛血清的低糖DMEM培养液调细胞浓度为9×103/ml,按100μL /孔体积接种于96孔板。实验分组:FLNa-siRNA转染组和HK对照组;FLNa-full质粒转染组和空质粒pcDNA3.1对照组。分别用不同浓度的EGF(4nM、20nM、100nM)刺激过夜,用MTT法检测细胞的增殖水平。
结果:
1.FLNa沉默后MDA-MB-231细胞增殖水平:在4nM、20nM、100nM浓度的EGF刺激下,FLNa-siRNA转染组细胞的增殖水平随刺激浓度的增加而增高, FLNa沉默后细胞的增殖水平(1.28±0.09、1.46±0.02、1.58±0.06)均低于对照组(1.55±0.12、1.62±0.06、1.68±0.12),差异有显著性(p<0.01),见Fig.3-1、Table 3-1。
2. FLNa过表达后MDA-MB-231的细胞增殖水平:在4nM、20nM、100nM浓度的EGF刺激下,FLNa-full质粒转染组细胞的增殖水平随刺激浓度的增加而增高,但FLNa-full质粒转染组细胞的增殖水平(1.14±0.08、1.28±0.11、1.51±0.03)高于对照组(1.09±0.17、1.21±0.17、1.25±0.05),差异有显著性(P<0.01),见Fig.3-2、Table 3-2。
结论:
1. FLNa在正常乳腺组织少量表达;FLNa的表达随浸润性乳腺癌分化程度的降低而增高,且与淋巴结转移有关;FLNa的表达与乳腺癌细胞的增殖能力呈正相关。
2. FLNa的表达可调控乳腺癌细胞EGFR的磷酸化,磷酸化的EGFR又通过MAPK增殖信号传导通路活化ERK,影响乳腺癌细胞的增殖。
3.沉默FLNa的表达导致乳腺癌细胞的增殖水平降低;FLNa的过表达则使乳腺癌细胞的增殖水平升高,表明FLNa可通过调控EGFR的活化影响乳腺癌的发生和发展。
Objective:Breast Carcinoma is the leading fetal cancer that affects the health of women now. With the development of economy and the improve- ment of peoples’life, the mortality of breast carcinoma is going up. Breast carcinoma recurrence and metastasis are the main death causes of these patients. However, the mechanism of occurrence and development remains obscure. Patients suffering from breast cancer have a poor prognosis because of deficiency in effective treatment measures in clinical practices.
EGFR family encompasses members of erbB-1/EGFR, erbB-2/ HER2/ neu, erbB-3 and erbB-4. They belong to tyrosine kinase receptor which can be activated by tyrosine phosphorylation. HER2 is unactived oncogene in normal correlating with regulating to growth and differentiation. Amplification and overexpression of HER2 have been examined in a deep-going way and associated with invasion and diffusion. Trastuzumab (Herceptin), a humanized monoclonal antibody, targets activated HER2 and is clinically effective in HER2-over-expressing breast cancers. However, Nineteen percent of breast carcinoma which has no EGFR, ER and PR, is prone to recurrence and metastasis. Total survival rate is lower than that of other breast carcino ma.These patients have worse prognisis. But effective targeted therapy has not been emerged now. Studies have shown that overexpression of EGFR exists in this kind of breast carcinoma whose phenotype is significantly correlated with EGFR and lymphnode metastasis.
EGFR distributes in the way of transmembrane controlling proliferation, differentiation, motility and survival. Appropriate amount expression in normal tissue is necessary for normal vital movement.Overexpression or strong activity of EGFR can promote proliferation system. The excessive proliferation and malignant phenotype will be emerged. Overexpression of EGFR has been in 35%～50 % breast carcinoma and correlates with prognosis. The expression of EGFR in metastasis is significantly higher than that of non-metastasis one. No recurrence and high survival rate result from negative expression of EGFR. Tyrosine phosphorylation is the key molecular event in the EGFR signaling pathway. The level of EGFR phosphorylation decides the prognosis of breast carcinoma. Higher phosphorylation of EGFR will lead to poor prognosis. However, the mechanisms accounting for the upregulation of EGFR activity are largely unknown. So searching the factors that affect EGFR activity is the key purpose and provides foundation for new target in tumor.
Filamins A (Filamin-1, FLNa) are large actin-binding and scaffolding proteins expressed widely in cytoplasm. FLNa can distribute in the way of transmembrane or localize to the nucleus. Vertebrate filamins are elongated dimeric V-shaped proteins with two large polypeptide chains. Each monomeric chain of filamins consists of an N-terminal actin-binding domain (ABD), a long rod-like domain of twenty-four repeated and two hinges. FLNa is endued with powerful functions because of special structure. FLNa stabilizes cortical three-dimensional F-actin networks, anchors cytoskeleton to cellular membranes by binding to transmembrane receptors and integrate cell architecture and functions signaling, which is essential for cell motility correlating with proliferation, migration, organ development and potential role in oncogenesis;Acting as cross-linking proteins, Filamins not only link various signaling proteins to the cytoskeleton,but also collect costimulus molecule of cytomembrane. So they can take part in many important signaling transduction and make cell response to external stimulus in time;
Overexpression of FLNa has been confirmed in many malignancy tumors. It correlates with migration, adhesion and invasion in lung and prostate carcinoma. High expression of FLNa exists in breast carcinoma, too. The expression of FLNa correlating with the malignancy degree and metastasis has been proved with immunohistochemistry and Flow cytometry. Fiori and Zhu et al have shown that FLNa can regulate the activity of EGFR in melanoma cells and the expressions of FLNa and EGFR in melanoma with highly metastatic potentiality are higher than that of lower one. Up to now, FLNa influencing EGFR phosphorylation or not has not been reported.
Thus, the phosphorylation level of EGFR and correlated signaling molecules in breast carcinoma cells were observed after FLNa siRNA and full-lengh FLNa transfection, then the relationship between FLNa and EGFR phosphorylation as well as the effection of FLNa on proliferation were detected by Immunoprecipitation and MTT, respectively. Analysis was performed aggregately about the role of FLNa regulating to EGFR plosphorylation in breast carcinoma genesis from the levels of tissue, cell and molecule. In this study, the putative connection between FLNa and EGFR plosphorylation was explored and would provide a new target for effective treatment of breast carcinoma.
Part one The expression of filaminA in invasive breast carcinoma and its relationship with clinicopathological features
Methods:
Streptavidin-biotin-peroxidase (SP) Immunohistochemistry and Flow Cytometry were applied to detect the expression of FLNa and PCNA in 46 cases invasive breast cancer with different differentiation, using normal breast tissue or benign breast hyperplasia as negative control.
Results:
1. FLNa expression in breast cancer cells
1.1 FLNa expression with immunohistochemical detection in breast cancer cells:
FLNa expression was mainly distributed in the cytoplasm of mammary epithelial cells, staining shallow was regarded as negative in normal breast tissue (Fig.1-1); however, FLNa positive expression was gradually increased with the reducing of differentiation in invasive breast cancer. There were significant differences between the results of different differentiations (P <0.05); FLNa expression in the metastasis group was significantly stronger than that of non-metastasis one, the differences between them was statistically significant (P <0.05). FLNa has nothing to do with the histologic types in invasive breast cancer cells. (Fig.1-2～4 and Table1-1)
1.2 FLNa expression with Flow cytometry in breast cancer cells:
There was little expression of FLNa in normal breast tissue. It was gradually increasing with the reducing of differentiation. The highest expres- sion was in poorly differentiated breast cancer. The expression of FLNa in poorly differentiated breast carcinomas (1.22±0.13) was higher than that of well differentiated ones (1.10±0.11); Metastasis group (1.21±0.11) was higher than that of non-metastasis group (1.11±0.10), too. It has nothing to do with the histological types. There was a statistical difference between two groups (P <0.05). (Fig.1-6 and Table1-2)
2. PCNA expression in breast cancer cells
2.1 The results of PCNA with immunohistochemical detection in breast cancer cells:
The positive expression of PCNA in nucleus of breast cancer cell was very similar to the expression tendency of FLNa. PCNA expression had also changed with the differentiation. PCNA expression showed strong positive expression in poorly differentiated invasive breast cancer. (Fig.1-5)
2.2 The results of PCNA with Flow Cytometry in breast cancer cells
The expression of PCNA increased with differentiation decreased. The expression of PCNA in poorly differentiated breast carcinoma (1.65±0.22) was significantly higher than that of well-differentiated ones (1.35±0.15); Metastasis group (1.56±0.16) was higher than that of non-metastasis group (1.42±0.15), too. There was a statistical difference between two groups (P <0.05). (Fig.1-7 and Table 1-3). The expression of PCNA was correlated positively with that of FLNa (P <0.05).
Part two Effect of FLNa on the EGFR phosphorylation in breast carcinoma Methods:
1. Effect of FLNa silence on EGFR phosphorylation in Human breast carcinoma.
Human breast carcinoma MDA-MB-231 cells were cultured at 37℃, 5%CO2 in DMEM media containing 10% fetal calf serum and divided into two groups: FLNa-siRNA group and HK group. Each group was stimulated for 0min, 5min and 10min by 20nM EGF. The expression levels of FLNa, EGFR, p-EGFR, ERK and p-ERK were assessed by western-blot at the end.
2. Effect of FLNa over expression on EGFR phosphorylation in Human breast carcinoma.
MDA-MB-231 cells were cultured and divided into two groups: FLNa-full group and pcDNA3.1 group. Each group was stimulated for 0min, 5min and 10min by 20nM EGF. The expression levels of FLNa, EGFR, p-EGFR, ERK and p-ERK were examined by western-blot at last.
3. FLNa regulating to EGFR phosphorylation was demonstrated by co- immunoprecipitation in human breast carcinoma.
MDA-MB-231 cells were cultured and divided into two groups: FLNa-siRNA group and HK group.Each group was stimulated for 0min and 10min by 20nM EGF. Then 4G10 or EGFR protein immuneprecipitation was performed. The expressions levels of EGFR and p-EGFR were assessed by western-blot at the end.
Results:
1. Effect of FLNa silence on EGFR phosphorylation in Human breast carcinoma.
(1)The expression of FLNa
After FLNa-siRNA transfection,the expressions of FLNa in FLNa- siRNA group (1.40±0.10, 1.27±0.06) were lower than that of control group of HK. (3.97±0.29, 3.33±0.06). There was a statistical difference between two groups(P<0.01).(Fig. 2-1, Table 2-1)
(2)The expression of phosphorylated EGFR
After stimulation of EGF at 5min and 10min, the expressions of phosphorylated EGFR in FLNa-siRNA group (0.73±0.01, 0.37±0.05) were lower than that of control group of HK (1.08±0.01, 1.30±0.06). There was statistical difference between two groups(P<0.01). (Fig. 2-2, Table 2-2)
(3) The expression of phosphorylated ERK
After stimulation of EGF at 5min and 10min, the expressions of phosphorylated ERK in FLNa-siRNA group (0.17±0.00, 0.12±0.04) were lower than that of control group of HK (0.32±0.02, 0.54±0.04). There was a statistical difference between two groups(P<0.01). (Fig. 2-3, Table 2-3).
2. Effect of FLNa over expression on EGFR phosphorylation in Human breast carcinoma.
(1)The expression of FLNa
After FLNa-full transfection, the expressions of FLNa in FLNa-full group (5.93±0.38, 6.07±0.32) were higher than that of control group of pcDNA3.1 (4.50±0.20, 4.60±0.17). There was a statistical difference between two groups (P<0.01). (Fig. 2-4, Table 2-4).
(2)The expression of phosphorylated EGFR
After stimulation of EGF at 5min and 10min, the expressions of phosphorylated EGFR in FLNa-full group ( 1.53±0.11,0.86±0.14) were higher than that of control group of pcDNA3.1(0.57±0.01, 0.44±0.02).There was statistical difference between two groups(P<0.01, P<0.05). (Fig. 2-5, Table 2-5).
(3) The expression of phosphorylated ERK after FLNa-full transfection
After stimulation of EGF at 5min and 10min, the expressions of phosphorylated ERK in FLNa-full group (1.83±0.07, 1.06±0.07) were higher than that of control group of pcDNA3.1 (0.74±0.01, 0.59±0.03). There was statistical difference between two groups(P<0.01). (Fig. 2-6, Table 2-6). 3. FLNa regulating to EGFR phosphorylation was proved by Immunoprecipi- tation in human breast carcinoma.
3.1The expressions of FLNa with Western blot detection after FLNa-siRNA transfection
After FLNa-siRNA transfection,the expressions of FLNa in FLNa- siRNA group (3.53±1.08,4.33±1.33) were lower than that of control group of HK (7.23±2.20,7.53±1.86). There was statistical difference between two groups(P<0.01). (Fig. 2-7, Table 2-7)
3.2 The expression of phosphorylated EGFR after EGFR IP:
After stimulation of EGF at 10min,the expression of phosphorylated EGFR in FLNa-siRNA group (1.09±0.09) was lower than that of control group of HK (2.32±0.22).There was statistical difference between two groups(P<0.01). (Fig. 2-8, Table 2-8)
3.3 The expression of EGFR after 4G10 IP:
After stimulation of EGF at 10min,the expression of EGFR in FLNa- siRNA group(0.42±0.01) was lower than that of control group of HK (1.07±0.01). There was statistical difference between two groups(P<0.01). (Fig. 2-9, Table 2-9)
Part Three Effect of FLNa on proliferation of breast carcinoma Methods:
MDA-MB-231 cells were cultured and transferred cells into 96 holes plate. MDA-MB-231 cells were divided into two groups: FLNa-siRNA and HK-siRNA transfection group;FLNa-full and pcDNA3.1 plasmid transfection group.EGF stimulating was done with concentration of 4nM, 20nM and 100nM on the two groups overnight. The MDA-MB-231 cells proliferation was examined by MTT after FLNa-siRNA and FLNa-full plasmid trans- fection.
Results:
1. MTT detection after FLNa-siRNA transfection
Under the EGF stimulation with concentration of 4nM, 20nM and 100nM, the proliferation level of FLNa-siRNA group (1.28±0.09, 1.46±0.02, 1.58±0.06 was lower than that of control group of HK(1.55±0.12, 1.62±0.06, 1.68±0.12).There was statistical difference between two groups (P<0.01).The proliferation level of two groups was increasing following concentration of EGF.
2. MTT detection after FLNa-full transfection
Under the EGF stimulation with concentration of 4nM, 20nM and 100nM, the proliferation level of FLNa-full group (1.14±0.08, 1.28±0.11,1.51±0.03) were higher than that of control group of pcDNA3.1 (1.09±0.17, 1.21±0.17, 1.25±0.05). There was statistical difference between two groups(P<0.01). The proliferation level of two groups was rising following concentration of EGF.
Conclusion:
1. The expression of FLNa is little in normal breast tissue. It is gradually increasing with the reducing of differentiation and correlated with lymphnode metastasis in breast carcinoma. The expression of FLNa is correlated positive- ly with proliferation of breast carcinoma.
2. The expression of FLNa can regulate EGFR phosphorylation which active the molecule of ERK through MAPK signaling pathway and effect the proliferation of breast carcinoma.
3. FLNa silence can lead to the proliferation level of breast carcinoma decreasing, while the overexpression of FLNa can lift the the proliferation level of breast carcinoma.The results show that FLNa can regulate phos- phorylation of EGFR and effect genesis and development of breast carcinoma.
引文
1陈建国,陆建华.国内外癌症防制现状[J].肿瘤,2007,27(9): 755-760
2 Feng Y Y, Walsh C A. The many faces of filamin: A versatile molecularscaffold for cell motility and signaling [J].Nature Cell Biology,2004,6(11):1034-1038
3 Keshamouni V G, Michailidis G, Grasso C S, et al. Differential protein expression profiling by iTRAQ-2DLC-MS/MS of lung cancer cells undergoing epithelial-mesenchymal transition reveals a migratory/invasive phenoltype[J].J Proteome Res,2006,5(5),1143-1154
4 Elston E W, Ellis I O. Method for grading breast cancer [J]. J ClinPathol, 1993, 46(2):189-190
5田斌,孙涛,牛虎,等. PTEN,PCNA,IV型胶原在乳腺癌中的表达及其临床意义[J].第四军医大学学报[J],2008,29(5):457-460
6 Nakamura F, Osborn T M, Hartemink C A, et al. Structural basis of filaminA functions [J]. J Cell Biol, 2007, 179(5):1011-1025
7 Kolahi K S, Mofrad M R. Molecular mechanics of filamin's rod domain [J]. Biophys J,2008 ,94(3):1075-1083
8 Bachmann A S,Howard J P,Vogel C W, et al. Actin-binding protein filamin A is displayed on the surface of human neuroblastoma cells[J]. Cancer Sci, 2006, 97(12):1359-1365
9 Zhou X, Borén J, Akyürek L M.Filamins in cardiovascular development [J].Trends Cardiovasc, 2007, 17(7):222-229
10 Huang C , Wu Z, Hujer K M, et al. Silencing of filamin A gene expression inhibits Ca2+-sensing receptor signaling [J]. FEBS Letters, 2006,580(7): 1795-1800
11李孟圈,鲍俊涛,李靖若,等.MTA1蛋白在乳腺癌浸润与转移中作用的研究[J]肿瘤,2007,27(8):655-657
12焦南林,王娟,郑杰.骨桥蛋白下调对乳腺癌细胞生物学行为及MMP-2表达的影响[J].肿瘤,2007,27(12):939-943
13杨银龙,翟羽,邵志敏.乳腺癌转移抑制基因1对乳腺癌细胞运动能力的影响[J].肿瘤,2008,28(3):207-210
14 Fiori J L, Zhu T N,O’Connell M P,et al.Filamin A modulates kinase activation and intracellular trafficking of epidermal growth factor recap tors in human melanoma cell lines[J]. Endocrinology, 2009, 150(6): 2551- 2560
15 Kim H, Sengupta A ,Glogauer M ,et al. Filamin A regulates cellSpreading and survival via beta1 integrins [J].Exp Cell Res,2008,314(4): 834-846
16 Klaile E, Müller M M,Kannicht C, et al. CEACAM1 functionally interacts with filamin A and exerts a dual role in the regulation of cell migration[J]. J Cell Sci,2005,118 (23):5513-5524
17 McDonough W S,Tran N L,Berens M E.Regulation of glioma cell migra tion by serine-phosphorylated p311 [J].Neoplasia, 2005, 7(9): 862- 872
18 Ohta1 Y, Hartwig1 J H,Stosse T P. FilGAP, a Rho-and ROCK-Regulated GAP for Rac binds filamin A to control actin remodeling [J]. Nature Cell Biology, 2006, 8(8):803-813
1 Zhou AX, Hartwig JH, Akyürek LM. Filamins in cell signaling, transcription and organ development.Trends Cell Biol, 2010 , 20(2) : 113- 123
2 Fiori JL, Zhu TN, O'Connell MP,et al. Filamin A Modulates Kinase Activation and Intracellular Trafficking of Epidermal Growth Factor Receptors in Human Melanoma Cells. Endocrinology, 2009, 150(6): 2551-2560
3 Zhou Q, Shaw PG, Davidson NE. Inhibition of histone deacetylase suppresses EGF signaling pathways by destabilizing EGFR mRNA in ER-negative human breast cancer cells.Breast Cancer Res Treat, 2009, 117 (2):443-451
4 L?nne GK, Masoumi KC, Lennartsson J, et al. Protein kinase Cdelta supports survival of MDA-MB-231 breast cancer cells by suppressing the ERK1/2 pathway. J Biol Chem, 2009, 284(48):33456-65.
5 Keshamouni VG, Michailidis G, Grasso CS,et al. Differential protein expression profiling by iTRAQ-2DLC-MS/MS of lung cancer cells undergoing epithelial-mesenchymal transition reveals a migratory/ invasive phenotype. J Proteome Res, 2006 , 5(5):1143-1154
6 Bedolla RG, Wang Y, Asuncion A, et al. Nuclear versus cytoplasmic localization of filamin A in prostate cancer: immunohistochemical correlation with metastases. Clin Cancer Res, 2009, 15(3):788-796
7 Alper O, Stetler-Stevenson WG, Harris LN,et al. Novel anti-filamin-A antibody detects a secreted variant of filamin-A in plasma from patients with breast carcinoma and high-grade astrocytoma. Cancer Sci, 2009, 100(9):1748-1756
8 Morrison DK, Davis RJ.Regulation of MAP kinase signaling modules by scaffold proteins in mammals. Annu Rev Cell Dev Biol, 2003,19:91-118
9 Normanno N, De Luca A, Bianco C, S,et al. Epidermal growth factor receptor (EGFR) signaling in cancer. Gene, 2006, 366:2-16
10 Lichtenberger BM, Tan PK, Niederleithner H, et al. Autocrine VEGF signaling synergizes with EGFR in tumor cells to promote epithelial cancer development. Cell, 2010 Jan,140(2):268-279
11 Liu Y, Ji R, Li J,et al. Correlation effect of EGFR and CXCR4 and CCR7 chemokine receptors in predicting breast cancer metastasis and prognosis.J Exp Clin Cancer Res, 2010, 29(1):16. [Epub ahead of print]
12 De Luca A, Carotenuto A, Rachiglio A,et al. The role of the EGFR signaling in tumor microenvironment. J Cell Physiol, 2008,214 (3): 559- 567
13 Yotsumoto F, Sanui A, Fukami T, et al. Efficacy of ligand-based targeting for the EGF system in cancer. Anticancer Res, 2009 Nov; 29 (11): 4879- 4885
14 Lurje G, Lenz HJ.EGFR signaling and drug discovery. Oncology, 2009; 77(6):400-410
15 Wang J, Barnes RO, West NR, et al. Jab1 is a target of EGFR signaling in ER alpha-negative breast cancer. Breast Cancer Res, 2008; 10(3):R51
16 Ghayad SE, Vendrell JA, Larbi SB, Endocrine resistance associated with activated ErbB system in breast cancer cells is reversed by inhibiting MAPK or PI3K/Akt signaling pathways. Int J Cancer, 2010 , 126 (2): 545- 562
17 Ioachim E, Kamina S, Athanassiadou S, et al. The prognostic significance of epidermal growth factor receptor (EGFR), C-erbB-2, Ki-67 and PCNA Expression in breast cancer. Anticancer Res. 1996,16(5B):3141-3147
18 Zhang M, Breitwieser GE.High affinity interaction with filamin A protects against calcium-sensing receptor degradation. J Biol Chem, 2005, 280(12): 11140-11146
19 Orton RJ, Adriaens ME, Gormand A,et al.Computational modelling of cancerous mutations in the EGFR/ERK signalling pathway. BMC Syst Biol, 2009 ,5(3):100
20 Alokail MS.Transient transfection of epidermal growth factor receptor gene into MCF7 breast ductal carcinoma cell line. Cell Biochem Funct, 2005,23(3):157-161
21 Hirsch DS, Shen Y, Wu WJ. Growth and motility inhibition of breast cancer cells by epidermal growth factor receptor degradation is correlated with inactivation of Cdc42. Cancer Res, 2006 ,66(7):3523-3530
22 Magkou C, Nakopoulou L, Zoubouli C, et al.Expression of the epidermalgrowth factor receptor (EGFR) and the phosphorylated EGFR in invasive breast carcinomas. Breast Cancer Res, 2008, 10(3):R49.
23 Lammering G, Valerie K, Lin PS, et al. Radiation-induced activation of a common variant of EGFR confers enhanced radioresistance. Radiother Oncol, 2004 ,72(3):267-273
24 Fan WH, Lu YL, Deng F, et al. EGFR antisense RNA blocks expression of the epidermal growth factor receptor and partially reverse the malignant phenotype of human breast cancer MDA-MB-231 cells. Cell Res, 1998 , (1):63-71
25 Duffy A, Kummar S.Targeting mitogen-activated protein kinase kinase (MEK) in solid tumors. Target Oncol, 2009 , 4(4):267-273
26 Jo M, Thomas KS, Takimoto S, et al. Urokinase receptor primes cells to proliferate in response to epidermal growth factor. Oncogene, 2007, 26 (18):2585-2594
27 Cortes-Reynosa P, Robledo T, Salazar EP.Epidermal growth factor promotes epidermal growth factor receptor nuclear accumulation by a pathway dependent on cytoskeleton integrity in human breast cancer cells. Arch Med Res, 2009,40(5):331-338
28 Irma Onoprishvili , Solav Ali , Matthew L.et al. Filamin A Mutant Lacking Actin-Binding Domain Restores Mu Opioid Receptor Regulation in Melanoma Cells. Neurochem Res, 2008, 33:2054-2061
29 Ravid D, Chuderland D, Landsman L, et al. Filamin A is a novel caveolin-1-dependent target in IGF-I-stimulated cancer cell migration. Exp Cell Res, 2008, 314(15):2762-2773
30 Cáceres M, Guerrero J, Martínez J.Overexpression of RhoA-GTP induces activation of the Epidermal Growth Factor Receptor, dephosphorylation of focal adhesion kinase and increased motility in breast cancer cells. Exp Cell Res, 2005,309(1):229-238
31 Lev DC, Kim LS, Melnikova V, et al. Dual blockade of EGFR and ERK1/2 phosphorylation potentiates growth inhibition of breast cancer cells, Br J Cancer. 2004, 91(4):795-802
32 K. E. Kasza, F. Nakamura, S. Hu, etal. Filamin A Is Essential for Active Cell Stiffening but not Passive Stiffening under External Force. Biophysical Journal, 2009,96:4326-4335
33 Komaletdinova FM, Pinaev GP. The filamin in cell signaling.Tsitolo- giia,2006; 48(11):924-934
34 Hugh Kima, Anita Senguptab, Michael Glogauera, et al.Filamin A regulates cell spreading viaβ1 integrins. Experimental Cell Research, 2008, 314: 834-846
35 Kimura F, Iwaya K, Kawaguchi T, et al. Epidermal growth factor- dependent enhancement of invasiveness of squamous cell carcinoma of the breast. Cancer Sci, 2010. [Epub ahead of print]
36 Bonine-Summers AR, Aakre ME, Brown KA, et al.Epidermal growth factor receptor plays a significant role in hepatocyte growth factor mediated biological responses in mammary epithelial cells. Cancer Biol Ther, 2007,6(4):561-570
1 Fei Han, Donghua Gu, Qi Chen, et al. Caveolin-1 Acts as a Tumor Suppressor by Down-Regulating pidermal Growth Factor Receptor- Mitogen-Activated Protein Kinase Signaling Pathway in Pancreatic Carcinoma Cell Lines. Pancreas, 2009,38(7):766-774
2 Zhang M, Breitwieser GE.High affinity interaction with filamin A protects against calcium-sensing receptor degradation. J Biol Chem, 2005,280 (12):11140-11146
3 Fiori JL, Zhu TN, O'Connell MP,et al. Filamin A Modulates Kinase Activation and Intracellular Trafficking of Epidermal Growth FactorReceptors in Human Melanoma Cells. Endocrinology, 2009, 150 (6): 2551-2560
4 Woo MS, Ohta Y, Rabinovitz I,et al. Ribosomal S6 kinase (RSK) regulates phosphorylation of filamin A on an important regulatory site.Mol Cell Biol. 2004,24(7):3025-3035
5 Scott MG, Pierotti V, Storez H, et al.Cooperative regulation of extra- cellular signal-regulated kinase activation and cell shape change by filamin A and beta -arrestins. Mol Cell Biol, 2006,26(9):3432-3445
6 Zhong Z, Yeow WS, Zou C, et al.Cyclin D1/cyclin-dependent kinase 4 interacts with filamin A and affects the migration and invasion potential of breast cancer cells. Cancer Res. 2010,70(5):2105-2114
7 Li C, Yu S, Nakamura F,et al. Binding of pro-prion to filamin A disrupts cytoskeleton and correlates with poor prognosis in pancreatic cancer.J Clin Invest. 2009,119(9):2725-2736
8 Nakamura F, Heikkinen O, Pentik?inen OT,et al.Molecular basis of filamin A-FilGAP interaction and its impairment in congenital disorders associated with filamin A mutations. PLoS One. 2009;4(3):e4928
9 Lad Y, Jiang P, Ruskamo S, et al. Structural basis of the migfilin-filamin interaction and competition with integrin beta tails.J Biol Chem. 2008, 283 (50):35154-35163
1 Wang K, Ash JF, Singer SJ.Filamin, a new high-molecular-weight protein found in smooth muscle and non-muscle cells[J]. Cell Biology, 1975, 72(11): 4483-4486
2 Huang C, Wu Z, Hujer KM, et al.Silencing of filamin A gene expression inhibits Ca2+-sensing receptor signaling [J]. FEBS Letters, 2006, 580(7): 1795-1800.
3 Feng Y, Walsh CA.The many faces of filamin: A versatile molecular scaffold for cell motility and signaling [J]. Nature Cell Biology.2004, 6(11):1034-1038.
4 Bachmann AS, Howard JP, Vogel CW. Actin-binding protein filamin A is displayed on the surface of human neuroblastoma cells [J]. Cancer Sci, 2006 ,97(12) 1359-1365
5 Zhou X, Borén J, Akyürek LM. Filamins in cardiovascular development [J].Trends Cardiovasc Med, 2007,17(7):222-229
6 Loy CJ, Sim KS, Yong EL.Filamin-A fragment localizes to the nucleus to regulate androgen receptor and coactivator functions[J]. PNAS ,2003, 100(8):4562-4567
7 Nakamura F, Osborn TM, Hartemink CA. Structural basis of filamin A functions [J]. J Cell Biol, 2007,179(5):1011-1025
8 Kolahi KS, Mofrad MR. Molecular mechanics of filamin's rod domain[J]. Biophys J, 2008,94(3):1075-1083
9 Hj?lm G, MacLeod RJ, Kifor O, et al. Filamin-A Binds to the Carboxyl- terminal Tail of the Calcium-sensing Receptor, an Interaction That Participates in CaR-mediated Activation of Mitogen-activated Protein Kinase[J].J Biol Chem, 2001,276(37): 34880-34887
10 Fiori J L, Zhu T N, O’Connell M P, et al. Filamin A modulates kinase activation and intracellular trafficking of epidermal growth factor receptors in human melanoma cell lines[J]. Endocrinology, 2009, 150(6):2551-2560
11 Uribe R, Jay D. A review of actin binding proteins: new perspectives[J]. Mol Biol Rep,2009,36(1):121-125
12 Lu H, Guo X, Meng X, et al. The BRCA2-interacting Protein BCCIP Functions in RAD51 and BRCA2 Focus Formation and Homologous Recombinational Repair[J].Molecular and Cellular Biology, 2005, 25 (5): 1949-1957
13 Meng X, Yuan Y, Maestas A,et al. Recovery from DNA Damage-induced G2 Arrest Requires Actinbinding Protein FilaminA/Actin-binding Protein 280[J].The Journal of Biological Chemistry, 2004,279 (7): 6098-6105
14 Browne KA, Johnstone RW, Jans DA,et al. Filamin (280-kDa Actin- binding Protein) Is a Caspase Substrate and Is Also Cleaved Directly by the Cytotoxic T Lymphocyte Protease Granzyme B during Apoptosis[J]. The Journal of Biological Chemistry, 2000,275 (50): 39262- 39266
15 Conway RE, Petrovic N, Li Z,et al.Prostate-specific membrane antigen regulates angiogenesis by modulating integrin signal transduction[J]. Mol Cell Biol, 2006,26(14): 5310-5324
16 Ghosh A, Heston WD. Heston.Tumor Target Prostate Specific Membrane Antigen (PSMA) and its Regulation in Prostate Cancer.Journal of Cellular Biochemistry 2004,91:528-539
17 Hayashi K, Altman A.Filamin A is Required for T Cell Activation Mediated by Protein Kinase C-theta[J].The Journal of Immunology.2006, 177(3):1721-1728
18 Tavano R, Contento RL, Baranda SJ,et al. CD28 interaction with filaminA controls lipid raft accumulation at the T-cell immunological synapse [J]. Nat Cell Biol, 2006,8(11):1270-1276
19 Kim EJ, Park JS, Um SJ. Filamin A negatively regulates the transcrip- tional activity of p73alpha in the cytoplasm [J]. Biochem Biophys Res Commun, 2007, 362(4):1101-1106
20 Johnson GL, Nakamura K.The c-Jun Kinase/Stress-activated Pathway: Regulation, Function and Role in Human disease[J].Biochim Biophys Acta, 2007,1773(8):1341-1348
21 Leonardi A, Ellinger-Ziegelbauer H, Franzoso G,et al. Physical and Functional Interaction of Filamin (Actin-binding Protein-280) and Tumor Necrosis Factor Receptor-associated Factor 2[J]. J Biol Chem, 2000, 275 (1): 271-278
22 Jiménez-Baranda S, Gómez-Moutón C, Rojas A,et al .Filamin-A regulates actin-dependent clustering of HIV receptor s [J]. Nat Cell Biol, 2007, 9(7):838-846
23 Klaile E, Müller MM, Kannicht C,et al. CEACAM1 functionally interacts with filamin A and exerts a dual role in the regulation of cell migration [J].Journal of Cell Science, 2005, 118 (23):5513-5524
24 McDonough WS, Tran NL, Berens ME. Regulation of Glioma Cell Migration by Serine- Phosphorylated P311[J].Neoplasia,2005,7(9): 862- 872
25 Keshamouni VG, Michailidis G, Grasso CS,et al. Differential Protein Expression Profiling by iTRAQ-2DLC-MS/MS of Lung Cancer Cells Undergoing Epithelial-Mesenchymal Transition Reveals a Migratory/ Invasive Phenotype[J]. Journal of Proteome Research, 2006, 5(5): 1143- 1154
26 Ohta Y, Hartwig JH, Stossel TP. FilGAP, a Rho- and ROCK-regulated GAP for Rac binds filamin A to control actin remodeling [J].Nature Cell Biology, 2006,8(8):803-814
27 Yulia Shifrin, Pamela D. Arora, et al. The Role of FilGAP-Filamin AInter- actions in Mechanoprotection. Molecular Biology of the Cell, 2009, 20: 1269-1279
28 Kim H, Sengupta A, Glogauer M,et al. Filamin A regulates cell spreading and survival via beta1 integrins. Experimental Cell Research [J].2008, 314(4):834-846
29 Zhu TN, He HJ, Kole S,et al. Filamin A-mediated down-regulation of the exchange factor Ras-GRF1 correlates with decreased matrix metallo- proteinase-9 expression in human melanoma cells[J].J Biol Chem, 2007, 282(20):14816-14826
30 Michael P. O’Connell, Jennifer L, et al. Wnt5A Activates the Calpain- Mediated Cleavage of Filamin A.J Invest Dermatol. 2009, 129(7): 1782- 1789
31 Michael Maceyka, ergio E, Alvarez,et al. Filamin A Links Sphingosine Kinase 1 and Sphingosine-1-Phosphate Receptor 1 at Lamellipodia To Orchestrate Cell Migration. MOLECULAR AND CELLULAR BIOLOGY, 2008, 28(18): 5687-5697
32 Keshamouni VG, Michailidis G, Grasso CS,et al. Differential protein expression profiling by iTRAQ-2DLC-MS/MS of lung cancer cells undergoing epithelial-mesenchymal transition reveals a migratory/ invasive phenotype. J Proteome Res. 2006,5(5):1143-1154
33 Ravid D, Chuderland D, Landsman L,et al.Filamin A is a novel caveolin-1 -dependent target in IGF-I-stimulated cancer cell migration. Exp Cell Res. 2008, 314(15):2762-2773
34 Ricardo Uribe .David Jay. A review of actin binding proteins: new perspectives. Mol Biol Rep, 2009, 36:121-125
35 Rajasekaran AK, Anilkumar G, Christiansen JJ. Is prostate-specific membrane antigen a multifunctional protein? Am J Physiol Cell Physiol. 2005,288(5):C975-981
36 Howard Cukier, Yun Li,Jonathan M,et al. Cyclin B1/Cdk1 binds and phosphorylates Filamin A and regulates its ability to cross-link actinq. FEBS Letters, 2007, 581:1661-1672
1曹亚.肿瘤分子生物学研究进展.国外医学·生理、病理科学与临床分册, 2005,25(1):1-4
2严景华,叶棋浓,黄翠芬.乳腺癌易感基因BRCA1的研究进展.遗传HEREDITAS(Beijing)2004,26(3):367-372
3 Tripathi MK, Misra S, Khedkar SV,et al.Regulation of BRCA2 gene expression by the SLUG repressor protein in human breast cells. J BiolChem. 2005,280(17):17163-17171
4 Bernard-Gallon DJ, Satih S,et al.Phytoestrogens regulate the expression of genes involved in different biological processes in BRCA2 knocked down MCF-7, MDA-MB-231 and MCF-10a cell lines. Oncol Rep. 2010, 23(3): 647-653
5 Chiappetta G, Ferraro A, Botti G, et al. FRA-1 protein overexpression is a feature of hyperplastic and neoplastic breast disorders. BMC Cancer 2007,25(7):17
6 Weber F, Shen L, Fukino K,et al. Total-Genome Analysis of BRCA1/2- Related Invasive Carcinomas of the Breast Identifies Tumor Stroma as Potential Landscaper for Neoplastic Initiation. The American Journal of Human Genetics,2006 June,78:961-972
7 White MF. Insulin signaling in health and disease. Science 2003, Dec 5;302(5651):1710-1711
8 Alper O, Stetler-Stevenson WG, Harris LN,et al. Novel anti-filaminA antibody detects a secreted variant of filamin-A in plasma from patients with breast carcinoma and high-grade astrocytoma. Cancer Sci. 2009, 100 (9):1748-1756
9 Zhang M, Breitwieser GE.High affinity interaction with filamin A protects against calcium-sensing receptor degradation. J Biol Chem. 2005,280 (12): 11140-11146
10 Zhong Z, Yeow WS, Zou C, et al.Cyclin d1/cyclin-dependent kinase 4 interacts with filamin a and affects the migration and invasion potential of breast cancer cells. Cancer Res. 2010, 70(5):2105-:2114
11 Conway K, Parrish E, Edmiston SN, et al.Risk factors for breast cancer characterized by the estrogen receptor alpha A908G (K303R) mutation. Breast Cancer, Research,2007,9(3):R36
12 Li H, Weinstein IB.Protein kinase C beta enhances growth and expression of cyclin D1 in human breast cancer cells. Cancer Res.2006, 66(23): 11399-11408
13 Siriwardana G, Bradford A, Coy D,et al.Autocrine/paracrine regulation ofbreast cancer cell proliferation by growth hormone releasing hormone via Ras, Raf, and mitogen-activated protein kinase. Mol Endocrinol. 2006 , 20(9):2010-2019
14 Malkas LH, Herbert BS, Abdel-Aziz W,et al. A cancer- associated PCNA expressed in breast cancer has implications as a potential biomarker. PNAS,2006,103(51):19472-19477
15 Schultz-Norton JR, Gabisi VA, Ziegler YS, et al.Interaction of estrogen receptor alpha with proliferating cell nuclear antigen. Nucleic Acids Research,2007,35(15):5028-5038
16 Beresford MJ, Wilson GD, Makris A.Measuring proliferation in breast cancer:practicalities and applications.Breast Cancer Research,2006,8(6): 216-316
17 Zaczek A, Brandt B, Bielawski KP.The diverse signaling network of EGFR, HER2, HER3 and HER4 tyrosine kinase receptors and the consequences for therapeutic approaches. Histol Histopathol. 2005 Jul;20(3):1005-1015
18 Kaya H, Erbarut I, Ozkan N,et al.Immunoexpression of HER family, neuregulin, MAPK and AKT in invasive ductal carcinomas of the breast. Eur J Gynaecol Oncol. 2008;29(4):350-356
19 Magkou C, Nakopoulou L, Zoubouli C,et al.Expression of the epidermal growth factor receptor (EGFR) and the phosphorylated EGFR in invasive breast carcinomas. Breast Cancer Res. 2008;10(3):R49.
20 Park SS, Kim JE, Kim YA,et alCaveolin-1 is down-regulated and inversely correlated with HER2 and EGFR expression status in invasive ductal carcinoma of the breast. Histopathology. 2005 ,47(6):625-630
21 Han F, Zhu HG.Over-expression of caveolin-1 inhibits proliferation and invasion of pancreatic carcinoma cells in vitro. Zhonghua Zhong Liu Za Zhi, 2009,31(10):732-737
22 Malik FA, Sanders AJ, Kayani MA,et al. Effect of expressional alteration of KAI1 on breast cancer cell growth, adhesion, migration and invasion. Cancer Genomics Proteomics. 2009 ,6(4):205-13
23 Crawford NP,Qian X, Ziogas A, et al. Rrp1b, a New Candidate Susceptibi- lity Gene for Breast Cancer progression and Metastasis. PLOS Genet, 2007,3(11):2296-2311
24 Grunewald TG, Kammerer U,Kapp M,et al. Nuclear localization and cytosolic overexpression of LASP-1 correlates with tumor size and nodal-positivity of human breast carcinoma.BMC Cancer 2007,7:198
25 Chang Y, Zuka M, Perez-Pinera P,etal.Secretion of pleiotrophin stimulates breast cancer progression through remodeling of the tumor micro environment. PNAS,2007,104(26):10888-10893
26 Fujita M, Khazenzon NM, Bose S,et al.Overexpression ofβ1- chain- containing laminins in capillary basement membranes of human breast cancer and its metastases. Breast Cancer Research,2005, 7(4): 411- 421
27 Dong M, How T, Kirkbride KC, et al. The typeⅢTGF-βreceptor suppresses breast cancer progression.The Journal of Clinical Investigation, 2007, 117(1):206-217
28 Yi CH, Smith DJ, West WW, et al. Loss of Fibulin-2 Expression Is Associated with Breast Cancer Progression. The American Journal of Pathology, 2007,170(5):1535-1545
29 Yang S, Du J, Wang Z, et al.BMP-6 promotes E-cadherin expression through repressing delta EF1 in breast cancer cells. BMC Cancer, 2007, 7:211
30 Draffin JE, McFarlane S, Hill A, et al.CD44 potentiates the adherence of metastatic prostate and breast cancer cells to bone marrow endothelial cells. Cancer Res. 2004,64(16):5702-5711
31 Zhou X, Liu Y, You J, Myosin light-chain kinase contributes to the proliferation and migration of breast cancer cells through cross-talk with activated ERK1/2. Cancer Lett. 2008,270(2):312-327
32王震,刘乾,陈琦,等.NDRG1基因与乳腺癌转移的关系及其转染对乳腺癌细胞株增殖及侵袭力的影响.中华病理学杂志,2006,35(6):333- 338.
33柴伟,张国建,张云昌,等. NDRG-1、WWOX及P53基因在胃癌组织中的表达及临床意义.昆明医学院学报2009,30(5):37-42
34柴丽,石林祥,房林,等.KiSS-1基因及其受体在乳腺癌中的表达及临床意义.中华实验外科杂志,2005,22(9):1068-1070
35 SerajM J,SamantR S,VerderameM F,et al.Functi onal evidence for a novel human breast carcinomametastasis suppressor, BRMS1, encoded at chromos ome 11q13 [J]. Cancer Res,2000,60 (11):2764
36 CicekM, Samant R S, KinterM, et al. Identficati on of metastasisass ociated proteins through protein analysis ofmetastaticMDA-MB-435 cells [ J ]. Clin Exp Metastasis,2004, 21(2):149
37 Lee TH, Seng S, Sekine M, et al.Vascular Endothelial Growth Factor Mediates Intracrine Survival in Human Breast Carcinoma Cells through Internally Expressed VEGFR1/FLT1.PLoS Medicine, 2007,4(6): 1101- 1116.
38 Wang J, Barnes RO, West NR, Jab1 is a target of EGFR signaling in ER alpha-negative breast cancer. Breast Cancer Res. 2008; 10(3):R51
39 King JA, Ofori-Acquah SF, Stevens T, et al. Activated leukocyte cell adhesion molecule in breast cancer:prognostic indicator.Breast Cancer Res,2004,6(5):R478-R487
40 Fabre-Lafay S,Monville F,Garrido-Urbani S, et al.Nectin-4 is a new histo- logical and serological tumor associated marker for breast cancer. BMC Cancer, 2007,7:73
41阎志胜,刘建民.转化生长因子β1和细胞周期蛋白E在肾透明细胞癌中的表达和意义.山西医药杂志(J),2009 ,38(5):387-389
42 He Wenshan,HUANG Tao,WANG Haijiu.Expression of Cyclin E and Its Relationship with the Prognosis of Patients with Breast Carcinoma.The Chinese German Journal Clinical Oncology,2006,5(4):275-278
2 Feng Y Y, Walsh C A. The many faces of filamin: A versatile molecularscaffold for cell motility and signaling [J].Nature Cell Biology,2004,6(11):1034-1038
3 Keshamouni V G, Michailidis G, Grasso C S, et al. Differential protein expression profiling by iTRAQ-2DLC-MS/MS of lung cancer cells undergoing epithelial-mesenchymal transition reveals a migratory/invasive phenoltype[J].J Proteome Res,2006,5(5),1143-1154
4 Elston E W, Ellis I O. Method for grading breast cancer [J]. J ClinPathol, 1993, 46(2):189-190
5田斌,孙涛,牛虎,等. PTEN,PCNA,IV型胶原在乳腺癌中的表达及其临床意义[J].第四军医大学学报[J],2008,29(5):457-460
6 Nakamura F, Osborn T M, Hartemink C A, et al. Structural basis of filaminA functions [J]. J Cell Biol, 2007, 179(5):1011-1025
7 Kolahi K S, Mofrad M R. Molecular mechanics of filamin's rod domain [J]. Biophys J,2008 ,94(3):1075-1083
8 Bachmann A S,Howard J P,Vogel C W, et al. Actin-binding protein filamin A is displayed on the surface of human neuroblastoma cells[J]. Cancer Sci, 2006, 97(12):1359-1365
9 Zhou X, Borén J, Akyürek L M.Filamins in cardiovascular development [J].Trends Cardiovasc, 2007, 17(7):222-229
10 Huang C , Wu Z, Hujer K M, et al. Silencing of filamin A gene expression inhibits Ca2+-sensing receptor signaling [J]. FEBS Letters, 2006,580(7): 1795-1800
11李孟圈,鲍俊涛,李靖若,等.MTA1蛋白在乳腺癌浸润与转移中作用的研究[J]肿瘤,2007,27(8):655-657
12焦南林,王娟,郑杰.骨桥蛋白下调对乳腺癌细胞生物学行为及MMP-2表达的影响[J].肿瘤,2007,27(12):939-943
13杨银龙,翟羽,邵志敏.乳腺癌转移抑制基因1对乳腺癌细胞运动能力的影响[J].肿瘤,2008,28(3):207-210
14 Fiori J L, Zhu T N,O’Connell M P,et al.Filamin A modulates kinase activation and intracellular trafficking of epidermal growth factor recap tors in human melanoma cell lines[J]. Endocrinology, 2009, 150(6): 2551- 2560
15 Kim H, Sengupta A ,Glogauer M ,et al. Filamin A regulates cellSpreading and survival via beta1 integrins [J].Exp Cell Res,2008,314(4): 834-846
16 Klaile E, Müller M M,Kannicht C, et al. CEACAM1 functionally interacts with filamin A and exerts a dual role in the regulation of cell migration[J]. J Cell Sci,2005,118 (23):5513-5524
17 McDonough W S,Tran N L,Berens M E.Regulation of glioma cell migra tion by serine-phosphorylated p311 [J].Neoplasia, 2005, 7(9): 862- 872
18 Ohta1 Y, Hartwig1 J H,Stosse T P. FilGAP, a Rho-and ROCK-Regulated GAP for Rac binds filamin A to control actin remodeling [J]. Nature Cell Biology, 2006, 8(8):803-813
1 Zhou AX, Hartwig JH, Akyürek LM. Filamins in cell signaling, transcription and organ development.Trends Cell Biol, 2010 , 20(2) : 113- 123
2 Fiori JL, Zhu TN, O'Connell MP,et al. Filamin A Modulates Kinase Activation and Intracellular Trafficking of Epidermal Growth Factor Receptors in Human Melanoma Cells. Endocrinology, 2009, 150(6): 2551-2560
3 Zhou Q, Shaw PG, Davidson NE. Inhibition of histone deacetylase suppresses EGF signaling pathways by destabilizing EGFR mRNA in ER-negative human breast cancer cells.Breast Cancer Res Treat, 2009, 117 (2):443-451
4 L?nne GK, Masoumi KC, Lennartsson J, et al. Protein kinase Cdelta supports survival of MDA-MB-231 breast cancer cells by suppressing the ERK1/2 pathway. J Biol Chem, 2009, 284(48):33456-65.
5 Keshamouni VG, Michailidis G, Grasso CS,et al. Differential protein expression profiling by iTRAQ-2DLC-MS/MS of lung cancer cells undergoing epithelial-mesenchymal transition reveals a migratory/ invasive phenotype. J Proteome Res, 2006 , 5(5):1143-1154
6 Bedolla RG, Wang Y, Asuncion A, et al. Nuclear versus cytoplasmic localization of filamin A in prostate cancer: immunohistochemical correlation with metastases. Clin Cancer Res, 2009, 15(3):788-796
7 Alper O, Stetler-Stevenson WG, Harris LN,et al. Novel anti-filamin-A antibody detects a secreted variant of filamin-A in plasma from patients with breast carcinoma and high-grade astrocytoma. Cancer Sci, 2009, 100(9):1748-1756
8 Morrison DK, Davis RJ.Regulation of MAP kinase signaling modules by scaffold proteins in mammals. Annu Rev Cell Dev Biol, 2003,19:91-118
9 Normanno N, De Luca A, Bianco C, S,et al. Epidermal growth factor receptor (EGFR) signaling in cancer. Gene, 2006, 366:2-16
10 Lichtenberger BM, Tan PK, Niederleithner H, et al. Autocrine VEGF signaling synergizes with EGFR in tumor cells to promote epithelial cancer development. Cell, 2010 Jan,140(2):268-279
11 Liu Y, Ji R, Li J,et al. Correlation effect of EGFR and CXCR4 and CCR7 chemokine receptors in predicting breast cancer metastasis and prognosis.J Exp Clin Cancer Res, 2010, 29(1):16. [Epub ahead of print]
12 De Luca A, Carotenuto A, Rachiglio A,et al. The role of the EGFR signaling in tumor microenvironment. J Cell Physiol, 2008,214 (3): 559- 567
13 Yotsumoto F, Sanui A, Fukami T, et al. Efficacy of ligand-based targeting for the EGF system in cancer. Anticancer Res, 2009 Nov; 29 (11): 4879- 4885
14 Lurje G, Lenz HJ.EGFR signaling and drug discovery. Oncology, 2009; 77(6):400-410
15 Wang J, Barnes RO, West NR, et al. Jab1 is a target of EGFR signaling in ER alpha-negative breast cancer. Breast Cancer Res, 2008; 10(3):R51
16 Ghayad SE, Vendrell JA, Larbi SB, Endocrine resistance associated with activated ErbB system in breast cancer cells is reversed by inhibiting MAPK or PI3K/Akt signaling pathways. Int J Cancer, 2010 , 126 (2): 545- 562
17 Ioachim E, Kamina S, Athanassiadou S, et al. The prognostic significance of epidermal growth factor receptor (EGFR), C-erbB-2, Ki-67 and PCNA Expression in breast cancer. Anticancer Res. 1996,16(5B):3141-3147
18 Zhang M, Breitwieser GE.High affinity interaction with filamin A protects against calcium-sensing receptor degradation. J Biol Chem, 2005, 280(12): 11140-11146
19 Orton RJ, Adriaens ME, Gormand A,et al.Computational modelling of cancerous mutations in the EGFR/ERK signalling pathway. BMC Syst Biol, 2009 ,5(3):100
20 Alokail MS.Transient transfection of epidermal growth factor receptor gene into MCF7 breast ductal carcinoma cell line. Cell Biochem Funct, 2005,23(3):157-161
21 Hirsch DS, Shen Y, Wu WJ. Growth and motility inhibition of breast cancer cells by epidermal growth factor receptor degradation is correlated with inactivation of Cdc42. Cancer Res, 2006 ,66(7):3523-3530
22 Magkou C, Nakopoulou L, Zoubouli C, et al.Expression of the epidermalgrowth factor receptor (EGFR) and the phosphorylated EGFR in invasive breast carcinomas. Breast Cancer Res, 2008, 10(3):R49.
23 Lammering G, Valerie K, Lin PS, et al. Radiation-induced activation of a common variant of EGFR confers enhanced radioresistance. Radiother Oncol, 2004 ,72(3):267-273
24 Fan WH, Lu YL, Deng F, et al. EGFR antisense RNA blocks expression of the epidermal growth factor receptor and partially reverse the malignant phenotype of human breast cancer MDA-MB-231 cells. Cell Res, 1998 , (1):63-71
25 Duffy A, Kummar S.Targeting mitogen-activated protein kinase kinase (MEK) in solid tumors. Target Oncol, 2009 , 4(4):267-273
26 Jo M, Thomas KS, Takimoto S, et al. Urokinase receptor primes cells to proliferate in response to epidermal growth factor. Oncogene, 2007, 26 (18):2585-2594
27 Cortes-Reynosa P, Robledo T, Salazar EP.Epidermal growth factor promotes epidermal growth factor receptor nuclear accumulation by a pathway dependent on cytoskeleton integrity in human breast cancer cells. Arch Med Res, 2009,40(5):331-338
28 Irma Onoprishvili , Solav Ali , Matthew L.et al. Filamin A Mutant Lacking Actin-Binding Domain Restores Mu Opioid Receptor Regulation in Melanoma Cells. Neurochem Res, 2008, 33:2054-2061
29 Ravid D, Chuderland D, Landsman L, et al. Filamin A is a novel caveolin-1-dependent target in IGF-I-stimulated cancer cell migration. Exp Cell Res, 2008, 314(15):2762-2773
30 Cáceres M, Guerrero J, Martínez J.Overexpression of RhoA-GTP induces activation of the Epidermal Growth Factor Receptor, dephosphorylation of focal adhesion kinase and increased motility in breast cancer cells. Exp Cell Res, 2005,309(1):229-238
31 Lev DC, Kim LS, Melnikova V, et al. Dual blockade of EGFR and ERK1/2 phosphorylation potentiates growth inhibition of breast cancer cells, Br J Cancer. 2004, 91(4):795-802
32 K. E. Kasza, F. Nakamura, S. Hu, etal. Filamin A Is Essential for Active Cell Stiffening but not Passive Stiffening under External Force. Biophysical Journal, 2009,96:4326-4335
33 Komaletdinova FM, Pinaev GP. The filamin in cell signaling.Tsitolo- giia,2006; 48(11):924-934
34 Hugh Kima, Anita Senguptab, Michael Glogauera, et al.Filamin A regulates cell spreading viaβ1 integrins. Experimental Cell Research, 2008, 314: 834-846
35 Kimura F, Iwaya K, Kawaguchi T, et al. Epidermal growth factor- dependent enhancement of invasiveness of squamous cell carcinoma of the breast. Cancer Sci, 2010. [Epub ahead of print]
36 Bonine-Summers AR, Aakre ME, Brown KA, et al.Epidermal growth factor receptor plays a significant role in hepatocyte growth factor mediated biological responses in mammary epithelial cells. Cancer Biol Ther, 2007,6(4):561-570
1 Fei Han, Donghua Gu, Qi Chen, et al. Caveolin-1 Acts as a Tumor Suppressor by Down-Regulating pidermal Growth Factor Receptor- Mitogen-Activated Protein Kinase Signaling Pathway in Pancreatic Carcinoma Cell Lines. Pancreas, 2009,38(7):766-774
2 Zhang M, Breitwieser GE.High affinity interaction with filamin A protects against calcium-sensing receptor degradation. J Biol Chem, 2005,280 (12):11140-11146
3 Fiori JL, Zhu TN, O'Connell MP,et al. Filamin A Modulates Kinase Activation and Intracellular Trafficking of Epidermal Growth FactorReceptors in Human Melanoma Cells. Endocrinology, 2009, 150 (6): 2551-2560
4 Woo MS, Ohta Y, Rabinovitz I,et al. Ribosomal S6 kinase (RSK) regulates phosphorylation of filamin A on an important regulatory site.Mol Cell Biol. 2004,24(7):3025-3035
5 Scott MG, Pierotti V, Storez H, et al.Cooperative regulation of extra- cellular signal-regulated kinase activation and cell shape change by filamin A and beta -arrestins. Mol Cell Biol, 2006,26(9):3432-3445
6 Zhong Z, Yeow WS, Zou C, et al.Cyclin D1/cyclin-dependent kinase 4 interacts with filamin A and affects the migration and invasion potential of breast cancer cells. Cancer Res. 2010,70(5):2105-2114
7 Li C, Yu S, Nakamura F,et al. Binding of pro-prion to filamin A disrupts cytoskeleton and correlates with poor prognosis in pancreatic cancer.J Clin Invest. 2009,119(9):2725-2736
8 Nakamura F, Heikkinen O, Pentik?inen OT,et al.Molecular basis of filamin A-FilGAP interaction and its impairment in congenital disorders associated with filamin A mutations. PLoS One. 2009;4(3):e4928
9 Lad Y, Jiang P, Ruskamo S, et al. Structural basis of the migfilin-filamin interaction and competition with integrin beta tails.J Biol Chem. 2008, 283 (50):35154-35163
1 Wang K, Ash JF, Singer SJ.Filamin, a new high-molecular-weight protein found in smooth muscle and non-muscle cells[J]. Cell Biology, 1975, 72(11): 4483-4486
2 Huang C, Wu Z, Hujer KM, et al.Silencing of filamin A gene expression inhibits Ca2+-sensing receptor signaling [J]. FEBS Letters, 2006, 580(7): 1795-1800.
3 Feng Y, Walsh CA.The many faces of filamin: A versatile molecular scaffold for cell motility and signaling [J]. Nature Cell Biology.2004, 6(11):1034-1038.
4 Bachmann AS, Howard JP, Vogel CW. Actin-binding protein filamin A is displayed on the surface of human neuroblastoma cells [J]. Cancer Sci, 2006 ,97(12) 1359-1365
5 Zhou X, Borén J, Akyürek LM. Filamins in cardiovascular development [J].Trends Cardiovasc Med, 2007,17(7):222-229
6 Loy CJ, Sim KS, Yong EL.Filamin-A fragment localizes to the nucleus to regulate androgen receptor and coactivator functions[J]. PNAS ,2003, 100(8):4562-4567
7 Nakamura F, Osborn TM, Hartemink CA. Structural basis of filamin A functions [J]. J Cell Biol, 2007,179(5):1011-1025
8 Kolahi KS, Mofrad MR. Molecular mechanics of filamin's rod domain[J]. Biophys J, 2008,94(3):1075-1083
9 Hj?lm G, MacLeod RJ, Kifor O, et al. Filamin-A Binds to the Carboxyl- terminal Tail of the Calcium-sensing Receptor, an Interaction That Participates in CaR-mediated Activation of Mitogen-activated Protein Kinase[J].J Biol Chem, 2001,276(37): 34880-34887
10 Fiori J L, Zhu T N, O’Connell M P, et al. Filamin A modulates kinase activation and intracellular trafficking of epidermal growth factor receptors in human melanoma cell lines[J]. Endocrinology, 2009, 150(6):2551-2560
11 Uribe R, Jay D. A review of actin binding proteins: new perspectives[J]. Mol Biol Rep,2009,36(1):121-125
12 Lu H, Guo X, Meng X, et al. The BRCA2-interacting Protein BCCIP Functions in RAD51 and BRCA2 Focus Formation and Homologous Recombinational Repair[J].Molecular and Cellular Biology, 2005, 25 (5): 1949-1957
13 Meng X, Yuan Y, Maestas A,et al. Recovery from DNA Damage-induced G2 Arrest Requires Actinbinding Protein FilaminA/Actin-binding Protein 280[J].The Journal of Biological Chemistry, 2004,279 (7): 6098-6105
14 Browne KA, Johnstone RW, Jans DA,et al. Filamin (280-kDa Actin- binding Protein) Is a Caspase Substrate and Is Also Cleaved Directly by the Cytotoxic T Lymphocyte Protease Granzyme B during Apoptosis[J]. The Journal of Biological Chemistry, 2000,275 (50): 39262- 39266
15 Conway RE, Petrovic N, Li Z,et al.Prostate-specific membrane antigen regulates angiogenesis by modulating integrin signal transduction[J]. Mol Cell Biol, 2006,26(14): 5310-5324
16 Ghosh A, Heston WD. Heston.Tumor Target Prostate Specific Membrane Antigen (PSMA) and its Regulation in Prostate Cancer.Journal of Cellular Biochemistry 2004,91:528-539
17 Hayashi K, Altman A.Filamin A is Required for T Cell Activation Mediated by Protein Kinase C-theta[J].The Journal of Immunology.2006, 177(3):1721-1728
18 Tavano R, Contento RL, Baranda SJ,et al. CD28 interaction with filaminA controls lipid raft accumulation at the T-cell immunological synapse [J]. Nat Cell Biol, 2006,8(11):1270-1276
19 Kim EJ, Park JS, Um SJ. Filamin A negatively regulates the transcrip- tional activity of p73alpha in the cytoplasm [J]. Biochem Biophys Res Commun, 2007, 362(4):1101-1106
20 Johnson GL, Nakamura K.The c-Jun Kinase/Stress-activated Pathway: Regulation, Function and Role in Human disease[J].Biochim Biophys Acta, 2007,1773(8):1341-1348
21 Leonardi A, Ellinger-Ziegelbauer H, Franzoso G,et al. Physical and Functional Interaction of Filamin (Actin-binding Protein-280) and Tumor Necrosis Factor Receptor-associated Factor 2[J]. J Biol Chem, 2000, 275 (1): 271-278
22 Jiménez-Baranda S, Gómez-Moutón C, Rojas A,et al .Filamin-A regulates actin-dependent clustering of HIV receptor s [J]. Nat Cell Biol, 2007, 9(7):838-846
23 Klaile E, Müller MM, Kannicht C,et al. CEACAM1 functionally interacts with filamin A and exerts a dual role in the regulation of cell migration [J].Journal of Cell Science, 2005, 118 (23):5513-5524
24 McDonough WS, Tran NL, Berens ME. Regulation of Glioma Cell Migration by Serine- Phosphorylated P311[J].Neoplasia,2005,7(9): 862- 872
25 Keshamouni VG, Michailidis G, Grasso CS,et al. Differential Protein Expression Profiling by iTRAQ-2DLC-MS/MS of Lung Cancer Cells Undergoing Epithelial-Mesenchymal Transition Reveals a Migratory/ Invasive Phenotype[J]. Journal of Proteome Research, 2006, 5(5): 1143- 1154
26 Ohta Y, Hartwig JH, Stossel TP. FilGAP, a Rho- and ROCK-regulated GAP for Rac binds filamin A to control actin remodeling [J].Nature Cell Biology, 2006,8(8):803-814
27 Yulia Shifrin, Pamela D. Arora, et al. The Role of FilGAP-Filamin AInter- actions in Mechanoprotection. Molecular Biology of the Cell, 2009, 20: 1269-1279
28 Kim H, Sengupta A, Glogauer M,et al. Filamin A regulates cell spreading and survival via beta1 integrins. Experimental Cell Research [J].2008, 314(4):834-846
29 Zhu TN, He HJ, Kole S,et al. Filamin A-mediated down-regulation of the exchange factor Ras-GRF1 correlates with decreased matrix metallo- proteinase-9 expression in human melanoma cells[J].J Biol Chem, 2007, 282(20):14816-14826
30 Michael P. O’Connell, Jennifer L, et al. Wnt5A Activates the Calpain- Mediated Cleavage of Filamin A.J Invest Dermatol. 2009, 129(7): 1782- 1789
31 Michael Maceyka, ergio E, Alvarez,et al. Filamin A Links Sphingosine Kinase 1 and Sphingosine-1-Phosphate Receptor 1 at Lamellipodia To Orchestrate Cell Migration. MOLECULAR AND CELLULAR BIOLOGY, 2008, 28(18): 5687-5697
32 Keshamouni VG, Michailidis G, Grasso CS,et al. Differential protein expression profiling by iTRAQ-2DLC-MS/MS of lung cancer cells undergoing epithelial-mesenchymal transition reveals a migratory/ invasive phenotype. J Proteome Res. 2006,5(5):1143-1154
33 Ravid D, Chuderland D, Landsman L,et al.Filamin A is a novel caveolin-1 -dependent target in IGF-I-stimulated cancer cell migration. Exp Cell Res. 2008, 314(15):2762-2773
34 Ricardo Uribe .David Jay. A review of actin binding proteins: new perspectives. Mol Biol Rep, 2009, 36:121-125
35 Rajasekaran AK, Anilkumar G, Christiansen JJ. Is prostate-specific membrane antigen a multifunctional protein? Am J Physiol Cell Physiol. 2005,288(5):C975-981
36 Howard Cukier, Yun Li,Jonathan M,et al. Cyclin B1/Cdk1 binds and phosphorylates Filamin A and regulates its ability to cross-link actinq. FEBS Letters, 2007, 581:1661-1672
1曹亚.肿瘤分子生物学研究进展.国外医学·生理、病理科学与临床分册, 2005,25(1):1-4
2严景华,叶棋浓,黄翠芬.乳腺癌易感基因BRCA1的研究进展.遗传HEREDITAS(Beijing)2004,26(3):367-372
3 Tripathi MK, Misra S, Khedkar SV,et al.Regulation of BRCA2 gene expression by the SLUG repressor protein in human breast cells. J BiolChem. 2005,280(17):17163-17171
4 Bernard-Gallon DJ, Satih S,et al.Phytoestrogens regulate the expression of genes involved in different biological processes in BRCA2 knocked down MCF-7, MDA-MB-231 and MCF-10a cell lines. Oncol Rep. 2010, 23(3): 647-653
5 Chiappetta G, Ferraro A, Botti G, et al. FRA-1 protein overexpression is a feature of hyperplastic and neoplastic breast disorders. BMC Cancer 2007,25(7):17
6 Weber F, Shen L, Fukino K,et al. Total-Genome Analysis of BRCA1/2- Related Invasive Carcinomas of the Breast Identifies Tumor Stroma as Potential Landscaper for Neoplastic Initiation. The American Journal of Human Genetics,2006 June,78:961-972
7 White MF. Insulin signaling in health and disease. Science 2003, Dec 5;302(5651):1710-1711
8 Alper O, Stetler-Stevenson WG, Harris LN,et al. Novel anti-filaminA antibody detects a secreted variant of filamin-A in plasma from patients with breast carcinoma and high-grade astrocytoma. Cancer Sci. 2009, 100 (9):1748-1756
9 Zhang M, Breitwieser GE.High affinity interaction with filamin A protects against calcium-sensing receptor degradation. J Biol Chem. 2005,280 (12): 11140-11146
10 Zhong Z, Yeow WS, Zou C, et al.Cyclin d1/cyclin-dependent kinase 4 interacts with filamin a and affects the migration and invasion potential of breast cancer cells. Cancer Res. 2010, 70(5):2105-:2114
11 Conway K, Parrish E, Edmiston SN, et al.Risk factors for breast cancer characterized by the estrogen receptor alpha A908G (K303R) mutation. Breast Cancer, Research,2007,9(3):R36
12 Li H, Weinstein IB.Protein kinase C beta enhances growth and expression of cyclin D1 in human breast cancer cells. Cancer Res.2006, 66(23): 11399-11408
13 Siriwardana G, Bradford A, Coy D,et al.Autocrine/paracrine regulation ofbreast cancer cell proliferation by growth hormone releasing hormone via Ras, Raf, and mitogen-activated protein kinase. Mol Endocrinol. 2006 , 20(9):2010-2019
14 Malkas LH, Herbert BS, Abdel-Aziz W,et al. A cancer- associated PCNA expressed in breast cancer has implications as a potential biomarker. PNAS,2006,103(51):19472-19477
15 Schultz-Norton JR, Gabisi VA, Ziegler YS, et al.Interaction of estrogen receptor alpha with proliferating cell nuclear antigen. Nucleic Acids Research,2007,35(15):5028-5038
16 Beresford MJ, Wilson GD, Makris A.Measuring proliferation in breast cancer:practicalities and applications.Breast Cancer Research,2006,8(6): 216-316
17 Zaczek A, Brandt B, Bielawski KP.The diverse signaling network of EGFR, HER2, HER3 and HER4 tyrosine kinase receptors and the consequences for therapeutic approaches. Histol Histopathol. 2005 Jul;20(3):1005-1015
18 Kaya H, Erbarut I, Ozkan N,et al.Immunoexpression of HER family, neuregulin, MAPK and AKT in invasive ductal carcinomas of the breast. Eur J Gynaecol Oncol. 2008;29(4):350-356
19 Magkou C, Nakopoulou L, Zoubouli C,et al.Expression of the epidermal growth factor receptor (EGFR) and the phosphorylated EGFR in invasive breast carcinomas. Breast Cancer Res. 2008;10(3):R49.
20 Park SS, Kim JE, Kim YA,et alCaveolin-1 is down-regulated and inversely correlated with HER2 and EGFR expression status in invasive ductal carcinoma of the breast. Histopathology. 2005 ,47(6):625-630
21 Han F, Zhu HG.Over-expression of caveolin-1 inhibits proliferation and invasion of pancreatic carcinoma cells in vitro. Zhonghua Zhong Liu Za Zhi, 2009,31(10):732-737
22 Malik FA, Sanders AJ, Kayani MA,et al. Effect of expressional alteration of KAI1 on breast cancer cell growth, adhesion, migration and invasion. Cancer Genomics Proteomics. 2009 ,6(4):205-13
23 Crawford NP,Qian X, Ziogas A, et al. Rrp1b, a New Candidate Susceptibi- lity Gene for Breast Cancer progression and Metastasis. PLOS Genet, 2007,3(11):2296-2311
24 Grunewald TG, Kammerer U,Kapp M,et al. Nuclear localization and cytosolic overexpression of LASP-1 correlates with tumor size and nodal-positivity of human breast carcinoma.BMC Cancer 2007,7:198
25 Chang Y, Zuka M, Perez-Pinera P,etal.Secretion of pleiotrophin stimulates breast cancer progression through remodeling of the tumor micro environment. PNAS,2007,104(26):10888-10893
26 Fujita M, Khazenzon NM, Bose S,et al.Overexpression ofβ1- chain- containing laminins in capillary basement membranes of human breast cancer and its metastases. Breast Cancer Research,2005, 7(4): 411- 421
27 Dong M, How T, Kirkbride KC, et al. The typeⅢTGF-βreceptor suppresses breast cancer progression.The Journal of Clinical Investigation, 2007, 117(1):206-217
28 Yi CH, Smith DJ, West WW, et al. Loss of Fibulin-2 Expression Is Associated with Breast Cancer Progression. The American Journal of Pathology, 2007,170(5):1535-1545
29 Yang S, Du J, Wang Z, et al.BMP-6 promotes E-cadherin expression through repressing delta EF1 in breast cancer cells. BMC Cancer, 2007, 7:211
30 Draffin JE, McFarlane S, Hill A, et al.CD44 potentiates the adherence of metastatic prostate and breast cancer cells to bone marrow endothelial cells. Cancer Res. 2004,64(16):5702-5711
31 Zhou X, Liu Y, You J, Myosin light-chain kinase contributes to the proliferation and migration of breast cancer cells through cross-talk with activated ERK1/2. Cancer Lett. 2008,270(2):312-327
32王震,刘乾,陈琦,等.NDRG1基因与乳腺癌转移的关系及其转染对乳腺癌细胞株增殖及侵袭力的影响.中华病理学杂志,2006,35(6):333- 338.
33柴伟,张国建,张云昌,等. NDRG-1、WWOX及P53基因在胃癌组织中的表达及临床意义.昆明医学院学报2009,30(5):37-42
34柴丽,石林祥,房林,等.KiSS-1基因及其受体在乳腺癌中的表达及临床意义.中华实验外科杂志,2005,22(9):1068-1070
35 SerajM J,SamantR S,VerderameM F,et al.Functi onal evidence for a novel human breast carcinomametastasis suppressor, BRMS1, encoded at chromos ome 11q13 [J]. Cancer Res,2000,60 (11):2764
36 CicekM, Samant R S, KinterM, et al. Identficati on of metastasisass ociated proteins through protein analysis ofmetastaticMDA-MB-435 cells [ J ]. Clin Exp Metastasis,2004, 21(2):149
37 Lee TH, Seng S, Sekine M, et al.Vascular Endothelial Growth Factor Mediates Intracrine Survival in Human Breast Carcinoma Cells through Internally Expressed VEGFR1/FLT1.PLoS Medicine, 2007,4(6): 1101- 1116.
38 Wang J, Barnes RO, West NR, Jab1 is a target of EGFR signaling in ER alpha-negative breast cancer. Breast Cancer Res. 2008; 10(3):R51
39 King JA, Ofori-Acquah SF, Stevens T, et al. Activated leukocyte cell adhesion molecule in breast cancer:prognostic indicator.Breast Cancer Res,2004,6(5):R478-R487
40 Fabre-Lafay S,Monville F,Garrido-Urbani S, et al.Nectin-4 is a new histo- logical and serological tumor associated marker for breast cancer. BMC Cancer, 2007,7:73
41阎志胜,刘建民.转化生长因子β1和细胞周期蛋白E在肾透明细胞癌中的表达和意义.山西医药杂志(J),2009 ,38(5):387-389
42 He Wenshan,HUANG Tao,WANG Haijiu.Expression of Cyclin E and Its Relationship with the Prognosis of Patients with Breast Carcinoma.The Chinese German Journal Clinical Oncology,2006,5(4):275-278